CN118620969B

CN118620969B - A two-stage biological method for producing xylitol using hemicellulose hydrolysate

Info

Publication number: CN118620969B
Application number: CN202411121164.XA
Authority: CN
Inventors: 李培清; 徐瑶; 汤海斌; 高京伟; 苏飞虎
Original assignee: Zhejiang Rongrui Technology Co ltd
Current assignee: Zhejiang Rongrui Technology Co ltd
Priority date: 2024-08-15
Filing date: 2024-08-15
Publication date: 2025-03-04
Anticipated expiration: 2044-08-15
Also published as: CN118620969A

Abstract

The invention provides a method for producing xylitol by using hemicellulose hydrolysate two-stage biological method, which comprises the steps of taking hemicellulose hydrolysate as a raw material, carrying out first-stage fermentation culture in a fermentation culture medium inoculated with fermentation engineering bacteria until no L-arabinose residues exist in the fermentation liquid to obtain fermentation reaction liquid I, adding glucose feed and inducer into the fermentation reaction liquid I, and continuing to carry out second-stage fermentation culture to obtain a fermentation product containing xylitol. The method utilizes the way of producing xylitol by the metabolism of L-arabinose constructed in fermentation engineering bacteria and the time node control of the fermentation process, so that both L-arabinose and D-xylose can be fully utilized and converted into xylitol, the two-stage biological process for producing xylitol by taking hemicellulose hydrolysate as raw materials is realized, the yield of xylitol in the hemicellulose hydrolysate is obviously improved, and a new xylitol production process with better popularization is provided for the xylitol production industry.

Description

Method for producing xylitol by hemicellulose hydrolysate two-stage biological method

Technical Field

The invention relates to the technical field of biology, in particular to a method for producing xylitol by using a hemicellulose hydrolysate two-stage biological method.

Background

Xylitol (Xylitol) is a kind of natural pentose alcohol (molecular formula C ₅H₁₂O₅, molecular weight 152.15), white powdery crystal substance, which is an intermediate product of normal metabolism of human body, and is also present in various fruits, yeasts, moss, seaweed, mushrooms and vegetables, but in lower content. At present, xylitol has wide application in foods, medicines, cosmetics and health care products, and the application with the largest consumption is used as a natural sweetener in the food industry. With the improvement of the living standard of people, obesity becomes a common problem which puzzles the public. Consumers are increasingly favoured with low-sugar low-calorie foods, and xylitol has caries preventing effect, so that the xylitol is increasingly applied to food additives and the demand is increased.

The xylitol is produced by catalytic hydrogenation under the conditions of chemical high temperature and high pressure mainly by using xylose from hemicellulose hydrolysate as a raw material. The high preparation cost promotes the selling price of the xylitol and becomes a main factor for restricting the wider application of the xylitol. The biological method for producing xylitol is a novel process for preparing xylitol, has mild reaction conditions and lower production cost, and is expected to replace the traditional chemical hydrogenation production process.

In recent years, attention has been paid to the use of the materials at home and abroad. The xylitol is produced by a biological method mainly by utilizing microorganisms such as bacteria, fungi, yeast or recombinant genetic engineering bacteria, and the like, and different biomass raw materials are used for fermentation to obtain xylitol products. For example, chinese patent application publication No. CN104357339A discloses a candida tropicalis, which is used for producing xylitol by fermenting with high-concentration xylose as the only carbon source, wherein the concentration of xylitol can reach 20-65 g/L, and the concentration of residual sugar is lower than 5%. The Chinese patent publication No. CN110982850B discloses an Aspergillus oryzae genetically engineered bacterium, which takes 50 g/L xylan as the only carbon source, and the concentration of xylitol produced is 13.5 g/L, and the yield and the productivity are 0.27 g/g and 0.16 g/L/h respectively. The Chinese patent publication No. CN106661540B discloses recombinant Pichia pastoris, which takes 250 g/L glucose monohydrate as a raw material to produce 120 g/L xylitol and 5 g/L ribitol, wherein the yield and the productivity of the xylitol are respectively 0.48 g/g and 1.81 g/L/h.

The biomass raw material with the largest five-carbon sugar in nature is hemicellulose, and the preparation of xylitol by hemicellulose hydrolysate is the most economical scheme. Patent applications with publication numbers of CN108949839A and CN111440830A disclose techniques for preparing xylitol by fermenting corncob hydrolysate (hemicellulose hydrolysate) with escherichia coli genetic engineering bacteria. However, hemicellulose is a polysaccharide having a main chain of 1,4- β -D-xylopyranose and 4-oxymethyl-glucopyranose aldehyde acid as a branched chain, and may further contain L-furanosyl group as a branched chain, and is linked to the main chain of xylo-oligosaccharide. Therefore, hemicellulose hydrolysate mainly contains D-xylose (D-Xylose) and various miscellaneous sugars such as Glucose (Glucose), L-arabinose (L-Arabinose) and the like. In particular to the L-arabinose which can generate L-arabitol after being metabolized by microorganisms and enter the subsequent working section of xylitol production, thereby increasing the refining difficulty of xylitol products and increasing the manufacturing cost of xylitol products. Therefore, development of genetically engineered bacteria and fermentation process thereof is urgently needed to accelerate metabolism rate of mixed sugar, avoid production of impurity L-arabitol, and improve yield and productivity of xylitol.

Disclosure of Invention

The invention provides a method for producing xylitol by using a hemicellulose hydrolysate two-stage biological method, which utilizes a way for producing xylitol by metabolism of L-arabinose constructed in fermentation engineering bacteria and time node control of fermentation process, so that the L-arabinose can be fully utilized and converted into xylitol, a two-stage biological method xylitol production process using the hemicellulose hydrolysate as a raw material is realized, the yield of xylitol in the hemicellulose hydrolysate is obviously improved, and a new xylitol production process with better popularization is provided for the xylitol production industry.

The specific technical scheme is as follows:

the invention provides a method for producing xylitol by using hemicellulose hydrolysate two-stage biological method, which comprises the following steps:

(1) Taking hemicellulose hydrolysate as a raw material, and performing first-stage fermentation culture in a fermentation culture medium inoculated with fermentation engineering bacteria until no L-arabinose residue exists in the fermentation liquid to obtain fermentation reaction liquid I;

(2) And (3) adding glucose feed and an inducer into the fermentation reaction liquid I, and continuing to perform second-stage fermentation culture to obtain a fermentation product containing xylitol.

The method of the invention utilizes the expression regulation of functional genes in the fermentation engineering bacteria and the control of glucose feed supplement and inducer addition time to divide the fermentation stage of fermenting hemicellulose hydrolysate by the fermentation engineering bacteria to produce xylitol into two stages, wherein in the first stage, cells are rapidly increased in value and metabolize L-arabinose to produce xylitol, and in the second stage, cells are induced to produce xylose reductase, so that xylose and glucose are co-metabolized to produce xylitol.

Further, the fermentation engineering bacteria comprise host cells containing xylose reductase XR genes and target genes coexpressed in the host cells, wherein the target genes comprise L-arabinose isomerase genes araA, D-psicose-3-epimerase genes dpe and L-xylulose reductase genes lxr.

