CN118325753A - Yarrowia lipolytica utilizing formic acid obtained by alanine-glyoxylate aminotransferase and preparation method thereof - Google Patents

Abstract

The invention discloses a yarrowia lipolytica utilizing formic acid obtained by alanine-glyoxylate transaminase and a preparation method thereof. Transcriptome sequencing is performed on wild type yarrowia lipolytica which is domesticated in a laboratory and tolerates high concentration of formate, and after the transcriptome analysis is assisted by flux distribution data predicted by a metabolic network model, a formate metabolic pathway from the endogenous source of the yarrowia lipolytica strain, namely a serine dual cycle pathway which depends on glyoxylate and threonine driving (gSer-tSer dual cycle) is discovered. The invention carries out single factor over-expression test on the core gene in the pathway in wild yarrowia lipolytica W29, can obviously increase the biomass of the strain in formate culture medium, and increases the amplification by 10% -50%. Since this pathway is a complete autocatalytic cycle, the net reaction is the conversion of one molecule of formic acid and one molecule of carbon dioxide or bicarbonate to one molecule of glycine, and this route can be used to produce glycine and its derivatives.

Description

Yarrowia lipolytica utilizing formic acid obtained by alanine-glyoxylate aminotransferase and preparation method thereof

The application relates to yarrowia lipolytica with the original application number 202410420032.0 and the name of yarrowia lipolytica for formic acid utilization and a preparation method thereof, and the application is a divisional application of an application patent application of 2024, 4 and 9.

Technical Field

The invention belongs to the fields of synthetic biology and genetic engineering, and particularly relates to yarrowia lipolytica utilizing formic acid obtained by alanine-glyoxylate aminotransferase and a preparation method thereof.

Background

Formic acid and methanol are known as liquid sunlight and can be prepared by electrochemical reduction of carbon dioxide. Compared with the combustibility of methanol, formic acid and salts thereof are safer and easier to store. And the formic acid assimilating strain can avoid the strong toxicity of formaldehyde, which is a toxic intermediate of the methanol nutrition strain. Therefore, an efficient formic acid biological utilization technical route is designed and excavated, and the efficient formic acid biological utilization technical route is applied to the development of strains capable of utilizing formic acid for synthesizing target products, and has definite low-carbon properties and important application prospects.

Currently, formate-rich strains are largely divided into two classes, methyl-rich strains that naturally have a formate assimilation route and engineered formate assimilation strains. The synthesis of methyl nutrition type bacterial strains constructed by integrating formic acid assimilating elements and routes in natural bacteria into bacterial strains easy to edit and grow rapidly, such as escherichia coli, pseudomonas, saccharomyces cerevisiae and the like, is increasingly the most main obtaining means of the methyl nutrition type bacterial strains. However, the formate assimilation route is very rare, mainly the natural glycine reduction pathway, the natural serine cycle, the artificially modified serine cycle, and the process of producing pyruvic acid by using acetyl-CoA to assimilate formate under anaerobic conditions. Glycolysis, the tricarboxylic acid cycle and pentose phosphate pathways are well-recognized central metabolic pathways in carbohydrate substrate utilization, the former two metabolic flux distributions being particularly dominant. However, depending on the environment and growth phase of the cells, there may be some difference or even the opposite result. In methanotrophic bacteria, the pentose phosphate pathway tends to be more dominant due to the change in substrate uptake, cells need retrograde regenerated glucose as a component of the cell wall, etc., and the TCA cycle may be partially bypassed due to the change in energy demand. Thus, in formate-utilizing strains, the type of central metabolism is likely to also exist in a process that is distinct from the traditional carbohydrate metabolism process. How to present and utilize these specific backbone routes in a network of intricate cells is of great importance to the low carbon biological manufacturing process under the aim of dual carbon.

