Construction and application of escherichia coli strain for synthesizing alanine by using methanol as raw material

Abstract

The application discloses a recombinant Escherichia coli strain and application thereof, and the strain can synthesize alanine by taking methanol or a one-carbon compound as a raw material. On the premise of taking a chassis strain with strong methanol tolerance and utilization capacity as a basis, the recombinant strain comprises the following genetic modifications: (1) knocking out a by-product synthesis related gene, such as a phosphoacetyltransferase gene; (2) knocking out an alanine metabolism related gene, such as an alanine racemase gene; (3) enhancing the expression or function of an alanine transport protein; and (4) introducing an alanine dehydrogenase coding gene to realize the synthesis of alanine. The methanol utilization efficiency of the modified strain is significantly improved, the strain can efficiently convert methanol into various target products, and the strain shows a good application prospect in the fields of biological manufacturing and food raw material production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered strain capable of synthesizing L-alanine and / or D-alanine from methanol or other one-carbon compounds, its construction method, and its application. Background Technology

[0002] Alanine (C3H7NO2) is an aliphatic nonpolar α-amino acid with two enantiomers, L- and D-, both of which have significant applications in medicine, food, feed, biosynthesis, and scientific research. In medicine, L-alanine is a key component of amino acid infusions and compound preparations, used as an adjunct treatment for hepatic encephalopathy and for nutritional support in chronic nephritis. It is also an intermediate in the synthesis of anti-tuberculosis drugs and vitamin B6. In the food industry, it is widely used as a flavor enhancer and nutritional fortifier in beverages, condiments, and baked goods, helping to improve flavor stability and storage performance, meeting national food safety standards. In feed, adding L-alanine can improve feed conversion ratio and growth performance in livestock and aquatic animals, reducing breeding costs and aligning with current policies to reduce and replace soybean meal. In biosynthesis and scientific research, alanine serves as an important substrate or regulator in metabolic engineering and synthetic biology, participating in the regulation of microbial cell wall synthesis and can be used in the preparation of chiral materials, polypeptide bioproducts, and related biochemical research. D-alanine also has unique and important applications. The pharmaceutical field is its primary application area. As a key chiral raw material for the synthesis of antibacterial drugs (such as D-cycloserine), peptide drugs, and chiral drug intermediates, it can be used to treat tuberculosis and nervous system diseases. In the food and feed industry, D-alanine can be used as a flavor modifier and animal nutritional supplement to improve product taste and promote growth. Furthermore, in the biomedical materials field, D-alanine can be used to prepare biodegradable polyester polymers and chiral catalysts, playing an important role in asymmetric synthesis and new material development. In terms of the market, the global L-alanine market size was approximately US$165 million (approximately RMB 1.174 billion) in 2024, with an urgent need for efficient industrial production. Global demand for D-alanine is projected to reach 8,000–10,000 tons in 2025, with a market size of approximately US$90 million to US$100 million, of which the pharmaceutical sector accounts for over 60%.

[0003] Traditional methods for producing L-alanine mainly include enzymatic conversion and fermentation. Enzymatic conversion uses L-aspartic acid as a substrate, employing microorganisms with L-aspartic acid-β-decarboxylase activity. The cells or cell suspensions are immobilized as biocatalysts, catalyzing the production of L-alanine. However, the raw material L-aspartic acid used in enzymatic conversion is obtained from the cleavage of fumaric acid. The main raw material for fumaric acid production is petroleum, a non-renewable resource, which significantly increases the cost of L-alanine production via enzymatic methods. D-alanine production methods mainly fall into three categories: chemical synthesis, microbial fermentation, and enzymatic synthesis. Chemical synthesis is a mature process but involves many steps and insufficient optical purity. Enzymatic synthesis of D-alanine, involving a two-enzyme continuous catalytic system and a two-step lactic acid conversion process, is still immature. Microbial fermentation for L-alanine or D-alanine production primarily uses carbohydrates such as glucose and sucrose as raw materials, utilizing traditional fermentation strains (such as Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis) for biosynthesis. Microbial fermentation is simple, has mild reaction conditions, and is suitable for large-scale industrial production. However, carbon source cost is one of the main cost components of microbial fermentation. Carbohydrates such as glucose and sucrose are greatly affected by fluctuations in grain yield and price, posing a risk to food security and limiting the scale expansion of the industry.

