CN102719467A - Method for biosynthesizing fatty alcohol by using fatty acyl ACP (acyl carrier protein) reductase - Google Patents

Abstract

The invention discloses a method for biosynthesizing fatty alcohols by using fatty acyl ACP reductase. The method is to introduce the gene of fatty acyl carrier protein reductase into the body of the host cell, and express the gene in the body of the host cell, thereby transforming heterotrophic microorganisms or Fatty acid metabolic pathways in heterotrophic cells to produce fatty alcohols. The invention further improves the yield of fatty alcohol through metabolic transformation. The present invention realizes the synthesis of fatty alcohols in microorganisms through biosynthesis. This technology is a renewable and low-consumption technology, and this environmentally friendly production method can reduce the consumption of scarce resource oil, and does not need to regenerate Fatty alcohol can be directly produced by chemical transformation, which has great application prospects in industry.

Description

A kind of method utilizing fatty acyl ACP reductase to biosynthesize fatty alcohol

technical field

The invention belongs to the fields of renewable energy, biomass energy and chemical raw material production, and specifically relates to a method for producing fatty alcohol by transforming the fatty acid metabolism pathway of heterotrophic microorganisms.

Background technique

Fatty alcohols are one of the raw materials of surfactants used in detergents, and are widely used in detergents, skin care products, cosmetics, and pharmaceuticals. Fatty alcohol was first produced from spermaceti, and the resulting mixed fatty alcohol became sulfate after sulfonation and neutralization, which is the earliest anionic detergent . Afterwards, coconut oil, palm oil and tallow with relatively rich sources were developed and utilized as raw materials, and the fatty acids obtained by hydrolysis were then reduced to alcohols, collectively referred to as natural fatty alcohols. After the development of the petrochemical industry, the fatty alcohols produced from petroleum products as raw materials are called synthetic fatty alcohols. The total fatty alcohol consumption in the world is about 2.5 million tons. By the end of the 20th century, the ratio of natural alcohol to synthetic alcohol was roughly 4.9:5.1. In recent years, due to the continuous rise in the price of petroleum raw materials, the increasing demand for fatty alcohols in the Americas and Asia, and people's preference for natural raw material fatty alcohols, the demand for natural fatty alcohols has increased. The price of fatty alcohol has reached as high as 2,000 US dollars per ton. At present, many countries in the world still need to rely on imports to meet the demand for fatty alcohol. However, fatty alcohols that rely on petroleum products as raw materials also consume a large amount of petroleum reserves. Today, as resources are increasingly scarce, this development method will gradually be eliminated. Therefore, a more environmentally friendly and renewable production method is needed. If it is possible to heterologously express fatty alcohol synthase, ie, fatty acyl carrier protein reductase, in the modified fatty acid metabolic pathway, a large amount of fatty alcohol can be produced by microbial fermentation.

