Yarrowia lipolytica genetically engineered bacteria for producing erythritol by glycerol

Abstract

The application discloses a Yarrowia lipolytica gene engineering bacterium for producing erythritol by glycerol, and the Yarrowia lipolytica gene engineering bacterium is obtained by random insertion mutation mediated by non-homologous end connection, and strains with high cell growth and high erythritol yield are screened. Further, the gene engineering bacterium overexpresses glycerol dehydrogenase gene GCY3 or ARA1 and dihydroxyacetone kinase gene DAK2 of a glycerol metabolism dihydroxyacetone pathway. Further, the gene engineering bacterium overexpresses transketolase gene TKL1 and transaldolase gene TAL1 of a non-oxidative pentose phosphate pathway. The application further discloses a construction method and application of the gene engineering bacterium. The superior gene engineering bacterium obtained by constructing a mutant library has the characteristics of tolerating high-concentration glycerol, and the substrate conversion rate and glycerol utilization rate are significantly higher than those of a starting strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a genetically engineered *Yersinia lipolytica* strain that produces erythritol using glycerol. Background Technology

[0002] Erythritol (R,S-1,2,3,4-butanetetrol, CAS No. 149-32-6), molecular formula C4H 10 O4, as a novel sweetener, has the following advantages: 1. Refreshing taste and low sweetness; 2. High stability; 3. Significant endothermic effect when dissolved in water; 4. Relatively low hygroscopicity; 5. Solubility changes significantly with temperature. Because it is not metabolized by bacteria that cause tooth decay, it is non-cariogenic and has been shown to reduce dental plaque. Erythritol has also been shown to be a free radical scavenger with antioxidant properties. Through available metabolic, toxicological, and clinical studies in animals and humans, it has been found that long-term, high-dose intake of erythritol is safe for humans. Furthermore, as an important C4 platform chemical, erythritol can be used in chemical reactions such as hydrogen reduction (hydrodeoxygenation) with suitable catalyst components (active metals, additives, and carriers) to produce butadiene, 1,4-butanediol, 2,5-dihydrofuran, and tetrahydrofuran. Currently, erythritol is used in the food industry, daily chemical industry, pharmaceutical field, and large-scale consumer chemical industry.

[0003] The main production methods for erythritol include chemical synthesis and microbial fermentation. However, chemical synthesis methods are plagued by complex reactions, high costs, harsh reaction conditions, low product conversion rates, and difficulties in subsequent purification. Therefore, microbial fermentation is the primary method used in actual industrial production. Utilizing industrial byproducts as carbon sources for fermentation is an important research area in the field of biotechnology, aligning with current policies on green manufacturing and a circular, renewable economy. The production of biodiesel generates a large amount of glycerol as a byproduct; effectively utilizing and converting glycerol through microbial technology will greatly promote the development of a circular economy.

[0004] Yarrowia lipolytica ( Yarrowia lipolyticaAs an unconventional yeast tolerant to hyperosmolar conditions, it can spontaneously produce erythritol as its own osmoprotectant under hyperosmolar conditions and has a wide range of substrate utilization capabilities, making it a recognized safe (GARS) strain. Endogenous DNA double-strand breaks (DSBs) occur frequently in many biological processes, such as DNA replication, three-dimensional genome folding, and gene transcription. In *Yarrowia lipolytica*, the vast majority of DSBs are repaired through non-homologous end joining (NHEJ), and NHEJ can be used to achieve random integration of endogenous or exogenous genes into the *Yarrowia lipolytica* genome. Therefore, this randomness of insertion allows for the rapid construction of mutant libraries of *Yarrowia lipolytica*, providing a novel modification strategy for the construction of efficient cell factories.

[0005] Glycerol catabolism relies on two pathways: the G3P pathway, which uses glycerol-3-phosphate as an intermediate metabolite, and the DHA pathway, which uses dihydroxyacetone as an intermediate metabolite. In the G3P pathway, glycerol is converted to glycerol-3-phosphate by glycerol kinase, which consumes one molecule of ATP. Glycerol-3-phosphate is then converted to dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase. In the DHA pathway, glycerol is first converted to cytotoxic dihydroacetone by glycerol dehydrogenase, which then consumes one molecule of ATP by dihydroxyacetone kinase to generate dihydroacetone phosphate (DHAP). The glycerol metabolite dihydroacetone phosphate is then converted to glyceraldehyde-3-phosphate by triose phosphate isomerase, entering the gluconeogenesis, glycolysis, and tricarboxylic acid cycle pathways. Currently, there are few reports on the production of erythritol from glycerol, and studies on enhancing glycerol metabolism in *Yarrowia lipolytica* have largely focused on the G3P pathway, neglecting the potential impact of the DHA pathway on glycerol metabolism. Therefore, it is urgent to explore the key genes in the DHA metabolism pathway of glycerol in Yersinia lipolytica and to develop genetically engineered Yersinia lipolytica strains that utilize glycerol to produce erythritol. Summary of the Invention