[0004] Methanol and formaldehyde, as typical one-carbon compounds, are highly promising and inexpensive carbon sources and metabolic intermediates in the field of biosynthesis, playing a crucial role in the microbial transformation and preparation of high-value-added chemicals. Methanol is widely available and inexpensive, generated from resources such as natural gas and coalbed methane, or prepared through the hydrogenation reduction of carbon dioxide, aligning with the resource recycling requirements under the "dual carbon" goal. Formaldehyde, as a core intermediate in methanol metabolism, can be rapidly integrated into the central metabolic pathway of microorganisms, providing the carbon skeleton and energy support for the synthesis of target products. Utilizing microbial transformation technology to directionally regulate the metabolic flow of methanol and formaldehyde to synthesize amino acid products such as L-alanine or D-alanine not only overcomes the cost and food security limitations of traditional carbohydrate carbon sources but also achieves the high-value utilization of one-carbon resources, becoming a key research direction in the field of synthetic biology. Therefore, developing engineered strains that efficiently convert methanol into one-carbon compounds, synthesize alanine in high yields, have low by-product accumulation, and exhibit excellent genetic stability is of great technological and industrial significance. This breakthrough addresses the core issues in existing technologies, such as low utilization efficiency of one-carbon substrates, insufficient yield of target products, by-product interference, and poor genetic stability. It is crucial for promoting the low-cost industrial production of alanine and realizing the high-value utilization of one-carbon resources.

[0005] Escherichia coli, as a rapidly growing industrial cell factory with a mature and sophisticated genetic operating system, is widely considered to be the most promising microbial substrate for industrialization. Based on this, if a methanol-based alanine synthesis pathway is constructed using preliminarily modified E. coli with methanol utilization and tolerance as a host, it is expected to enable flexible switching between other carbon sources during fermentation, reducing production costs while supporting continuous strain iteration and process optimization. This strategy represents a highly feasible industrialization technology route. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention provides, in a first aspect, a recombinant *Escherichia coli* strain capable of producing L-alanine or D-alanine from methanol as a carbon source. The methanol feedstock is derived from carbon dioxide via electrocatalytic conversion. Thus, the recombinant *E. coli* strain can synthesize L-alanine or D-alanine from carbon dioxide as a starting material, through a methanol intermediate.

[0007] Furthermore, the recombinant Escherichia coli possesses the following metabolic pathway:

[0008] (S1) Convert methanol to fructose-6-phosphate;

[0009] (S2) Convert pyruvate to L-alanine or D-alanine;

[0010] Furthermore, the metabolic pathway (S1) is catalyzed by methanol dehydrogenase, 3-hexylose-6-phosphate synthase and 6-phosphate-3-hexose isomerase, thereby converting 1 molecule of methanol and 1 molecule of xylulose-5-phosphate into 1 molecule of fructose-6-phosphate.

[0011] The metabolic pathway (S2) is catalyzed by L-alanine dehydrogenase or D-alanine dehydrogenase, thereby converting 1 molecule of pyruvate into 1 molecule of L-alanine or 1 molecule of D-alanine.

[0012] The 5-phospholipid xylulose can be replenished using the gluconeogenesis and pentose phosphate pathways naturally present in Escherichia coli, and the 3-phosphoglyceraldehyde can be further converted into pyruvate using the glycolysis pathway naturally present in Escherichia coli.

[0013] Furthermore, the recombinant Escherichia coli has the following characteristics (A1) or (A2):

[0014] (A1) Contains an L-alanine dehydrogenase gene, and / or contains a substance that enhances the expression of the L-alanine dehydrogenase gene, and / or contains a substance that enhances the activity or content of L-alanine dehydrogenase; and, the alanine is L-alanine.

[0015] (A2) Contains a D-alanine dehydrogenase gene, and / or contains a substance that enhances the expression of the D-alanine dehydrogenase gene, and / or contains a substance that enhances the activity or content of D-alanine dehydrogenase; and the alanine is D-alanine.