The method of the invention utilizes fermentation engineering bacteria to convert hemicellulose hydrolysate to produce xylitol, and the method is divided into two stages, wherein the first stage is used for completely converting L-arabinose into xylitol under the action of L-arabinose isomerase, D-psicose-3-epimerase, L-xylulose reductase and NADH expressed by the fermentation engineering bacteria, and the second stage is used for completely converting unreacted xylose in the hemicellulose hydrolysate into xylitol under the action of xylose reductase induced and expressed by the fermentation engineering bacteria. After the two stages are finished, the conversion rate of the raw materials is more than 99%, and the fermentation product has no impurity sugar residue and no L-arabitol. The method simplifies the refining process of xylitol, and improves the production efficiency and product yield of xylitol production from hemicellulose hydrolysate.

Further preferably, the nucleotide sequence of the gene araA is shown as SEQ ID NO.1, the nucleotide sequence of the gene dpe is shown as SEQ ID NO.4, and the nucleotide sequence of the gene lxr is shown as SEQ ID NO. 6.

Further preferably, the nucleotide sequence of the 5' untranslated region of the gene araA is shown as SEQ ID NO. 3.

In the invention, the L-arabinose isomerase is selected from L-arabinose isomerase gene araA from E.coli W3110, the nucleotide sequence of which is shown as SEQ ID NO.1, the amino acid sequence of which is shown as SEQ ID NO.2, and the 5' untranslated region (5 ' UTR) of which is modified to obtain mutated 5' UTR sequence (abbreviated as araA17 in the case of the invention) the nucleotide sequence of which is shown as SEQ ID NO. 3. The D-psicose-3-epimerase is selected from D-psicose-3-epimerase gene dpe derived from Rhizobium sp.RM, the nucleotide sequence is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 5. The L-xylulose reductase is selected from L-xylulose reductase gene lxr of Brettanomyces Brettanomyces bruxellensisAWRI1499, the nucleotide sequence is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 7.

Further, the mode of co-expression of the target gene in the host cell is one of the following:

(A) Co-expression in the form of a recombinant plasmid in a host cell;

(B) Is inserted into the host cell genome in the form of an expression cassette for co-expression.

The means for co-expressing the genes araA, dpe and lxr include plasmid co-expression and genome integration, and more preferably, the recombinant plasmid is pBAD18.

Further, in (B), the desired gene is inserted into a non-coding region of the host cell genome, or the ptsG gene, ptsF gene, xylA and xylB gene are randomly replaced by the genes araA, dpe and lxr.

The genome integration type co-expression is to replace glucose transport, metabolism related genes ptsG and ptsF and xylose metabolism related genes xylA and xylB in the engineering bacterium genome with an arabinose inducible promoter pBAD and araA, dpe and lxr gene co-expression cassettes respectively, so as to integrate the genes into the engineering bacterium genome. In order to improve the co-expression efficiency, the gene can be integrated into the engineering bacterium genome in a multi-copy mode, and the gene is integrated into the engineering bacterium genome in a 3-copy mode in the invention as an example.

Wherein, the substitution of ptsG slows down CCR effect, improves the co-metabolism efficiency of glucose and other saccharides, and the metabolism of glucose generates a cofactor NADPH, thereby ensuring the supply of NADPH when xylose reductase catalyzes the reduction of xylose into xylitol. Substitution of ptsF can reduce extracellular to intracellular transport of xylitol, and substitution of xylA and xylB can block downstream catabolic pathways of xylose. Meanwhile, the expression of the replaced L-arabinose isomerase gene (araA 17), D-psicose-3-epimerase gene (dpe 1) and L-xylulose reductase gene (lxr 8) strengthens the efficiency of producing xylitol by L-arabinose metabolism.

Furthermore, the nucleotide sequence of the ptsG gene is shown as SEQ ID NO.20, and the nucleotide sequence of the ptsF gene is shown as SEQ ID NO. 21.

Furthermore, the xylA gene and the xylB gene are connected together, and when the genes are replaced, the two genes are replaced together, and the two genes of the xylA gene and the xylB gene are combined to be called xylAB gene, and the nucleotide sequence of the xylAB gene is shown as SEQ ID NO. 22.

The host cell adopted by the method can be microorganisms such as escherichia coli, corynebacterium glutamicum, bacillus subtilis, pichia pastoris, saccharomyces cerevisiae and the like, and further the host cell provided by the invention is escherichia coli. Still further, the host cell provided by the invention is E.coli W3110.

In order to further enhance the yield of L-arabinose metabolism and xylitol production, the invention further knocks out related genes araB, araD and lyxK of other L-arabinose metabolism paths.

Thus, further, at least one of the araB gene, araD gene and lyxK gene is knocked out in the genome of the host cell.

Further, the nucleotide sequence of the gene araB is shown as SEQ ID NO.12, the nucleotide sequence of the gene araD is shown as SEQ ID NO.13, and the nucleotide sequence of the gene lyxK is shown as SEQ ID NO. 14.

Since the L-arabinose metabolism xylitol production pathway needs to consume the cofactor NADH, in order to improve the efficiency of L-arabinose metabolism xylitol production, the invention integrates a glycerol transporter gene glpF and a glycerol kinase gene glpK coexpression cassette and a glycerol triphosphate dehydrogenase gene glpD expression cassette into an untranslated region of an engineering bacterium genome so as to strengthen the glycerol metabolism xylitol production cofactor NADH.

Further, at least one of the glycerol transporter gene glpF, the glycerol kinase gene glpK and the glycerol triphosphate dehydrogenase gene glpD is inserted into the genome non-coding region of the host cell.

Further, the nucleotide sequence of the gene glpF is shown as SEQ ID NO.9, the nucleotide sequence of the gene glpK is shown as SEQ ID NO.10, and the nucleotide sequence of the gene glpD is shown as SEQ ID NO. 11.

In theory, the xylose reductase adopted by the invention can be a xylose reductase gene which can be functionally expressed in engineering bacteria from any source and has corresponding catalytic activity.

Further preferably, the xylose reductase gene is a xylose reductase gene derived from Candida boidinii, candida gracilis Candida tenuis CBS 4435, candida tropicalis Candida tropicalisIFO 0618, rhodotorula mucilaginosa Rhodotorula mucilaginosa, neurospora crassa Neurospora crassa, kluyveromyces lactis Kluyveromyces lactis and aspergillus niger Aspergillus niger, which are abbreviated as XR1 gene, XR4 gene, XR5 gene and XR6 gene in this order, and NCBI accession numbers are AF451326.3, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, Q9P8R5.1, respectively. And the promoter used for expressing xylose reductase may be any inducible promoter other than arabinose promoter, and further preferably, the inducible promoter is rhamnose promoter, anhydrotetracycline promoter or lactose promoter.

Further, the nucleotide sequence of the inducible promoter of the XR gene is one of the sequences shown in SEQ ID NO. 15-19.

The hemicellulose hydrolysate is obtained by hydrolyzing biomass containing rich hemicellulose, the biomass containing rich hemicellulose is corncob, papermaking short fibers, bagasse, straw, rice husk or the like, and the hemicellulose hydrolysate at least contains L-arabinose, glucose and xylose.

Further, the mass ratio of the L-arabinose to the glucose to the xylose is 5% -6%, 2% -5% and 35% -40%.

Further, in the step (1), the conditions of the first-stage fermentation culture are that the temperature is 18-37 ℃, the pH value is 6.0-8.0, and the dissolved oxygen is controlled to be maintained at 0-50% by using a dissolved oxygen meter under the initial fermentation condition after inoculation.

Further, in the step (1), the fermentation medium includes hemicellulose hydrolysate containing L-arabinose, glucose and xylose, respectively, nitrogen source (yeast powder or peptone), carbon source (glycerol or glucose), inorganic salt (such as phosphate), etc.