Patent application CN117363499a discloses a method for producing single cell proteins using industrial wastewater, sodium acetate, sodium formate to culture yarrowia lipolytica, wherein sodium formate is only used as a very low content component (0.5-2 g/L) of the culture medium, only achieving very limited availability of formate and not improving the strain's tolerance to formate. Patent application CN116622612a discloses a method for obtaining formate-tolerant saccharomyces cerevisiae by adopting plasma mutagenesis technology, which has high mortality rate and difficult evaluation of potential influence on phenotype of surviving strain, and the formed strain has great defect risk in the subsequent expanding application process. Patent application CN115521880A discloses a method for constructing transgenic formate nutritional yeast, which is realized by introducing exogenous genes into the yeast, and limits the application of the strain in the fields of single cell proteins, single cell grease and the like. Moreover, none of the above methods provides only a method for obtaining strains, and does not provide a new tool, a new route and a new enzyme element that can migrate applications, and the expansibility and reusability are greatly reduced.

Disclosure of Invention

According to the method for efficiently and quickly obtaining the formic acid nutritional yarrowia lipolytica, the dependence of the strain on glucose, xylose and other carbohydrate carbon sources is eliminated, the methyl nutritional attribute of the strain is furthest molded, and meanwhile, enough strain groups for continuous evolution can be accumulated, so that the methyl nutritional yarrowia lipolytica which can endure high-concentration formic acid and takes the formic acid as a main or unique carbon source is efficiently and quickly obtained, and is used as a base synthesis chassis of single-cell protein, single-cell grease and engineering formic acid-based products. Meanwhile, through analyzing the metabolic basis of the strain, a core enzyme element and a metabolic route with functions are obtained, so that the method is applied to improving the formic acid utilization capacity of the strain and the synthesis capacity of the derivative biobased product. The present invention has been finally developed.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

The invention also provides a yarrowia lipolytica with formic acid utilization, which is obtained by over-expressing a gene coding for a catalytic enzyme involved in one or more links of a serine synthesis cycle gSer cycle driven by glyoxylate and a serine cycle tSer cycle driven by threonine in the yarrowia lipolytica under formate culture conditions:

Wherein, gSer loops are as follows: 1) formic acid is catalyzed by trifunctional tetrahydrofolate formylase/methylene-tetrahydrofolate dehydrogenase/methylene tetrahydrofolate cyclohydrolase to form 5, 10-methylene tetrahydrofolate, 2) 5, 10-methylene tetrahydrofolate and glycine are catalyzed by serine hydroxymethyltransferase to form serine, 3) serine is catalyzed by serine deaminase to form pyruvic acid, 4) pyruvic acid is catalyzed by pyruvate carboxylase to fix carbon dioxide to form oxaloacetic acid, 6) oxaloacetic acid and acetyl-CoA are catalyzed by citrate synthase to form citric acid, 7) citric acid is converted to isocitric acid by cis-aconitate synthase and then is decomposed to glyoxylic acid and succinic acid by isocitrate lyase, 8) glyoxylic acid is catalyzed by alanine-glyoxylate transaminase to form glycine, 9) succinic acid is reacted in cascade catalyzed by succinate dehydrogenase, fumaric acid hydratase and malate dehydrogenase to regenerate oxaloacetic acid; tSer loop links: 1) the formation of 5, 10-methylene tetrahydrofolate by catalysis of trifunctional tetrahydrofolate formylase/methylene-tetrahydrofolate dehydrogenase/methylene tetrahydrofolate cyclohydrolase, 2) the formation of serine by catalysis of serine hydroxymethyltransferase with 5, 10-methylene tetrahydrofolate, 3) the conversion of serine to pyruvate by catalysis of serine deaminase, 4) the formation of oxaloacetate by catalysis of pyruvate carboxylase, 5) the conversion of oxaloacetate to threonine by cascade catalysis of glutamate-oxaloacetate transaminase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine kinase and threonine synthase, 6) the cleavage of threonine to acetaldehyde and glycine by threonine aldolase, 7) the catalytic conversion of acetaldehyde to acetyl-CoA synthase or bifunctional acetyl-CoA hydrolase/succinyl-CoA: acetate-CoA transferase.

Preferably, one or more of the following coding genes and their isozymes are overexpressed: trifunctional tetrahydrofolate formative enzyme/methylene-tetrahydrofolate dehydrogenase/methylene tetrahydrofolate cyclohydrolase, formate dehydrogenase-1, acetyl-CoA hydrolase/succinyl-CoA: acetate-CoA transferase, acetaldehyde dehydrogenase-3, alanine-glyoxylate transaminase, isocitrate lyase-1, isocitrate lyase-2, threonine aldolase, pyruvate carboxylase, serine deaminase-1 and serine deaminase-2.