[0016] Furthermore, the recombinant Escherichia coli also has the following characteristics:

[0017] (A3) Contains a methanol dehydrogenase gene, and / or contains substances that enhance the expression of the methanol dehydrogenase gene, and / or contains substances that enhance the activity or content of methanol dehydrogenase;

[0018] (A4) Contains the 3-hexylose-6-phosphate synthase gene, and / or contains substances that enhance the expression of the 3-hexylose-6-phosphate synthase gene, and / or contains substances that enhance the activity or content of 3-hexylose-6-phosphate synthase;

[0019] (A5) Contains a gene for 3-phosphate-3-hexose isomerase, and / or contains substances that enhance the expression of the gene for 3-phosphate-3-hexose isomerase, and / or contains substances that enhance the activity or content of 3-phosphate-3-hexose isomerase;

[0020] (A6) Does not contain the phosphorylacetyltransferase gene, and / or does not contain substances with phosphorylacetyltransferase activity;

[0021] (A7) Does not contain the pyruvate formate lyase gene, and / or does not contain substances with pyruvate formate lyase activity;

[0022] (A8) Does not contain the aldosterone dehydrogenase gene, and / or does not contain substances with aldosterone dehydrogenase activity;

[0023] (A9) Does not contain the alanine racemase gene, and / or does not contain any substance with alanine racemase activity;

[0024] (A10) Contains an alanine transporter gene, and / or contains substances that enhance the expression of the alanine transporter gene, and / or contains substances that enhance the activity or content of the alanine transporter.

[0025] Furthermore, the recombinant Escherichia coli is prepared by performing at least the following steps on Escherichia coli, which serves as the recipient bacterium:

[0026] (M1) Knockout of the phosphorylacetyltransferase gene pta;

[0027] (M2) Knock out the pyruvate formate lyase gene pflB;

[0028] (M3) Knockout of the aldol dehydrogenase gene adhE;

[0029] (M4) Knockout of the alanine racemase gene alar;

[0030] (M5) enhances the alanine transporter gene alaE;

[0031] (M6) Introduce an alanine dehydrogenase gene, specifically an L-alanine dehydrogenase gene GsldhA and / or a D-alanine dehydrogenase gene Stdadh, wherein when the L-alanine dehydrogenase gene is introduced, the product is L-alanine; when the D-alanine dehydrogenase gene is introduced, the product is D-alanine.

[0032] The *Escherichia coli* serving as the recipient bacterium is a methanol-efficient *E. coli* strain, which can be an *E. coli* strain that has been artificially modified to possess a methanol utilization metabolic pathway. In some specific embodiments of the present invention, the *E. coli* serving as the recipient bacterium is *E. coli* strain GL05 (accession number: CGMCC No. 35139), which already has the ability to express the methanol dehydrogenase gene *mdh*, the 3-hexylose-6-phosphate synthase gene *hps*, and the 6-phosphate-3-hexose isomerase gene *phi*.

[0033] Furthermore, the sequence of the L-alanine dehydrogenase gene GsldhA is shown in SEQ ID NO.1, and the sequence of the protein it encodes is shown in SEQ ID NO.2;

[0034] The sequence of the D-alanine dehydrogenase gene Stdadh is shown in SEQ ID NO.3, and the sequence of the protein it encodes is shown in SEQ ID NO.4.

[0035] The sequence of the methanol dehydrogenase gene mdh is shown in SEQ ID NO.5, and the sequence of the protein it encodes is shown in SEQ ID NO.6.

[0036] The sequence of the 3-hexylose-6-phosphate synthase gene hps is shown in SEQ ID NO.7, and the sequence of the protein it encodes is shown in SEQ ID NO.8;

[0037] The sequence of the 6-phosphate-3-hexose isomerase gene phi is shown in SEQ ID NO.9, and the sequence of the protein it encodes is shown in SEQ ID NO.10;

[0038] The sequence of the phosphorylated acetyltransferase gene pta is shown in the NCBI database under GeneID: 946778 (2026.4.23), and the sequence of the protein it encodes is shown in the NCBI database under NP_416800.1 (2025.12.9).

[0039] The sequence of the pyruvate formate lyase gene pflB is shown in the NCBI database under GeneID: 945514 (2026.4.23), and the sequence of the protein it encodes is shown in the NCBI database under NP_415423.1 (2025.12.9).

[0040] The sequence of the aldol dehydrogenase gene adhE is shown in the NCBI database under GeneID: 945837 (2026.4.23), and the sequence of the protein it encodes is shown in the NCBI database under NP_415757.1 (2025.12.9).