Further, in the step (2), the addition amount of the glucose feed is 10% -50% of xylose by the mass of xylose in the semi-hydrolysate, the inducer is one of lactose, rhamnose or anhydrotetracycline, and the addition amount of the inducer is 1 g/L-10 g/L by the initial fermentation volume.

Preferably, the inducer is lactose.

In the step (2), the fermentation culture condition of the second stage is that the temperature is 18-37 ℃, the pH value is 6.0-8.0, and the dissolved oxygen is maintained to be 0-50% based on 100% of the dissolved oxygen in the initial fermentation culture condition after inoculation.

In the step (1), the OD ₆₀₀ of the fermentation engineering bacteria inoculated to the fermentation medium is 0.6-0.8, and the inoculation amount is 1-15% based on the initial fermentation volume of the fermentation engineering bacteria in the tank.

In the invention, a 15L fermentation tank is used as a fermentation container, and fermentation engineering bacteria are inoculated into a fermentation medium containing hemicellulose hydrolysate for two-stage fermentation;

the fermentation medium in the step (1) at least comprises a carbon source, a nitrogen source and inorganic salt, wherein the addition amount of the carbon source is 10-50 g/L, the addition amount of the nitrogen source is 5-30 g/L, the carbon source is preferably glycerol, and the addition amount of the glycerol is preferably more than 0.6 times of the mass of arabinose based on the initial fermentation volume;

In the step (2), the xylose content in the hydrolysate is taken as a reference, the adding amount of the glucose feed is 10-50% of that of xylose by mass percent, the inducer is one of lactose, rhamnose or anhydrotetracycline, and the adding amount of the inducer is 1-g/L-10 g/L by volume percent by taking the initial fermentation volume as a reference.

Further, in the first stage, the components and the concentrations of the components in a fermentation medium are Na ₂HPO₄6～10 g/L,KH₂PO₄ -5 g/L, glycerol 6-15 g/L, yeast powder 10-30 g/L and peptone 5-15 g/L, the final concentration of xylose in hemicellulose hydrolysate is 100-150 g/L, the fermentation culture temperature is 18-37 ℃ and the fermentation culture time is 10-20 h;

in the second stage, the fermentation reaction liquid I has no L-arabinose, the addition concentration of an inducer is 3-5 g/L, the addition concentration of glucose is 10-75 g/L, the fermentation culture temperature is 18-37 ℃ and the fermentation culture time is 20-60 h.

Compared with the prior art, the invention has the following beneficial effects:

(1) The two-stage biological fermentation method for producing xylitol has simple and convenient process, and the xylose reductase gene expressed in the used genetically engineered bacteria has wide sources and does not need to mutate and reform the substrate selectivity.

(2) The conversion rate of L-arabinose and xylose of the genetically engineered bacterium fermented hemicellulose hydrolysate exceeds 99%, no impurity sugar remains, and no L-arabitol exists in the product, so that the problems of complex process, high separation difficulty and high cost in the xylitol refining process are successfully solved.

(3) The invention can fully convert the substrate L-arabinose in the hemicellulose hydrolysate into xylitol during fermentation of the genetically engineered bacteria, fully utilizes the impurity sugar in the hemicellulose hydrolysate, and further improves the yield of xylitol.

Drawings

FIG. 1 is a schematic diagram of metabolic pathways for producing xylitol by using a hemicellulose hydrolysate two-stage biological method according to the invention.

FIG. 2 is a schematic diagram of the metabolic pathway of L-arabinose to xylitol.

FIG. 3 is a graph showing the process of producing xylitol by fermenting hemicellulose hydrolysate with Escherichia coli fermentation engineering bacteria in example 6.

FIG. 4 is a HPLC detection chart of a standard sample in example 6, wherein 11.6 min out of the peak and 12.7 min out of the peak and 12.7 and min out of the peak and 18.1 and min out of the peak and 21.2 and min out of the peak are respectively D-glucose, D-xylose, L-arabinose and xylitol.

FIG. 5 is an HPLC detection chart of hemicellulose hydrolysate fermentation 0 h in example 6, wherein 11.6 min peaks at D-glucose, 12.7 min peaks at D-xylose, and 14.4 min peaks at L-arabinose.

FIG. 6 is an HPLC detection chart of hemicellulose hydrolysate fermentation 48 h in example 6, wherein 12.7 min peaks at D-xylose and 21.0 min peaks at xylitol.

FIG. 7 is an HPLC detection chart of hemicellulose hydrolysate fermentation 60 h in example 6, wherein 20.7. 20.7 min shows xylitol as a peak.

Detailed Description

The invention is described in further detail below with reference to the drawings and the detailed description.

The experimental methods in the invention are all conventional methods unless otherwise specified, and the gene cloning operation can be specifically found in the "molecular cloning Experimental guidelines" by J.Sam Broker et al. The kit for gene manipulation was purchased from TAKARA company, and the reagents used were xylose, arabinose, glucose, xylitol, and arabitol were purchased from national pharmaceutical chemicals Co.

Sugar alcohol detection, high performance liquid chromatography measurement, AGILENT HI-Plex Ca chromatographic column (7.7 mm ×300 mm) with flow rate of 0.6 mL/min and column temperature of 85deg.C.

The names of the genes involved in the present invention are explained as follows:

ptsG, glucose phosphotransferase system enzyme II component gene;

ptsF, fructose phosphotransferase system enzyme II component gene;

xylA, xylose isomerase gene;

xylB xylulokinase gene:

an araA L-arabinose isomerase gene;

dpe D-psicose-3-epimerase gene;

lxr L-xylulose reductase gene;

araB, ribulokinase gene;

araD, L-ribulose-5-phosphate-4-epimerase gene;

lyxK L-xylulokinase gene;

glpF, glycerol transporter gene;

glpK: glycerol kinase gene;

glpD glycerol-3-phosphate dehydrogenase gene.

In E.coli, two genes of xylA gene and xylB gene are joined together, and the two genes are knocked out together at the time of gene knockout, and in the following examples, the two genes of xylA gene and xylB gene are collectively referred to as xylAB gene.

In the following examples, the final concentration of kanamycin in the medium was 50. Mu.g/mL, the final concentration of spectinomycin in the medium was 100. Mu.g/mL, and the final concentration of chloramphenicol in the medium was 25. Mu.g/mL.

In the following examples, xylitol was produced by a two-stage biological method using hemicellulose hydrolysate, and the specific manner is shown in fig. 1.

The primer sequence information used in the following examples is shown in Table 1.

TABLE 1 primer list

EXAMPLE 1 construction of pathway for producing xylitol by metabolizing L-arabinose

1. Construction of engineering bacteria for knockout of araB, araD and lyxK in genome

Referring to the method of literature （Multigene Editing in theEscherichia coliGenome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl Environ Microbiol, 2015）, knockout of the araB, araD, lyxK isogenes as shown in FIG. 2 in E.coli genome was performed to eliminate or alleviate catabolism of L-ribulose and L-xylulose, which are intermediate metabolites of the L-arabinose metabolic pathway.

(1) Construction of araB Gene knockout Strain E.coli W3110. DELTA. AraB

PCR amplification, in which ara-U-F and ara-U-R, ara-D-F and ara-D-R are used as primers, E.coli W3110 genome is used as template amplification fragments, each of which is 500 bp, glue is recovered, and two recovered 500 bp fragments are used as primers, and the recovered two recovered 500 bp fragments are used as template amplification to obtain a repair template fragment of about 1000 bp, and glue is recovered.

