CN101665801B - Method for enhancing reverse resistance of microorganisms - Google Patents

Abstract

The invention discloses a method for regulating and controlling the metabolism of microorganisms and enhancing the reverse resistance of the microorganisms. In the method provided by the invention, poly-hydroxy fatty acid ester is synthesized in the microorganisms by introducing the relevant gene of a synthesis approach of the poly-hydroxy fatty acid ester into the microorganisms. By applying the method into various microbial production bacteria, the metabolism approach of the production bacteria can be effectively influenced, the reverse resistance of the bacteria is increased, and the production efficiency of biological engineering products comprising hyaluronic acid, pyruvic acid, ergosterine, beer, carnine, elastolytic enzymes, carotenoid, glutamine, lysine, phenylalanine, gluconic acid, amylase, alcohol, glycerol, twelve-carbon dibasic acid, fifteen-carbon dibasic acid, entobakterin powder, vernine, glutathione, erythrol, D-ribose, 1,3-propylene glycol and the like is enhanced.

Description

A method of improving microbial stress resistance

This application is a divisional application with the application number 200510068113.6, the application date is April 26, 2005, and the invention name is "A Method for Regulating Microbial Metabolism and Improving Microbial Stress Resistance". the

technical field

The invention relates to a method for regulating microbial metabolism and improving microbial stress resistance in the field of biotechnology.

Background technique

Polyhydroxyalkanoate (Polyhydroxyalkanoate or PHA) is a kind of intracellular polyester that can be synthesized by many types of bacteria. It exists mainly as a storage material of intracellular carbon source and energy in organisms (Lemoigne M.Products of dehydration and of polymerization of -hydroxybutyric acid. Bull. Soc. Chem. Biol., 1926, 8: 770-782). The general structural formula of PHA is as follows:

(Formula I)

Where n can be 1, 2, 3 or 4, when n=1, it means 3-hydroxy fatty acid ester; m is the degree of polymerization; R is a variable side chain group, such as saturated or unsaturated, straight chain or side chain Alkyl chains of different chain lengths and substituents.

According to the length of the PHA side chain, PHA can be divided into three types:

(1) Short-chain PHA (short-chain-length PHA, scl PHA), such as polyhydroxybutyrate (polyhydroxybutyrate, PHB), a copolymer of hydroxybutyrate and hydroxyvalerate [poly(hydroxybutyrate-co-hydroxyvalerate ), PHBV], the monomer contains 3-5 carbon atoms.

(2) Medium-chain-length PHA (mcl PHA), such as polyhydroxyhexanoate (PHHx) and polyhydroxyoctanoate (PHO), the monomer contains more than 6 carbon atoms. Multiple functional groups can be contained in mcl PHA, such as double bonds (Huigberts GNM, Eggink G, Waard P, Huisman GW and Witholt B. Pseudomonas putitaKT2442cultivated on glucose accumulates poly-β-hydroxyalkanoates consisting of saturated and unsaturated. ., 1992, 58: 536-544), triple bond, halogen (Doi Y and Abe C. Biosynthesis and characterization ofa new bacterial copolyester of 3-hydroxyalkanoates and 3-hydroxy-w-chloroalkanoates. Macromolecules, 1990, 23: 2705-3707 ), phenol (FritzscheK, Lenz RW and Fuller RC. An unusual bacterial polyesters with a phenyl pendant group. Macromol Chem, 1990, 191: 1957-1968) and cyanide (Schulz C, Wolk S, Lenz RW and Fuller RC. Growth and tionpolyester produce by Pseudomonas oleovorans on branchedoctanoic acid substrates. Macromolecules.1995, 27:6358-6362) and so on. Most mcl PHAs are random copolymers of two or more monomers.

(3) PHAs containing short-chain and medium-long-chain monomers are mostly random copolymers of two or more monomers (LageveenRG, Huisman GW, Preusting H, Ketelaar H, Eggink G and Witholt B. Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl. En viron. Microbiol, 1988, 54: 2924-2932).

PHA is of great significance for bacteria to adapt to changes in the external environment and survive. Taking PHB as an example, PHB in some bacterial cells can slow down the degradation of important intracellular components-RNA and protein under starvation conditions, thereby increasing the resistance of bacteria to survival adversity. In addition, in some Bacillus species, although PHB is not required for spore formation, PHB can be used as an energy and carbon source during spore formation to increase the chances of survival of these bacteria. In addition, PHB can also provide the necessary carbon and energy sources for the cyst formation of certain nitrogen-fixing bacteria. Studies on the nitrogen fixation of Rhizobium sp.ORS 571 and the formation and degradation of PHB have shown that when the oxygen concentration in nodules increases, PHB can protect nitrogenase from damage through its own degradation (Anderson AJ and Dawes EA.Occurense, Metabolism , Metabolic Role, and Industrial Use of Bacterial Polyhydroxyalkanoates. Microb. Rev., 1990, 54: 450-472).

PHA can also serve as a marker for the environment. Studies on microorganisms in river bay sediments have shown that by comparing the synthesis ratios of phospholipids and PHA, the degree of influence of external factors on sediments can be monitored. Moreover, many bacteria that can synthesize PHA are screened out in activated sludge and an environment rich in carbon source organic matter. Studies on psychrophilic marine microorganisms isolated from places severely polluted by crude oil have shown that most of them can accumulate PHA in cells under nitrogen-limited conditions (Alvarez HM, Pucci OH and Steinbüchel A. Lipid storage compounds in marine bacteria. Appl. Microbiol. Biotechnol., 1997, 47: 132-139). This shows that most bacteria in these carbon-rich environments have the ability to convert excess carbon sources into PHA for storage.

PHB is a carbon source and energy reserve in bacterial cells. But it has recently been found to be present in the membranes of prokaryotes and eukaryotes as well. In prokaryotes, it exists in the plasma membrane; in eukaryotes, the ratio is the largest in mitochondria and microsomal membranes. Compared with the high-molecular-weight polymers in the cell, the PHB molecules present on the membrane are small, only 100-200 monomers. Studies of this small molecular weight PHB suggest that it may function as an ion channel in the membrane. Recently, a relatively large amount of this small molecule PHB has also been found in human blood cells. This provides a theoretical basis for the use of PHB to encapsulate drugs for directional quantitative release and the application of PHA in medical treatment.

As an ecological biopolymer material, PHA can not only be promoted and applied in the plastics industry, and become a new type of biodegradable plastic, but also because of its special properties, such as biocompatibility, optical activity, piezoelectric properties, gas barrier properties, etc., and many other properties that have not yet been discovered, which may be applied in some high value-added fields. The research on PHA has become an application field in which basic theory and practical application are closely combined.

The synthesis pathways of different types of PHA in bacteria are different. Currently, there are three known synthesis pathways (Anderson AJ and Dawes EA. Occurense, Metabolism, Metabolic Role, and Industrial Use of Bacterial Polyhydroxyalkanoates. Microb. Rev., 1990, 54: 450-472; Lee SY. Bacterial Polyhydroxyalkanoates. Biotech. Bioeng. 1996, 49: 1-14):

1. Take Wautersia eutropha (formerly called Ralstonia eutropha, also called Alcaligeneseutrophus) as a representative to directly synthesize PHB pathway by acetyl-CoA: its PHB synthetase system is encoded by pha synthesis operon phbCAB (SEQ ID NO: 1) gene, Contains three enzymes: β-ketothiolase (β-ketothiolase), NADPH-dependent acetoacetyl-CoA reductase (acetoacyl-CoA reductase) and PHB synthase (PHBsynthase); It is encoded by the phbC gene and is located on the same operon. The intracellular metabolic product acetyl-CoA is catalyzed by β-ketothiolase (PhaA) to form acetoacetyl-CoA, and then reduced to ( R)-hydroxybutyryl-CoA, (R)-hydroxybutyryl-CoA is polymerized into PHB under the action of PHB synthetase. This process needs to consume NADPH and produce NADP.

2. Fatty acid de novo synthesis pathway represented by Pseudomonas aeruginosa: the synthesis of PHA from this pathway requires the catalysis of two enzymes: 3-hydroxyacyl-acyl transfer protein coenzyme A transferase (3-Hydroxyacyl-ACP-CoA transferase) encoded by phaG ) and PHA synthase encoded by phaC. The intermediate product 3-hydroxyacyl-acyl transfer protein in the fatty acid de novo synthesis pathway is catalyzed by 3-hydroxyacyl-acyl transfer protein coenzyme A transferase (PhaG) to form the precursor 3-hydroxyacyl-CoA of PHA, which is then catalyzed by PHA synthase polymerized into PHA. The same pathway has the synthetic pathway of Pseudomonas putita, and the Genbank accession number of the gene encoding PhaG of Pseudomonas putita is AF052507.

3. The β-oxidation pathway represented by Pseudomonas oleovorans: the synthesis of PHA from this pathway requires the action of two enzymes: (R)-enoyl-hydratase ((R)-enoyl-CoA hydratase) encoded by phaJ and phaC Encoded PHA synthase. The intermediate product of fatty acid β-oxidation, enoyl-acyl transfer protein, is catalyzed by (R)-enoyl-hydratase (PhaJ) to form the precursor of PHA, 3-hydroxyacyl-CoA, which is then polymerized into PHA by PHA polymerase. The same pathway has the synthetic pathway of Aeromonas hydrophila (Aeromonas hydrophila), and the Genbank accession number of the PHA synthase operon (PHA synthase operon) encoding gene of Aeromonas hydrophila is AY093685, including PhaJ and the gene encoding PhaC.

The study of fatty acid synthesis in Pseudomonas putida by ¹³ C-NMR showed that de novo fatty acid synthesis and β-oxidation are independent of PHA synthesis (Huijberts GN, De Rijk TC, De Waard P and Eggink G..13 C nuclear magnetic resonance studies of Pseudomonas putidas fatty acids metabolic routes involved i

In the metabolic pathway of PHA, there are both energy metabolism (NAD/NADH, NADP/NADPH, etc.) The process of changing matter. Since PHA can widely exist in various cells, this also provides a basis for using the PHA synthesis mechanism as a regulatory means to regulate its energy and material levels in various microorganisms, thereby affecting metabolites.

Contents of the invention

One object of the present invention is to provide a method for regulating the metabolism of microorganisms.

The method for regulating microbial metabolism provided by the present invention is to synthesize polyhydroxyalkanoate in microorganisms; the synthesis of said polyhydroxyalkanoate is achieved by introducing related genes of polyhydroxyalkanoate synthesis pathway in said microorganism Achieved.

The metabolism includes energy and substance metabolism.

The relevant genes of the polyhydroxyalkanoate synthesis pathway are selected from any one of the following 1), 2) and 3):

1) phbCAB gene, 2) phaG and phaC genes, 3) phaJ and phaC genes.

The phbCAB gene preferably has the DNA sequence of SEQ ID NO: 1 in the sequence listing; the PhaG gene is preferably AF052507, the phaC gene is preferably from AY093685, and the phaJ gene is preferably from AY093685.

The relevant genes of the PHA synthesis pathway can be introduced into host microorganisms by conventional means in the art, for example, expression vectors such as plasmids, cosmids, phages, etc. can be utilized to be introduced into host microorganisms by means of transformation, transduction, conjugation, etc.; Protoplast fusion and other means to achieve.

The regulation of microbial metabolism is the regulation of the ability of microorganisms to produce fermentation products.

The microorganisms are bacteria, archaea and fungi; the fermentation products include organic acids, alcohols, antibiotics, amino acids and vitamins.