[0041] The sequence of the alanine racemase gene arr is shown in the NCBI database under GeneID: 948564 (2026.4.23), and the sequence of the protein it encodes is shown in the NCBI database under NP_418477.1 (2025.12.9).

[0042] The sequence of the alanine transporter gene alaE is shown in the NCBI database under GeneID: 947147 (2026.4.23), and the sequence of the protein it encodes is shown in the NCBI database under NP_417156.1 (2025.12.9).

[0043] Secondly, the present invention provides a recombinant *Escherichia coli* strain that uses methanol as a carbon source to produce L-alanine or D-alanine, wherein the recombinant *Escherichia coli* strain, based on the recombinant *Escherichia coli* strain described in the first aspect, further lacks the following metabolic pathway:

[0044] (S3) Convert pyruvate into acetyl-CoA.

[0045] Furthermore, the metabolic pathway (S3) is catalyzed by pyruvate dehydrogenase.

[0046] Furthermore, the recombinant Escherichia coli described in the second aspect, based on the characteristics of the first aspect (A1)-(A10), further possesses the following characteristics:

[0047] (B1) Does not contain the pyruvate dehydrogenase gene, and / or does not contain substances with pyruvate dehydrogenase activity;

[0048] (B2) Contains the propionyl-CoA carboxylase gene, and / or contains substances that enhance the expression of the propionyl-CoA carboxylase gene, and / or contains substances that have propionyl-CoA carboxylase activity.

[0049] (B3) Contains a methylmalonyl-CoA epimerase gene, and / or contains a substance that enhances the expression of the methylmalonyl-CoA epimerase gene, and / or contains a substance with methylmalonyl-CoA epimerase activity.

[0050] (B4) Contains the methylmalonyl-CoA mutase gene, and / or contains substances that enhance the expression of the methylmalonyl-CoA mutase (MCM) gene, and / or contains substances with methylmalonyl-CoA mutase activity.

[0051] Furthermore, the recombinant Escherichia coli described in the second aspect is prepared by performing at least the following steps on the recombinant Escherichia coli described in the first aspect:

[0052] (M7) Knock out the pyruvate dehydrogenase gene; specifically, the pyruvate dehydrogenase E1 subunit gene aceE.

[0053] (M8) Introduce the propionyl-CoA carboxylase gene SpccB;

[0054] (M9) The methylmalonyl-CoA epimerase gene Pmce was introduced;

[0055] (M10) Introduce methylmalonyl-CoA mutase genes; the methylmalonyl-CoA mutase genes are specifically the methylmalonyl-CoA mutase large subunit gene PmcmL and the methylmalonyl-CoA mutase small subunit gene PmcmS.

[0056] Furthermore, the sequence of the pyruvate dehydrogenase E1 subunit gene aceE is shown in the NCBI database GeneID:944834 (2026.6.3), and the sequence of the protein it encodes is shown in the NCBI database NP_414656.1 (2025.12.9);

[0057] The sequence of the propionyl-CoA carboxylase gene SpccB is shown in SEQ ID NO.11, and the sequence of the protein it encodes is shown in SEQ ID NO.12.

[0058] The sequence of the methylmalonyl-CoA epimerase gene Pmce is shown in SEQ ID NO.13, and the sequence of the protein it encodes is shown in SEQ ID NO.14.

[0059] The sequence of the methylmalonyl-CoA mutase large subunit gene PmcmL is shown in SEQ ID NO.15, and the sequence of the protein it encodes is shown in SEQ ID NO.16.

[0060] The sequence of the methylmalonyl-CoA mutase small subunit gene PmcmS is shown in SEQ ID NO.17, and the sequence of the protein it encodes is shown in SEQ ID NO.18.

[0061] Thirdly, the present invention provides the use of recombinant Escherichia coli as described in either the first or second aspect in the preparation of alanine.

[0062] Fourthly, the present invention provides a method for producing alanine, which utilizes the recombinant Escherichia coli described in either the first or second aspect, and obtains it through cell culture; specifically, it includes the steps of culturing the strain in a fermentation medium and collecting alanine from the fermentation product; wherein the fermentation medium uses methanol as a carbon source.