[0006] The purpose of this invention is to optimize the synthetic pathway of Yersinia lipolytica to produce erythritol using glycerol through metabolic engineering, thereby obtaining a genetically engineered Yersinia lipolytica strain with optimized performance for producing erythritol using glycerol.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a genetically engineered *Yersinia lipolytica* strain that produces erythritol using glycerol. This *Yersinia lipolytica* strain is obtained by screening for strains with significantly improved cell growth and erythritol production through random insertion mutations mediated by non-homologous end linkages.

[0008] According to the present invention, the random insertion mutation is to... loxp-ura3-loxp Linear gene fragments were introduced into Yersinia lipophila.

[0009] Furthermore, the preservation number of the genetically engineered *Yarrowia lipolytica* strain is CCTCC NO:M 20221813.

[0010] A second aspect of the present invention provides a genetically engineered *Yersinia lipolytica* strain that produces erythritol from glycerol, which is based on the aforementioned *Yersinia lipolytica* strain and overexpresses a glycerol dehydrogenase gene involved in the dihydroxyacetone metabolism pathway. GCY3 or ARA1 and dihydroxyacetone kinase gene DAK2 .

[0011] A third aspect of the present invention provides a genetically engineered *Yersinia lipolytica* strain that produces erythritol from glycerol, which is based on the aforementioned *Yersinia lipolytica* strain and overexpresses a transketolase gene of the non-oxidative pentose phosphate pathway. TKL1 and transaldase gene TAL1 .

[0012] A fourth aspect of the present invention provides a method for constructing the above-mentioned Yersinia lipophila genetically engineered strain, comprising the following steps:

[0013] S1: Will loxp-ura3-loxp Linear gene fragments were introduced into Yersinia lipophila to construct a random insertion mutant library of Yersinia lipophila.

[0014] S2: Single clones with high osmotic tolerance were screened from the mutant library and subjected to multiple shake-flask fermentations to screen strains with significantly improved cell growth and erythritol production.

[0015] A fifth aspect of the present invention provides a method for constructing the above-mentioned genetically engineered *Yarrowia lipophila* strain, comprising the following steps:

[0016] S1: Will loxp-ura3-loxp Linear gene fragments were introduced into Yersinia lipophila to construct a random insertion mutant library of Yersinia lipophila.

[0017] S2: Screening monoclonal strains with high osmotic tolerance from mutant libraries for multiple shake-flask fermentations to screen strains with significantly improved cell growth and erythritol production;

[0018] S3: Endogenous Yeast Extract from the lipophilic yeast. DAK2 and GCY3 ,or DAK2 and ARA1 Two genes were introduced into the *Yersinia lipolytica* genetically engineered strain obtained in S2 to produce the *Yersinia lipolytica* genetically engineered strain.

[0019] A sixth aspect of the present invention provides a method for constructing the above-mentioned Yersinia lipophila genetically engineered strain, comprising the following steps:

[0020] S1: Will loxp-ura3-loxp Linear gene fragments were introduced into Yersinia lipophila to construct a random insertion mutant library of Yersinia lipophila.

[0021] S2: Screening monoclonal strains with high osmotic tolerance from mutant libraries for multiple shake-flask fermentations to screen strains with significantly improved cell growth and erythritol production;

[0022] S3: Endogenous Yeast Extract from the lipophilic yeast. DAK2 and GCY3 ,or DAK2 and ARA1 Two genes were introduced into the *Yersinia lipolytica* genetically engineered strain obtained in S2 to obtain a new *Yersinia lipolytica* genetically engineered strain.

[0023] S4: Endogenous Yeast Rice (Yeast Rice) TAL1 and TKL1 The gene combination was integrated into the *Yersinia lipolytica* genetically engineered strain obtained from S3 to obtain a new *Yersinia lipolytica* genetically engineered strain.