Michael et al. purified the fatty acyl CoA reductase of jojoba in 2000, and proved that the gene - jojoba far can reduce fatty acids to fatty alcohols. This work was published in "Plant Physiology" (James G. Metz, Michael R. Pollard 1 , Lana Anderson, Thomas R. Hayes, and Michael W. Lassner. 2000. Purification of a jojoba embryo fatty acyl-Coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed. Vol. 122: 635–644). Tan et al. expressed the fatty acyl-CoA reductase FAR from jojoba, mouse, Arabidopsis, etc. in the cyanobacteria Syn-LY2 strain lacking the ability to synthesize fatty alcohols, and found that jojoba and one of them, Arabidopsis, were expressed heterologously. FAR cyanobacteria are capable of synthesizing fatty alcohols, whereas cyanobacteria heterologously expressing several other FARs are unable to produce fatty alcohols. This work was published in "Metabolic Engineering" (Tan X , Yao L, Lu X. et al. 2010. Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metabolic Engineering 13 (2011) 169–176). Professor Liu Tiangang and others developed a "cell free" system to study the rate-limiting steps in the fatty acid metabolism pathway and guide how to increase the fatty acid content in microorganisms. This work was published in "Metabolic Engineering" (Tiangang Liu , Harmit Vora , Chaitan Khosla. 2010. Quantitative analysis and engineering of fatty acid biosynthesis in E. coli . Metabolic Engineering 12 :378–386). In 2011, researchers cloned and identified a fatty acyl carrier protein reductase DPW involved in the development of monocot rice anthers and pollen exine. The study revealed that the DPW protein is located in the plastid, and the recombinant protein can be a fatty acyl carrier protein as a substrate , reducing sixteen fatty acids to their corresponding fatty alcohols, thus participating in the synthesis of anthers and pollen exines, and experiments such as genetic complementation proved that DPW has a conserved biological function in the dicotyledonous Arabidopsis (Shi J, Tan H, Yu XH, Liu Y, Liang W, Ranathunge K, et al. Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase. The Plant cell 2011;23:2225-46). In 2010, Andreas Schirmer et al. published a work in Science, which proved that the co-expression of a gene sequence PCC7942-orf1594 ( AAR gene) and PCC7942-orf1593 from cyanobacteria PCC7942 can produce aliphatic alkanes (Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB. Microbial biosynthesis of alkanes. Science 2010;329:559-62). Fatty acyl carrier protein reductase genes are easy to optimize, and there are many kinds of microorganisms that can be genetically manipulated, and the fatty acid synthesis and metabolism pathway is a necessary condition for the survival of each microorganism, and the current research has a deep understanding of this pathway. The quick steps are basically clear, which is conducive to the transformation. As we all know, due to the rapid growth of heterotrophic microorganisms or heterotrophic cells and the need for exogenous supplementary nutrients, it is convenient to increase their production capacity by supplementing the medium or adding certain nutrients, so that they can achieve large-scale industrial production in industry . And because it must rely on artificial nourishment, each stage of its production can be strictly controlled to prevent its wanton growth and cause an uncontrollable situation. At present, there have been many successful examples in the industry of using heterotrophic microorganisms to produce and obtain products through fermentation. Using fatty acyl carrier protein reductase genes to produce fatty alcohols by modifying their own metabolic pathways in heterotrophic microorganisms has great application prospects.

Contents of the invention

The object of the present invention is to provide a method for biosynthesizing fatty alcohols using fatty acyl ACP reductase, transforming its fatty acid metabolism pathways and expressing foreign proteins in heterotrophic microorganisms or heterotrophic cells to produce important chemical raw materials—fatty alcohols .

To achieve the above object, the present invention provides a method for biosynthesizing fatty alcohols using fatty acyl ACP reductase, which is to introduce the gene of fatty acyl carrier protein reductase into heterotrophic microorganisms or heterotrophic cells, and induce expression.

The fatty acyl carrier protein reductase gene encodes the protein shown in SEQ ID No.2 or SEQ ID No.4. Alternatively, the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.4 is substituted, replaced and/or increased by one or several amino acids, and the protein derived from the DPW protein and the AAR protein has the same activity as the DPW protein and the AAR protein . Preferred are DPW gene and AAR gene.

The heterotrophic microorganisms or heterotrophic cells of the present invention include mammalian cells, plant cells, insect cells, yeast cells, fungal cells, filamentous fungal cells, bacterial cells and cyanobacterial cells.

In addition, the method of the present invention also includes improving the yield of fatty alcohol by modifying the metabolic pathway of heterotrophic microorganisms or heterotrophic cells or optimizing the fatty acyl carrier protein reductase gene according to its codon preference.

In addition, the method of the present invention also includes performing tolerance screening on heterotrophic microorganisms transformed with the fatty acyl carrier protein reductase gene to obtain dominant strains, thereby increasing the yield of fatty alcohols.

Wherein, the modified heterotrophic microorganism metabolic pathway is a fatty acid metabolic pathway in the microorganism, including: a pathway for increasing fatty acid production or reducing fatty acid production for the production of fatty alcohols, for example: introducing the pMSD8 plasmid into Escherichia coli and overexpressing fatty acetyl-CoA carboxylase, increase the fatty acid content used to produce fatty alcohols; change the way in which fatty acid metabolic pathway intermediates in the microorganism are used to produce fatty alcohols, for example: knock out the fadE gene in Escherichia coli to accumulate more fatty acid metabolic pathway intermediates Fatty acyl carrier protein of the substance, so as to obtain more fatty alcohols; change the pathway of fatty acid metabolism pathway derivatives in the microorganism to produce fatty alcohols.

Wherein, the strain transformation includes: using fatty alcohol tolerance to screen and transform the tolerant strains obtained.