Constructing an ara-Target plasmid, namely, PCR (polymerase chain reaction) amplifying a fragment of about 2.1 kb by using ara-Target-F and ara-Target-R as primers and pTargetF-cadA as a template, and constructing to obtain the ara-Target plasmid after DpnI digestion.

Electrotransformation competent cell preparation a) E.coli single colonies were picked up in 5mL LB tube medium, incubated at 37℃at 200 rpm to an OD600 of about 0.6, b) bacterial cells were split into 1.5 mL tubes each, centrifuged at 4℃at 4000 rpm at 10min, the supernatant discarded, C) 1mL pre-chilled sterilized 10% glycerol was added, gently resuspended, centrifuged at 4℃at 4000 rpm at 10min, the supernatant carefully discarded, this step was repeated 2 more times, d) resuspended with 100. Mu.L 10% glycerol.

Coli W3110 was prepared using the above described electrotransformation competent preparation method, the pCas plasmid was electrotransformed (electrotransformation conditions: 2.5 kV,200Ω, 25. Mu.F) into competent cells, single colonies of W3110/pCas were picked up in 5mL LB tubes containing kanamycin and 10mM arabinose, cultured at 37℃to an OD600 of about 0.6, and then W3110/pCas electrotransformation competent was prepared using the above described method.

Electrotransformation the ara repair template fragment and ara-Target plasmid are electrotransformed into W3110/pCas competent cells, spread on LB plate containing spectinomycin and kanamycin, cultured overnight at 37 ℃, single colony is grown and colony PCR verification is carried out by using specific primer.

The plasmid is lost, colony PCR is selected to verify as positive single colony, the colony is inoculated into an LB test tube containing kanamycin, meanwhile, rhamnose with the final concentration of 10 mM ℃ is added for overnight culture at 37 ℃, bacterial liquid in the test tube is inoculated into an antibiotic-free LB test tube containing 10 g/L sucrose for overnight culture at 37 ℃, bacterial liquid in the test tube is directly streaked on an antibiotic-free LB plate for overnight culture at 37 ℃, the single colony is selected the next day and transferred onto an LB plate containing spectinomycin or kanamycin respectively, and if the colony cannot grow, the strain pCas and ara-Target plasmids are lost, and the strain W3110 delta araB is obtained.

(2) Construction of araD Gene knockout Strain E.coli W3110 delta araBAD

PCR amplification, in which the araD-U-F and araD-U-R, araD-D-F and araD-D-R are used as primers, E.coli W3110 genome is used as template to amplify fragments, each of which is 500 bp, and glue is recovered, and the araD-U-F and araD-D-R are used as primers, and two recovered 500 bp fragments are used as templates to amplify the recovered repair template fragments of about 1000 bp, and glue is recovered.

Construction of araD-Target plasmid the araD-Target plasmid was constructed by PCR amplification of a fragment of about 2.1 kb with araD-Target-F and araD-Target-R as primers and pTargetF-cadA as template and DpnI digestion.

E.coli W3110 ΔaraB competence was prepared by the electrotransformation competence preparation method described above, pCas plasmids were electrotransformed into competent cells, E.coli W3110 ΔaraB/pCas single colonies were picked up in 5 mL LB tubes containing kanamycin and 10mM arabinose, cultured at 37℃to an OD600 of about 0.6, and E.coli W3110 ΔaraB/pCas competence was prepared by the method described above.

Electrotransformation the araD repair template fragment and araD-Target plasmid were electrotransformed into W3110 delta araB/pCas competent cells, plated on LB plates containing spectinomycin and kanamycin, cultured overnight at 37℃and single colonies were grown for colony PCR validation using specific primers.

Plasmid loss two plasmids pCas and ptsF-N20 were deleted by the method described above to give E.coli W3110. DELTA. AraBAD strain.

(3) Construction of lyxK Gene knockout Strain E.coli W3110 DeltaaraBAD Delta lyxK

PCR amplification, namely amplifying fragments by taking lyxK-U-F and lyxK-U-R, lyxK-D-F and lyxK-D-R as primers and E.coli W3110 genome as templates, respectively carrying out 500 bp and glue recovery, and amplifying two recovered fragments of 500 bp by taking lyxK-U-F and lyxK-D-R as primers to obtain a repair template fragment of about 1000 bp and glue recovery.

LyxK-Target plasmid construction by PCR amplification of about 2.1 kb fragment with lyxK-Target-F and lyxK-Target-R as primers and pTargetF-cadA as template, and DpnI digestion to construct lyxK-Target plasmid.

E.coli W3110. DELTA. AraBAD competence was prepared by the electrotransformation competence preparation method described above, pCas plasmids were electrotransformed into competent cells, E.coli W3110. DELTA. AraBAD/pCas single colonies were picked up in 5 mL LB tubes containing kanamycin and 10 mM arabinose, cultured at 37℃to an OD600 of about 0.6, and E.coli W3110. DELTA. AraBAD/pCas competence was prepared by the method described above.

Electrotransformation, namely electrotransformation of lyxK repair template fragment and lyxK-Target plasmid into E.coli W3110 delta BAD/pCas competent cells, coating on LB plate containing spectinomycin and kanamycin, culturing at 37 ℃ overnight, and carrying out colony PCR verification on single colony by using specific primers.

Plasmid loss by removing pCas and lyxK-Target two plasmids by the method described above, the E.coli W3110 DeltaaraBAD Delta lyxK strain was obtained.

2. Enhancement of the glycerol-metabolizing cofactor NADH pathway (insertion of glpF, glpK and glpD into the E.coli genome)

The engineering bacteria E.coliW3110 delta araBAD delta lyxK or the engineering bacteria E.coliW3110 obtained in the step (3) are taken as starting strains, and a CRISPR/Cas9 method is utilized to integrate a glycerol transporter gene glpF and a glycerol kinase gene glpK coexpression cassette and a glycerol triphosphate dehydrogenase gene glpD expression cassette into an untranslated region of an escherichia coli genome, so that W3110 delta araBAD delta lyxK: (glpF-glpK):: glpD or W3110:: (glpF-glpK): glpD are constructed and obtained.

Example 2

1. Plasmid co-expression of araA, dpe, lxr genes

The plasmids pBAD18-kan (containing the arabinose inducible promoter Para, SEQ ID No. 8), pBAD18-dpe1 (containing the gene dpe, SEQ ID No. 4), pBAD18-lxr8 (containing the gene lxr, SEQ ID No. 6) and pBAD18-araA17 (containing the gene araA and the mutation 5'UTR,SEQ ID NO.1, SEQ ID No. 3) were extracted, respectively, the plasmids pBAD18-kan, ecoRI and SphI were digested with restriction endonucleases EcoRI and SphI, the plasmids pBAD18-dpe1, bamHI and XbaI were digested with XbaI, the plasmids pBAD18-lxr8, xbaI and SphI were digested with plasmid pBAD18-ara17, and 4 DNA fragments were electrophoresed and recovered. The 4 fragments were ligated with T4 ligase to construct the plasmid pBAD18-dpe 1-lxr-araA 17.