Specifically, the over-expression is realized by introducing an expression plasmid carrying a coding gene into yarrowia lipolytica, and a specific plasmid is taken as a starting plasmid, wherein the plasmid pCQ contains PGPD promoter and TPEX20 terminator.

More specifically, the coding gene is derived from yarrowia lipolytica.

The invention provides application of yarrowia lipolytica in synthesizing glycine or derivative products thereof.

Further, the derivative product is one or more of serine, methylated glycine, betaine, and glyphosate.

Specifically, the raw material required for synthesizing glycine or its derivative product is formic acid, carbon dioxide or bicarbonate. More specifically, the yarrowia lipolytica is cultured with formic acid as the primary or sole carbon source at the time of application.

Transcriptome sequencing was performed on wild type yarrowia lipolytica which was domesticated in the laboratory and tolerated to high formate concentrations, and after assisted transcriptomic analysis by flux distribution data predicted by metabolic network model, a formate metabolic pathway was found from the endogenous source of yarrowia lipolytica strain, gSer-tSer double cycle (serine double cycle pathway dependent on glyoxylate and threonine drive). The invention carries out single factor over-expression test on 14 core genes in the pathway in wild yarrowia lipolytica W29, wherein most (11/14) can obviously increase the biomass of the strain in formate culture medium, and the amplification is 10% -50%. Since this pathway is a complete autocatalytic cycle, the net reaction is the conversion of one molecule of formic acid and one molecule of carbon dioxide (or bicarbonate) to one molecule of glycine, and this route can be used to produce glycine and its derivatives.

Drawings

FIG. 1 is a representation of the growth of an evolved strain. Panel a shows the growth curve of an evolved strain with 6.6% sodium formate added to YEP medium; panel b shows the maximum specific growth rate and the time required to reach the maximum specific growth rate for each strain in panel a; panel c shows the growth curve of the evolved strain with 6.6% sodium formate added to the SC medium; d, graph c shows the maximum specific growth rate of each strain and the time required to reach the maximum specific growth rate; panel e shows the maximum dry weight of each strain cultured with 6.6% sodium formate added to the SC medium; FIG. f shows the growth curve of W29 in different media.

FIG. 2 shows the growth curve and formate utilization of the evolutionarily dominant strain. Growth curve and formic acid consumption curve of a strain grown at 100 h in SC medium, wherein the additional carbon source is 10 g/L formic acid: wherein, the wild strain W29 is shown in the graph a, the adaptive strain M33-3 is shown in the graph b, the evolved strain M25-14 assisted by a base editing tool is shown in the graph c, the evolved strain M25-70 assisted by the base editing tool is shown in the graph d, and the formic acid consumption of the four strains at 24, 48 and 72 h is shown in the graph e.

FIG. 3 shows the results of transcriptome analysis of Y. Panel a shows metabolic pathways and transcript levels involved in gene formation of formate utilization central pathways; panel b shows transcript levels of the endogenous 9 formate dehydrogenases (Fdh) in the strain; panel c shows the transcript levels of genes associated with formate metabolic pathways.

FIG. 4 is a model predicted metabolic pathway for yarrowia lipolytica to regenerate biomass using formate. The numbers represent metabolic flux; the full names of enzymes and metabolites are shown in tables 4 and 5.

FIG. 5 shows the results of an overexpression test of key genes of the formate assimilation module. Wherein, panels a and b show OD ₆₀₀ after 48: 48 h culture in SC medium containing 10 g/L sodium formate, and panel c shows OD ₆₀₀ after 48: 48 h culture in YEP medium containing 66: 66 g/L sodium formate, and Control represents Control strain carrying empty plasmid.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

EXAMPLE 1 construction of genomic Global perturbation tool plasmid and Yeast transformation