The present invention also provides a heterotrophic microorganism or heterotrophic cell, which is a heterotrophic microorganism or heterotrophic cell transformed with a fatty acyl carrier protein reductase gene. And the preferred heterotrophic microorganisms or heterotrophic cells obtained through the above-mentioned transformation approach.

The fatty acyl carrier protein reductase gene encodes the protein shown in SEQ ID No.2 or SEQ ID No.4. Alternatively, the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.4 is substituted, replaced and/or increased by one or several amino acids, and the protein derived from the DPW protein and the AAR protein has the same activity as the DPW protein and the AAR protein . Preferred are DPW gene and AAR gene.

The advantage of the present invention is that the introduction of exogenous genes in heterotrophic microorganisms transforms its own fatty acid metabolism pathways to achieve the purpose of producing fatty alcohols as industrial raw materials, without excessive chemical synthesis reactions, which reduces environmental pollution and reduces environmental pollution. depletion of oil reserves. At the same time, due to the fast growth of most heterotrophic microorganisms, it is convenient for genetic manipulation, and their anti-pollution performance is excellent, and their growth speed can be adjusted manually to prevent their wanton growth from damaging the environment. Various factors indicate that it is practical to transform heterotrophic microorganisms for industrial production. feasible.

Description of drawings

Figure 1 is a schematic diagram of the designed and constructed pRL108 expression vector.

Fig. 2 is a schematic diagram of the designed and constructed pFASR expression vector.

Fig. 3 is a graph of GC-MS results after the culture of Escherichia coli RL6 was induced by IPTG and subjected to fatty alcohol extraction. C15:0 means pentadecanol; C14:0 means tetradecanol; C16:0 means hexadecanol.

Fig. 4 is a graph of GC-MS results after the culture of Escherichia coli RL7 induced by IPTG and extracted with fatty alcohol. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 5 is a GC-MS result graph of Escherichia coli RL8 induced by IPTG and its culture subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 6 is a graph showing the GC-MS results of the culture of Escherichia coli RL9 induced by IPTG after fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 7 is a graph of GC-MS results after the culture of Escherichia coli RL10 induced by IPTG was subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 8 is a graph showing the GC-MS results of the culture of Escherichia coli RL11 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 9 is a GC-MS result graph of the culture of Escherichia coli RL12 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 10 is a graph of GC-MS results of the culture of Escherichia coli RL13 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 11 is a graph of GC-MS results of Escherichia coli RL14 induced by IPTG and its culture subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 12 is a GC-MS result graph of the culture of Escherichia coli RL15 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 13 is a GC-MS result graph of the culture of Escherichia coli RL16 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Fig. 14 is a GC-MS result graph of the culture of Escherichia coli RL17 induced by IPTG and subjected to fatty alcohol extraction. C15:0 represents pentadecanol; C14:0 represents tetradecanol; C16:0 represents hexadecanol; C16:1 represents Δ9-hexadecanol; C18:1 represents Δ9-octadecanol.

Figure 15 shows the results of Escherichia coli RL15 and RL17 after the fed-batch fermentation test.

the

Detailed ways

The object of the present invention is achieved through the following measures:

An exogenous gene—fatty acyl carrier protein reductase is introduced into the heterotrophic microorganisms to catalyze the reduction of its own fatty acyl carrier protein to form fatty alcohols.

The introduced fatty acyl carrier protein reductase reductase is the DPW gene derived from rice and the AAR gene derived from cyanobacteria PCC7942.

As a kind of heterologous microorganism, Escherichia coli has a clear background of genetic manipulation, fast growth and mild culture conditions, and is one of the preferred strains for fermentation production. In the examples, Escherichia coli BL21 (DE3) or MG1655 (DE3) were selected ΔrecAΔendA was used as the production strain, and its expression plasmid pET28 was selected and transformed into pRL108 vector expressing DPW gene and pFASR vector expressing AAR gene.

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention.

Example 1

The published DPW gene sequence (SEQ ID No.1, synthesized by GenScript Biotechnology Co., Ltd.) was cloned into the pET28 vector through Eco RI and Xho Ⅰ restriction sites to construct a good plasmid Named pRL108.

