Method for producing glycerol and lipid by open fermentation of lignocellulose hydrolysate

Abstract

The invention discloses a construction method of engineering bacteria for producing glycerol and lipid by efficiently fermenting lignocellulose hydrolysate, and belongs to the technical field of microorganisms. The invention provides a method for producing glycerol and lipid by fermenting lignocellulose hydrolysate by using Candida glycinogenes which is resistant to high osmotic pressure as a production strain. According to the present invention, with the Candida glycyinogenes engineering strain Cg-AAGD constructed by using the method, the virus-free lignocellulose hydrolysate (containing about 175 g / L of glucose and 25 g / L of xylose) can be fermented to produce 82.2 g / L of glycerol and 7.3 g / L of lipid; according to the method, the lignocellulose hydrolysate is selected as a raw material, the purpose of deep resource utilization of agricultural waste is achieved, the related technological operation flow is relatively simple and convenient, and the requirement for production equipment is easy to achieve.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a method for producing glycerol and lipids from open fermentation of lignocellulose hydrolysate. BACKGROUND

[0002] In today's society, sustainable development and resource recycling are highly concerned. Lignocellulose is a large amount of waste in agricultural production, which is rich in polysaccharide components such as cellulose and hemicellulose. However, most of the lignocellulose is not reasonably utilized at present, which not only causes resource waste, but also brings certain environmental burden.

[0003] Glycerol (glycerol) as a key platform compound is widely used in food, cosmetics, medicine and many other industries. Lipids (triglycerides) also show great application potential in the fields of biological energy and nutritional health. The traditional biological production of glycerol and lipids relies on high-priced substrates, which is high in cost and does not meet the requirements of environmental protection and sustainable development. The production of glycerol from lignocellulose hydrolysate can reduce the production cost, and the glycerol can accumulate lipids in the later stage of fermentation. The fermentation broth and the cells are fully utilized, which is more energy-saving and environmentally friendly.

[0004] Candida glycerinogenes CCTCC M 93018 is a high-osmotic-tolerant yeast that can tolerate the toxicity of lignocellulose hydrolysate. It has the ability to produce glycerol in high-osmotic and high-toxicity environments. The harsh environment of high-osmotic and high-toxicity also provides the basis for open fermentation. In addition, open fermentation does not require sterilization, which can reduce the cost of sterilization equipment and energy. The production of glycerol can also improve the production capacity of lipids, so that the glycerol in the fermentation broth and the lipids in the cells can be fully utilized. The technical problem solved by the present application is to develop a method for open fermentation of lignocellulose hydrolysate to produce glycerol and oil, and the cells and fermentation broth can be fully utilized at the same time. SUMMARY

[0005] In view of the above deficiencies of the prior art, the present application provides an innovative method for open fermentation to produce glycerol and lipids using lignocellulose hydrolysate as raw material, which aims to solve the problem that the cells and fermentation broth cannot be fully utilized at the same time during fermentation.

[0006] The first technical solution of the present application is an engineered bacterium for efficiently producing lipids, wherein the engineered bacterium takes Candida glycerinogenes CCTCC M 93018 as a host, acetyl-CoA synthetase gene ACS1, acetyl-CoA carboxylase gene ACC1, 3-phosphoglycerate dehydrogenase gene GPD1 derived from Candida glycerinogenes, and diacylglycerol acyltransferase gene DGA1 derived from Yarrowia lipolytica are integrated into the host to construct the engineered bacterium for efficiently producing lipids from lignocellulose hydrolysate.

[0007] In some embodiments, the acetyl-CoA synthetase amino acid sequence is shown in SEQ ID NO. 1; the acetyl-CoA carboxylase amino acid sequence is shown in SEQ ID NO. 2; the 3-phosphoglycerate dehydrogenase amino acid sequence is shown in SEQ ID NO. 3; and the diacylglycerol acyltransferase amino acid sequence is shown in SEQ ID NO. 4.

[0008] In some embodiments, the acetyl-CoA synthetase nucleotide sequence is shown in SEQ ID NO. 5; the acetyl-CoA carboxylase nucleotide sequence is shown in SEQ ID NO. 6; the 3-phosphoglycerate dehydrogenase nucleotide sequence is shown in SEQ ID NO. 7; and the diacylglycerol acyltransferase nucleotide sequence is shown in SEQ ID NO. 8.