All strains used in the present invention are shown in Table 1, in which the construction and transformation of plasmids were carried out using E.coli DH5a as cloning host, and yarrowia lipolytica strain W29 was derived from ARS Culture Collection (NRRL). Genomic global perturbation tool plasmid pCQ was constructed by isothermal Assembly (Gibson Assembly) method. First, starting from a laboratory-stored pMY19 plasmid (comprising the ampicillin resistance gene Amp ^R, the plasmid replication element CEN adapted to yarrowia lipolytica, the replication element (ori) sequence adapted to E.coli and the defective supply back gene URA 3), URA3 was replaced with Hygromycin B resistance gene HygB, thereby forming plasmid pCQ. The above backbone fragments were obtained by PCR amplification using primers CQ-063 and CQ-064, including the yarrowia lipolytica Hygromycin B (HygB) resistance gene, the Autonomously Replicating Sequence (ARSs) ORI-CEN, the replicating element (ORI) sequence of E.coli and the ampicillin resistance gene (AmpR). The oleic acid-inducible promoter PPOX2 (accession number GB: CP017558.1 in NCBI) and the yarrowia lipolytica-derived helicase MCM5 gene (accession number GB: CP028448.1 in NCBI) were amplified from the genomic DNA of W29 using primer pairs CQ-065, CQ-066 and CQ-068, CQ-069. Sequence PmCDA in the genomic global perturbation tool plasmid was a cytidine deaminase gene from sea eel, which was codon optimized synthesized via GENEWIZ (Azenta life sciences company, tianjin, china) according to the preference of y. The constructed plasmid was verified by DNA sequencing (GENWIZ, china). The yeast transformation adopts a lithium acetate method. Finally, the transformants developed a single clone on YPD agar medium containing 200 μg/L HygB, indicating successful transformation of the genomic global perturbation tool plasmid in wild-type yarrowia lipolytica W29. The detailed information of plasmids, primers and genes is shown in tables 2-3 and SEQ ID No.1.

Table 1. Yeast strains mentioned in the present invention.

TABLE 2 plasmids used in the present invention

TABLE 3 primers used in the present invention

TABLE 4 names and Gene numbering of enzymes

TABLE 5 full and abbreviations for metabolites

Example 2 cultivation and adaptive laboratory evolution of yarrowia lipolytica

The present invention uses YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) or YPD medium supplemented with 200 μg/mL HygB to culture yeast cells and yeast cells harboring genomic global perturbation tool plasmid pCQ or vector plasmid pCQ 24. Formate tolerance tests were performed using YEP (10 g/L yeast extract and 20 g/L peptone) and Synthetic Complete (SC) medium (1.5 g/L yeast nitrogen-free nitrogen source without amino acids or ammonium sulfate, 5 g/L ammonium sulfate, 36 mg/L myo-inositol and 2 g/L amino acid mixture). Different amounts of sodium formate (10 g/L, 20 g/L, 40 g/L, 60 g/L and 80 g/L, respectively) were added to the medium with or without 20 g/L glucose. Laboratory adaptive evolution experiments used YEP medium (YEP-F medium) supplemented with 200. Mu.g/mLHygB sodium formate. To increase the tolerance of the cells, the supplemental sodium formate concentration was increased stepwise from 40 g/L to 66 g/L. YEP and SC media supplemented with 66 g/L sodium formate were used to evaluate formate availability. To overexpress the key genes in formate assimilating strains (constructed in plasmids 29-32), the present invention used YEP medium supplemented with 66 g/L sodium formate and 200. Mu.g/mL HygB, and modified SC medium supplemented with 10 g/L formic acid and 200. Mu.g/mL HygB (sodium glutamate instead of ammonium sulfate). The medium used for formate consumption determination was SC medium supplemented with 10 g/L formate. To overexpress the key genes in formate assimilating strains (carried in plasmids 29-32), we used YEP medium supplemented with 66 g/L sodium formate and 200. Mu.g/mL HygB, and modified SC medium supplemented with 10 g/L formic acid and 200. Mu.g/mL HygB (sodium glutamate instead of ammonium sulfate). The medium used for formate consumption determination was SC medium supplemented with 10 g/L sodium formate. Coli strains were grown in LB medium (LB, 20 g/L DifcoTM LB Broth Miller) containing 100 mg/L of ampicillin at 37 ℃. When agar plates were prepared, 20 g/L of BactoTM-agar was added. The growth condition of the strain is estimated by carrying out real-time on-line monitoring on a 48-hole deep hole plate through a high-flux microorganism growth curve analysis system (Gering, tianjin in China), and the oscillation frequency is 800 rpm.