Knockout of the fatty acyl carrier protein dehydrogenase fadE gene in BL21 (DE3) can reduce the consumption of fatty acyl-CoA, thereby accumulating more fatty acyl carrier protein and fatty acyl carrier protein for the production of fatty alcohols. In this example, the Escherichia coli BL21 (DE3) in which the fadE gene was knocked out was named TL101. Knockout of the fatty acyl-CoA synthetase fadD gene in TL101 can interrupt the formation of fatty acyl-CoA, so that only fatty acyl carrier protein is synthesized in the cell. In this example, according to the principle of homologous recombination, a knockout plasmid was constructed to knock out the fadD gene in TL101. Through two primers pRL1-S (TTAA GCATGC GAAGATTTTA CTGCGGATAT T ( Sph Ⅰ)) and pRL1-AS (ATAT GGATCC GCGTTAAGTC AGTCGTC ( Bam HI)) PCR amplification of a left arm of about 1 kb—that is, the upstream sequence of the fadD gene, Through the other two primers pRL2-S (ATAT GGATCC TTCTTCACCT CTAAAATGCG T ( Bam HI)) and pRL2-AS (ATAT GAGCTC GATGAAAACG GTATCTGGC (Sac Ⅰ) ) to amplify a right arm of about 1 kb—that is, the downstream sequence of the fadD gene, The two gene sequences were spliced and cloned on the thermosensitive plasmid pMAK705, and the knockout plasmid pRL103 was successfully constructed. The constructed knockout plasmid pRL103 was introduced into E. coli TL101 competent cells. Since the knockout plasmid pRL103 contains a sequence homologous to the E. coli genome, using the principle of homologous recombination, this sequence can be combined with fadD on the E. coli genome. The same sequence before and after the gene undergoes two homologous recombination, so that the fadD gene can be knocked out. In addition, the temperature-sensitive nature of the knockout plasmid pRL103 was used to carry out repeated temperature-changing cultures, allowing it to recombine and lose itself, so that the suspected correct recombinant bacteria were screened out using antibiotic sensitivity tests. Two primer pairs (KS: AAGCGAAACCCCATGACATC; KAS: GTCGATGTGAACGGTTTTC) were used for colony PCR verification, and the Escherichia coli that successfully knocked out fadD on the basis of Escherichia coli TL101 was named RL100 and kept.

The plasmid pMSD8 constructed by Professor John Cronan can overexpress fatty acetyl-CoA carboxylase in Escherichia coli (Mark S. Davis, Jose' Solbiati, and John E. Cronan, Jr. 2000. Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in Escherichia coli. THE JOURNAL OF BIOLOGICAL CHEMISTRY .275(37):28593–28598.). In this example, the present invention uses this plasmid to increase the production of fatty alcohols.

TL101 was screened for fatty alcohol tolerance with LB medium containing dodecanol, tetradecanol and hexadecanol, and the concentration of fatty alcohols (fatty alcohols) was gradually increased from 80 mg/L. Increase to a final concentration of 1.2 g/L fatty alcohols (fatty alcohols) to screen for TL101, and finally continue to screen for one week under the condition of the highest concentration of 1.2 g/L fatty alcohols (fatty alcohols) concentration, will eventually survive The cell was kept and named RL101.

The pRL108 was transformed into BL21(DE3), and the successful transformant was obtained by kanamycin screening, which was named RL6.

Transform pRL108 into MG1655(DE3) ΔrecAΔendA , and use kanamycin to screen the successful transformant, which was named RL7.

Transform pRL108 into TL101, and use kanamycin to select a successful transformant, which was named RL8.

Transform pRL108 into RL101, and use kanamycin to select a successful transformant, which was named RL9.

The pRL108 was transformed into RL100, and the successful transformant was selected by karimycin, which was named RL10.

pMSD8 and pRL108 were co-transformed into RL101, and the successful transformants were screened with kanamycin and carbenicillin, which were named RL11.

RL6, RL8 and RL10 were inoculated in 5 mL LB medium containing corresponding antibiotics and cultured at 37 °C for about 12 h, then transferred to 300 mL LB medium containing corresponding antibiotics and continued to culture at 37 °C for about 90 min. When the OD reached 0.6, 0.25mM IPTG was added to induce expression, and after continuing to culture at 37°C for 12h, it was then transferred to 30°C for 6 hours.