[0009] In some embodiments, the acetyl-CoA synthetase ACS1, the acetyl-CoA carboxylase gene ACC1, the 3-phosphoglycerate dehydrogenase gene GPD1, and the diacylglycerol acyltransferase gene DGA1 are sequentially integrated into the 5.8S rDNA site on the genome of Candida glycerinogenes CCTCC M 93018.

[0010] The second technical solution provided by the present application is a method for constructing an engineered bacterium for efficiently metabolizing lignocellulose hydrolysate to produce glycerolipids, which comprises the following steps:

[0011] (1) sequentially connecting acetyl-CoA synthetase gene ACS1 derived from Candida glycerinogenes, acetyl-CoA carboxylase gene ACC1, 3-phosphoglycerate dehydrogenase gene GPD1, and diacylglycerol acyltransferase gene DGA1 derived from Yarrowia lipolytica to the two enzyme cutting sites of pURGAPU plasmid BamH I and Not I to construct a recombinant expression vector, and linearizing the recombinant expression vector with Hind III;

[0012] (2) using lithium acetate transformation method to transform the linearized recombinant expression vector into the genome of Candida glycerinogenes CCTCC M 93018 to construct the engineered strain Cg-AAGD for efficiently producing glycerol and lipids from lignocellulose hydrolysate.

[0013] The third technical solution of the present application is a method for producing glycerol and lipids, which comprises using the engineered strain of the first technical solution to ferment the non-detoxified lignocellulose hydrolysate as a carbon source to obtain glycerol and lipids.

[0014] In some embodiments, the method for preparing the lignocellulose hydrolysate comprises the following steps:

[0015] (1) corn cob is crushed into particles, dried to constant weight after passing through a 40-mesh sieve;

[0016] (2) under the condition of solid-liquid mass ratio of 1:5-1:20, the dried cellulose obtained in step (1) is placed in a 0.5-3.0% sulfuric acid solution, and high-temperature reaction is carried out in a 110-130°C sterilization pot for 1-4h for hydrolysis;

[0017] (3) after hydrolysis, the hydrolyzed solid cellulose is filtered, washed with water for two times, and dried to constant weight;

[0018] (4) enzymatic hydrolysis: the pH value of the system is adjusted to 3.5-7.0 using 0.001-0.01M citric acid-sodium citrate buffer; the cellulase dosage is 2-20 FPU / g lignocellulose (DM), and the enzyme hydrolysis solid load is 5-25% (w / v); after enzyme hydrolysis at 25-40°C, 200-400rpm for 60-96h, lignocellulose hydrolysate is obtained by centrifugation, and the prepared hydrolysate is concentrated to obtain non-detoxified lignocellulose hydrolysate for fermentation.

[0019] In some embodiments, the final concentration of glucose in the fermentation system is 180g / L.

[0020] In some embodiments, the fermentation conditions are as follows: the inoculation amount is 1-10%, the fermentation temperature is 25-37°C, the rotation speed is 300-500rpm, and the fermentation time is 60-84h.

[0021] The fourth technical solution provided by the application is a method for improving the production of lipids by Candida glycerinogenes using acetic acid, wherein acetyl-CoA synthetase ACS1, acetyl-CoA carboxylase ACC1, 3-phosphoglycerate dehydrogenase GPD1 derived from Candida glycerinogenes and diacylglycerol acyltransferase DGA1 derived from Yarrowia lipolytica are integrated and expressed on the genome of Candida glycerinogenes CCTCC M 93018.

[0022] In some embodiments, the acetyl-CoA synthetase amino acid sequence is shown in SEQ ID NO. 1; the acetyl-CoA carboxylase amino acid sequence is shown in SEQ ID NO. 2; the 3-phosphoglycerate dehydrogenase amino acid sequence is shown in SEQ ID NO. 3; and the diacylglycerol acyltransferase amino acid sequence is shown in SEQ ID NO. 4.