[0063] The beneficial technical effects achieved by this invention are as follows:

[0064] This invention provides a recombinant Escherichia coli that can grow and synthesize a variety of chemicals using one-carbon feedstocks such as methanol. Taking the synthesis of L-alanine or D-alanine as an example, the application value is explained in detail. This establishes a biotechnological route for synthesizing a variety of chemicals using inexpensive one-carbon feedstocks, which can be widely applied to the large-scale production of chemicals such as L-alanine and D-alanine. Preservation Instructions

[0065] (1) Bacterial strain name: Escherichia coli

[0066] Latin name: Escherichia coli

[0067] Strain number: GL05

[0068] Preservation Institution: CGMCC - China General Microbiological Culture Collection Center

[0069] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0070] Deposit date: July 8, 2025

[0071] CGMCC Registration Number: CGMCC No. 35139 Attached Figure Description

[0072] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0073] Figure 1 The graph shows the yield of L-alanine and D-alanine synthesized by engineered strains of Escherichia coli using methanol as a raw material. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0076] In the following embodiments, the *Escherichia coli* strain used as the recipient bacterium may be GL05. The *E. coli* GL05 strain is further derived from the *E. coli* MG1655 strain. The GL05 strain already includes the following characteristics: the methanol dehydrogenase gene *mdh*, the 3-hexylose-6-phosphate synthase gene *hps*, and the 6-phosphate-3-hexose isomerase gene *phi*. *E. coli* GL05 is deposited at the China General Microbiological Culture Collection Center (CGMCC) (accession number CGMCC No. 35139). For specific construction methods, please refer to the applicant's patent application (application number: 202610394842.2; application date: March 29, 2026).

[0077] In the following examples, plasmid pKD46 (CGSC#: 7739) is a product of the E. coli Genetic Depository at Yale University, USA (CGSC). The sequence information of plasmid pYB1k is described in patent CN119799606A. The sequence information of plasmid pSCre is described in patent CN120485085A. Plasmid pYB1k contains an arabinose promoter for the expression of exogenous genes. Both pKD46 and pSCre plasmids contain the temperature-sensitive origin of replication oriR101, which replicates normally at 30°C but is automatically lost when cultured above 37°C. Plasmid pKD46 contains Gam, Exo, and Beta proteins for mediating syngeneic expression, while plasmid pSCre contains Cre recombinase for the elimination of selection marker genes. The sequence of plasmid pUC57 can be found in GenBank: Y14837.1 (February 11, 1999) in the NCBI database. The pUC57 plasmid is a cloning vector. During the artificial gene synthesis process, the synthesized gene is directly constructed into the EcoRV site of the pUC57 plasmid.

[0078] T4 DNA Ligase (Catalog No. M0202V), BbsⅠ-HF (Catalog No. R3539S), and DpnⅠ (Catalog No. R0176L) were purchased from NEB. The DNA gel rapid purification kit (Catalog No. EG101-02) was purchased from TransGen.

[0079] The LB medium in the following examples was prepared as follows: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride were dissolved in deionized water, and the volume was adjusted to 1 L. The solution was then autoclaved at 121°C for 20 min at pH 7.0. An additional 15 g of agar powder was added to the solid medium.

[0080] The inorganic salt culture medium in the following examples was prepared as follows: 1.7 g citric acid, 5 g yeast extract, 9 g KH2PO4, 4 g (NH4)2HPO4, 20 g glucose, and 0.6 g MgSO4·7H2O were dissolved in deionized water, and the volume was adjusted to 1 L. The pH was 7.0, and the medium was autoclaved at 112°C for 30 min.

[0081] The fermentation medium composition and final concentrations in the following examples are as follows: Na₂HPO₄: 25 mM, KH₂PO₄: 25 mM, NH₄Cl: 50 mM, MgSO₄: 2 mM, methanol: 5 g / L. During the conversion process, methanol can be added to a final concentration of 5 g / L.