RL7, RL9, and RL11 were inoculated in 5 mL LB medium containing corresponding antibiotics and cultured at 30 °C for about 12 h, then transferred to 300 mL LB medium containing corresponding antibiotics and continued to culture at 30 °C for about 12 h. After 120 min, when the OD reached 0.6, 0.25 mM IPTG was added to induce expression, and culture was continued at 30°C for 18 hours.

After induction of expression for 18 h, 100 mL of the culture was taken for fatty alcohols extraction.

The extraction method of RL6, RL8 and RL10 was as follows: add 100 μL of 10 mg/mL pentadecyl alcohol as an internal reference to 100 mL of culture to make the final concentration 10 mg/L, and add 2 mL of n-decane. Then add 200 mL of organic liquid A (hexane:isopranol=3:2), extract vigorously for 10 minutes, let stand for 10 minutes, remove the lower aqueous solution, then add 180 mL of sodium sulfate solution B, continue vigorously extracting for 10 minutes, and then After standing for 10 minutes, the lower aqueous solution was removed, and the upper organic layer was transferred to a 500 mL round bottom flask for rotary evaporation. Conditions are: 50°C water bath, pressure 50 psi. After the organic liquids n-hexane and isopropanol have been evaporated (n-decane cannot be rotary evaporated under this condition), transfer the n-decane solution containing the product into a sample bottle with a built-in tube.

The extraction method of RL7, RL9 and RL11 was as follows: add 100 μL of 10 mg/mL pentadecanol to 100 mL of culture as an internal reference, then add 200 mL of solution A (hexane:isopranol=3:2), extract vigorously for 10 min, and then statically Set aside for 10 min, remove the lower aqueous solution, then add 180 mL of solution B (12 g sodium sulfate dissolved in 180 mL of water), continue to extract vigorously for 10 min, then let stand for 10 min, remove the lower aqueous solution, transfer the upper organic layer to into a 500 mL round bottom flask for rotary evaporation. Conditions: 40°C water bath, pressure 250 psi. After the organic liquid was evaporated, 3 mL of n-hexane was used to dissolve the product on the inner wall of the round-bottomed flask three times, and then the 9 mL of liquid was transferred to a 25 mL round-bottomed flask, and rotary evaporation was carried out under the same conditions. After the organic layer evaporated, dissolve the product in the round bottom flask with 300 μL of n-hexane, and transfer it into a sample bottle with a built-in tube.

The processed samples were subjected to GC-MS (gas chromatography-mass spectrometry) detection. GC-MS is Agilent's 5975C/7890A system, the column used is HP-INNOWax, the helium flow rate is 1 mL/min, the injection volume is 1 μL, the split ratio is 10:1, and the programmed temperature of RL6, RL8 and RL10 is: 150 ℃ for 2 minutes, increase 5 ℃ per minute to 240 ℃, keep for 5 minutes. The program temperature of RL7, RL9 and RL11 is: 50°C for 2 minutes, increasing by 10°C every minute to 240°C, and maintaining for 10 minutes.

Experimental results: Fatty alcohols were produced in all six strains, and the products included tetradecyl alcohol, hexadecanol and octadecenol, and the production reached the highest in Escherichia coli RL11, about 10.5 mg/L. This example fully proves the feasibility of utilizing DPW protein to produce fatty alcohol.

Example 2

The published AAR gene sequence was optimized and fully synthesized (SEQ ID No.3, synthesized by Jinweizhi Biotechnology Co., Ltd.), and cloned into the pET28 vector through Nco Ⅰ and Bam HI two restriction sites, The constructed plasmid was named pFASR.

The pFASR was transformed into BL21(DE3), and a successful transformant was obtained by screening with kanamycin, which was named RL12.

The pFASR was transformed into MG1655(DE3) ΔRECAΔENDA , and a successful transformant was obtained by screening with kanamycin, which was named RL13.

The pFASR will be transformed into TL101, and the successful transformants obtained by kanamycin screening will be named RL14.

The pFASR was transformed into RL101, and a successful transformant was obtained by kanamycin screening, which was named RL15.

The pFASR and pMSD8 were co-transformed into TL101, and the successful transformant was screened by karimycin, which was named RL16.

The pFASR and pMSD8 were co-transformed into RL101, and the successful transformant was selected by kanamycin and carbenicillin, which was named RL17.