[0023] The fifth technical solution provided by the application is the application of the engineered bacteria of the first technical solution, the method of the second technical solution, the method of the third technical solution or the method of the fourth technical solution in the industrial production of glycerol and lipids.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) The high-osmotic-pressure-resistant Candida glycerinogenes strain Cg-AAGD constructed by the application can produce glycerol and lipids simultaneously, and both the fermentation broth and the bacterial cells can be fully utilized, which is economical, green and environmentally friendly.

[0026] (2) The high-osmotic-pressure-resistant Candida glycerinogenes strain Cg-AAGD can be used for open fermentation of non-detoxified lignocellulose hydrolysate containing 200 g / L glucose and xylose, and has good resistance to high osmotic pressure in the hydrolysate, thereby saving costs.

[0027] (3) The high-osmotic-pressure-resistant Candida glycerinogenes strain Cg-AAGD has good resistance to acetic acid and can directly ferment acetic acid to produce lipids.

[0028] (4) The high-osmotic-pressure-resistant Candida glycerinogenes strain Cg-AAGD has the characteristic of high glycerol yield, and the strain can generate 82.2 g / L of glycerol and 0.44 g / g of lipids using lignocellulose hydrolysate, which has industrialization potential.

[0029] In summary, the present application provides a method for producing glycerol and lipids by fermenting lignocellulose hydrolysate using the C. glycerinogenes strain as the production strain, and the cell and fermentation broth can be fully utilized. The C. glycerinogenes strain Cg-AAGD constructed by the present application can utilize the lignocellulose hydrolysate (containing about 175 g / L of glucose and 25 g / L of xylose) without detoxification to produce 82.2 g / L of glycerol and 0.44 g / g of lipids. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The schematic diagram for constructing the recombinant plasmid.

[0031] Figure 2 The lipid production of different strains under the condition of acetic acid as the carbon source.

[0032] Figure 3 The fermentation of the engineering strain in the unsterilized and sterilized non-detoxified corncob hydrolysate at the shake flask level.

[0033] Figure 4 The production of glycerol by the engineering strain in the 5L fermenter under the condition of fermenting the non-detoxified corncob hydrolysate.

[0034] Figure 5 The production of lipids by the engineering strain in the 5L fermenter under the condition of fermenting the non-detoxified corncob hydrolysate. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application, and are not used to limit the present application.

[0036] The culture media involved in the following examples are as follows:

[0037] YPD culture medium: 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose.

[0038] MM culture medium: 6.7 g / L of YNB (amino acid-free yeast nitrogen source), 10 g / L of yeast powder, 20 g / L of peptone, and 20 g / L of glucose.

[0039] YMJ culture medium: 2 g / L of urea, 5 g / L of corn steep liquor (4 g / L of acetic acid or 100 g / L of glucose).

[0040] CCB culture medium: lignocellulose hydrolysate (175 g / L of glucose and 25 g / L of xylose), 2 g / L of urea, and 5 g / L of corn steep liquor.

[0041] The detection methods involved in the following examples are as follows:

[0042] The concentrations of glucose, xylose, glycerol, acetic acid, and furfural in the culture medium were detected by HPLC (high performance liquid chromatograph). The chromatographic column was an amino HPX-87H column (Bio-Rad), the column temperature was 60°C; the mobile phase was 5 mmol / L H2SO4, the flow rate was 0.6 mL / min; the injection volume was 20 μL, and glucose, xylose, and glycerol were detected by a differential detector RI-101 (Shodex), and acetic acid was detected by a UV detector (Dionex).

[0043] Method for detecting biomass (cell dry weight):

[0044] The culture solution to be tested was placed in a centrifuge tube, repeatedly centrifuged and washed with water, dried at 105°C, and then weighed.

[0045] Method for extracting lipids:

[0046] The dried cell sample was treated with 4 mol / L hydrochloric acid in a water bath at 100°C for 10 min, and then extracted twice with chloroform / methanol (2:1 by volume). After washing with a 0.1% sodium chloride solution, the organic phase was dried at 105°C overnight, and the total lipids were obtained by the difference method.

[0047] The primer sequences involved in the following examples and comparative examples are shown in Table 1.