Wherein, the microorganism can be Streptococcus zooepidemicus (Streptococcus zooepidemicus), preferably Streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC 39920, and the fermentation product is lactic acid and hyaluronic acid;

The microorganism can be Torulopsis glabrata, preferably Torulopsis glabrata CCTCCM202019, and the fermentation product is pyruvate;

The microorganism can be Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae IFFI 01338, and the fermentation product is ergosterol;

The microorganism can be Bacillus subtilis (Bacillus subtilis), preferably Bacillus subtilis (Bacillus subtilis) ATCC 19162, and the fermentation product is inosine;

The microorganism can be Bacillus alcalophilus, preferably Bacillus alcalophilus Ya-B, and the fermentation product is elastase;

The microorganism can be Rhodotorula glutinis, preferably Rhodotorula glutinis NCIM 3353, and the fermentation product is carotenoid;

The microorganism can be Corynebacterium glutamicum (Corynebacterium glutamicum), preferably Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032, and the fermentation product is glutamine;

The microorganism may be Brevibacterium lactofermentum, preferably Brevibacterium lactofermentum ATCC 31269, and the fermentation product is lysine;

The microorganism can be Escherichia coli (Escherichia.coli), preferably Escherichia coli (Escherichia.coli) ATCC 31882, and the fermentation product is phenylalanine;

The microorganism can be Pseudomonas fluorescens (Pseudomonas fluorescens), preferably Pseudomonas fluorescens AS 1.55, and the fermentation product is gluconic acid;

The microorganism can be Bacillus subtilis (Bacillus subtilis), preferably Bacillus subtilis ATCC 21556, and the fermentation product is α-amylase;

The microorganism can be Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae ACCC 2063, and the fermentation product is ethanol;

The microorganism can be Candida krusei (Candida krusei), preferably Candida krusei (Candida krusei) ACCC 2196, and the fermentation product is glycerol;

The microorganism can be Candida tropicalis (Candida tropicalis), preferably Candida tropicalis (Candida tropicalis) UH22248, and the fermentation product is dodecanedioic acid;

The microorganism may be Candida tropicalis, preferably Candida tropicalis T _25-14 , and the fermentation product is pentadecanedioic acid;

The microorganism can be Bacillus subtilis (Bacillus subtilis), preferably Bacillus subtilis ATCC 19220, and the fermentation product is guanosine;

The microorganism can be Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae KY6186, and the fermentation product is glutathione;

The microorganism can be ToruLopsis sp, preferably ToruLopsis sp B845, and the fermentation product is erythritol;

The microorganism can be bacillus pumilus (Bacillus pumilus), preferably bacillus pumilus (Bacillus pumilus) ATCC 31095, and the fermentation product is D-ribose;

The microorganism can be Clostridium butyricum (Clostridium butyricum), preferably Clostridium butyricum (Clostridium butyricum) ATCC 8260, and the fermentation product is 1,3-propanediol;

The microorganism can be Pseudomonas fluorescens (Pseudomonas fluorescens), preferably Pseudomonas fluorescens AS 1.55, and the fermentation product is gluconic acid;

The microorganism can be Bacillus subtilis (Bacillus subtilis), preferably Bacillus subtilis (Bacillus subtilis) ATCC 19162, and the fermentation product is inosine;

The microorganism can be Zymomonas mobilis (Zymomonas mobilis), preferably Zymomonas mobilis NRRL B-4286, and the fermentation product is ethanol.

Another object of the present invention is to provide a method for improving the stress resistance of microorganisms.

The method for improving the stress resistance of microorganisms provided by the present invention is to synthesize polyhydroxyalkanoate in microorganisms; the synthesis of said polyhydroxyalkanoate is by introducing the relevant pathway of polyhydroxyalkanoate synthesis in said microorganisms genetically accomplished.

The relevant genes of the polyhydroxyalkanoate synthesis pathway are selected from any one of the following 1), 2) and 3): 1) phbCAB gene, 2) phaG and phaC genes, 3) phaJ and phaC genes.

The phbCAB gene preferably has the DNA sequence of SEQ ID NO: 1 in the sequence listing; the PhaG gene is preferably AF052507, the phaC gene is preferably from AY093685, and the phaJ gene is preferably from AY093685.

Wherein, in the method, the microorganism can be Saccharomyces cerevisiae, preferably Saccharomyces cerevisiae ACCC 2063, and the adversity stress is cold stress or heat stress;

The microorganism can be Escherichia coli (Escherichia coli), preferably Escherichia coli (Escherichia coli) JM109, and the adversity stress is heavy metal ion stress; the heavy metal ion can be Hg ²⁺ ;

The microorganism is Zymomonas mobilis (Zymomonas mobilis), preferably Zymomonas mobilis NRRL B-4286, and the adversity stress is low pH stress or high osmotic pressure stress;

The microorganism can be Lactobacillus acidophilus (Lactobacillus acidophilus), preferably Lactobacillus acidophilus (Lactobacillus acidophilus) ATCC 53671, and the adversity stress is low pH stress;

The microorganism can be bacillus, preferably Bacillus thuringiensis (Bacillus thuringiensis), especially preferably Bacillus thuringiensis (Bacillus thuringiensis) ACCC 10068;

The microorganism can be a bacillus, preferably a bacillus (Bacillus sp) L-23; the adversity stress is crude oil stress.

In the invention, the metabolism of the microorganism itself is regulated by introducing the PHA synthesis gene, and the resistance to adversity stress is improved.

The PHA synthesis mechanism includes the PHB pathway directly synthesized by acetyl-CoA represented by Wautersia eutropha and Rhodospirillum rubrum; the de novo synthesis pathway of fatty acid represented by Pseudomonas aeruginasa, the fatty acid β-oxidation pathway represented by Pseudomonas oleovorans, etc.

In the method for regulating microbial metabolism and improving microbial stress resistance of the present invention, the related gene of PHA synthesis can be cloned into the plasmid vector for the target bacteria, and the constructed plasmid can be introduced into the target bacteria to obtain recombinant bacteria, and fermented and recombined Bacteria to obtain desired products or improve stress resistance. The genes related to PHA synthesis can be on the plasmid or on the chromosome.

In order to improve the transformation efficiency, the method of introducing the recombinant plasmid expression vector into the target bacteria is preferably the electroporation method, but other commonly used methods in the field of bioengineering can also be used, including heat shock method, protoplast transformation method and conjugation transformation method.

The present invention adjusts the intracellular NAD(P)/NAD(P)H metabolism and the flow direction of intermediate metabolites such as acetyl-CoA and pyruvate by introducing the PHA synthesis pathway into the microbial recombinant production bacteria to obtain the desired fermentation product An adjustable synthetic environment is provided, and the accumulation of PHA can improve the tolerance of the recombinant production bacteria to harsh environments, especially the feedback inhibition of the product, which is also conducive to improving the fermentation yield.

The present invention uses the PHA synthesis pathway to regulate microbial metabolism, improve the production efficiency of microbial fermentation products, and further improve the stress resistance of microorganisms. The method provided by the invention is to construct a plasmid containing a gene related to PHA synthesis, and introduce it into a corresponding bacterial strain for expression. The bacterial strains can be various microbial producers. Applying the method of the present invention to various microbial production bacteria can effectively affect the metabolic pathways of the production bacteria, increase the stress resistance of the bacteria, and improve Protease, Carotenoids, Glutamine, Lysine, Phenylalanine, Gluconic Acid, Amylase, Alcohol, Glycerin, Dodecanedioic Acid, Pentadecanedioic Acid, Bacillus thuringiensis Powder, Guanosine, Production efficiency of glutathione, erythritol, D-ribose, 1,3-propanediol and other bioengineered products.

Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements in form and details, and these are all within the protection scope of the present invention, such as using The nucleic acid molecules obtained by the principle of sub-degeneracy or the principle of base complementarity are basically the same as the related gene functions of the polyhydroxyalkanoate synthesis pathway of the present invention. Amino acid insertions and/or deletions and/or substitutions at the sites of action can result in genes encoding proteins or polypeptides with substantially the same function to achieve the purpose of the present invention and are considered to fall within the protection scope of the present invention.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are all mass percentages unless otherwise specified.

The term "PHA" used in the present invention refers to polyhydroxyalkanoate synthesized by microorganisms, its structural general formula is shown in formula (I), and the PHA molecular formula included is such as polyhydroxybutyric acid (PHB), 3-hydroxy Copolymers of butyric acid and 3-hydroxyvaleric acid PHBV, copolymers of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid PHBHHx and medium- and long-chain monomers such as 3-hydroxyhexanoic acid HHx to 3-hydroxydodecanoic acid 3- Monopolymer or copolymer of HDD,.

The term "related genes of the PHA synthesis pathway" used in the present invention refers to, in the PHA synthesis pathway, the genes corresponding to various related synthetases and regulatory proteins that have participated in the entire synthesis process of PHA; for example, the PHA synthase gene phaC , 3-hydroxyacyl-acyl transfer protein coenzyme A transferase gene phaG, (R)-enoyl-hydratase gene phaJ gene, etc.

The term "stress resistance" used in the present invention refers to the ability of microorganisms to survive in an environment that deviates from its optimal growth conditions, such as higher or lower temperature, higher or lower pH, higher or lower Lower osmotic pressure, higher feedback inhibition, presence of toxic substances, etc.

The term "microorganism" used in the present invention includes bacteria, archaea, fungi (such as yeast) and the like.

The term "fermentation product" used in the present invention refers to all products obtained through microbial fermentation or transformation, such as organic acids, alcohols, other alcohols, antibiotics, amino acids, vitamins, etc.

Embodiment 1, expressing the phbCAB gene of Wautersia eutropha in Streptococcus zooepidemicus affects the production of lactic acid and hyaluronic acid

Bacteria: Streptococcus zooepidemicus ATCC 39920

1) Under standard polymerase chain reaction conditions, pBHR68 plasmid (Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbüchel A.A sensitive, viable- colony staining method using Nile red for direct screening of bacteria that accumulatepolyhydroxyalka

2) Prepare competent cells of Streptococcus zooepidemicus, and transfer the plasmid pEUHBNOI into Streptococcus zooepidemicus by electroporation.

Preparation of Competent Cells:

Cultivate Streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC 39920 in a medium containing THYB (Todd-Hewitt broth) (purchased from OXOID) 36.4g/L and yeast extract 0.5% for 12h; then inoculate it into 50mL fresh THYB In the medium, the D530 after inoculation should not exceed 0.05, and it is enough to cultivate until OD530=0.25; and add hyaluronidase 0.4 mg/mL half an hour before the end of the culture;

4°C, 8000g, centrifuge for 10 minutes, discard the supernatant, and resuspend the cells with 20mL 0.5mmol/L sucrose solution;

4°C, 8000g, centrifuge for 10 minutes, discard the supernatant, resuspend the cells with 1mL 0.5mmol/L sucrose solution, centrifuge again, discard the supernatant; resuspend the cells in 250μL 0.5mmol/L sucrose solution containing 10% glycerol , according to the volume used, packed into Eppendorf tubes; quickly stored at -80 ℃.

Electroporation of Streptococcus zooepidemicus

Add 500-5000ng of plasmid pEUHBNOI to 200μL of competent cells, and ice-bath for 10 minutes; transfer the mixed system to a 2mm electric shock cup for electric shock, and set the voltage to 2.5kv;

Use 1mL of ice-cold THYB to transfer the mixed system to 10mL of THYB, ice-bath for 30-60 minutes; then culture at 37°C for 1 hour; then at 14°C, 6000g, centrifuge for 10 minutes, resuspend the bacteria in 1mL of THYB and spread on Transformants were screened on the resistance plate to obtain recombinant Streptococcus zooepidemicus (Streptococcus zooepidemicus ATCC 39920 containing pEUHBNOI).

3) Fermentation of recombinant Streptococcus zooepidemicus

The composition of the seed medium is sucrose: 20g/L, yeast powder: 10g/L, beef extract: 10g/L, MgSO ₄ 7H ₂ O: 2g/L, MnSO ₄ 4H ₂ O: 0.1g/L, KH ₂ PO ₄ : 2g/L, trace element solution: 1mL/l, seed buffer: 40mL/l. The components of the fermentation medium are yeast powder: 20g/L, Na ₂ HPO ₄ 12H ₂ O: 6.2g/L, MgSO ₄ 7H ₂ O: 2g/L, K ₂ SO ₄ 1.3g/L, FeSO ₄ 7H ₂ O: 5mg/L, trace element solution: 2.5mL/l, sucrose: 70g/L. The trace element solution contains CaCl ₂ : 2g/L, MnSO ₄ 4H ₂ O: 24mg/L, ZnCl ₂ : 46mg/L, CuSO ₄ 5H ₂ O: 19mg/L; the seed buffer contains Na ₂ HPO ₄ . 12H ₂ O: 36.76 g/L, NaH ₂ PO ₄ ·12H 2 O: 15.98 g/L, NaHCO ₃ : 12.5 g/L.

Pick up Streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC 39920 and recombinant strains on the plate with an inoculation loop, insert them into 500mL Erlenmeyer flasks with 150mL seed medium respectively, and cultivate them on a shaker at 200rpm for 14-16 hours. The culture temperature was set at 37° C.; then the seeds were inserted into a fermenter (3 liters of liquid in a 7.5 liter fermenter (NBS Bioflo 3000, NJ, USA)) at an inoculum size of 10% (volume ratio). The temperature of the fermenter is controlled at 37°C, the pH value is 7.0, and the ventilation rate is 5L/min. The initial stirring speed is 200rpm. When the dissolved oxygen drops to 0, the speed is increased to 500rpm. When the dissolved oxygen drops to 0 again, the speed is adjusted to 650rpm, and maintained until the end of fermentation. Streptococcus zooepidemicus ATCC 39920 is fermented for 14 hours, and the Bacteria fermented for 16 hours. The contents of hyaluronic acid and lactic acid in the fermentation medium were determined respectively. The results showed that the hyaluronic acid (Hyaluronic acid, HA) and lactic acid yields of Streptococcus zooepidemicus (Streptococcus zooepidemicus) ATCC 39920 fermented for 14 hours were 5.4g/L and 65g/L, respectively. The 14-hour hyaluronic acid and lactic acid production of the recombinant bacteria were 5.6g/L and 38g/L, and the lactic acid production decreased by 42%; the 16-hour production was 7.3g/L and 41g/L, and the hyaluronic acid production increased by 34%. Lactic acid production decreased by 37%. Lactic acid synthesis is the main way for Streptococcus zooepidemicus to obtain intracellular oxidative power (NAD) under hypoxic conditions, so the bacteria produce a large amount of lactic acid during fermentation. The introduction of the PHB synthesis pathway provides cells with an exogenous NAD regeneration pathway, which can reduce the pressure on the lactic acid pathway, greatly reduce the lactic acid synthesis, and greatly reduce the lactic acid, so that more carbon sources can flow to other metabolic pathways. Thus, the production of hyaluronic acid is also improved to a certain extent.