[0024] A seventh aspect of the present invention provides the application of the above-mentioned Yersinia lipolytica genetically engineered strain in the production of erythritol.

[0025] The present invention has the following beneficial effects:

[0026] 1. This invention obtains superior genetically engineered bacteria with the characteristic of tolerating high concentrations of glycerol by constructing a mutant library and screening them. The substrate conversion rate and glycerol utilization rate are significantly higher than those of the starting strain.

[0027] 2. This invention overexpresses key genes in the potential DHA glycerol catabolism pathway. DAK2 , GCY3 and ARA1 This greatly improves the rate at which Yersinia lipophila utilizes extracellular glycerol.

[0028] 3. This invention overexpresses key genes in the non-oxidative pathway of pentose phosphate. TAL1 and TKL1 This allows for the influx of as much carbon flux as possible into the erythritol synthesis pathway, thereby significantly increasing erythritol production. Attached Figure Description

[0029] Figure 1 The metabolic pathway by which Yersinia lipophila produces erythritol using glycerol as a substrate.

[0030] Figure 2 The image shows the results of erythritol production by the mutant T-09 obtained through random insertion mutation mediated by the non-homologous end joining pathway (NHEJ) in Example 1.

[0031] Figure 3 The image shows the results of erythritol production by strains JT-17 and JT-18 in Example 2.

[0032] Figure 4 The image shows the results of erythritol production by fermentation using JT-21 and JT-22 in Example 3.

[0033] Figure 5 This is a summary diagram of the shake-flask fermentation results of the engineered strain of Yersinia lipophila in Example 4.

[0034] Storage

[0035] The genetically engineered *Yarrowia lipolytica* strain T-09 obtained in this invention was deposited with the China Center for Type Culture Collection on November 24, 2022, with accession number CCTCC NO:M 20221813. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. It should be understood that the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] The sources of biological materials in the following examples are:

[0039] 1. Yarrowia lipolytica ( Yarrowia lipolytica The starting strain was Yersinia lipophila Polf (ATCC MYA-2613).

[0040] 2. Plasmid pINA1312- DAK2 - GCY3 pINA1312- DAK2 - ARA1 and pINA1269- TAL1 - TKL1The preparation method described in Schwartz, C., Shabbir-Hussain, M., Frogue, K., Blenner, M., Wheeldon, I. 2017. Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth Biol, 6(3), 402-409.

[0041] 3. Plasmids pINA1312 and pINA1269 were prepared according to the method described in Nicand, JM; Madzak, C.; van den Broek, P.; Gysler, C.; Duboc, P.; Niederberger, P.; Gaillardin, C. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res. 2002, 2, 371-379.

[0042] 4. The plasmid pUB4-CRE was prepared according to the method described in Fickers, P., Le Dall, MT, Gaillardin, C., Thonart, P., and Nicaud, JM (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J. Microbiol. Methods 55, 727-737.

[0043] Example 1: Construction of strain T-09

[0044] 1.1 Using primers P1 and P2, a linear gene fragment was amplified using pINA1312 plasmid as a template. loxp-ura3- loxp (SEQ ID NO.11):

[0045] P1: ataacttcgt atagcataca ttatacgaag ttattgtttc tcggt;

[0046] P2: ataacttcgt ataatgtatg ctatacgaag ttatactaaa tttat.

[0047] 1.2. Using the non-homologous end joining pathway (NHEJ), the material obtained in step 1.1 is... loxp-ura3-loxp Linear gene fragments were integrated into *Yersinia lipolytica* Polf to obtain a random insertion mutant library of *Yersinia lipolytica*. Single colonies with larger colonies were selected from plates and subjected to two fermentation trials for validation, resulting in a genetically engineered *Yersinia lipolytica* strain, T-09, with significantly improved performance.

[0048] The transformation was performed using the Frozen EZ Yeast Transformation II kit. TM (Purchased from ZymoResearch) Follow the instructions in the kit's manual.

[0049] The genetically engineered *Yarrowia lipolytica* strain T-09 can produce erythritol using glycerol as a carbon source, with a yield reaching 26 g / L. Figure 2 As shown.

[0050] The obtained Yeast Extract T-09 was deposited with the China Center for Type Culture Collection on November 24, 2022, with accession number CCTCC NO:M 20221813.