[0048] Table 1: Primers (5'-3')

[0049]

[0050]

[0051] Construction of engineered strain Cg-AAGD

[0052] (1) Construction of recombinant vector

[0053] The target gene fragments were obtained by PCR using the primers in Table 1, and the acetyl-CoA synthetase gene ACS1 (nucleotide sequence as shown in SEQ ID NO. 5) from Candida glycerinogenes, the acetyl-CoA carboxylase gene ACC1 (nucleotide sequence as shown in SEQ ID NO. 6), the 3-phosphoglycerate dehydrogenase gene GPD1 (nucleotide sequence as shown in SEQ ID NO. 7), and the diacylglycerol acyltransferase gene DGA1 (nucleotide sequence as shown in SEQ ID NO. 8) from Yarrowia lipolytica were sequentially connected to the BamH I and Not I enzyme cutting sites of the pURGAP plasmid to obtain the recombinant plasmid pURGAP-ACS1-ACC1-GPD1-DGA1. The vector was named pURGAP-AAGD, and the plasmid map is shown in Figure 1

[0054] The PCR reaction system involved was 50 μL, including 25 μL Prime STAR Max Premix (2x), 10 pmol primers (the primers involved are shown in Table 1), 100 ng template, and distilled water to 50 μL. The primers were synthesized by Yixin Biotechnology (Shanghai) Co., Ltd., and the rest were purchased from TaKaRa Company.

[0055] The PCR amplification conditions were as follows: denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 1 kb / min, 30-35 cycles, and extension at 72℃ for 10 min.

[0056] (2) Construction of recombinant bacteria

[0057] The recombinant expression vector pURGAP-AAGD obtained in step (1) was linearized into a DNA fragment using Hand III.

[0058] The linearized recombinant expression vector was transformed into the 5.8S rDNA site (nucleotide sequence as shown in SEQ ID NO. 12) of the Candida glycerinogenes CCTCC M 93018 genome using the lithium acetate transformation method, and was named Cg-AAGD.

[0059] Example 2: Detection of the ability of the engineered bacteria Cg-AAGD to produce lipids by fermentation of acetic acid

[0060] The specific steps are as follows:

[0061] (1) One loop of the engineered bacteria Cg-AAGD in Example 1 was picked and inoculated into YPD seed culture medium, and was cultured at 30℃ and 200 rpm for 12 h to obtain a seed solution.​

[0062] (2) Respectively, the seed liquid is inoculated into 50 mL YMJ (4 g / L acetic acid) fermentation medium at a inoculation amount of 5% (v / v), the fermentation temperature is controlled at 30°C, the rotation speed is 200 rpm, and the fermentation time is 96 h. The OD 600 and the acetic acid concentration in the sample, the acetic acid concentration is detected by high performance liquid chromatography, and the lipid yield is detected by the difference method.

[0063] The specific results are shown in Table 1. Figure 2 The lipid yield of the unmodified wild strain accounts for 36.3% of the dry weight of the bacteria, and the lipid yield of the engineering strain Cg-AAGD accounts for 44.7% of the dry weight of the bacteria.

[0064] Example 3: Fermentation of sterilized and unsterilized corn cob hydrolysate by production strain

[0065] (1) One ring of the engineering strain Cg-AAGD of Example 1 is selected and inoculated into YMJ (100 g / L glucose) seed culture medium, which is cultured at 30°C and 200 rpm for 18 h to obtain seed liquid.

[0066] (2) The seed liquid is inoculated into 30 mL CCB fermentation medium (total sugar 200 g / L) at a inoculation amount of 5% (v / v), the fermentation temperature is controlled at 32°C, the rotation speed is 100-150 rpm, the fermentation time is 84-120 h, the glucose, xylose, glycerol, acetic acid and furfural concentrations in the fermentation broth are detected by high performance liquid chromatography, and the lipid yield is detected by the difference method.

[0067] Un-detoxified CCB fermentation medium: concentrated hydrolysate (glucose 175 g / L, xylose 25 g / L), urea 2 g / L, corn syrup 5 g / L, and the concentrated cellulose hydrolysate is diluted to 1 L.

[0068] Preparation of sterilized lignocellulose hydrolysate: the un-detoxified CCB fermentation medium is placed in a sterilization pot and sterilized at 121°C for 20 min.

[0069] The compositions of unsterilized and sterilized hydrolysate are shown in Table 2.