Wherein, hyaluronic acid content assay adopts Bitter-Muir's method (Bitter T., Muri HM.A modifieduronic acid carbazol reaction.Anal.Biochem.1962,4:330-334), the measurement sample preparation method of fermented liquid is as follows: a ) Dilute 1mL of fermentation broth to 4mL accurately, and centrifuge at 6000rpm for 10 minutes; b) Take 1mL of supernatant and add it to 4mL absolute ethanol, shake, and centrifuge at 6000rpm for 5 minutes; c) Discard the supernatant, add 2mL of 0.2mmol/ LNaCl, place, shake occasionally, until finally dissolved; d) Add 4 mL of absolute ethanol, shake, 6000 rpm centrifugation for 5 minutes; e) Discard the supernatant, and dissolve the precipitate with 2 mL of deionized water as a sample for HA determination. Determination of lactate concentration: adopt HPLC (SpectroSYSTEM P2000, Thermo Separation Products, USA) method. Refractive detector, ion exchange column Aminex HPX-87H, 300mm×7.8mm, mobile phase is 0.005mmol/L sulfuric acid solution, flow rate 0.50mL/min. Before testing, take 0.2mL fermentation sample, accurately dilute to 4mL, centrifuge at 10000rpm/min for 5 minutes, remove the supernatant, and filter it with a 0.45μm filter membrane as a chromatographic sample.

Example 2, expression of phbCAB gene in Torulopsis glabrata affects pyruvate production

Strain: Torulopsis glabrata CCTCCM202019

Medium: slant and seed medium (L): glucose 20g/L, peptone 10g/L, KH ₂ PO ₄ 1g/L, MgSO ₄ 7H2O 0.15g/L, agar 20g/L (for slant), pH 5.5 .

Fermentation medium: glucose 80g/L, peptone 15g/L (nitrogen content 12%), KH ₂ PO ₄ 5g/L, KCl 5g/L, MgSO ₄ 7H2O 0.18g/L, niacin 4mg/L, hydrochloric acid Thiamine 20 μg/L, pyridoxine hydrochloride 100 μg/L, biotin 10 μg/L, riboflavin 50 μg/L, pH 5.0.

After plasmid pBHR68 was digested with BamHI and EcoRI, the obtained 5.2 kb fragment was inserted between the BamHI and EcoRI recognition sites of plasmid pPIC9K (purchased from Invitrogen) to obtain plasmid pPICHB. This plasmid was transferred into Torulopsis glabrata CCTCCM202019 by electroporation to obtain a recombinant strain (Torulopsis glabrata CCTCCM202019 containing pPICHB) for fermentation experiments.

Torulopsis glabrata (Torulopsis glabrata) CCTCCM202019 and recombinant bacteria were inoculated into the seed medium (50mL/500mL Erlenmeyer flask) cultured on a slant, respectively, and cultured at 30°C and 200rpm for 12 hours. ) inoculum size into the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, a temperature of 30°C, an aeration rate of 2L/min, a stirring speed of 700rpm, and a pH of 5.0 controlled with 5mol/L KOH. When the residual sugar was reduced to 4%, 400g/L glucose solution was fed at a certain rate to carry out fed-batch culture, and the fermentation time was 48 hours.

After 2 mL of fermentation broth was centrifuged at 8000 g for 5 minutes, the content of pyruvate in the supernatant was determined by lactate dehydrogenase method (Lamperecht W, Heinz F.In: Methods of enzymatic analysis, ed. Bergmeyer, H.U.VCH, Weinheim, 1984, 6: pp570-577). Cells were washed twice with deionized water and then dried in vacuum. The results showed that the pyruvate production of the recombinant bacteria was 68g/L, which was about 31% higher than that of Torulopsis glabrata CCTCCM202019 (Torulopsis glabrata) CCTCCM202019's 52g/L. The pathway of glycolysis to produce pyruvate consumes NAD, and the metabolism of pyruvate in cells is the process of synthesizing NAD. Therefore, a large amount of pyruvate accumulation in cells requires a high oxidative environment. The introduction of the phbCAB gene can provide cells with oxidative power, thereby increasing the production of pyruvate.

Embodiment 3, expression phbCAB gene influences ergosterol production in Saccharomyces cerevisiae

Strain: Saccharomyces cerevisiae (Saccharomyces cerevisiae) IFFI 01338 (preservation number of the Culture Center of the Institute of Microbiology, Chinese Academy of Sciences)

Slope, seed medium and fermentation medium are: glucose 40g/L, peptone 20g/L, yeast powder 10g/L, agar 20g/L (for slope), pH 5.5.

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Saccharomyces cerevisiae IFFI 01338 by lithium acetate transformation to obtain recombinant bacteria (Saccharomyces cerevisiae IFFI 01338 containing pPICHB) for fermentation experiments.

Saccharomyces cerevisiae IFFI 01338 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), cultivated at 30°C and 200rpm for 12 hours, and then inoculated with 10% (v /v) The inoculum is introduced into the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, a temperature of 30°C, a ventilation rate of 2L/min, an automatic control of dissolved oxygen at 15%, and a pH of 5.5. After 10 hours of fermentation, feed was fed once every 6 hours, 40 g of glucose was added, and geneticin 0.2 g/L was added to the recombinant culture medium. After 30 hours of fermentation, the fermentation was terminated. The determination of ergosterol content refers to the literature (Xu Xuping, Li Huizhen, She Chenxing, Xie Hualing, Lin Zhijun. Research on the optimization of fermentation conditions of ergosterol-producing bacteria Torullopsis famata. Journal of Biology, 2002, 19: 15-17).

The measurement results show that the ergosterol content of the recombinant bacteria is 1.2%, which is 33% higher than the 0.9% of Saccharomycescerevisiae IFFI 01338. Studies have shown that the synthesis of ergosterol has the characteristics of oxygen metabolism, and aerobic conditions are conducive to its synthesis. Therefore, the introduction of phbCAB gene provides cells with more oxidative power, thereby promoting the synthesis of ergosterol in cells.

Example 4, expressing phbCAB gene in Saccharomyces cerevisiae improves the stress resistance of yeast

Strains: Saccharomyces cerevisiae ACCC 2063

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Saccharomyces cerevisiae ACCC 2063 by lithium acetate transformation to obtain recombinant bacteria (Saccharomyces cerevisiae ACCC 2063 containing pPICHB) for fermentation experiments.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) ACCC 2063 and recombinant bacteria cultured on fresh slopes were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively, cultivated at 30°C and 200rpm for 12 hours, and then inoculated with 10% (v /v) The inoculum is introduced into the new medium. The medium components are: glucose 40g/L, peptone 20g/L, yeast powder 10g/L, agar 20g/L (for slant), pH 5.5. The seeds of Saccharomyces cerevisiae (Saccharomyces cerevisiae) ACCC2063 and the recombinant bacteria were divided into three groups before being inserted into the new medium, one group was placed at 48°C for 1 hour, one group was placed at -80°C for 48 hours, and the other group was left untreated. deal with. Then the seeds were respectively inserted into the new medium, and their growth curves were measured. Calculate the difference in lag time between untreated and treated seeds. The results showed that the activity of the seeds of the recombinant bacteria was not greatly reduced after cold or heat treatment, that is, the lag phase time was not much different from that of untreated seeds. However, the seed activity of Saccharomyces cerevisiae ACCC 2063 was greatly affected by cold or heat treatment. After heat treatment, the time difference of Saccharomyces cerevisiae ACCC 2063 is 7 hours, the time difference of recombinant bacteria is 1 hour, and the difference of the two is 6 hours. That is to say, cells accumulating PHA have better tolerance to harsh environments and stronger vitality. This is of great significance to the brewing industry, because the death of yeast during fermentation will affect fermentation and bring unpleasant taste, so strong strains have better application prospects.

Example 5. Expression of phbCAB in Bacillus subtilis affects the biosynthesis of inosine

Bacterial species: Bacillus subtilis ATCC 19162.

The composition of the seed medium is glucose 20g/L, yeast powder 15g/L, peptone 10g/L, corn solution 7g/L, sodium chloride 2.5g/L, urea 2g/L, pH 7.0. The composition of the fermentation medium is glucose 140g/L, corn liquid 16g/L, yeast powder 16g/L, urea 9g/L, ammonium sulfate 16g/L, magnesium sulfate heptahydrate 4g/L, dipotassium hydrogen phosphate 5g/L, carbonic acid Calcium 20g/L.

The plasmid pEUHB obtained according to the method of Example 1 was transferred into Bacillus subtilis (Bacillus subtilis) ATCC 19162 by electrotransformation to obtain recombinant bacteria (Bacillus subtilis (Bacillus subtilis) ATCC 19162 containing pEUHB) for fermentation experiments.

Bacillus subtilis (Bacillus subtilis) ATCC 19162 and recombinant bacteria cultured on fresh slopes were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively. (v/v) The inoculum size was inserted into the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, automatically controls the temperature at 35°C, pH 6.5, stirring speed at 600rpm, and ventilation at 1L/min. Stop the fermentation until the inosine production no longer increases, and the fermentation time is 68 hours. The inosine content was determined by HPLC, the mobile phase was 0.5% dipotassium hydrogen phosphate, the flow rate was 1.2mL/min, the chromatographic column was a HypersilODS C18 reverse-phase column, and the detection wavelength was 254nm. The results of HPLC measurement showed that the glycoside yield of Bacillus subtilis (Bacillus subtilis) ATCC 19162 was 19g/L, and the yield of the recombinant strain was 24g/L, an increase of 26%. Among intracellular metabolic pathways, the monophosphate hexose pathway that provides precursors for inosine synthesis consumes a large amount of NADP, while the regulatory enzyme 6-phosphate glucose dehydrogenase from glucose 6-phosphate to pentose phosphate pathway is regulated by NADPH Feedback inhibition, thus reduction of NADPH can promote the conversion of glucose to pentose phosphate pathway. In general, inosine synthesis is a biochemical metabolic process that requires a large amount of oxidative power, and high NADP/NADPH levels can effectively promote inosine synthesis. The introduction of the phbCAB gene can fulfill this requirement, thus significantly increasing the production of inosine.

Embodiment 6, expressing phbCAB gene in alkalophilic bacillus influences elastase production

Bacterial species: Bacillus alcalophilus Ya-B (Takagi H, Tsai YC, Nakamori S, Yamasaki M. Improved Production and Recovery of Alkaline Elastase from Alkalophilic Bacillus Strains by a Change of Medium Composition. Biosci1 Biotech 5 Biochem, 5 1591-1592)

The composition of the seed medium is glucose 10g/L, peptone 5g/L, yeast powder 5g/L, dipotassium hydrogen phosphate 1g/L, magnesium sulfate heptahydrate 0.2g/L, sodium chloride 5g/L, sodium carbonate 10g/L . The composition of the fermentation medium is glucose 20g/L, bean cake powder 5g/L, yeast powder 2.5g/L, dipotassium hydrogen phosphate 0.75g/L, magnesium sulfate heptahydrate 0.2g/L, sodium chloride 20g/L, sodium carbonate 10g/L.

The pEUHB obtained in Example 1 was transformed into Bacillus alcalophilus Ya-B by electrotransformation to obtain a recombinant strain (Bacillus alcalophilus Ya-B containing pEUHB) for fermentation experiments. Recombinant bacteria were obtained for fermentation experiments.

Bacillus alcalophilus Ya-B and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), and cultured at 37°C and 250rpm for 12 hours Afterwards, insert the fermentation medium (50mL/500mL Erlenmeyer flask) with 1% (v/v) inoculation amount, cultivate for 48 hours at 37°C and 250rpm, and stop the fermentation. The fermentation broth was centrifuged at 8000g for ten minutes, the supernatant was collected, and the ammonium sulfate was used for graded salting out (30%-65% saturation), and the precipitate was dissolved in 0.05mol/l pH 9.0 boric acid buffer to obtain a crude enzyme solution. Add 1mL 0.05mol/L pH 9.0 boric acid buffer solution to 1mL crude enzyme solution and 20mg lichenin-elastin respectively, and incubate in a water bath at 55°C for one hour, shaking from time to time. The reaction was terminated with 2 mL of 0.7 mol/L pH 6.0 phosphate buffer, centrifuged at 8000 g for 10 minutes, and the absorbance of the supernatant was measured at 590 nm. After 20mg lichenin-elastin is completely hydrolyzed, half of the light absorption value at 590nm is 10 enzyme activity units.