The plasmid was electrotransferred into strain E.coliW3110, E.coliW3110. DELTA. AraBAD. DELTA. lyxK, or E.coli W3110. DELTA. AraBAD. DELTA. lyxK: (glpF-glpK): glpD, to obtain strain E.coli W3110/pBAD18-dpe 1-lxr-araA 17, or E.coli W3110. DELTA. AraBAD. DELTA. lyxK/pBAD18-dpe 1-lxr-araA 17, or E.coli W3110. DELTA. AraBAD. DELTA. lyxK: (glpF-glpK): glpD/pBAD18-dpe1-lxr8-araA17.

2. AraA, dpe, lxr genomic co-expression (optimization of xylose and glucose xylitol production pathway)

According to xylose metabolism pathway and global regulation thereof in escherichia coli, a series of engineering bacteria are constructed on the basis of strain E.coliW3110, E.coliW3110 delta araBAD delta lyxK or E.coli W3110 delta araBAD delta lyxK (glpF-glpK) glpD, glucose transport and metabolism related genes ptsG and ptsF are replaced by dpe, lxr and araA17 gene expression cassettes respectively, and xylose metabolism related genes xylAB is replaced by dpe1, lxr8 and araA17 gene expression cassettes.

Wherein, the substitution of ptsG slows down CCR effect, improves the co-metabolism efficiency of glucose and other saccharides, the substitution of ptsF can reduce the transportation of xylitol from outside to inside, and the substitution of xylA and xylB can block the downstream catabolic pathway of xylose. Meanwhile, the expression of the 3 copies of the L-arabinose isomerase gene (araA 17), the D-psicose-3-epimerase (dpe 1) and the L-xylulose reductase (lxr 8) is replaced, so that the efficiency of producing xylitol by the metabolism of the L-arabinose is enhanced. The replacement operation of ptsF, ptsG, xylA and xylB genes in the escherichia coli genome is carried out by adopting a CRISPR/Cas9 gene editing method.

(1) Replacement of ptsG Gene

PCR amplification, wherein ptsG-u-F and ptsG-u-R, ptsG-d-F and ptsG-d-R are respectively used as primers, E.coli W3110 genome is used as a template amplified fragment, each of the primers is 500 bp, glue is recovered, ATX-F and ATX-R are used as primers, plasmid/pBAD 18-dpe 1-lxr-araA 17 is used as a template amplified fragment, about 5.1 kb is recovered, ptsG-u-F and ptsG-d-R are used as primers, three DNA fragments obtained by recovery are used as template amplified to obtain a repair template fragment of about 6.1 bp, and glue is recovered.

Construction of ptsG-N20 plasmid by PCR amplification of about 2.2 kb fragment with ptsG-N20-F and ptsG-N20-R as primers and pTargetF-cadA as template and DpnI digestion.

Electrotransformation competent cell preparation, a) E.coli single colonies were picked up in 5mL LB tube medium, incubated at 37℃with 200 rpm until OD ₆₀₀ was about 0.6, b) bacterial cells were split up 1.5mL per tube using sterilized 2mL EP tubes, centrifuged at 4℃with 4000 rpm at 10 min, the supernatant discarded, C) 1mL pre-chilled sterilized 10% glycerol was added, gently resuspended, centrifuged at 4℃with 4000 rpm at 10 min, the supernatant carefully discarded, this step was repeated 2 more times, d) resuspended with 100. Mu.L 10% glycerol. E.coli W3110 competent E.coli W3110 ΔaraBAD Δ lyxK competent E.coli W3110 ΔaraBAD Δ lyxK: (glpF-glpK): glpD competent cell was prepared by the above described electrotransformation method, pCas plasmid was electrotransformed (electrotransformation condition: 2.5 kV,200Ω, 25. Mu.F) into competent cell, E.coli W3110/pCas single colony, E.coli W3110 ΔaraBAD Δ lyxK/pCas single colony, E.coli W3110 ΔaraBAD Δ lyxK: (glpF-glpK): (glpF-pCas single colony) was cultured in an LB tube containing kanamycin and 10 mM arabinose to OD 2 of about 0.6, E.coli E.W 3110/pCas single colony was selected, E.coli W3110 ΔaraBAD Δ lyxK/pCas single colony, E.coli W3110 ΔaraBAD Δ lyxK were prepared by the above described method.

Electrotransformation the ptsG repair template fragment and ptsG-N20 plasmid were electrotransformed into E.coli W3110 DeltaaraBAD Delta lyxK: (glpF-glpK): glpD/pCas competent cells, plated onto LB plates containing spectinomycin and kanamycin, cultured overnight at 37℃and single colonies were grown for colony PCR verification using specific primers.

Plasmid loss, the colony PCR is checked to be positive single colony, inoculated into LB test tube containing kanamycin, and added with 10 mM rhamnose with final concentration, and cultured overnight, the bacterial liquid in the test tube is inoculated into a non-resistant LB test tube containing 10 g/L sucrose, and cultured overnight at 37 ℃, the bacterial liquid in the test tube is directly streaked on a non-resistant LB plate, and cultured overnight at 37 ℃, the single colony is respectively transferred onto a spectinomycin-containing or kanamycin LB plate the next day, if neither growth is possible, the pCas and ptsG-N20 plasmids are lost, and E.coli W3110 ΔptsG (araA 17-dpe-lxr 8) strain, E.coli W3110 ΔaraBAD Δ lyxK ΔptsG (araA 17-dpe-368) strain, E.coli W3110 ΔBAD Δ lyxK ΔglsG (araG 17-84) strain (araglA 17-84: pF) strain are obtained.

(2) Replacement of ptsF Gene

PCR amplification, namely taking ptsF-u-F and ptsF-u-R, ptsF-d-F and ptsF-d-R as primers, taking a W3110 genome as a template amplified fragment, respectively carrying out 500 bp and glue recovery, taking ATX-F and ATX-R as primers, taking a plasmid/pBAD 18-dpe 1-lxr-araA 17 as a template amplified fragment to carry out 5.1 kb and glue recovery, taking three DNA fragments obtained by recovery as the template amplified fragments to obtain a repair template fragment of about 6.1 bp, and carrying out glue recovery.

Construction of ptsF-N20 plasmid by PCR amplification of about 2.2 kb fragment with ptsF-N20-F and ptsF-N20-R as primers and pTargetF-cadA as template and DpnI digestion.

E.coli W3110 ΔptsG was prepared using the electrotransformation competent preparation method described above: (araA 17-dpe 1-dpe 8) competent, E.coli W3110. DELTA. AraBAD. DELTA dpe. DELTA. PtsG: (araA 17-dpe 1-dpe 8) competent, E.coli W3110. DELTA. AraBAD. DELTA dpe. DELTA. PtsG: the method comprises the steps of (araA 17-dpe) conducting electrotransformation of dpe plasmids into competent cells, picking E.collW 3110 delta ptsG: (araA 17-dpe) single colonies, E.collW 3110 delta araBAD delta dpe-dpe single colonies, E.collW 3110 delta araBAD delta dpe delta ptsG: (araA 17-dpe) delta air-post-PK): (glaF-dpe single colonies, and preparing E.collW 3110 delta post-G (delta A17-dpe) delta dpe-post after post.

Electrotransformation, namely electrotransformation of ptsF repair template fragment and ptsF-N20 plasmid into E.coliW3110△ptsG::(araA17-dpe1-lxr8)/pCas、E.coliW3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)/pCas、E.coliW3110△araBAD△lyxK△ptsG::(araA17-dpe1-lxr8)::(glpF-glpK)::glpD/pCas competent cells, coating on LB plate containing spectinomycin and kanamycin, culturing at 37 ℃ overnight, and carrying out colony PCR verification on single colony by using specific primers.