[0070] Table 2: Composition of unsterilized and sterilized CCB hydrolysate

[0071]

[0072] The results in Table 2 show that sterilization causes the content of fermentable sugars glucose and xylose in CCB hydrolysate to decrease by 6.5 g / L and 3.0 g / L, and the total aldehyde content to increase by 1.08 g / L. As Figure 3As shown, the lipid and glycerol production of the CCB hydrolysate after sterilization was 6.4 g / L and 70.7 g / L, respectively, and the lipid and glycerol production of the unsterilized CCB hydrolysate was 6.9 g / L and 76.1 g / L, respectively. Figure 3 The results show that sterilization reduces the lipid and glycerol production of the CCB hydrolysate, and unsterilized open fermentation is more suitable for glycerol and lipid production.

[0073] Example 4: Glycerol and lipid production of the production strain in a 5L fermenter

[0074] (1) One loop of the engineered bacteria Cg-AAGD of Example 1 was picked and inoculated into YPD seed culture medium, and cultured at 30°C, 200 rpm for 18 h to obtain a first-stage seed solution.

[0075] (2) The first-stage seed solution was inoculated into concentrated CCB medium with a total sugar content of about 100 g / L at a seeding amount of 1% (v / v), and cultured at 30°C, 200 rpm for 24 h to obtain a second-stage seed solution.

[0076] (3) The prepared second-stage seed solution was inoculated into concentrated CCB medium (unconcentrated cellulose hydrolysate prepared by the method of step 1 of Example 3) with a total sugar concentration of 200 g / L at a seeding amount of 5% (v / v), and cultured in a 5L fermenter at a temperature of 30-35°C, an aeration amount of 1-1.5vvm, a rotation speed of 500 rpm, and a natural pH.

[0077] Preparation of concentrated CCB medium with a total sugar concentration of 200 g / L: concentrated corncob hydrolysate (total sugar concentration of 200 g / L, glucose 175 g / L, xylose 25 g / L), urea 2 g / L, corn steep liquor 5 g / L, and the concentrated corncob hydrolysate was used to make up to 1 L.

[0078] It was found that the engineered bacteria Cg-AAGD of the present application could utilize non-detoxified corncob hydrolysate for combined production of glycerol and lipids, with a glycerol production of 82.2 g / L (as shown in the results of Figure 4 and a lipid production of 0.44 g / g (as shown in the results of Figure 5 .

[0079] Comparative Example 1

[0080] The overexpression of the 3-phosphoglycerate dehydrogenase gene GPD1 derived from Candida glycerinogenes in Example 1 was omitted, and the other steps were according to Example 1 to obtain the strain Cg-AAD.

[0081] The overexpression of diacylglycerol acyltransferase gene DGA1 from Yarrowia lipolytica in Example 1 was omitted, and other steps refer to Example 1 to obtain strain Cg-AAG.

[0082] The 3-phosphoglycerate dehydrogenase gene GPD1 from Candida glycerinogenes in Example 1 was replaced by 3-phosphoglycerate dehydrogenase gene ScGPD1 (Gene ID: 851539) from Saccharomyces cerevisiae, and other steps refer to Example 1 to obtain strain Cg-AAScGD.

[0083] The 3-phosphoglycerate dehydrogenase gene GPD1 from Candida glycerinogenes in Example 1 was replaced by 3-phosphoglycerate dehydrogenase gene YlGPD1 (Gene ID: 2906777) from Yarrowia lipolytica, and other steps refer to Example 1 to obtain strain Cg-AAYlGD.

[0084] The diacylglycerol acyltransferase gene DGA1 from Yarrowia lipolytica in Example 1 was replaced by diacylglycerol acyltransferase gene ScDGA1 (Gene ID: 854419) from Saccharomyces cerevisiae, and other steps refer to Example 1 to obtain strain Cg-AAGScD.

[0085] The diacylglycerol acyltransferase gene DGA1 from Yarrowia lipolytica in Example 1 was replaced by diacylglycerol acyltransferase gene CgDGA1 (SEQ ID NO. 9) from Candida glycerinogenes, and other steps refer to Example 1 to obtain strain Cg-AAGCgD.

[0086] The cultivation and detection were performed according to the steps in Example 2, and the lipid production of different strains was shown in Table 3.