After 48 hours of fermentation and cultivation, the enzyme activity of the recombinant bacteria was 198 U/mL, which was 12.5% higher than that of Bacillus alcalophilus (Bacillus alcalophilus) 176 U/mLYa-B, and the recombinant bacteria had higher elastase production. Studies have shown that high dissolved oxygen is beneficial to the synthesis of elastase, and its synthesis is a process that requires oxidative power. The introduction of phbCAB gene can just provide more oxidative power for cells, which is beneficial to the synthesis of elastase.

Example 7, expressing phbCAB gene in Rhodotorula saccharomyces affects carotenoid production

Strains: Rhodotorula glutinis NCIM 3353 (National Center for Industrial Microbiology Preservation of India)

The composition of the slant medium is: glucose 35g/L, malt extract 3g/L, yeast powder 2g/L, KH ₂ PO ₄ 3g/L, K ₂ HPO ₄ 3g/L, MgSO ₄ 7H2O 0.2g/L, agar 20g/L, pH 6.0.

The liquid medium is: glucose 25g/L, yeast powder 10g/L, KH ₂ PO ₄ 2g/L, K ₂ HPO ₄ 2g/L, pH 6.0.

The pPICHB obtained in Example 2 was transformed into Rhodotorula glutinis NCIM 3353 by lithium acetate transformation to obtain recombinant bacteria (Rhodotorula glutinis NCIM 3353 containing pPICHB) for fermentation experiments.

Red yeast (Rhodotorula glutinis) NCIM 3353 and recombinant bacteria cultured on fresh slopes were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively. /v) The inoculum is transferred into fresh liquid culture medium (100 mL/500 mL Erlenmeyer flask), cultured at 28° C. and 240 rpm for 72 hours. The fermentation broth was centrifuged at 3000rpm for ten minutes to collect the bacterial cells, washed twice with deionized water, then freeze-dried and weighed. Add 5mL of 3mol/L HCl per gram of bacteria, soak at room temperature for 1 hour, boil in a boiling water bath for 4 minutes, cool rapidly, centrifuge at 3000g for 15 minutes, discard the supernatant, wash the precipitate twice with deionized water, and then add 3mL of acetone , shake at room temperature for 30 minutes, and then centrifuge at 3000g for 20 minutes. The supernatant is the carotenoid extract. After diluting the extract, measure the absorbance value at 475nm, and calculate the carotene content according to the following formula: carotene content (μg /g bacterial cells)=A ₄₇₅ ×V×D/0.16w. A475 is the absorbance value of carotene, V is the amount of solvent used for extraction (mL), D is the dilution factor of the sample, w is the amount of bacteria (g), and 0.16 is the molar extinction coefficient of carotene.

The results showed that the content of carotenoids in Rhodotorula glutinis NCIM 3353 was 76 μg/g, while that of the recombinant strain was 92 μg/g, an increase of 21%. High oxidative environment is conducive to the synthesis of carotenoids. The introduction of phbCAB gene can provide cells with oxidative power, thereby promoting the synthesis of carotenoids.

Embodiment 8, expressing phbCAB in Corynebacterium glutamicum species affects glutamine synthesis

Bacterial species: Corynebacterium glutamicum ATCC 13032.

Seed medium and fermentation components are: glucose monohydrate 50g/L, (NH ₄ ) ₂ SO ₄ 7.5g/L, NaCl 2g/L, CH ₃ COONa·2H ₂ O 3g/L, CaCl ₂ ·2H ₂ O 0.1 g/L, K ₂ HPO ₄ 3H ₂ O 8g/L, KH ₂ PO ₄ 2g/L, urea 5g/L, MgSO ₄ 7H ₂ O 0.5g/L, salt solution 10mL/l, biotin 6μg/L L, Vitamin B1 1mg/L, Kanamycin 20mg/L. The salt solution is: MnSO ₄ 7H ₂ O 20mg, Na ₂ B ₄ O ₇ 10H ₂ O 2mg, (NH ₄ ) ₅ MO ₇ O ₂₄ 4H ₂ O 1mg, FeCl ₂ 6H ₂ O 20mg, ZnSO ₄ · 7H ₂ O 5 mg, CuSO ₄ · 5H ₂ O 2 mg, FeSO ₄ · 7H ₂ O 250 mg, dissolved in 50 mL of 1mol/L HCl.

The plasmid pEUHB obtained in Example 1 was transferred to Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 by electrotransformation to obtain recombinant bacteria (corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 containing pEUHB) and carried out Fermentation experiments.

Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), cultured at 30°C and 200rpm for 12 hours, then 5 % (v/v) of the inoculum added to the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, automatic temperature control at 30°C, pH 6.5, stirring speed at 600rpm, ventilation rate at 1L/min, fermentation for 48 hours, stop fermentation, and measure glutamine content in the fermentation broth. Glutamine was determined using Xinhua No. 3 filter paper at room temperature by the unsaturated method, and the developing agent was a mixture of n-propanol and concentrated ammonia water with a volume ratio of 5:2, and the ascending method was used. Weigh the glutamine standard substance and make solutions with different mass concentrations such as 1%, 1.5%, 2%, 2.5%, 3%, etc., spot 1 μL of each solution, and centrifuge the fermentation broth to take 1 μL of the supernatant for spotting. After the analysis is completed, air-dry the filter paper, develop color with 0.5g/L ninhydrin acetone solution, and dry at 105°C for 5 minutes. Purple-red amino acid spots appear on the filter paper. 38 (0.1g/L CuSO ₄ ·5H ₂ O and 75% ethanol mixed solution) was eluted for 30 minutes, then the light absorption was measured at 520nm, and the glutamine content in the fermentation broth was obtained from the standard curve. The results showed that the glutamine output of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 was 11 g/L, and that of the recombinant strain was 14 g/L, which was increased by about 27%. The introduction of phbCAB gene can reduce the metabolism of pyruvate to lactic acid, promote the conversion of pyruvate into acetyl-CoA, and the precursor of glutamine is the intermediate product of the tricarboxylic acid cycle, which can provide more glutamine synthesis. more precursors, thus increasing the yield.

Example 9. Expression of phbCAB in Brevibacterium lactofermentum affects lysine production

Strains: Brevibacterium lactofermentum ATCC 31269.

The composition of the slant medium is: beef extract 0.3%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.2-7.4.

The composition of the seed medium is: glucose 2%, KH ₂ PO ₄ 0.1%, (NH ₄ ) ₂ SO ₄ 1%, MgSO ₄ ·7H ₂ O 0.04%, FeSO ₄ ·7H ₂ O 1mg/L, MnSO ₄ ·H ₂ O 0.8mg/L, biotin 5μg/L, VB 120mg/L, bean cake hydrolyzate 5%, pH 7.0.

The composition of the fermentation medium is: glucose 14%, KH ₂ PO ₄ 0.1%, (NH ₄ ) ₂ SO ₄ 4%, MgSO ₄ ·7H ₂ O 0.04%, FeSO ₄ ·7H ₂ O 1mg/L, MnSO ₄ ·H ₂ O 0.8mg/L, biotin 5μg/L, vitamin B1 20mg/L, bean cake hydrolyzate 5%, calcium carbonate 2%, pH 7.0.

The plasmid pEUHB obtained in Example 1 was transformed into Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC 31269 by electroporation to obtain recombinant bacteria (Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC 31269 containing pEUHB) for fermentation experiments.

Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC 31269 and recombinant bacteria cultured on fresh slant were taken respectively, inoculated in primary seed medium, filled with 30 mL of liquid in a 500 mL shaker flask, and cultivated on a shaker at 30°C and 110 rpm for 12 hours. Inoculate 2mL of primary seeds into 30mL of secondary seed medium, and culture on a shaker at 30°C and 110rpm for 10-12 hours. Then take 1 mL of the secondary seeds and inoculate them into the fermentation medium, fill a 500 mL shake flask with a volume of 20 mL, culture at 30° C. and 110 rpm for 72 hours. The lysine content was measured by the ninhydrin colorimetric method: the fermented liquid was centrifuged at 3500g for 15 minutes, 0.1mL of the supernatant was taken and diluted to 100mL, and 1mL of the diluted solution was added to 4mL of the ninhydrin reagent (take 1.0g of ninhydrin hydrate, Add 75 mL of ethylene glycol methyl ether, and add another 1.34 g of CuCl ₂ 2H ₂ O into 25 mL of pH 1.3 citric acid buffer, mix the two solutions, add 100 mL of distilled water to obtain ninhydrin solution), boil water bath After heating for 20 minutes, measure the absorbance value at 478nm after rapid cooling. The lysine content was determined according to the standard curve.

The results showed that the lysine yield of Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC 31269 was 53g/L, while the yield of the recombinant strain was 58g/L, about 10% increased. In anabolism, there are three pathways for the synthesis of oxaloacetate, the precursor of lysine, but they are all related to the metabolism of pyruvate and NADP/NADPH. Therefore, the introduction of phbCAB gene can affect the production of lysine.

Example 10, expressing phbCAB gene in Escherichia coli affects the production of phenylalanine

Bacterial species: Escherichia coli (Escherichia.coli) ATCC 31882.

The composition of the seed medium is: peptone 1%, yeast powder 0.5%, NaCl 1%, glucose 2%, pH 7.5.

The components of the fermentation medium are: Na ₂ HPO ₄ ·12H ₂ O: 20g/L, sodium citrate 6g/L, sodium glutamate 0.4g/L, tyrosine 0.6g/L, glucose 20g/L.

Feed medium: CaCl ₂ ·2H ₂ O 0.6g/L, tyrosine 500mg/L, glucose 500g/L, MgSO ₄ ·7H ₂ O 1g/L, vitamin B1500mg/L, ammonia water 28%.

The pBHR68 plasmid was transformed into Escherichia coli (Escherichia.coli) ATCC 31882 by electroporation to obtain recombinant bacteria (Escherichia.coli ATCC 31882 containing pBHR68) for fermentation experiments.

Take Escherichia coli (Escherichia.coli) ATCC 31882 and recombinant bacteria cultured on fresh slant respectively, inoculate them into seed culture medium (50mL/500mL Erlenmeyer flask) respectively, cultivate them at 37°C and 200rpm for 12 hours, then add 5% (v/v) The inoculum size was inserted into the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, automatically controls the temperature at 38.5°C, pH 7.0, dissolved oxygen at 20%, and controls the glucose concentration at 1.5% by feeding, and the fermentation time is 48 hours. The method for determining the content of phenylalanine is as follows: take 2 μL of the supernatant of the fermentation broth and spot it on the filter paper, place it in the chromatographic fluid for one-way upward analysis for 16 hours, spray it with ninhydrin to develop the color after drying, and use the standard amino acid as the control , cut off the colored spots after chromatography, place in 65% ethanol solution for decolorization for 2 hours, measure light absorption at 520nm, amino acid amount=standard amino acid solution concentration×measured fermentation broth optical density/standard amino acid solution optical density.

The results showed that Escherichia coli (Escherichia.coli) ATCC 31882 was fermented to obtain a concentration of phenylalanine of 8.2 g/L, while the concentration of the recombinant bacterium product was 10.6 g/L, an increase of 29%. For the amino acid synthesis of Escherichia coli, better results can be achieved if global metabolic regulation is used. The introduction of phbCAB gene, an effective regulation is to enhance the pentose phosphate pathway, which is beneficial to the synthesis of amino acids.

Example 11. Expression of phbCAB gene in Pseudomonas fluorescens affects gluconic acid production

Bacteria: Pseudomonas fluorescens AS 1.55

The composition of the slant medium is: beef extract 0.5%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.0.

The composition of the seed medium is: 2% glucose, 1% corn steep liquor, 0.2% urea, 0.2% KH ₂ PO ₄ , 0.05% MgSO ₄ ·7H ₂ O, pH 7.0.

The composition of the fermentation medium is: starch hydrolysis sugar 14%, corn steep liquor 1.5%, light calcium carbonate 4%, pH 6.7.

After the plasmid pBHR68 was digested with HindIII and BamHI, it was inserted into the plasmid pBBR1MCS2 (Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM. Four new derivatives of the broad-host-range cloning vector pBBR1MCS , carrying differentantibiotic-resistance cassettes.Gene.1995,166:175-176) between the recognition sites of HindIII and BamHI, the plasmid pBBRHB was obtained. The plasmid was transformed into Pseudomonas fluorescens AS 1.55 by electroporation to obtain recombinant bacteria (Pseudomonas fluorescens AS 1.55 containing pBBRHB) for fermentation experiments.

Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.55 and recombinant bacteria cultured on fresh slant were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively. % (v/v) of the inoculum added to the fermentation medium. A 500-mL fermentation bottle was filled with 50 mL of liquid, and cultured at 30° C. and 230 rpm for 72 hours. The content of ketogluconic acid in the fermentation broth is determined by optical polarimetry: the content of ketogluconic acid in the fermentation broth = the absolute value of the optical rotation of the fermentation broth at 25° C./0.88.

The content of ketogluconic acid in the fermentation broth of Pseudomonas fluorescens (Pseudomonas fluorescens) K1005 is 13.4g/L, and the output of the recombinant bacteria is 16g/L, which has increased by about 19%. Tolerance, better growth, resulting in higher conversion rates.