Plasmid loss by removing pCas and ptsF-N20 plasmids using the methods described above, E.coliW3110△ptsG△ptsF::2(araA17-dpe1-lxr8),E.coliW3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8),E.coliW3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD strain was obtained.

(3) Replacement of xylAB gene

PCR amplification, namely, taking xylAB-u-F and xylAB-u-R, xylAB-d-F and xylAB-d-R as primers, taking a W3110 genome as a template amplified fragment, respectively carrying out 500 bp and glue recovery, taking ATX-F and ATX-R as primers, taking a plasmid/pBAD 18-dpe 1-lxr-araA 17 as a template amplified fragment to carry out 5.1 kb and glue recovery, taking xylAB-u-F and xylAB-d-R as primers, obtaining three recovered DNA fragments as templates, amplifying to obtain a repair template fragment of about 6.1 bp, and carrying out glue recovery.

The xylAB-N20 plasmid is constructed by taking xylAB-N20-F and xylAB-N20-R as primers and pTargetF-cadA as a template, amplifying a fragment of about 2.2 kb by PCR, and constructing after DpnI digestion.

E.coli W3110 ΔptsG ΔptsF was prepared using the electrotransport competent preparation method described above: 2 (araA 17-dpe1-lxr 8) competent, E.coli W3110 ΔaraBAD Δ lyxK ΔptsG ΔptsF: 2 (araA 17-dpe 1-dpe 8) competent, E.coli W3110 ΔaraBAD Δ3932 ΔptsG ΔptsF: the single colony of E.collW 3110 ΔptsF dpe (araA 17-dpe)/dpe (glpF dpe single colony of E.collW 3110 ΔptsF dpe (araA 17-dpe)/dpe single colony of E.collW 3110 ΔarBAD Δ3932 ΔptsF (araA 17-dpe) single colony of E.collW3110 ΔarBAD Δ3932/ΔptsF (araA 17-dpe) single colony of E.collW3110 ΔarBAD Δ3932 ΔptsF (araA 17-dpe) single colony of E.collA2 (glpF-dpe) single colony of glpD/dpe single colony was prepared in a test tube containing kanamycin and 10mM arabinose, and the method of E.collW3110 ΔW2/ΔW2 (ΔA17.A2) was prepared at a 3.ΔW2/dpe. DELTA.1-dpe. Delta.1/dpe. Delta. Mu.1.3.1/dpe. Delta. Colla2. Delta. Sol2. Dpe. Delta. Sol2. GlaP.1-dpe. GlaP. Dpe) was used.

Electrotransformation, namely electrotransformation of xylAB repair template fragment and xylAB-N20 plasmid into E.coliW3110△ptsG△ptsF::2(araA17-dpe1-lxr8)/pCas,E.coliW3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)/pCas,E.coliW3110△araBAD△lyxK△ptsG△ptsF::2(araA17-dpe1-lxr8)::(glpF-glpK)::glpD competent cells, coating on LB plate containing spectinomycin and kanamycin, culturing at 37 ℃ overnight, and carrying out colony PCR verification on single colony by using specific primers.

Plasmid loss by removing pCas and xylAB-N20 by the method described above, E.coli W3110 ptsG.DELTA.ptsF.DELTA.xylAB: 3 (araA 17-dpe 1-lxr), or E.coliW3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8),E.coliW3110△araBAD△lyxK△ptsG△ptsF△xylAB::3(araA17-dpe1-lxr8)::(glpF-glpK)::glpD strain was obtained.

EXAMPLE 3 recombinant efficient expression of xylose reductase

(1) Comparison of xylose reductase from different sources

The method comprises the steps of selecting 11 wild type XR genes from different sources, carrying out escherichia coli codon optimization and total gene synthesis by Nanjing Jinsri biotechnology limited company, constructing a plasmid pET-28a, introducing the plasmid pET-28a into escherichia coli E.coli BL21 genetic engineering for shake flask fermentation and enzyme production, obtaining a xylose reductase enzyme library, and respectively measuring the enzyme activity of xylose reductase with L-arabinose as a substrate (the product is L-arabitol) and the enzyme activity of xylose with D-xylose as the substrate (the product is xylitol), wherein the results are shown in Table 2. Reference to shake flask fermentation enzyme production operations Evolution in reverse: engineering a D-xylose-specific xylose reductase. Chembiochem, 2008, 9(8): 1213-5.

TABLE 2 sources of xylose reductase and results of enzyme Activity measurements

(2) Construction of lactose-induced metabolic gene engineering bacteria for recombinant expression of xylose reductase

A) 11 genes for xylose reductase of different origin were reconstructed onto plasmid pSU2718 and the P _lac promoter was constructed upstream of the XR gene.

B) Coli E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD electrotransformation competence;

c) Adding 2-3 mu L of xylose reductase expression plasmid into the prepared electrotransformation competent cells in ice bath 5min for electrotransformation;

d) And finally, uniformly coating the strain in an LB plate containing chloramphenicol, and placing the strain in a 37 ℃ incubator for culturing overnight to obtain lactose-induced xylose reductase gene recombinant expression metabolic engineering bacteria:

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR1 (abbreviated as P1);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR2 (abbreviated as P2);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR3 (abbreviated as P3);

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR4 (abbreviated as P4);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR5 (abbreviated as P5);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR6 (abbreviated as P6);

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR7 (abbreviated as P7);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR8 (abbreviated as P8);

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR9 (abbreviated as P9);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR10 (abbreviated as P10);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR11 (abbreviated as P11).

According to the method, XR plasmids induced by the 11P _lac promoters are respectively transferred into E.coli W3110 delta ptsG delta ptsF delta xylAB::3 (araA 17-dpe1-lxr 8) strains to obtain strains P12-P22.

According to the method, the XR plasmids induced by the 11P _lac promoters are respectively transferred into E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr) strains to obtain strains P23-P33.

(3) Shaking flask fermentation experiment

A) The 33 successfully constructed monoclonal engineering bacteria are respectively picked up and cultured in a 5 mL LB-cm culture medium at 37 ℃ under 220 rpm to obtain seed liquid after 8-12 h.

B) The bacterial liquid is inoculated into a shake flask fermentation medium with an inoculum size of 2 percent, and is cultured at 37 ℃ until the OD is 1-2.

C) Adding proper amounts of L-arabinose, glycerol, xylose and glucose mother liquor into each shake flask fermentation medium, so that the concentration of the L-arabinose in the fermentation liquor is 2.5 g/L, the concentration of the glycerol is 2.5 g/L, the concentration of the xylose is 20 g/L, the concentration of the glucose is 10 g/L, and continuously culturing at 37 ℃ after substrate addition for 3-4 h until all the L-arabinose is converted into xylitol. After fermentation culture, the concentration of the fermentation substrate L-arabinose and the concentration of the product xylitol are detected, and the conversion rate of the L-arabinose is calculated.

D) After complete consumption of L-arabinose, an appropriate amount of lactose was added to each flask as inducer and the final lactose concentration in the broth was 1 g/L. After substrate addition, the culture was induced at 30℃for 24-48 h. After fermentation culture, the concentration of xylose as a fermentation substrate and the concentrations of xylitol and L-arabitol as products are detected, and the conversion rate of xylose is calculated.

The results of shake flask fermentation experiments of 33 metabolic engineering bacteria which use P _lac as a promoter and contain xylose reductase genes of different sources are shown in Table 3.