[0051] Example 2: Construction of strains JT-17 and JT-18

[0052] 2.1 Removal of *Yarrowia lipolyticis* genetically engineered strain T-09 (CCTCC NO: M 20221813) obtained in Example 1 hurray3 The specific experimental procedures for screening and labeling are as follows:

[0053] The plasmid pUB4-CRE was transformed into the genetically engineered *Yarrowia lipolytica* strain T-09 and placed on a YPD selection plate supplemented with 800 mg / L hygromycin B. Colonies were picked from the plate for verification. Further verification was then performed. hurray3 The filter markers were successfully removed.

[0054] 2.2 Constructing expression cassette plasmids for endogenous genes in Yersinia lipophila, as detailed below:

[0055] Endogenous Yeast lipase DAK2 Gene (SEQ ID NO.1), promoter P U4TEF (SEQ ID NO.9) and terminator T XPR2 (SEQ ID NO.10) Expression cassette P was obtained via overlap PCR.U4TEF - DAK2 -T XPR2 Sequence fragments;

[0056] Endogenous Yeast lipase GCY3 Gene (SEQ ID NO.2), promoter P U4TEF (SEQ ID NO.9) and terminator T XPR2 (SEQ ID NO.10) Expression cassette P was obtained via overlap PCR. U4TEF - GCY3 -T XPR2 Sequence fragments;

[0057] Endogenous Yeast lipase ARA1 Gene (SEQ ID NO.3), promoter P U4TEF (SEQ ID NO.9) and terminator T XPR2 (SEQ ID NO.10) Expression cassette P was obtained via overlap PCR. U4TEF - ARA1 -T XPR2 Sequence fragment.

[0058] P is assembled using the Gibson assembly method. U4TEF - DAK2 -T XPR2 and P U4TEF - GCY3 -T XPR2 The expression cassette was seamlessly cloned into plasmid pINA1312, resulting in plasmid pINA1312- DAK2 - GCY3 Its sequence is shown in SEQ ID No. 6;

[0059] P is assembled using the Gibson assembly method. U4TEF - DAK2 -T XPR2 and P U4TEF - ARA1 -T XPR2 The expression cassette was seamlessly cloned into plasmid pINA1312, resulting in plasmid pINA1312- DAK2 - ARA1 Its sequence is shown in SEQ ID No. 7.

[0060] 2.3 Constructing genetically engineered strains JT-17 and JT-18, as detailed below:

[0061] The plasmid pINA1312- obtained in step 2.2 DAK2 - GCY3 through Note After linearization by I enzyme digestion, it was transformed into a solution for removing... hurray3 The engineered strain JT-17 was obtained from the selected marker Yersinia lipophila T-09.

[0062] It has been verified that the genome of *Yarrowia lipolyticis* JT-17 has been successfully integrated with... DAK2 Genes and GCY3 Gene.

[0063] The genetically engineered *Yarrowia lipolytica* strain JT-17 can produce erythritol using glycerol as a carbon source, with a yield reaching 47.1 g / L. Figure 3 As shown.

[0064] The plasmid pINA1312- obtained in step 2.2 DAK2 - ARA1 through Note After linearization by I enzyme digestion, it was transformed into a solution for removing... hurray3 The engineered strain JT-18 was obtained from the selected marker Yersinia lipophila T-09.

[0065] It has been verified that the genome of the genetically engineered *Yarrowia lipophila* strain JT-18 has been successfully integrated with... DAK2 and ARA1 Gene.

[0066] The genetically engineered *Yarrowia lipolytica* strain JT-18 can produce erythritol using glycerol as a carbon source, achieving a yield of 37.0 g / L. Figure 3 As shown.

[0067] Example 3: Construction of strains JT-21 and JT-22

[0068] 3.1 Construct the expression cassette sequence fragment of the endogenous gene in *Yarrowia lipophila*, as follows:

[0069] Endogenous Yeast lipase TAL1 Gene (SEQ ID NO.5), promoter P U4TEF (SEQ ID NO.9) and terminator T XPR2 (SEQ ID NO.10) Expression cassette P was obtained via overlap PCR. U4TEF - TAL1 -T XPR2 Sequence fragments;

[0070] Endogenous Yeast lipase TKL1 Gene (SEQ ID NO.4), promoter P U4TEF (SEQ ID NO.9) and terminator T XPR2 (SEQ ID NO.10) Expression cassette P was obtained via overlap PCR. U4TEF - TKL1 -T XPR2 Sequence fragment.