Example 12, the expression of phbCAB gene in Bacillus subtilis affects the synthesis of α-amylase

Bacterial species: Bacillus subtilis ATCC 21556.

The composition of the seed medium is (300 mL): 1 g of glucose, 1 g of beef extract, 1 g of peptone, 1 g of NaCl, 1 g of KH ₂ PO ₄ , 1 g of starch, and pH 7.0.

The components of the fermentation medium are (200 mL): 1 g of beef extract, 1 g of peptone, 1 g of NaCl, 1 g of KH ₂ PO ₄ , 1 g of NaH ₂ PO ₄ , and 3 g of starch.

The method of lithium acetate transformation of the plasmid pEUHB obtained according to the method of Example 1 was transferred into Bacillus subtilis (Bacillus subtilis) ATCC 21556 to obtain recombinant bacteria (Bacillus subtilis (Bacillus subtilis) ATCC 21556 containing pEUHB) for fermentation experiments.

Bacillus subtilis (Bacillus subtilis) ATCC 21556 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), cultivated at 32°C and 200rpm for 12 hours, then added with 10% ( v/v) The inoculum was transferred into the fermentation medium (50 mL/500 mL Erlenmeyer flask), cultured at 32° C. and 200 rpm for 72 hours, and the enzyme activity of α-amylase was measured. Determination of enzyme activity: take 5mL of fermentation broth, centrifuge at 4000g for 15 minutes, take the supernatant and react with 5-acetal-maltoheptose-p-nitrobenzene, the enzyme activity unit is defined as: 1 μmol starch decomposed in 1 minute at 37°C is Glucose is 1 enzyme activity unit.

The results showed that the enzyme activity in the fermentation broth of Bacillus subtilis ATCC 21556 was 67U/L, while that of the recombinant bacteria was 76U/L. The fermentation broth of the recombinant bacteria has higher amylase activity, which may be because the phbCAB gene leads to changes in the metabolic pathways in the cells, which tends to favor the direction of cell growth and improves the ability of cells to use starch as a carbon source , which increases the amylase production rate of the cells.

Example 13. Expression of phbCAB gene in Saccharomyces cerevisiae affects alcohol production

Strains: Saccharomyces cerevisiae ACCC 2063.

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Saccharomyces cerevisiae ACCC 2063 by lithium acetate transformation to obtain recombinant bacteria (Saccharomyces cerevisiae ACCC 2063 containing pPICHB) for fermentation experiments.

Preparation of solid medium: Measure 200mL of 5Bx wort into a 500mL beaker, add 4g of agar in a boiling state, stir continuously until it is completely melted, put it in a test tube for autoclaving, and then put it on a slope; preparation of liquid medium: measure Take 110mL of 5Bx wort and put it into a 250mL Erlenmeyer flask, adjust the pH to 4.8-5.0, and then autoclave; preparation of fermentation medium: weigh 300g of starch and pour it into 1200mL of warm water at 40°C, stir evenly, add 0.2% calcium chloride, Adjust the pH to 6.0-6.2, weigh 1g of liquefied enzyme, and liquefy at 90°C-93°C for 30 minutes until the iodine solution does not change color. After liquefaction, quickly cool it to 58°C-60°C and adjust the pH value 4.4-4.6, add 1mL glucoamylase (100,000 units) for saccharification, stop when the sugar content of the starch hydrolyzate is 23Bx, add 2% corn steep liquor, boil for 1 hour, cool and filter, then adjust the pH to 4.8-5.0 , sterilized for use. During fermentation, the inoculation amount of 10% (v/v) is inserted into the fermentation medium, and the fermentation is incubated at 30° C. for 72 hours. The ethanol concentration is measured by high-performance liquid chromatography: the fermentation broth is centrifuged at 8000g for ten minutes, and the supernatant is filtered as a sample; the chromatographic column is a Beckman L2Spherogel ligand exchange column, and the mobile phase is 98% concentrated sulfuric acid: H ₂ O (volume ratio 0.5:1000), flow rate 1mL/min.

The results showed that the ethanol concentration of Saccharomyces cerevisiae (Saccharomyces cerevisiae) ACCC 2063 fermentation liquid was 15%, while the ethanol concentration of recombinant yeast was 20%, which increased by 33%. Synthesis can improve the tolerance of recombinant yeast to high ethanol environment, thereby increasing the production of ethanol.

Example 14. Expression of phbCAB gene in Candida krusei affects glycerol production

Species: Candida krusei ACCC 2196.

The composition of the slant medium is: glucose 50g/L, yeast extract 20g/L, calcium carbonate 5g/L, agar 20g/L.

The composition of the seed medium is: glucose 200g/L, urea 2g/L, corn steep liquor 7g/L.

The composition of the fermentation medium is: glucose 200g/L, urea 2g/L, corn steep liquor 6g/L.

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Candida krusei (Candida krusei) ACCC 2196 with lithium acetate to obtain recombinant bacteria (Candida krusei (Candidakrusei) ACCC 2196 containing pPICHB) ) for fermentation experiments.

Candida krusei (Candida krusei) ACCC 2196 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (40mL/500mL Erlenmeyer flask), cultivated at 30°C and 230rpm for 12 hours, then 10% (v/v) inoculum amount is inserted into the fermentation medium, the liquid volume of the 7.5L fermentation tank is 3L, the pH is automatically controlled to 5.5, the temperature is 30°C, the ventilation rate is 0.5L/min, the stirring speed is 450rpm, and the residual sugar is close to 0. Stop Fermentation, determination of glycerol content in fermentation broth. The glycerol content in the fermentation broth was detected by high performance liquid chromatography: the fermentation broth was centrifuged at 8000 g for ten minutes, and the supernatant was taken as a sample after filtration; the chromatographic column was L2Spherogel ligand exchange column of Beckman Company, and the mobile phase was 98% concentrated sulfuric acid: H ₂ O (The volume ratio is 0.5:1000), the flow rate is 1mL/min.

The results showed that the final glycerol concentration of Candida krusei (Candida krusei) ACCC 2196 was 62g/L, while that of the recombinant strain was 75g/L, an increase of 21%. During the fermentation process, a considerable part of the carbon source will flow to ethanol synthesis, which reduces the yield of glycerol. The ethanol pathway is a pathway that provides oxidative power for cells. The introduction of the phbCAB gene can reduce the pressure on the ethanol pathway, promote glycolysis, and allow more carbon sources to flow to glycerol synthesis, increasing the yield of glycerol.

Example 15, the expression of phbCAB gene in Candida tropicalis affects the synthesis of dodecanedioic acid

Strain: Candida tropicalis UH22248 (Ren Gang, Chen Yuantong, Research on the fermentation of dodecane dibasic acid, Chinese Journal of Bioengineering, 2000, 16: 198-202).

The components of the slant medium are: 10Bx wort juice, 1.5% agar powder.

The composition of the seed medium is: 5g of yeast extract, 3g of corn steep liquor, 5g of sucrose, 8g of KH ₂ PO ₄ , add water to 1L for sterilization, add 0.3% urea and 5% heavy wax when inoculating.

The fermentation medium is: yeast extract 2g, corn steep liquor 1g, sucrose 2g, KH ₂ PO ₄ 8g, NaCl 1g, add water to 100mL for sterilization, add n-dodecane 20%, urea 0.12%, heavy wax 3.3%, pH 7.3.

The plasmid pPICHB obtained according to the method in Example 2 was transformed into Candida tropicalis UH22248 by lithium acetate transformation to obtain recombinant bacteria (Candida tropicalis UH22248 containing pPICHB) for fermentation experiments.

Inoculate Candida tropicalis UH22248 and recombinant strains cultured on a slant for 48 hours into the seed medium (100mL/500mL shake flask), and after culturing at 30°C and 180rpm for 48 hours, inoculate 3mL to 100mL of fermentation culture culture medium at 30°C and 180rpm for 4 days, and the fermentation ended. Take 15ml of fermentation broth, adjust the pH value to 3.0 with 6mol/L HCl, add 120mL of ether, shake 100 times, let it stand for 30 minutes, then pour out 40mL of ether extract, air-dry in a fume hood to obtain a white solid, and then use 95% Dissolve in neutral hot ethanol, add a drop of phenolphthalein, titrate with standard NaOH solution, record the NaOH used, and calculate the yield of dodecanedioic acid.

The results showed that the recombinant bacteria (dodecanedioic acid production of 92g/L) showed higher dodecanedioic acid production than Candida tropicalis (Candidatropicalis) UH22248 (dodecanedioic acid production of 78g/L). acid production, the fermentation broth is relatively viscous, and the cells are in an environment with a low dissolved oxygen level. The introduction of the phbCAB gene can provide the cells with oxidative power under such conditions, and it may also inhibit the fatty acid oxidation pathway, thereby increasing The production of dibasic acids in the fermentation broth.

Example 16, the expression of phbCAB gene in Candida tropicalis affects the synthesis of pentadecanedioic acid

Species: Candida tropicalis T _25-14 (Chen Yuantong, Hao Xiuzhen, Pang Yuechuan, Research on the fermentation of pentacarbon dibasic acid, Acta Microbiology, 1995, 35: 433-437).

The components of the slant medium are: 10Bx wort juice, 1.5% agar powder.

The composition of the seed medium is: yeast extract 5g/L, corn steep liquor 3g/L, sucrose 5g/L, KH ₂ PO ₄ 8g/L for sterilization, and then add 0.3% urea and 5% heavy wax when inoculating.

The fermentation medium is: yeast extract 2g/L, corn steep liquor 1g/L, sucrose 1g/L, KH ₂ PO ₄ 8g/L, NaCl 1g/L, add water to 100mL for sterilization, add n-pentadecane when inoculating 20%, urea 0.12%, pH 7.5.

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Candida tropicalis (Candida tropicalis) T 25-14 by the method of lithium acetate transformation to obtain recombinant bacteria (Candida tropicalis (Candida _{tropicalis) T 25-14 containing} pPICHB ) for fermentation experiments.

The seeds of Candida tropicalis T _25-14 and recombinant strains cultured on the slant for 48 hours were respectively inoculated into the seed medium (50 mL/500 mL shake flask), and after culturing at 30 °C and 220 rpm for 48 hours, 5 The % (v/v) inoculum was inoculated into the fermentation medium (50 mL/500 mL shake flask), and cultured at 30° C. and 220 rpm for 72 hours. Take 15ml of fermented liquid, adjust the pH value to 3.0 with 6mol/l HCl, add 120ml of diethyl ether and mix evenly, place until the layers are separated, remove the water layer, release the diethyl ether extract, air-dry in a fume hood to obtain a white solid, and then use 95% Dissolve in neutral hot ethanol, add a drop of phenolphthalein, titrate with standard NaOH solution, record the NaOH used, and calculate the yield of dibasic acid.

The results showed that the dibasic acid production of Candida tropicalis T _25-14 was 36g/L, and that of the recombinant strain was 43g/L, which was increased by 19%. The fermentation broth of the recombinant bacteria is relatively viscous, and the cells are in an environment with a low dissolved oxygen level. The introduction of the phbCAB gene can provide the cells with oxidative power under this condition, and it may also inhibit the fatty acid oxidation pathway, thereby increasing the Dibasic acid production in fermentation broth.

Embodiment 17, the influence of expressing phbCAB gene on spore formation in Bacillus thuringiensis

Bacteria: Bacillus thuringiensis ACCC 10068.

The plasmid pEUHB obtained according to the method of Example 1 was transferred into Bacillus thuringiensis ACCC 10068 by electrotransformation to obtain recombinant bacteria (Bacillus thuringiensis ACCC 10068 containing pEUHB) for fermentation experiments.

Bacillus thuringiensis (Bacillus thuringiensis) ACCC 10068 and recombinant bacteria cultured on fresh slopes were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), cultivated at 25°C and 220rpm for 12 hours, and added with 1% (v/v) The inoculum was transferred into the fermentation medium (50mL/500mL Erlenmeyer flask), and fermented at 25°C and 220rpm for 36 hours. The medium components are: beef extract 0.3%, peptone 0.5%, pH 7.2. After fermentation, the spore content was measured with a hemocytometer.

It can be seen by microscopic observation that the bacterial volume (10.2*10 ⁹ /ml) and spore number (1.5*10 ⁹ /ml) of the recombinant bacteria are higher than those of Bacillus thuringiensis ACCC 10068 (bacterial volume 9.1*10 ⁹ /ml, the number of spores is 1.1*10 ⁹ /ml). Fermentation experiments showed that high dissolved oxygen was beneficial to spore formation. The introduction of phbCAB gene can promote sporulation.

Example 18. Expression of phbCAB in Bacillus subtilis affects guanosine synthesis

Bacterial species: Bacillus subtilis ATCC 19220.

The composition of the seed medium is glucose 20g/L, yeast powder 10g/L, peptone 10g/L, corn liquid 10g/L, sodium chloride 5g/L, urea 2g/L, monosodium glutamate 5g/L, pH 7.0-7.2.