[0082] Table 1. List of sequence fragments

[0083] Serial Number Segment Name Serial Number Segment Name SEQ ID NO.1 GsldhA SEQ ID NO.18 PmcmS encodes amino acid sequence SEQ ID NO.2 GsldhA encodes an amino acid sequence SEQ ID NO.19 LKL SEQ ID NO.3 Stdadh SEQ ID NO.20 P119 SEQ ID NO.4 Stdadh encodes an amino acid sequence SEQ ID NO.21 RBS1 SEQ ID NO.5 mdh SEQ ID NO.22 TrrnB SEQ ID NO.6 mdh encodes an amino acid sequence SEQ ID NO.23 pta-up SEQ ID NO.7 hps SEQ ID NO.24 pta-down SEQ ID NO.8 hps-encoded amino acid sequence SEQ ID NO.25 pflB-up SEQ ID NO.9 phi SEQ ID NO.26 pflB -down SEQ ID NO.10 phi-encoded amino acid sequence SEQ ID NO.27 adhE -up SEQ ID NO.11 SpccB SEQ ID NO.28 adhE -down SEQ ID NO.12 SpccB encodes the amino acid sequence SEQ ID NO.29 alr-up SEQ ID NO.13 Pmce SEQ ID NO.30 alr-down SEQ ID NO.14 Pmce encodes an amino acid sequence SEQ ID NO.31 alaE-up SEQ ID NO.15 PmcmL SEQ ID NO.32 alaE-down SEQ ID NO.16 PmcmL encodes amino acid sequence SEQ ID NO.33 aceE-up SEQ ID NO.17 PmcmS SEQ ID NO.34 aceE-down

[0084] Table 2. Primer sequence listing

[0085] Primer or sequence name Nucleotide sequence (5' to 3') Ec-pta-F gttttgtaacccgccaaatcggcggtaacg Ec-pta-R taaaaaaccggaaatagtgattatttccgg Ec-pta-YF agcaccgccagctgagctggcggtgtgaaa Kan-R ttctatcgccttcttgacgag Ec-pflB-F aaacgaccaccattaatggttgtcgaag Ec-pflB-R ctaaaaaaggccccactttcgtggagc Ec-pflB-YF ccggaaaatttttctcacctgacc Ec-adhE-F cgcactgactatactctcgtattcgagcag Ec-adhE-R ggtcaactaatccttaactgatcggcattg Ec-adhE-YF ttgattttcataggttaagcaaatcatcac Ec-alr-F cagtacgacgacgaataataattattttat Ec-alr-R ttaggattttattggccggataaggcattc Ec-alr-YF tggtcgcgcttcgacaactatgcggggccg Ec-alaE-F atagcgttacctcacccccaaactttcatt Ec-alaE-R cagaacagtaaacaaccatcgcgaacgtat Ec-alaE-YF gctactacatacaccgaattctctacgctt Ec-aceE-F ctttccggcgagagttcaatgggacaggtt Ec-aceE-R tcggtgatttcaacttcatcagccccgatg Ec-aceE-YF gcaactaaacgtagaacctgtcttattgag GsLADH-F1 cagcggcctggtgccgcgcggcagcctcgagatgaagatcggcattccaaaagaaatc GsLADH-R1 gagctcaccgaattcacctcatccgtgcaacaacgaatg StDADH-F1 cagcggcctggtgccgcgcggcagcctcgagatggacaagctgcgcgtggc StDADH-R1 gagctcaccgaattcaccctacaccagcttccggatccac pY-F ggtgaattcggtgagctcggtct pY-R ctcgaggctgccgcgcggcaccaggccgctg

[0086] The present invention specifically includes the following embodiments:

[0087] Example 1: Construction of recombinant Escherichia coli chassis strain

[0088] (1) Construction of Escherichia coli GAla01

[0089] Starting with Escherichia coli GL05, the phosphoacetyltransferase gene (pta) was knocked out to obtain the GLA01 strain. The specific steps are as follows:

[0090] (1-a) Preparation of the target fragment DC-pta

[0091] The artificially synthesized DNA fragment (Kangwei Century Company) consists of the following components from 5' to 3': pta-up (SEQ ID NO. 23), LKL (composed of lox66, Kan resistance gene, and lox71 from 5' to 3', as shown in SEQ ID NO. 19), and pta-down (SEQ ID NO. 24). Using Ec-pta-F / Ec-pta-R as primers, the synthesized DNA fragment was used as a template for PCR amplification to obtain the targeting fragment DC-pta.

[0092] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0093] The pKD46 plasmid was transformed into the originating *E. coli* GL05 using the calcium chloride conversion method. The culture was then carried out overnight at 30°C on LB agar plates containing ampicillin. Clones were selected to obtain *E. coli* GL05 / pKD46. *E. coli* GL05 / pKD46 was then transferred to 50 mL of liquid LB agar containing ampicillin and a final concentration of 0.2% L-arabinose, and cultured at 30°C and 220 rpm until OD500. 600 The nm value was 0.6. The cells were then collected by centrifugation at 3000g and 4℃ for 5 min. The cells were washed twice with 10 mL of pre-cooled 10% glycerol and finally resuspended in 200 μL of pre-cooled 10% glycerol to prepare competent cells.