As can be seen from Table 3, before induction, all L-arabinose is almost completely converted into xylitol by P1-P11 genetically engineered bacteria, wherein the conversion rate of the strain P5 is up to 99.89%, but the conversion rate of xylose is extremely low, P23-P33 also reaches a better L-arabitol conversion rate, but is lower than P1-P11, and P12-P22 has low efficiency of converting L-arabinose into xylitol due to the existence of a natural L-arabinose decomposition metabolic pathway. When L-arabinose is converted into xylitol, lactose further induces XR expression of different sources, thereby catalyzing xylose to produce xylitol. The efficiency of catalyzing xylose by 33 wild type XR from different sources is different, the efficiency of catalyzing xylose by most wild type XR is relatively high, and after the fermentation is induced at 30 ℃ for 24-48 h, almost all xylose is converted into xylitol by the P1-P11 strain, the xylose conversion rate is up to 99.95%, and no L-arabitol is produced.

TABLE 3 shaking flask fermentation results of wild type xylose reductase strain with P _lac as promoter

Example 4 engineering bacteria construction of different inducible promoters and shake flask fermentation

(1) Construction of different inducible promoter plasmids

Plasmids expressing XR-1, XR-5 and XR-6 were constructed from the 33 plasmids constructed in example 3 using P _lac as the promoter and from shake flask fermentation, respectively, by selecting the plasmids plac-XR-1, plac-XR-5 and plac-XR-6, respectively, and constructing plasmids expressing the different inducible promoters (rhamnose promoter P _rha, anhydrotetracycline promoter P _tet, lactose promoter P _tac or P _trc).

(2) Construction of genetically engineered bacteria of different inducible promoters

A) Coli E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD electrotransformation competence;

b) Carrying out electrotransformation on the prepared electrotransformation competent cells by using an ice bath 5min, and adding 2-3 mu L of plasmids expressed by XR;

c) And finally, uniformly coating the strain in an LB plate containing chloramphenicol, and placing the strain in a 37 ℃ incubator for culturing overnight, namely, the genetically engineered strain with different inducible promoters for expressing xylose reductase genes of different sources:

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/prha-XR1 (abbreviated as P34);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/prha-XR5 (abbreviated as P35);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/prha-XR6 (abbreviated as P36);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptet-XR1 (abbreviated as P37);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptet-XR5 (abbreviated as P38);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptet-XR6 (abbreviated as P39);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptac-XR1 (abbreviated as P40);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptac-XR5 (abbreviated as P41);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptac-XR6 (abbreviated as P42);

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptrc-XR1 (abbreviated as P43);

e.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptrc-XR5 (abbreviated as P44);

E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/ptrc-XR6 (abbreviated as P45).

(3) Shaking flask fermentation experiment of engineering bacteria with different inducible promoter genes

A) The P1, P5, P6, P34, P35, P36, P37, P38, P39, P40, P41, P42, P43, P44 and P45 monoclonal bacteria are respectively picked up in a 5mL LB-cm culture medium, and are subjected to shaking 220 rpm at 37 ℃ to be cultured for 8-12 h, so as to obtain seed liquid.

C) And adding a proper amount of L-arabinose, glycerol, xylose and glucose mother liquor into each shake flask fermentation medium, so that the concentration of the L-arabinose in the fermentation liquor is 2.5 g/L, the concentration of the glycerol is 2.5 g/L, the concentration of the xylose is 20 g/L and the concentration of the glucose is 10 g/L. After substrate addition, cultivation was continued at 37 ℃ for 3-4 h to complete conversion of L-arabinose to xylitol.

D) When L-arabinose is completely consumed, a proper amount of corresponding inducer is added into each shake flask, so that the final concentration of lactose inducer in fermentation broth is 1 g/L, the final concentration of rhamnose inducer is 10 mM, and the final concentration of anhydrotetracycline inducer is 1 g/L. After substrate addition, the culture was induced at 30℃for 24-48 h. After fermentation culture, the concentration of xylose and L-arabinose serving as fermentation substrates and the concentration of xylitol and L-arabitol serving as products are detected, and the conversion rate of xylose and arabinose is calculated.

The results of shake flask fermentation experiments of the genetically engineered bacteria in which the 15 different inducible promoters express wild-type xylose reductase genes of different sources are shown in Table 4.

As can be seen from Table 4, after the substrate L-arabinose is added and cultured (before induction), 15 genetically engineered bacteria can also completely convert all L-arabinose into xylitol, wherein the L-arabinose conversion rate of the strain P39 is as high as 99.38%, and after the L-arabinose is converted into xylitol, wild type XR expression from different sources is further induced by adding corresponding inducers, so that xylose is catalyzed to produce xylitol. Different inducers are used for inducing and expressing XR1, XR5 and XR6 to catalyze xylose with different efficiencies, wherein the efficiency of xylose reductase induced and expressed by lactose-induced promoters is higher than that of rhamnose and anhydrotetracycline-induced promoters, the conversion rate of engineering bacteria P6 xylose with P _lac as a promoter is highest and reaches 99.98%, the efficiency of xylose catalyzed by XR induced and expressed by rhamnose and anhydrotetracycline is lower, the conversion rate of xylose fermented by strain P15 with anhydrotetracycline as a promoter is 78.29%. In the shake flask fermentation experiments, no formation of L-arabitol was detected.

TABLE 4 shaking flask fermentation results of engineering bacteria with different inducible promoters

Example 5 optimization of the glycerol addition ratio in the Medium

According to the results of the shake flask fermentation in example 3, shake flask fermentation culture was performed using P6 (E.coli W3110. DELTA. AraBAD. DELTA. lyxK. DELTA.ptsG. DELTA.ptsF. DELTA.xylAB: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR 6) as the fermentation strain. The fermentation experiment conditions are the same as in example 3, wherein the variable of each shake flask fermentation experiment is the addition amount of glycerin, and the addition amount of glycerin is 0.5 g/L, 1. 1 g/L, 1.5 g/L, 2 g/L and 2.5 g/L respectively. The addition amount of other sugar was fixed, wherein the L-arabinose was 2.5 g/L, and the glycerol/L-arabinose (m/m) was 0.2, 0.4, 0.6, 0.8, 1.0, respectively. The shake flask fermentation results are shown in Table 5.

The conversion rate of L-arabinose increases with the increase of the glycerol addition ratio, the conversion rate of L-arabinose exceeds 99% when the glycerol addition ratio increases to 0.6, and the conversion rate of L-arabinose is not increased any more with the further increase of the glycerol addition ratio, which indicates that the coenzyme NADH provided by the fermentation strain through glycerol metabolism is enough for the metabolism consumption of L-arabinose when the mass concentration ratio of glycerol to L-arabinose in the fermentation system is 0.6.

TABLE 5 optimization of glycerol addition experimental results

EXAMPLE 6 preparation of xylitol by fermentation of hemicellulose hydrolysate by genetically engineered bacteria

P6 (E.coli W3110 DeltaaraBAD Delta lyxK DeltaptsG DeltaptsF DeltaxylAB:: 3 (araA 17-dpe 1-lxr): (glpF-glpK): glpD/plac-XR 6) is used as a fermentation strain, and corncob hydrolysate is used as a raw material for fermentation to prepare xylitol.

The fermentation specifically comprises the following steps:

a) The hemicellulose hydrolysate is concentrated to a certain concentration, and is added into a fermentation basic culture medium, so that the total concentration of xylose is about 130 g/L, the total concentration of glucose is about 17 g/L, the total concentration of L-arabinose is about 27 g/L, and the volume of the culture medium is 10L (15L fermentation tank).