The composition of the fermentation medium is glucose 120g/L, soybean cake hydrolyzate 50g/L, yeast powder 16g/L, ammonium sulfate 15g/L, magnesium sulfate heptahydrate 4g/L, dipotassium hydrogen phosphate 2g/L, monosodium glutamate 10g/L, Calcium carbonate 2g/L, pH 7.0-7.2.

The plasmid pEUHB obtained according to the method of Example 1 was transformed into Bacillus subtilis (Bacillus subtilis) ATCC 19220 by the method of lithium acetate transformation to obtain recombinant bacteria (Bacillus subtilis (Bacillus subtilis) ATCC 19220 containing pEUHB) for fermentation experiments.

Bacillus subtilis (Bacillus subtilis) ATCC 19220 and recombinant bacteria cultured on fresh slant were inoculated into seed medium (50mL/500mL conical flask) respectively, cultivated at 36°C and 140rpm for 10 hours, then added with 10% ( v/v) The inoculum was transferred into the fermentation medium (50 mL/500 mL Erlenmeyer flask), cultured at 36° C. and 220 rpm for 60 hours, and the content of guanosine was determined. The content of guanosine was determined by HPLC, the mobile phase was 0.5% dipotassium hydrogen phosphate, the flow rate was 1.2mL/min, the chromatographic column was a Hypersil ODS C18 reverse-phase column, and the detection wavelength was 254nm.

The results showed that the yield of Bacillus subtilis (Bacillus subtilis) ATCC 19220 guanosine was 16g/L, and the yield of the recombinant strain was 20g/L, which increased by 25%. In the intracellular metabolic pathway, the hexose monophosphate pathway that provides precursors for guanosine synthesis consumes a large amount of NADP, and high NADP/NADPH levels can effectively promote the synthesis of guanosine. The introduction of the phbCAB gene can fulfill this requirement, thus significantly increasing the production of guanosine.

Example 19. Expression of phbCAB gene in Saccharomyces cerevisiae affects glutathione production

Strain: Saccharomyces cerevisiae KY6186 (Sakato K, Tanaka H. Advanced control of glutathione fermentation process. Biotechnol Bioeng, 1992, 40: 904-912).

The composition of the slant medium is: wort juice 10g/L, yeast powder 3g/L, peptone 5g/L, glucose 10g/L, agar 20g/L, pH 7.0-7.2.

The composition of the seed medium is: glucose 30g/L, yeast powder 6g/L, diammonium hydrogen phosphate 3g/L, magnesium sulfate 0.8g/L, dipotassium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 1g/L, pH 7.0 -7.2.

The fermentation medium is: glucose 70g/L, yeast powder 15g/L, wort 60g/L, diammonium hydrogen phosphate 10g/L, magnesium sulfate 5g/L, honey 28g/L, corn steep liquor 16g/L, hydrogen phosphate Dipotassium 1g/L, potassium dihydrogen phosphate 1g/L, ZnCl ₂ 10mg/L, FeCl ₂ 6mg/L, CuCl ₂ 6mg/L, MnCl ₂ 6mg/L, pH 7.0-7.2.

The plasmid pPICHB obtained according to the method of Example 2 was transformed into Saccharomyces cerevisiae (Saccharomyces cerevisiae) KY6186 with lithium acetate to obtain recombinant bacteria (containing Saccharomyces cerevisiae (Saccharomyces cerevisiae) KY6186) for fermentation experiments.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) KY6186 and recombinant bacteria cultured on fresh slopes were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively, cultivated at 30°C and 180rpm for 24 hours, and then inoculated with 10% (v/ v) The inoculation amount is connected to the fermentation medium, the liquid volume in the 7.5L fermenter is 3L for fermentation, the temperature is automatically controlled at 30°C, the pH is 5.4, the ventilation rate is 2L/min, the speed is 200rpm for the first two hours, and then increased by 100rpm per hour until 600rpm, maintained until the end of fermentation, 36 hours to stop fermentation. Get the fresh yeast obtained by fermentation and wash it three times with distilled water, in 40% ethanol, extract for 2 hours at 30°C, centrifuge at 3000g, get the supernatant and use the ALLOXAN method (Shanghai Pharmaceutical Laboratory Clinical Biochemical Test (Volume 1), Shanghai, Shanghai) Science and Technology Press, 1979) Determination of glutathione content.

The results showed that the glutathione concentration of Saccharomyces cerevisiae (Saccharomyces cerevisiae) KY6186 fermentation liquid was 1.3 g/L, while the glutathione concentration of the recombinant bacteria was 1.6 g/L, which increased by 23%. Studies have shown that the reduction of ethanol synthesis is beneficial to the synthesis of glutathione, and ethanol synthesis is a process of consuming reducing power and gaining oxidative power. The introduction of phbCAB gene can inhibit the synthesis of ethanol and increase the production of glutathione.

Example 20, Effect of expressing phbCAB gene on erythritol production in Torulopsis sphaeroides

Bacterial species: ToruLopsis sp B845 (Wu Yan, Yang Xiaowei, Lu Maolin, Morphology and physiological characteristics of erythritol-producing strain B845, Biotechnology, 2002, 12: 20-21).

The composition of the slant medium is: glucose 10%, yeast extract 1%, urea 0.1%, agar 2%, pH 7.0-7.2.

The seeds and fermentation medium are: glucose 20%, yeast extract 0.5%, urea 0.1%, pH 7.0-7.2.

The plasmid pPICHB obtained according to the method of Example 2 was transferred into ToruLopsis sp B845 by electrotransformation to obtain recombinant bacteria (ToruLopsis sp B845 containing pPICHB) for fermentation experiments.

ToruLopsis sp B845 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (40mL/500mL Erlenmeyer flask), cultured at 30°C and 180rpm for 3 days, then added with 5% (v/ v) The inoculum was transferred into the fermentation medium (50 mL/500 mL Erlenmeyer flask), cultured at 30° C. and 180 rpm for 3 days, and the production of erythritol was measured. Erythritol production is measured by periodic acid oxidation method (Yuan Ye, Ying Xiangxian, Fan Guangxian, Zhuge Jian, direct determination of erythritol in fermentation broth by periodic acid oxidation method, Journal of Wuxi Light Industry University, 2000, 19: 72 -75). The results show that the erythritol yield of ToruLopsis sp B845 is 50g/L, and that of recombinant bacteria is 55g/L, which is about 10% higher. As an erythritol-producing bacterium, good hypertonic resistance is required and metabolic carbon source ability, and the introduction of phbCAB gene leads to the synthesis of intracellular PHA, which is conducive to improving the above-mentioned ability, so it also has a certain promotion effect on the synthesis of erythritol.

Example 21. Expression of phbCAB gene in Bacillus pumilus affects D-ribose production

Bacterial species: Bacillus pumilus ATCC 31095.

The composition of the seed medium is: glucose 20g/L, corn steep liquor 26g/L, dipotassium hydrogen phosphate 0.03g/L, potassium dihydrogen phosphate 0.01g/L, pH 6.7.

The composition of the fermentation medium is: glucose 180g/L, corn steep liquor 28g/L, ammonium sulfate 13g/L, manganese sulfate 0.05g/L, pH 7.0.

The plasmid pEUHB obtained according to the method of Example 1 was transformed into Bacillus pumilus ATCC 31095 by lithium acetate transformation to obtain recombinant bacteria (Bacillus pumilus ATCC 31095 containing pEUHB) for fermentation experiments.

Bacillus pumilus (Bacillus pumilus) ATCC 31095 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), cultivated at 37°C and 180rpm for 18-20 hours, with 10% The inoculum size of (v/v) was inserted into the fermentation medium. A 7.5L fermentation tank was filled with 3L of liquid, controlled temperature at 37°C, ventilation at 3L/min, stirring speed at 650rpm, and fermented for 72 hours to measure the D-ribose content in the fermentation broth. D-ribose is measured by the orcinol method: the fermentation broth is centrifuged at 5000g for 10 minutes, and the supernatant is diluted with distilled water to control the amount of ribose within the range of 10-90 g/3mL; take 3mL of the diluted solution and add 0.1% ferric chloride concentrated hydrochloric acid in turn Solution 3mL, 0.1% orcinol ethanol solution 0.3mL, shake well, bathe in boiling water for 40 minutes, measure the absorbance value at 670nm within 60 minutes after cooling with tap water, and calculate the ribose concentration through the standard curve. The results showed that the ribose concentration of Bacillus pumilus (Bacillus pumilus) ATCC 31095 was 55g/L, while that of the recombinant bacteria could reach 72g/L, which was an increase of 31%. The synthesis of ribose in cells is through the pentose phosphate pathway, which consumes a large amount of intracellular oxidative power, and meeting the metabolic needs of cells by increasing dissolved oxygen will promote the formation of spores, which is not conducive to the production of ribose through cell vegetative growth, so phbCAB The introduction of the gene can provide cells with a certain oxidizing power NADPH from the endogenous way, which is beneficial to the synthesis of ribose.

Example 22, expressing phbCAB in Escherichia coli improves bacterial tolerance to heavy metal ion Hg2+

Bacterial species: Escherichia coli JM109.

The basic medium of Escherichia coli JM109 is LB medium (g/100mL): peptone 1.0; yeast extract 0.5; NaCl 1.0, pH 7.0.

The basic medium of the recombinant bacteria is: add ampicillin to the LB medium to a final concentration of 60 μg/mL.

The plasmid pBHR68 was transformed into Escherichia coli (Escherichia coli) JM109 by electrotransformation method to obtain recombinant bacteria (Escherichia coli (Escherichia coli) JM109 containing pBHR68). Wild-type E.coli JM109 was used as a control.

Escherichia coli (Escherichia coli) JM109 and recombinant bacteria were respectively inserted into the solution of 1mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L, 8mg/L or 10mg/L HgCl ₂ In the respective basal medium, the culture conditions were as follows: 500 mL Erlenmeyer flask with 100 mL liquid volume, 37° C., 200 rpm, cultured for 24 hours, and measured the OD ₆₀₀ of the fermentation broth. The results showed that when the concentration of Hg ²⁺ was 4mg/L, the growth of the recombinant bacteria was inhibited to some extent, and the maximum OD ₆₀₀ dropped from 2.7 to 1.9; when the concentration of Hg ²⁺ rose to 10mg/L, the growth of the bacteria almost stopped completely. Experiments also showed that the original host strain E.coli JM109 without exogenous gene transformation could not grow at all when the Hg ²⁺ concentration was 2 mg/L. The transfer of PHB gene enhanced the tolerance of bacteria to mercury.

Under the same operating conditions, the two kinds of bacteria enriched mercury ions from the simulated wastewater with a concentration of 2.5mg/L Hg ²⁺ , and the final enrichment amount of the recombinant bacteria was 5.1mg/g cell dry weight, while the original E. Only 1.7mg/g cell dry weight. Among them, the dry weight of cells was measured by freeze-drying method.

Experiments have shown that the recombinant bacteria added with the PHB synthesis gene have a greater tolerance to Hg ²⁺ than Escherichia coli JM109, and can better survive in the medium containing heavy metal ions under the same conditions. grow in.

Example 23, expression of phbCAB in Zymomonas mobilis improves bacterial tolerance to high alcohol environments

Strain: Zymomonas mobilis NRRL B-4286.

The plasmid pBBRHB obtained according to the method of Example 11 was transferred to Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 by electroporation to obtain recombinant bacteria (Zymomonas mobilis (Zymomonas mobilis) NRRL containing pBBRHB B-4286) for fermentation experiments.

Seed medium components (g/L): glucose 100, peptone 10, yeast extract 15, pH 6.0.

Fermentation medium components (g/L): glucose 150, peptone 25, yeast extract 25, pH 4.5 or pH 6.0.

References for determination of ethanol mass concentration, bacterium concentration, and reducing sugar mass concentration in fermentation broth (Ingram LO, Gomez PF, Lai X, Moniruzzaman M, Wood BE, Yomano LP and York SW. Metabolic engineering of bacteria for ethanol production. Biotechnol. Bioeng. 1998, 58: 204-214)

Training conditions:

The bacterial strains preserved in the refrigerator were respectively inserted into the seed medium, and after 12 hours of cultivation, they were respectively inserted into the fermentation medium with an inoculation amount of 10% (v/v) to carry out static anaerobic ethanol fermentation.

Fermentation Erlenmeyer flask 250mL, liquid volume 100mL, fermentation pH 4.5 (recombinant bacteria), temperature 35°C, fermentation time 48 hours. Fermentation pH 4.5 and 6.0 of Zymomonas mobilis NRRL B-4286 were used as controls.

Experiments show that the ethanol production of Zymomonas mobilis NRRL B-4286 at pH 4.5 and 6.0 is 21.1g/L and 62.5g/L respectively, and the ethanol production of recombinant bacteria at pH 4.5 is 59.2g/L, It is close to the level of Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 at pH 6.0, indicating that the transfer of phbCAB gene can effectively improve the acid resistance of bacteria, and further optimization of fermentation conditions can realize the production of ethanol at a lower pH Efficient production.

Example 24, expressing phbCAB in Zymomonas mobilis improves the ability of bacteria to utilize glucose

Strain: Zymomonas mobilis NRRL B-4286.