The above six kinds of bacteria were inoculated in 5 mL LB medium containing corresponding antibiotics and cultured at 30°C for about 12 h, then transferred to 300 mL LB medium containing corresponding antibiotics and continued to culture at 30°C for about 120 min. When the OD reached 0.6, 0.25 mM IPTG was added to induce expression, and the culture was continued at 30°C for 18 hours.

After induction of expression for 18 h, 100 mL of the culture was taken for fatty alcohols extraction.

The extraction method is: add 100 μL of 10 mg/mL pentadecanol to 100 mL of culture as an internal reference, then add 200 mL of solution A (hexane:isopranol=3:2), extract vigorously for 10 minutes, and then let stand for 10 minutes to remove the lower layer After the aqueous solution, add 180 mL of solution B (12 g sodium sulfate dissolved in 180 mL of water), continue to extract vigorously for 10 min, and then let it stand for 10 min to remove the lower aqueous solution, and transfer the upper organic layer to a 500 mL round bottom The flask was rotovaped. Conditions: 40°C water bath, pressure 250 psi. After the organic liquid was evaporated, 3 mL of n-hexane was used to dissolve the product on the inner wall of the round-bottomed flask three times, and then the 9 mL of liquid was transferred to a 25 mL round-bottomed flask, and rotary evaporation was carried out under the same conditions. After the organic layer evaporated, dissolve the product in the round bottom flask with 300 μL of n-hexane, and transfer it into a sample bottle with a built-in tube.

The processed samples were subjected to GC-MS (gas chromatography-mass spectrometry) detection. GC-MS is Agilent's 5975C/7890A system, the column used is HP-INNOWax, the helium flow rate is 1 mL/min, the injection volume is 1 μL, the split ratio is 10:1, and the program temperature is: 50 ℃ for 2 min, every minute Increase the temperature by 10°C to 240°C and keep for 10 minutes.

Pick about 10 successful transformants RL101/pFASR or RL101/pMSD8/pFASR and culture them in 5 mL of M9 medium containing corresponding antibiotics. After culturing overnight at 30°C, transfer them to 1000 mL of M9 medium containing corresponding antibiotics After continuing to incubate at 30°C for 24 hours, transfer 1000 mL of the bacterial solution into sterile 80 mL centrifuge tubes in batches, centrifuge at 5000 rpm for 5 minutes to collect the bacterial cells, and finally mix all the bacterial cells with 50 mL of the corresponding antibiotic M9 The culture medium was suspended evenly and used as seed liquid for fermentation in tanks, and the scale of fermentation was 4 L. When the OD in the tank grows to about 6, supplement the feed medium at a rate of 0.24 mL/min. When the OD grows to about 13, add a final concentration of 0.25 mM IPTG for induction, and extract 90 mL of IPTG every 4 hours after induction. The cultures were quickly frozen with liquid nitrogen and stored in a -80°C freezer. Keep the dissolved oxygen above 80% before feeding. After feeding, the dissolved oxygen will naturally decrease due to the large growth of bacteria, so try to keep the dissolved oxygen as high as possible.

Method for extracting fatty alcohols from fermentation products: take the culture out of the -80 ℃ refrigerator, let it melt naturally, add 40 μL of 100 mg/mL carbon pentadecyl alcohol as an internal reference to 40 mL of culture, so that the final concentration is 100 mg/mL L, and add 2 mL of n-decane. Then add 200 mL of solution A (hexane:isopranol=3:2), extract vigorously for 10 minutes, let stand for 10 minutes, remove the lower aqueous solution, then add 180 mL of sodium sulfate solution B (12 g sodium sulfate dissolved in 180 mL water), continue to extract vigorously for 10 minutes, and then stand still for 10 minutes to remove the lower aqueous solution, and transfer the upper organic layer to a 500 mL round bottom flask for rotary evaporation. Conditions are: 50°C water bath, pressure 50 psi. After the organic liquids n-hexane and isopropanol have been evaporated (n-decane cannot be rotary evaporated under this condition), transfer the n-decane solution containing the product into a sample bottle with a built-in tube.

Detection method of fermentation product fatty alcohol: the extracted samples were detected by GCMS (gas chromatography mass spectrometry), and GCMS was Agilent 5975C/7890A system. The gas chromatographic column was HP-INNOwax column, the helium flow rate was 1 mL/min, and the split ratio was 10:1. The injection volume was 1 μL. The program temperature is: 50°C for 2 minutes, increasing by 10°C to 240°C per minute, and maintaining for 10 minutes.