The other components and the concentrations of the components in the fermentation basic culture medium are Na ₂HPO₄10 g/L, KH₂PO₄, g/L, glycerol 15, g/L, yeast powder 10, g/L and peptone 5, g/L.

In addition, 1L fermentation feed medium, glucose 500 g/L, was prepared.

B) The P6 seed solution (LB medium) is inoculated into the sterilized basic medium with an inoculation amount of 10-15%, and is cultured for about 12 h under the conditions that the aeration ratio is 0.8 vvm and the pH is 7.0 (controlled by fed-batch NH ₃•H₂ O) at 37 ℃ until the L-arabinose in the hemicellulose hydrolysate is completely consumed. The dissolved oxygen is kept between 20% and 40% by adjusting the rotating speed in the fermentation process.

C) After the L-arabinose is completely consumed, the fermentation temperature is reduced to 30 ℃ for continuous culture, and at the same time, lactose inducer with the final concentration of 5 g/L is added to induce the xylose reductase to express, and the feed medium is continuously added.

D) Samples were taken at intervals during the fermentation process, and the concentrations of the remaining substrates glucose, xylose and L-arabinose and the concentrations of the products xylitol and arabitol in the fermentation broth were measured.

The concentration change of each substance in the fermentation process is shown in fig. 3, the HPLC detection patterns of various standard samples are shown in fig. 4, the HPLC detection pattern of hemicellulose hydrolysate fermentation 0h is shown in fig. 5, the HPLC detection pattern after hemicellulose hydrolysate fermentation 48 h is shown in fig. 6, and the HPLC detection pattern after hemicellulose hydrolysate fermentation 60 h is shown in fig. 7. As can be seen from FIGS. 3 to 7, in the first stage of fermentation (fermentation 0-12 h), L-arabinose in the hemicellulose hydrolysate is continuously consumed and gradually converted into xylitol. After fermentation 12h, all of the L-arabinose in the hemicellulose hydrolysate had been converted to xylitol. In the second stage of fermentation, xylose reductase is expressed and catalyzes the reduction of xylose in hemicellulose hydrolysate to xylitol with the addition of lactose inducer. After fermentation culture of 60 h, all xylose in the hemicellulose hydrolysate is converted into xylitol, the xylose conversion rate is close to 100%, and no impurity sugar (glucose and L-arabinose) residues and no L-arabitol generation are detected basically. Finally obtaining fermentation liquor 11.5L, and the concentration of xylitol reaches 136.5 g/L. Since L-arabinose is also converted into xylitol, the yield of xylitol to xylose reaches 1.2 g/g, and the yield of xylitol reaches 2.3 g/L/h.

Claims

1. A two-stage biological method for producing xylitol using hemicellulose hydrolysate, comprising:

(1) using hemicellulose hydrolyzate as a raw material, performing a first stage fermentation culture in a fermentation medium inoculated with fermentation engineering bacteria until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I;

(2) After adding glucose feed and inducer to the fermentation reaction liquid I, the second stage of fermentation culture is continued to obtain a fermentation product containing xylitol;

The fermentation engineering bacteria include: a host cell containing a xylose reductase XR gene and a target gene co-expressed in the host cell; the target gene includes an L-arabinose isomerase gene araA , a D-psicose-3-epimerase gene dpe and an L-xylulose reductase gene lxr ;

The host cell is Escherichia coli;

At least one of the araB gene, the araD gene and the lyxK gene is knocked out in the genome of the host cell;

The promoter for driving the expression of the XR gene is an inducible promoter; the inducible promoter is a non-arabinose inducible promoter.

2. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that the nucleotide sequence of the gene araA is shown in SEQ ID NO.1; the nucleotide sequence of the gene dpe is shown in SEQ ID NO.4; and the nucleotide sequence of the gene lxr is shown in SEQ ID NO.6.

3. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that the target gene is co-expressed in the host cell in one of the following ways:

(A) Co-expression in host cells in the form of a recombinant plasmid;

(B) Inserted into the host cell genome in the form of an expression cassette for co-expression.

4. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 3, characterized in that, in (A), the original expression vector of the recombinant plasmid is pBAD18;

(B) The target gene is inserted into the non-coding region of the host cell genome; or the araA gene, the dpe gene and the lxr gene randomly replace the ptsG gene , the ptsF gene, the xylA gene and the xylB gene.

5. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate according to any one of claim 1, wherein the host cell is Escherichia coli W3110.

6. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate as claimed in claim 5, characterized in that at least one of the glycerol transporter gene glpF , the glycerol kinase gene glpK and the glycerol triphosphate dehydrogenase gene glpD is inserted into the non-coding region of the genome of the host cell.

7. The method for producing xylitol using a two-stage biological method of hemicellulose hydrolysate according to claim 6, characterized in that the nucleotide sequence of the gene glpF is shown in SEQ ID NO.9; the nucleotide sequence of the gene glpK is shown in SEQ ID NO.10; and the nucleotide sequence of the gene glpD is shown in SEQ ID NO.11.

8. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that the NCBI accession number of the XR gene is one of AF451326.3, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1 and Q9P8R5.1.

9. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that the inducible promoter of the XR gene is a rhamnose promoter, anhydrotetracycline promoter or lactose promoter.

10. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 9, characterized in that the nucleotide sequence of the inducible promoter of the XR gene is one of the sequences shown in SEQ ID NOs. 15-19.

11. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that the hemicellulose hydrolysate is obtained by hydrolyzing hemicellulose-containing biomass; the hemicellulose-containing biomass is corn cobs, papermaking short fibers, bagasse, straw or rice husks; and the hemicellulose hydrolysate contains at least L-arabinose, glucose and xylose.

12. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that in step (1), the conditions for the first stage fermentation culture are: temperature of 18-37°C, pH of 6.0-8.0; based on the dissolved oxygen under the initial fermentation conditions after inoculation being 100%, the dissolved oxygen is maintained between 0 and 50%.

13. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 1, characterized in that in step (1), the _OD600 of the fermentation engineering bacteria when inoculated into the fermentation medium is 0.6-0.8, and the inoculation amount is 1-15% based on the initial fermentation volume when loading.

14. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that in step (1), the fermentation medium comprises at least a carbon source, a nitrogen source and an inorganic salt; based on the initial fermentation volume, the amount of the carbon source added is 10 to 50 g/L in volume percentage; the amount of the nitrogen source added is 5 to 30 g/L.

15 . The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 14 , wherein the carbon source is glycerol.

16. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate according to claim 15, characterized in that the amount of glycerol added is more than 0.6 times the mass of arabinose.

17. The method for producing xylitol by two-stage biological method using hemicellulose hydrolysate as claimed in claim 11, characterized in that, in step (2), based on the xylose content in the hydrolysate, the amount of glucose feed added is 10% to 50% of the xylose in terms of mass percentage; the inducer is one of lactose, rhamnose or anhydrotetracycline; based on the initial fermentation volume, the amount of the inducer added is 1 g/L to 10 g/L in terms of volume percentage.

18. The method for producing xylitol using a two-stage biological method using hemicellulose hydrolysate as claimed in claim 1, characterized in that in step (2), the fermentation culture conditions of the second stage are: temperature of 18-37°C, pH of 6.0-8.0; based on the dissolved oxygen under the initial fermentation conditions after inoculation being 100%, the dissolved oxygen is maintained between 0 and 50%.