The plasmid pBBRHB obtained according to the method of Example 11 was transferred to Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 by electroporation to obtain recombinant bacteria (Zymomonas mobilis (Zymomonas mobilis) NRRL containing pBBRHB B-4286) for fermentation experiments.

Seed medium components (g/L): peptone 10, yeast extract 15, pH 6.0.

Fermentation medium components (g/L): peptone 25, yeast extract 25, pH 6.0, glucose. Wherein, the concentration of glucose is respectively 120, 150, 160, 180, 210 g/L gradient.

References for determination of ethanol mass concentration, bacterium concentration, and reducing sugar mass concentration in fermentation broth (Ingram LO, Gomez PF, Lai X, Moniruzzaman M, Wood BE, Yomano LP and York SW. Metabolic engineering of bacteria for ethanol production. Biotechnol. Bioeng. 1998, 58: 204-214)

Training conditions:

The strains stored in the refrigerator were respectively inoculated into the seed medium, and after 12 hours of cultivation, they were respectively inoculated into the fermentation medium with an inoculation amount of 10% by volume to carry out static anaerobic ethanol fermentation.

Fermentation Erlenmeyer flask 500mL, liquid volume 100mL, fermentation pH 6.0, temperature 35°C, fermentation time 48h. Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 and recombinant bacteria were cultured under different concentrations of glucose.

The experimental results showed that when the glucose concentration of Zymomonas mobilis NRRL B-4286 was 150g/L, the production of ethanol reached the maximum, which was 65g/L, while the recombinant bacteria produced ethanol when the glucose concentration increased to 210g/L. The yield has been on the rise, with a maximum of 95g/L. The introduction of the PHB synthesis pathway enhanced the bacteria's resistance to osmotic pressure, and maintained good growth even when the initial sugar concentration was high. The ethanol fermentation process of Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 has a higher utilization rate of glucose than yeast, but when the initial glucose concentration reaches a certain level, the ethanol production decreases instead. When the synthetic gene of PHB was transferred, the initial sugar concentration for fermentation to reach the maximum yield of ethanol was higher than that of Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286. It shows that the transfer of phbCAB is beneficial to improve the resistance of bacteria to osmotic pressure.

Example 25. Expression of phbCAB in Clostridium butyricum improves production of 1,3-propanediol (1,3-PDO) from glycerol fermentation

Bacterial species: Clostridium butyricum ATCC 8260.

The plasmid pEUHB obtained according to the method of Example 1 was transformed into Clostridium butyricum (Clostridium butyricum) ATCC 8260 with lithium acetate transformation method to obtain recombinant bacteria (clostridium butyricum (Clostridium butyricum) ATCC 8260 containing pEUHB) Conduct fermentation experiments.

The fermentation requires anaerobic culture, and the fermentation medium is: glycerol 6%, glucose 1%, corn steep liquor 2%, (NH ₄ ) ₂ SO ₄ 0.2%. The fermentation temperature is 34°C, and the initial pH is 6.5-7.

Training conditions:

Batch fermentation is divided into two steps. First, Clostridium butyricum ATCC 8260 and recombinant bacteria are respectively connected to 150 mL Erlenmeyer flasks with a liquid volume of 100 mL. Under anaerobic conditions, at 36 ° C, 100 rpm, Cultivate for 15 hours, then insert 10% (v/v) inoculum into a 250mL Erlenmeyer flask with a liquid volume of 200mL, seal with paraffin, and ferment at 34°C, 150rpm, and initial pH 6.8. Samples were taken regularly to determine the residual glycerol and the generation of 1,3-PDO.

The concentration of glycerol and 1,3-PDO in the fermentation broth was detected by gas chromatography. The chromatographic column was a large-diameter capillary tube, 0.5mm×20m, the column temperature was 170°C, the gasification chamber temperature was 260°C, the carrier gas was nitrogen, and the flow rate was 50mL/ min, quantified by internal standard method (internal standard substance is 1,62 hexanediol).

Product separation: After the fermentation broth is centrifuged, the supernatant is distilled in an oil bath to obtain 1,3-PDO product.

Biomass of bacterial growth: detected by colorimetry (OD ₆₅₀ nm).

The 1,3-propanediol yield of Clostridium butyricum ATCC 8260 was 15.7g/L, while that of the recombinant strain was 20.4g/L. The growth pH of Fusobacterium butyricum is between 6 and 7.5, and the by-products of its fermentation metabolism with glycerol as the substrate are mainly butyric acid and acetic acid, so the increase in acidity during the fermentation process will seriously affect the growth of the bacteria. Simultaneously inhibit 1,3-PDO generation. It can be seen that in order to obtain high-yield 1,3-PDO, the pH must be controlled between 6.5 and 7 during the fermentation process. If the synthetic gene of PHB is introduced into the bacterium, so that the tolerance of the bacterium to acidic conditions is enhanced, a higher 1,3-PDO yield can be obtained without adjusting the pH.

Example 26, expression of phbCAB in Bacillus improves the decomposition of crude oil under bacterial anaerobic conditions

Strain: Bacillus sp L-23 (Li Qingxin, Kang Congbao, Wang Hao, Zhang Changkai, Optimizing the fermentation conditions of Bacillus sp and preliminary research on enhancing oil recovery under indoor conditions, Industrial Microbiology, 2002 , 32: 28-31).

The acquisition of recombinant bacteria: the plasmid pEUHB obtained according to the method of Example 1 is transferred to Bacillus sp L-23 by the method of lithium acetate transformation to obtain recombinant bacteria (Bacillus sp L-23 containing pEUHB ) for fermentation experiments.

Seed medium (g/L): glucose 20, peptone 2, Na ₂ HPO ₄ 4, KH ₂ PO ₄ 2, MgSO ₄ 0.5, CaCl ₂ 0.005, pH 7.2.

Fermentation medium (g/L): Glucose 30, (NH ₄ ) ₂ SO ₄ 15, Na ₂ HPO ₄ 2, KH ₂ PO ₄ 2, MgSO ₄ 0.5, pH 7.2.

Insert Bacillus sp L-23 and recombinant strains into the seed medium respectively, and after culturing for 12 hours, insert them into the fermentation medium with an inoculation amount of 10% by volume fraction respectively, and ferment 500 mL of the Erlenmeyer flask, The liquid volume is 100mL, the temperature is 35°C, and the fermentation time is 48 hours.

Determination of Bacteria Concentration in Fermentation Broth

Take the culture solution and centrifuge it at 6000g for 30 minutes, make a bacterial suspension with an equal amount of distilled water, then dilute, measure the OD value of the bacterial suspension at 660nm with a 722-type spectrophotometer, and prepare a standard curve in advance to obtain the standard curve The equation, and then measure the bacterial concentration in the fermentation broth accordingly.

Crude oil viscosity reduction experiment: add different concentrations of Bacillus sp L-23 or recombinant bacterial fermentation broth to crude oil from different sources, incubate at 45°C for 24 hours, dehydrate with a centrifuge, and then measure with a viscometer Viscosity change, to find the viscosity reduction rate. For crude oils from different sources, the viscosity-reducing effect of bacteria is different, which may be caused by the different components of these crude oils. Crude oil is rich in hydrocarbons, colloids, asphaltenes and other substances, which make crude oil have a certain viscosity. When bacteria grow in crude oil, they can use components in crude oil as the carbon source for their growth. Therefore, there are two possibilities for the analysis of the reasons why they can reduce the viscosity of crude oil: one is to degrade the hydrocarbons and other substances in crude oil to make The viscosity of crude oil decreases; the second is that microorganisms use crude oil to produce some metabolites, and the effect of these products reduces the viscosity of crude oil.

Through the comparison of the wild bacteria (Bacillus sp L-23) and the recombinant bacteria transferred into the PHB synthetic gene, it can be seen that the viscosity-reducing effect of the recombinant bacteria on crude oil is more obvious. Taking the result of a crude oil as an example, The results showed that the growth of the recombinant bacteria in crude oil was better than that of Bacillus sp L-23, so the effect of reducing the viscosity of crude oil was better. This is because the transfer of the PHB synthesis gene makes the bacteria more resistant to osmotic pressure and can grow better in crude oil.

Table 1 Comparison of the viscosity-lowering effect of wild bacteria and recombinant bacteria

Example 27, expressing phbCAB in Lactobacillus acidophilus improves the survival rate of bacteria under acidic conditions

Species: Lactobacillus acidophilus (Lac tobacillus acidophilus) ATCC 53671.

The plasmid pEUHB obtained according to the method of Example 1 was transferred to Lactobacillus acidophilus (Lactobacillus acidophilus) ATCC 53671 by electroporation to obtain recombinant bacteria (Lactobacillus acidophilus (Lactobacillus acidophilus) ATCC 53671 containing pEUHB) for fermentation experiment .

The experimental method is as follows:

Strain subculture medium: 12.8g of skim milk powder was dissolved in 100mL of distilled water, and sterilized at 121°C for 15min.

Counting medium: beef extract 0.5%, peptone 1.0%, yeast extract 0.6%, glucose 0.5%, agar 1.8%, pH 6.8, sterilized at 121°C for 20 minutes.

Soybean juice preparation: Soak soybeans at room temperature (about 20°C) for 48 hours to fully swell the soybeans, refine the pulp (water: beans = 3:1), filter with two layers of gauze to remove the bean dregs. Sterilize at 115°C for 15 minutes.

Preparation of soybean juice with different pH values: Using lactic acid as a regulator, adjust the pH value of soybean juice to 4.0, 4.5, 5.0 or 5.5. The natural pH value of soybean juice is about 6.5. The preparation is carried out on a magnetic stirrer and monitored with a pH meter.

Preservation experiment: Lactobacillus acidophilus (Lactobacillus acidophilus) ATCC 53671 and recombinant bacteria were cultured in soybean juice medium (pH 6.5) for 48 hours, and then the bacterial liquid was added to different pH values (4.0, 4.5, 5.0, 5.5) In the sterilized soybean juice, the number of viable bacteria was adjusted to 1×10 ⁷ -10 ⁸ cfu/ml, stored in a refrigerator at 4°C for 10 weeks, and the change of the number of viable bacteria was detected. Among them, the counting method: according to the difference in the amount of bacteria in the sample, make a 10-fold serial concentration dilution, pour the plate, incubate at a constant temperature of 37°C for 72 hours, and count the number of colonies. Calculation of survival rate: Survival rate=the number of viable bacteria in the sample/the number of initial viable bacteria in the sample×100%.

The results showed that Lactobacillus acidophilus (Lactobacillus acidophilus) ATCC 53671 was preserved in soybean juice with different pH values, and the survival rate was different. The survival rate was the lowest (2%) at pH 4.0, and the highest (11%) at pH 5.0. When stored in pH 4.5-pH 5.5, the survival rate does not change significantly and is about 9%. The results of the 10-week storage experiment showed that the survival rate at pH 5.5 (9%) was only less than double that at pH 4.5 (8.2%). In terms of the number of viable bacteria, the number of viable bacteria at pH 4.0 can still be maintained above 1×10 ⁶ cfu/ml after 10 weeks, and the number of viable bacteria at pH 4.5-5.5 is above 1×10 ⁷ cfu/ml.

The survival rate of the recombinant bacteria did not change significantly at several pHs (4.0, 4.5, 5.0, 5.5) in the experiment, and they were all around 20%. The number of live bacteria was significantly higher than that of wild bacteria. The numbers were above 1×10 ⁷ cfu/ml. It indicated that the synthesis of PHB increased the number of viable bacteria under acidic conditions.

Recombinant Lactobacillus acidophilus has a higher survival rate and number of viable bacteria under lower pH value (4.0, 4.5) culture conditions, which is of great significance in production: on the one hand, it can improve the survival rate and make the preparation have a higher Viable Bacteria: On the other hand, a lower pH environment prevents bacterial contamination.

Example 28, the expression of genes related to the synthesis of PHA from the fatty acid β-oxidation pathway in Pseudomonas fluorescens affects the production of gluconic acid

Species: Pseudomonas fluorescens AS 1.55.

The composition of the slant medium is: beef extract 0.5%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.0.

The composition of the seed medium is: 2% glucose, 1% corn steep liquor, 0.2% urea, 0.2% KH ₂ PO ₄ , 0.05% MgSO ₄ ·7H ₂ O, pH 7.0.

The composition of the fermentation medium is: starch hydrolysis sugar 14%, corn steep liquor 1.5%, light calcium carbonate 4%, pH 6.7.

Under standard polymerase chain reaction conditions, the biosynthesis gene of Aeromonas caviae.J.Bacteriol., 1997, 179 : 4821-4830) as a template, using primers TAAGGTACCGAAGGAGAGCACATGAGCCA and TCTAAGCTTGGCTGATTGTGCCTGCGTG to amplify the phaCJ gene, and then after KpnI and HindIII digestion, insert it into the recognition site of the restriction endonuclease KpnI and HindIII of the plasmid pBBR1MCS2 to obtain a plasmid pBBRCJ. pBBRCJ was transformed into Pseudomonas fluorescens AS 1.55 by electrotransformation to obtain recombinant bacteria (Pseudomonas fluorescens AS 1.55 containing pBBRCJ) for fermentation experiments.