[0087] Table 3: The proportion of lipid production in dry cell weight of different engineering strains

[0088]

[0089] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. An engineered bacterium for efficiently producing lipids, characterized in that: The engineered bacteria uses Candida glycerinogenes CCTCC M 93018 as a host, and integrates the acetyl-CoA synthetase gene ACS1, the acetyl-CoA carboxylase gene ACC1, the glycerol-3-phosphate dehydrogenase gene GPD1 from Candida glycerinogenes, and the diacylglycerol acyltransferase gene DGA1 from Yarrowia lipolytica.

2. The engineered bacteria according to claim 1, characterized in that The amino acid sequence of the acetyl-CoA synthetase is shown in SEQ ID NO.1; the amino acid sequence of the acetyl-CoA carboxylase is shown in SEQ ID NO.2; the amino acid sequence of the 3-phosphoglycerate dehydrogenase is shown in SEQ ID NO.3; and the amino acid sequence of the diacylglycerol acyltransferase is shown in SEQ ID NO.

4.

3. The engineered bacteria according to claim 1, characterized in that The acetyl-CoA synthetase ACS1, the acetyl-CoA carboxylase gene ACC1, the 3-glycerol phosphate dehydrogenase gene GPD1, and the diacylglycerol acyltransferase gene DGA1 are sequentially integrated into the 5.8S rDNA site on the genome of Candida glycerologenes CCTCC M 93018.

4. A method for constructing an engineered bacterium for efficient lipid production, characterized in that: The method comprises the following steps: (1) The acetyl-CoA synthetase gene ACS1, acetyl-CoA carboxylase gene ACC1, and glycerol-3-phosphate dehydrogenase gene GPD1 from Candida glycerinogenes, and the diacylglycerol acyltransferase gene DGA1 from Yarrowia lipolytica were sequentially ligated between the BamHI and Not I restriction sites of the pURGAPU plasmid to construct a recombinant expression vector, which was linearized using Hind III. (2) The linearized recombinant expression vector was transformed into the genome of Candida glycerinogenes CCTCC M 93018 using the lithium acetate transformation method to construct an engineered bacterium that produces glycerol and lipids.

5. The construction method according to claim 4, characterized in that The nucleotide sequence encoding the acetyl-CoA synthetase is shown in SEQ ID NO.5; the nucleotide sequence encoding the acetyl-CoA carboxylase is shown in SEQ ID NO.6; the nucleotide sequence encoding the 3-phosphoglycerate dehydrogenase is shown in SEQ ID NO.7, and the nucleotide sequence encoding the diacylglycerol acyltransferase is shown in SEQ ID NO.

8.

6. A method for producing glycerol and lipids, characterized in that The method comprises the following steps: using the engineered bacteria according to any one of claims 1 to 3, and using undetoxified lignocellulose hydrolyzate as a carbon source to ferment and prepare glycerol and lipids.

7. The method according to claim 6, characterized in that In the fermentation system, the final concentration of glucose in the non-detoxified lignocellulose hydrolyzate is 180 g / L.

8. The method according to claim 6, characterized in that The fermentation conditions are as follows: inoculation amount of 1-10%, fermentation temperature of 25-37° C., rotation speed of 300-500 rpm, and fermentation time of 60-84 hours.

9. A method for improving the production of lipids using acetic acid by Candida glycerologenes, characterized in that: The method comprises integrating and expressing acetyl-CoA synthetase ACS1, acetyl-CoA carboxylase ACC1, glycerol-3-phosphate dehydrogenase GPD1 from Candida glycerinogenes and diacylglycerol acyltransferase DGA1 from Yarrowia lipolytica into the genome of Candida glycerinogenes CCTCC M 93018; The amino acid sequence of the acetyl-CoA synthetase is shown in SEQ ID NO.1; the amino acid sequence of the acetyl-CoA carboxylase is shown in SEQ ID NO.2; the amino acid sequence of the 3-phosphoglycerate dehydrogenase is shown in SEQ ID NO.3; and the amino acid sequence of the diacylglycerol acyltransferase is shown in SEQ ID NO.

4.

10. Use of the engineered bacteria according to any one of claims 1 to 3, or the method according to claim 4 or 5, or the method according to any one of claims 6 to 8, or the method according to claim 9 in the industrial production of glycerol and lipids.

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