Adaptive laboratory evolution method: adaptive laboratory evolution of wild-type yarrowia lipolytica W29 was performed in 15 mL culture tubes or 100mL flasks with 40 g/L formate and 200 μg/mL HygB of YEP medium added as starting medium. Cell culture was passaged once every 36 h, with an initial OD ₆₀₀ of 0.05. Initially, single colonies of yeast containing pCQ and pCQ plasmids were inoculated into YPD medium containing 200. Mu.g/mL HygB and cultured overnight at 30 ℃. Seed cultures with an inoculum size of 1% were then transferred to YEP-F medium (YEP medium, 40 g/L sodium formate and 200. Mu.g/mL HygB added) until a pre-log phase (about 12 h) was reached. Subsequently, 0.25% oleic acid was added to induce the expression of MCM 5-cytosine deaminase fusion genes. After growth editing 24 h, the yeast cells were transferred to 42 g/L sodium formate medium and the above steps were repeated. The sodium formate concentration was gradually increased in a gradient of 1-2 g/L and passaging was repeated until the OD ₆₀₀ of the medium was no longer significantly increased. The evolved bacterial cultures were then diluted and transplanted onto solid medium containing high concentrations of formate (SC-G medium containing 66G/L sodium formate) to screen strains capable of forming larger colonies. After cultivation in medium with formate as sole carbon source, dominant strains were selected for subsequent analysis according to the duration of the lag phase and the final biomass of the strain. For the confirmed target strain, plasmid removal experiments were performed using YPD medium. The evolved strain containing plasmid pCQ was cultured in YPD medium in two rounds of dilution (1:100). Single colony isolation was then performed on YPD agar plates. No single colony growth was found on YPD agar containing 200. Mu.g/mL HygB, whereas single colony growth was found on YPD agar, demonstrating successful plasmid elimination. To confirm that HygB plasmid pCQ25 contained in the strain had been deleted, the strain was inoculated into YPD medium supplemented with 200. Mu.g/mL HygB; strains lacking the pCQ plasmid were unable to grow in YPD medium containing HygB.

Wild type W29 strain was edited using the genomic global perturbation tool plasmid for yarrowia lipolytica constructed in example 1 above. The YEP medium is more favorable for accumulating yeast cells than the SC medium (f in fig. 1). Thus, adaptive evolution experiments first established formate-based evolution pressure using YEP-F medium (sodium formate added to YEP medium) and achieved adequate cell growth before starting random base editing. During the evolution period, a total of 25 rounds of switching were performed. In each round, cells grew to the exponential phase (about 12 h) in YEP-F, then 0.25% oleic acid was added as an inducer of editing, continuing to evolve 24 h. In each round, the formate concentration was gradually increased from 4% to 6.6% to gradually increase the tolerance of the cells. Finally, two improved strains resistant to formate were obtained by the genome global perturbation tool plasmid-assisted ALE method, designated M25-70 and M25-14, respectively. In addition, the control strain was obtained by a conventional laboratory adaptive evolution method under the same culture conditions by transferring an empty plasmid without editing function into W29, and was designated as M33-3. The results of the growth tests on the above strains showed that strains M25-70 and M25-14 showed a significant increase in toxicity to formate in the medium containing 6.6% sodium formate, with a delay period reduced by about 15 hours compared to the case of W29 (FIGS. 1 a, b). Furthermore, when these four strains were cultured in SC medium containing 6.6% sodium formate, M25-70 and M25-14 exhibited more significant growth, with an increase in OD ₆₀₀ of 39% and 37%, respectively, as compared to W29. In contrast, the adaptively evolved strain M33-3 increased only 17% (c in FIG. 1). Meanwhile, the maximum specific growth rates of M25-70 and M25-14 were increased by 49% and 37%, respectively, as compared to W29 (d in FIG. 1). These results were also consistent in YEP medium containing 6.6% sodium formate (b in fig. 1). Furthermore, the stem cell weight (DCW) of these two strains was increased by 45% and 42% respectively compared to the wild-type strain W29 in SC medium with 6.6% sodium formate as sole carbon source (e in fig. 1).