Experimental results: Fatty alcohols were produced in all six strains, and the products included tetradecyl alcohol, hexadecanol, hexadecenol and octadecenol. Escherichia coli RL15 and RL17 were subjected to fed-batch fermentation experiments, and the results showed that the yield of RL15 was up to 0.8 g/L, and the yield was 1.6 g/L/day; the yield of RL17 was up to 0.65 g/L, and the yield was 2g /L/day. This example fully proves the feasibility of using AAR protein to produce fatty alcohol, and the protein has great potential for industrial production of fatty alcohol.

the

Description of the sequence listing: SEQ ID No.1 and 2 are the DPW gene sequence and protein sequence respectively; SEQ ID No.3 and 4 are the optimized AAR gene sequence and protein sequence respectively; SEQ ID No.5&6 are the amplified fadD left The primers of arm; SEQ ID No.7 & 8 are primers for amplifying the right arm of fadD ; SEQ ID No. 9 & 10 are primers for RL100 positive clones.

Claims (12)

1. method of utilizing fatty acyl ACP reductase enzyme biosynthesizing Fatty Alcohol(C12-C14 and C12-C18), it is that the acyl carrier protein (ACP) reductase gene is imported in the host cell body, and makes this gene at this host cell expression in vivo.

2. method according to claim 1 is characterized in that, the said acyl carrier protein (ACP) reductase gene following albumen of encoding: the 1) albumen formed of aminoacid sequence shown in SEQ ID No.2 or the SEQ ID No.4; Or, 2) aminoacid insertion, replacement shown in SEQ ID No.2 or the SEQ ID No.4 and/or increase that one or several amino acid forms with 1) said albumen has the albumen of same function.

3. method according to claim 1 and 2 is characterized in that, the acyl carrier protein (ACP) reductase gene is cloned on expression vector, import in the corresponding host or insert in the genome of host cell, and abduction delivering.

4. method according to claim 1 and 2; It is characterized in that; The pathways metabolism that also comprises and degraded synthetic through engineered host cell lipid acid perhaps is optimized the acyl carrier protein (ACP) reductase gene according to the preferences of host cell codon, to improve the output of Fatty Alcohol(C12-C14 and C12-C18).

5. method according to claim 1 and 2 is characterized in that, also comprises carrying out tolerance screening to transforming acyl carrier protein (ACP) reductase gene heterotrophic microorganism, obtains dominant strain, thereby improves the output of Fatty Alcohol(C12-C14 and C12-C18).

6. method according to claim 1 and 2 is characterized in that, said host cell is selected from mammalian cell, vegetable cell, insect cell, yeast cell, fungal cell, filamentous fungal cells, bacterial cell and cyanobacteria cell.

7. method according to claim 4 is characterized in that, the host cell metabolic pathways of transformation be this host cell intravital about lipid acid synthetic and metabolic relational approach.

8. method according to claim 6 is characterized in that, the host cell metabolic pathways of transformation is to improve the output of lipid acid in this host cell body or reduce the approach of the output of lipid acid in order to the production Fatty Alcohol(C12-C14 and C12-C18).

9. method according to claim 4 is characterized in that, the host cell metabolic pathways of transformation is to change host cell body fat acid metabolic approach intermediate in order to produce the approach of Fatty Alcohol(C12-C14 and C12-C18).

10. method according to claim 4 is characterized in that, the heterotrophic microorganism pathways metabolism of transformation is to change this heterotrophic microorganism body fat acid metabolic approach verivate in order to produce the approach of Fatty Alcohol(C12-C14 and C12-C18).

11. heterotrophic microorganism or heterotrophic cell, it is heterotrophic microorganism or the heterotrophic cell who accepts to change the acyl carrier protein (ACP) reductase gene.

12. heterotrophic microorganism according to claim 11 is characterized in that, said acyl carrier protein (ACP) reductase gene does DPWGene or AARGene, its following albumen of encoding: the 1) albumen of the composition of aminoacid sequence shown in SEQ ID No.2 or the SEQ ID No.4; Or, 2) aminoacid insertion, replacement shown in SEQ ID No.2 or the SEQ ID No.4 and/or increase that one or several amino acid forms with 1) said albumen has the albumen of same function.

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