[0071] 3.2 Construct expression plasmids for endogenous genes in Yersinia lipophila, as detailed below:

[0072] P is assembled using the Gibson assembly method. U4TEF - TAL1 -T XPR2 and P U4TEF - TKL1 -T XPR2 The expression cassette was seamlessly cloned into plasmid pINA1269, resulting in plasmid pINA1269- TAL1 - TKL1 Its sequence is shown in SEQ ID No. 8.

[0073] 3.3. The plasmid pINA1269- obtained in step 3.2 is... TAL1 - TKL1 through BssH After linearization by enzyme II digestion, it was transformed into the genetically engineered yeast JT-17 to obtain the transformed strain JT-21.

[0074] It has been verified that the genome of the genetically engineered *Yarrowia lipophila* strain JT-21 has been successfully integrated with... TKL1 and TAL1 Gene.

[0075] The genetically engineered *Yarrowia lipolytica* strain JT-21 can produce erythritol using glycerol as a carbon source, achieving a yield of 53.1 g / L. Figure 4 As shown.

[0076] The plasmid pINA1269- obtained in step 3.2 TAL1 - TKL1 through BssH After linearization by enzyme II digestion, it was transformed into the genetically engineered yeast JT-18 to obtain the transformed strain JT-22.

[0077] It has been verified that the genome of the genetically engineered *Yarrowia lipophila* strain JT-22 has been successfully integrated with... TKL1 and TAL1 Gene.

[0078] The genetically engineered *Yarrowia lipolytica* strain JT-22 can produce erythritol using glycerol as a carbon source, achieving a yield of 39.0 g / L. Figure 4 As shown.

[0079] Example 4: Determination of erythritol production by the strain

[0080] The following strains of Yeast Extract (Polf), strain T-09 (CCTCC NO: M 20221813) prepared in Example 1, strains JT-17 and JT-18 prepared in Example 2, and strains JT-21 and JT-22 prepared in Example 3 were inoculated into 2 mL of YPG medium (which consists of 5% glycerol, 2% peptone, and 1% yeast extract, with the remainder being water, all percentages being by mass) and cultured for 24 hours. Then, the initial OD was used as the starting point. 600 The culture medium was inoculated at a rate of 0.4 g into 50 mL of EPF medium (which consists of 10% glycerol, 0.23% ammonium sulfate, 0.022% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.1% yeast extract, 2.5% sodium chloride, and 0.3% calcium carbonate, with the remainder being water; all percentages are by mass).

[0081] After 7 days of fermentation, erythritol was qualitatively and quantitatively analyzed using high-performance liquid chromatography (HPLC). The results are shown in [reference needed]. Figure 5 .

[0082] Figure 5 The results showed that strain JT-21 had a significantly improved ability to produce erythritol, with a yield of 53.1 g / L on day 7 of fermentation.

[0083] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A genetically engineered *Yersinia lipolytica* strain that produces erythritol from glycerol, characterized in that, Based on the genetically engineered *Yarrowia lipolyticis* strain with accession number CCTCC NO: M 20221813, the glycerol dehydrogenase gene of the dihydroxyacetone metabolism pathway was overexpressed. GCY3 and dihydroxyacetone kinase gene DAK2 or glycerol dehydrogenase gene ARA1 and dihydroxyacetone kinase gene DAK2 ,in: The glycerol dehydrogenase gene GCY3 The nucleotide sequence of the dihydroxyacetone kinase gene is shown in SEQ ID NO.

2. DAK2 The nucleotide sequence of the glycerol dehydrogenase gene is shown in SEQ ID NO.

1. ARA1 The nucleotide sequence is shown in SEQ ID NO.

3.

2. A genetically engineered *Yersinia lipolytica* strain that produces erythritol using glycerol, characterized in that, Based on the *Yersinia lipophila* genetically engineered strain described in claim 1, a transketase gene for the non-oxidative pentose phosphate pathway is overexpressed. TKL1 and transaldase gene TAL1 ,in: transketase gene TKL1 The nucleotide sequence is shown in SEQ ID NO.4, and the transaldolase gene is described. TAL1 The nucleotide sequence is shown in SEQ ID NO.

5.

3. The application of the genetically engineered *Yarrowia lipolytica* strain according to claim 1 or 2, characterized in that, Used in the production of erythritol.

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