EXAMPLE 3 formate consumption level test of evolved strains

The detailed method for formate concentration analysis is to take bacterial liquid 1mL in different time periods, centrifuge 5min at a rotating speed of 5,000 rpm, filter the supernatant by an organic nylon membrane, and fill the filtered supernatant into a sample bottle, and measure formate content by High Performance Liquid Chromatography (HPLC) (Waters, calif.) using a BioRad HPX 87H chromatographic column (9 μm,300 mm ×7.8 mm) and 5 mmol/L sulfuric acid as mobile phase. The fermentation cultures were collected at 16, 20, 24, 48 and 72h, respectively, and centrifuged at 5,000 rpm for 5 min. The flow rate was 0.5 mL/min, the sample injection amount was 10. Mu.L, the column temperature was 55deg.C, and detection was performed using a RID detector. W29 and three evolved strains M25-70, M25-14 and M33-3 were cultivated in SC medium containing 10 g/L sodium formate. The efficiency of these strains for formate utilization was assessed by measuring the change in formate concentration in the medium. As a result, as shown in FIG. 2, the two strains M25-70 and M25-14, which had been subjected to global perturbation tool plasmid-assisted evolution, exhibited more excellent formate consumption ability, and exhibited higher formate conversion rate and faster growth rate, compared to the wild-type W29 and the conventional ALE strain M33-3. When 48 h was cultured, strain M25-70 showed the highest sodium formate consumption of 1.778 g/L, which was a 50% increase in consumption compared to 1.186 g/L of wild type W29. Similarly, the sodium formate consumption of M25-14 was 1.581 g/L, which was increased by 33% on the basis of the wild-type W29. By 72h, the sodium formate consumption of strain M25-70 reached 1.742 g/L, whereas the sodium formate consumption of strain M25-14 reached a peak at 72h was 1.820 g/L, which was 58% increased compared to wild type W29.

Example 4 transcriptome sequencing and model-assisted transcriptome resolution

To fully understand global level changes in formate tolerance and optimized formate metabolism to y. Lipolytica under conditions affecting 6.6% sodium formate, we performed transcriptome sequencing of the dominant evolved population under 6.6% sodium formate influence and control group without formate addition. In order to obtain more referenceable data, the present example also adds the investigation of two suboptimal strains M25-35 and M25-59, in addition to the four strains W29, M33-3, M25-70 and M25-14 mentioned above. The newly added M25-35 and M25-59 strains were obtained using exactly the same methods and procedures as M25-70 and M25-14, with the only difference that they performed worse than the two evolutions of M25-70 and M25-14 in the growth phenotype test. The transcriptome analysis was performed by culturing 5 dominant strains and a wild-type control strain W29 in SC medium containing 66 g/L sodium formate at 30℃and 220: 220 rpm. Cells in mid-growth (18 h) were collected for RNA isolation. RNA preparation, library creation and sequencing were performed by Novogene company (Tianjin, china) using the Illumina Hiseq platform. The results indicate that 9 endogenous formate dehydrogenases (Fdh) show a collective up-regulation trend (b in fig. 3). Meanwhile, pyruvate carboxylase (Pyc) with carbon fixation function catalyzes the conversion of pyruvic acid into oxaloacetic acid) is up-regulated, which shows that the carbon fixation function in the strain is enhanced. In addition, some genes, such as LtaA, adh, ach, aarc, icl, sda and Agt, etc., were also up-regulated (c in fig. 3). To better visualize the association between these genes, flux distribution predictions were made for the strain's formate metabolic pathway using the metabolic network model iYali of y. Finally, a novel formate-centre metabolic pathway of yarrowia lipolytica under formate-specific culture conditions was discovered by mapping transcriptome data with a flux map, characterized by the concurrent synthesis of glycine from glyoxylic acid and threonine, followed by the combination of glycine with 5, 10-methylene tetrahydrofolate to serine, whereby deamination of serine to pyruvate is followed by carboxylation to oxaloacetic acid and to achieve communication and perfection of the overall metabolic process of the cell, the novel pathway disclosed by the transcriptome being named herein glyoxylic acid and threonine-enhanced serine double cycle (abbreviated gSer-tSer double cycle) (a in fig. 3).