Pseudomonas fluorescens (Pseudomonas fluorescens) AS 1.55 and recombinant bacteria cultured on fresh slant were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively. The % (v/v) inoculum was added to the fermentation medium respectively. A 500 mL fermentation bottle with a liquid volume of 50 mL was cultured at 30° C. and 230 rpm for 72 hours, and the content of ketogluconic acid in the fermentation liquid was determined. The content of ketogluconic acid in the fermentation broth is determined by optical polarimetry: the content of ketogluconic acid in the fermentation broth = the absolute value of the optical rotation of the fermentation broth at 25° C./0.88.

The results show that the content of ketogluconic acid in the fermentation broth of Pseudomonas fluorescens AS 1.55 is 13.6g/L, and the output of the recombinant bacteria is 15.8g/L, which is about 16% higher. The introduction of the phaCJ gene can The accumulation of PHA in the cells is beneficial to improve the tolerance of the bacteria, grow better, and obtain a higher conversion rate.

Example 29, the expression of genes related to the synthesis of PHA from fatty acid de novo synthesis pathway in Bacillus subtilis affects the biosynthesis of inosine

Bacterial species: Bacillus subtilis ATCC 19162.

The composition of the seed medium is glucose 20g/L, yeast powder 15g/L, peptone 10g/L, corn solution 7g/L, sodium chloride 2.5g/L, urea 2g/L, pH 7.0.

The composition of the fermentation medium is glucose 140g/L, corn liquid 16g/L, yeast powder 16g/L, urea 9g/L, ammonium sulfate 16g/L, magnesium sulfate heptahydrate 4g/L, dipotassium hydrogen phosphate 5g/L, carbonic acid Calcium 20g/L, pH 7.0.

Under standard polymerase chain reaction conditions, using the plasmid pQH-CG (Qiu Yuanzheng, Genetic engineering transformation of polyhydroxybutyrate hydroxyhexanoate biosynthetic pathway, Tsinghua University, 2005, doctoral dissertation.) as a template, with primers The phaGC gene was amplified by ATACGACTCACTATAGGGC and AGTGCCAGATCTCGCAACGCAATTAATG, then digested with BamHI and BglII, and inserted between the recognition sites of restriction endonucleases BglII and BamHI of plasmid pEU308 to obtain plasmid pEUGC. pEUGC was transformed into Bacillus subtilis (Bacillus subtilis) ATCC 19162 by the method of lithium acetate transformation to obtain recombinant bacteria (Bacillus subtilis (Bacillus subtilis) ATCC 19162 containing pEUGC) for fermentation experiments.

Bacillus subtilis (Bacillus subtilis) ATCC 19162 and recombinant bacteria cultured on fresh slopes were inoculated into seed medium (50mL/500mL Erlenmeyer flask) respectively. (v/v) The inoculum size was inserted into the fermentation medium. The 7.5L fermentation tank has a liquid volume of 3L, automatic temperature control of 35°C, pH 6.5, stirring speed of 600rpm, ventilation rate of 1L/min, and fermentation time of 68 hours (no increase in inosine production), and the content of inosine in the fermentation broth was determined. The inosine content was determined by HPLC, the mobile phase was 0.5% dipotassium hydrogen phosphate, the flow rate was 1.2mL/min, the chromatographic column was a Hypersil ODS C18 reverse-phase column, and the detection wavelength was 254nm. The results showed that the inosine yield of Bacillus subtilis (Bacillus subtilis) ATCC 19162 was 19.7g/L, and the yield of the recombinant strain was 24.9g/L, an increase of 26.4%. Among intracellular metabolic pathways, the monophosphate hexose pathway that provides precursors for inosine synthesis consumes a large amount of NADP, while the regulatory enzyme 6-phosphate glucose dehydrogenase from glucose 6-phosphate to pentose phosphate pathway is regulated by NADPH Feedback inhibition, thus reduction of NADPH can promote the conversion of glucose to pentose phosphate pathway. In general, inosine synthesis is a biochemical metabolic process that requires a large amount of oxidative power, and high NADP/NADPH levels can effectively promote inosine synthesis. The introduction of the phaG C gene can promote the de novo synthesis of fatty acids and increase the oxidative power in cells, thus significantly increasing the production of inosine.

Example 30. Expression of phbCAB gene in Zymomonas mobilis affects ethanol production

Strain: Zymomonas mobilis NRRL B-4286.

The composition of the slant medium is: glucose 10g/L, peptone 10g/L, yeast powder 15g/L, potassium dihydrogen phosphate 1g/L, ammonium sulfate 1g/L, magnesium sulfate heptahydrate 1g/L, NaCl 1g/L, agar 15g/L, pH 7.0.

The composition of the seed medium is: glucose 100g/L, yeast powder 15g/L, peptone 10g/L, pH 7.0.

The composition of the fermentation medium is: glucose 200g/L, yeast powder 15g/L, peptone 10g/L, pH 7.0.

The plasmid pBBRHB obtained according to the method of Example 11 was transferred to Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 by electroporation to obtain recombinant bacteria (Zymomonas mobilis (Zymomonas mobilis) NRRL containing pBBRHB B-4286) for fermentation experiments.

Zymomonas mobilis (Zymomonas mobilis) NRRL B-4286 and recombinant bacteria cultured on fresh slant were respectively inoculated into seed medium (50mL/500mL Erlenmeyer flask), and cultured at 30°C and 200rpm for 16 hours, The inoculum was respectively inserted into the fermentation medium with 10% (v/v) inoculum. A 500 mL fermentation bottle was filled with a liquid volume of 50 mL, cultured at 30° C. and 200 rpm for 72 hours, and the ethanol content in the fermentation liquid was measured. The ethanol concentration is measured by high-performance liquid chromatography: the fermentation broth is centrifuged at 8000g for ten minutes, and the supernatant is filtered as a sample; the chromatographic column is a Beckman L2Spherogel ligand exchange column, and the mobile phase is 98% concentrated sulfuric acid: H ₂ O (volume ratio 0.5:1000), flow rate 1mL/min.

The results showed that the ethanol concentration of the fermented liquid of wild bacteria was 14%, and that of recombinant bacteria was 18%, which was increased by 28%. The introduction of phbCAB gene could not only change the metabolic flow in cells, but also the synthesis of PHA could improve the bacteria's ability to treat high Tolerance of the ethanol environment, thereby increasing the production of ethanol.

sequence listing

<160>1

<210>1

<211>3884

<212>DNA

<213>Wautersia eutropha

<400>1

ataaagctta aggaggatgg cgaccggcaa aggcgcggca gcttccacgc aggaaggcaa 60

gtcccaacca ttcaaggtca cgccggggcc attcgatcca gccacatggc tggaatggtc 120

ccgccagtgg cagggcactg aaggcaacgg ccacgcggcc gcgtccggca ttccgggcct 180

ggatgcgctg gcaggcgtca agatcgcgcc ggcgcagctg ggtgatatcc agcagcgcta 240

catgaaggac ttctcagcgc tgtggcaggc catggccgag ggcaaggccg aggccaccgg 300

tccgctgcac gaccggcgct tcgccggcga cgcatggcgc accaacctcc catatcgctt 360

cgctgccgcg ttctacctgc tcaatgcgcg cgccttgacc gagctggccg atgccgtcga 420

ggccgatgcc aagacccgcc agcgcatccg cttcgcgatc tcgcaatggg tcgatgcgat 480

gtcgcccgcc aacttccttg ccaccaatcc cgaggcgcag cgcctgctga tcgagtcggg 540

cggcgaatcg ctgcgtgccg gcgtgcgcaa catgatggaa gacctgacac gcggcaagat 600

ctcgcagacc gacgagagcg cgtttgaggt cggccgcaat gtcgcggtga ccgaaggcgc 660

cgtggtcttc gagaacgagt acttccagct gttgcagtac aagccgctga ccgacaaggt 720

gcacgcgcgc ccgctgctga tggtgccgcc gtgcatcaac aagtactaca tcctggacct 780

gcagccggag agctcgctgg tgcgccatgt ggtggagcag ggacatacgg tgtttctggt 840

gtcgtggcgc aatccggacg ccagcatggc cggcagcacc tgggacgact acatcgagca 900

cgcggccatc cgcgccatcg aagtcgcgcg cgacatcagc ggccaggaca agatcaacgt 960

gctcggcttc tgcgtgggcg gcaccattgt ctcgaccgcg ctggcggtgc tggccgcgcg 1020

cggcgagcac ccggccgcca gcgtcacgct gctgaccacg ctgctggact ttgccgacac 1080

gggcatcctc gacgtctttg tcgacgaggg ccatgtgcag ttgcgcgagg ccacgctggg 1140

cggcggcgcc ggcgcgccgt gcgcgctgct gcgcggcctt gagctggcca ataccttctc 1200

gttcttgcgc ccgaacgacc tggtgtggaa ctacgtggtc gacaactacc tgaagggcaa 1260

cacgccggtg ccgttcgacc tgctgttctg gaacggcgac gccaccaacc tgccggggcc 1320

gtggtactgc tggtacctgc gccacaccta cctgcagaac gagctcaagg taccgggcaa 1380

gctgaccgtg tgcggcgtgc cggtggacct ggccagcatc gacgtgccga cctatatcta 1440

cggctcgcgc gaagaccata tcgtgccgtg gaccgcggcc tatgcctcga ccgcgctgct 1500

ggcgaacaag ctgcgcttcg tgctgggtgc gtcgggccat atcgccggtg tgatcaaccc 1560

gccggccaag aacaagcgca gccactggac taacgatgcg ctgccggagt cgccgcagca 1620

atggctggcc ggcgccatcg agcatcacgg cagctggtgg ccggactgga ccgcatggct 1680

ggccgggcag gccggcgcga aacgcgccgc gcccgccaac tatggcaatg cgcgctatcg 1740

cgcaatcgaa cccgcgcctg ggcgatacgt caaagccaag gcatgacgct tgcatgagtg 1800

ccggcgtgcg tcatgcacgg cgccggcagg cctgcaggtt ccctcccgtt tccatgaaa 1860

ggactacaca atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt 1920

tggcggctcg ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc 1980

gctggagcgc gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct 2040

gaccgccggt tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc 2100

gatggtgccg gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct 2160

ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa 2220

catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc 2280

caagctggtc gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat 2340

gggcatcacc gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga 2400

gttcgccgtc ggctcgcaga acaaggccga agccgcgcag aaggccggca agtttgacga 2460

agagatcgtc ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga 2520

cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga 2580

caaggccggc acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt 2640

ggtggtgatg tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa 2700

gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc 2760

caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac ctggacctga tggagatcaa 2820

cgaggccttt gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa 2880

ggtcaatgtg aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg 2940

tatcctggtg acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc 3000

gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aaggaagggg 3060

ttttccgggg ccgcgcgcgg ttggcgcgga cccggcgacg ataacgaagc caatcaagga 3120

gtggacatga ctcagcgcat tgcgtatgtg accggcggca tgggtggtat cggaaccgcc 3180

atttgccagc ggctggccaa ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg 3240

ccgcgccgcg aaaagtggct ggagcagcag aaggccctgg gcttcgattt cattgcctcg 3300

gaaggcaatg tggctgactg ggactcgacc aagaccgcat tcgacaaggt caagtccgag 3360

gtcggcgagg ttgatgtgct gatcaacaac gccggtatca cccgcgacgt ggtgttccgc 3420

aagatgaccc gcgccgactg ggatgcggtg atcgacacca acctgacctc gctgttcaac 3480

gtcaccaagc aggtgatcga cggcatggcc gaccgtggct ggggccgcat cgtcaacatc 3540

tcgtcggtga acggggcagaa gggccagttc ggccagacca actactccac cgccaaggcc 3600

ggcctgcatg gcttcaccat ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc 3660

aacacggtct ctccgggcta tatcgccacc gacatggtca aggcgatccg ccaggacgtg 3720

ctcgacaaga tcgtcgcgac gatcccggtc aagcgcctgg gcctgccgga agagatcgcc 3780

tcgatctgcg cctggttgtc gtcggtttct cgaccggcgc cgacttctcg 3840

ctcaacggcg gcctgcatat gggctgacct gccggatcga taac 3884

Claims (2)

1. A method for improving microbial stress resistance is to synthesize polyhydroxyalkanoate in microorganisms; the synthesis of said polyhydroxyalkanoate is by introducing the relevant gene of polyhydroxyalkanoate synthesis pathway in said microorganism realized; Described microorganism is Zymomonas mobilis (Zymomonas mobilis); The relevant gene of the polyhydroxyalkanoate synthesis pathway is the phbCAB gene, and the base sequence of the phbCAB gene is shown in SEQ ID NO: 1; The adversity stress is low pH stress or high osmotic pressure stress. 2. The method according to claim 1, characterized in that: said Zymomonas mobilis is Zymomonas mobilis NRRL B-4286.