Example 5 reverse metabolic engineering overexpression pathway Gene to elevate Strain growth level

In this example, 14 genes in the pathway or directly related to the pathway function were selected, and a single factor overexpression experiment was performed in the wild-type W29 strain, and the application effect of the pathway was further tested. Among them, the gene fragments Ftl1, ftl2, shmt1, shmt2, fdh-1, ach, adh-3, agt, icl-1, icl-2, ltaA, pyc, sda-1 and Sda-2 were amplified and purified from the W29 genome by PCR. The genes are carried by plasmids pCQ, pCQ, pCQ, 31, pCQ, pCQ, 49, pCQ50, pCQ, pCQ, pCQ, pCQ, pCQ, pCQ, pCQ and pCQ respectively, and the plasmids are constructed by means of standard restriction enzyme digestion and ligation.

These PCR products were designed with NheI and XhoI cleavage sites at the 5 'and 3' ends, respectively, and cloned into plasmid pCQ containing PGPD promoter and TPEX20 terminator. As shown in Table 2, the backbone in pCQ was amplified from plasmid pCQ, which retained the resistance markers ampicillin resistance gene (Amp ^R), hygromycin resistance gene (HygB) and replicative element (CEN), on the basis of which the PGPD promoter and TPEX20 terminator were added, and a multiple cloning site region (MCS) was inserted between them to facilitate integration of the foreign gene by means of cleavage-ligation. The 14 representative genes described above may cover the core reaction steps involved in the gSer-tSer double cycle, the purpose of the test being to further demonstrate that this pathway is a key pathway for formate assimilation, since it inevitably involves a rate limiting step that has a significant impact on growth. The above constructed strain was inoculated into a 48-well plate, cultured at 800 rpm,30℃in SC basal medium containing 10 g/L sodium formate to which 100 mg/L of ampicillin was added, and OD ₆₀₀ levels of the strain were recorded in real time by a Jieling microorganism growth Curve Analyzer (Tianjin, china). The results show that the over-expression of most of the genes (11/14) in the test significantly improves the growth level of cells in formic acid culture medium (a and b in FIG. 5) with the increasing range of 10% -50%, which strongly confirms that gSer-tSer double circulation plays a key role. For three Ftl2, shmt1 and Shmt2 genes that were not able to elevate cellular biomass by overexpression, they were also down-regulated in the transcriptome. Four strains overexpressing Ftl and Shmt were tested again in YEP medium containing 66 g/L sodium formate and found to be consistent with the expression of the above-described SC basal medium containing 10 g/L sodium formate (FIG. 5 c). The analytical reasons are that Ftl is taken as the primary step of formic acid assimilation, and the Ftl1 gene and the isozyme Ftl2 are not dominant in the real function; the reason why the slight down regulation and over expression of Shmt genes have no obvious promotion effect on growth is that the gSer-tSer double-cycle operation provides a large amount of glycine for cells, and Shmt taking glycine as a substrate can reduce the pressure of cell resources through down regulation and simultaneously maintain the original total catalytic activity, so that the cell requirements are met. Taken together, the above results strongly demonstrate that gSer-tSer double circulation enhances the utilization of yarrowia lipolytica strain formate to promote growth and functions to provide more adequate levels of amino acid loading for cells.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (8)

1. A recombinant yarrowia lipolytica, which is characterized in that it is obtained by over-expression of the following genes or their isozymes in yarrowia lipolytica: the gene codes for alanine-glyoxylate aminotransferase.

2. The formate-utilized yarrowia lipolytica of claim 1, wherein the over-expression is effected by introducing into the yarrowia lipolytica an expression plasmid carrying the encoding gene.

3. The formate-utilized yarrowia lipolytica of claim 2 wherein the expression plasmid is plasmid pCQ containing PGPD promoter and TPEX20 terminator.

4. The formate-utilized yarrowia lipolytica of claim 2, wherein the encoding gene is derived from yarrowia lipolytica.

5. Use of yarrowia lipolytica as claimed in any one of claims 1 to 4 for the synthesis of glycine or a derivative product thereof.

6. The use of claim 5, wherein the derivative is one or more of serine, methylated glycine, betaine, and glyphosate.

7. The use according to claim 6, wherein the starting material for the synthesis of glycine or its derivative is formic acid, carbon dioxide or bicarbonate.

8. The use of claim 7, wherein the yarrowia lipolytica is cultured with formic acid as the primary or sole carbon source.