[0094] (1-c) Transformation and identification of recombinant Escherichia coli strains

[0095] 50 ng of the target fragment DC-pta was added to 50 μL of the above-mentioned competent cells and electroporated using a suitable electroporator. Immediately afterward, 1 mL of LB medium was added, and the cells were incubated at 30°C and 120 r / min for 1 h. The cells were then plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Single clones were selected for PCR verification. PCR amplification was performed using Ec-pta-YF / Kan-R primers. A positive result was indicated by an amplified target band of approximately 1300 bp, and the positive clone was named GLA01-kan. Sequencing analysis showed that the relevant target sequence on the genome of GLA01-kan was correct, and the pta gene region was replaced with the LBL sequence. GLA01-kan was cultured overnight at 42°C to eliminate the temperature-sensitive plasmid pKD46.

[0096] The pSCre plasmid was transformed into the aforementioned GLA01-kan strain, which had pKD46 eliminated, using the calcium chloride transformation method. The strain was incubated overnight at 30°C on LB agar plates containing streptomycin and 0.2% L-arabinose, and the kanamycin resistance fragment was eliminated using the Cre recombinase on the pSCre plasmid. The culture was then incubated overnight at 42°C to eliminate the temperature-sensitive pSCre plasmid. The resulting plates were then streaked onto LB agar plates and incubated overnight at 37°C. Single colonies were picked, and strains that could not grow on LB agar containing kanamycin were selected and named GLA01. This bacterium is an *Escherichia coli* strain with the phosphoacetyltransferase gene (pta) knocked out.

[0097] (2) Obtaining strains GLA02, GLA03, GLA04, GLA05, and Gala06

[0098] Using the same method as in Example 1 (1), strains Gala02, Gala03, Gala04, Gala05, and Gala06 were obtained sequentially from strain Gala01. The construction process for each strain was exactly the same as in Example 1, except that different starting strains, targeting fragments, amplification primers, and identification primers were used. The starting strains, targeting fragments, amplification primers, identification primers, and strain information involved in each step of the construction process from GL05 to Gala06 are detailed in Table 3. The fragment sequences and primer sequences are shown in Tables 1 and 2, respectively.

[0099] Table 3 Construction process of GAla01-GAla06

[0100] Step number Starting strain Target practice segment name The target shooting sequence: 5'-3' consists of the following segments in sequence (sequence information is shown in Table 1). Modify target Amplification primers Identification primers Obtain strain 1 GL05 DC-pta pta-up, LKL, pta-down Knockout of the phosphoacetyltransferase gene (pta) Ec-pta-F / Ec-pta-R Ec-pta-YF / KAN-R GAla01 2 GAla01 DC-pflB pflB-up, LKL (, pflB-down) Knockout of the pyruvate formate lyase gene (pflB) Ec-pflB-F / Ec-pflB -R Ec-pflB-YF / KAN-R GAla02 3 GAla02 DC-adhE adhE -up, LKL, adhE -down Knockout of the aldol dehydrogenase gene (adhE) Ec-adhE-F / Ec-adhE -R Ec-adhE-YF / KAN-R GAla03 4 GAla03 DC-alr alr -up, LKL, alr -down Knockout of the alanine racemase gene (alr) Ec-adhE-F / Ec-adhE -R Ec-alr-YF / KAN-R GAla04 5 GAla04 ZQ-alaE alaE-up, LKL, P119, alaE-down Replace the promoter of the L-alanine transporter gene (alaE) with the P119 promoter. Ec-alaE-F / Ec-alaE -R Ec-alaE-YF / KAN-R GAla05 6 GAla05 ZQ-aceE aceE-up, LKL, P119, PmcmL, RBS, PmcmS, TrrnB, P119, SpccB, RBS, Pmce, TrrnB, aceE-down The pyruvate dehydrogenase E1 subunit gene (aceE) was knocked out and replaced with an expression cassette containing the large subunit gene (PmcmL), the small subunit gene (PmcmS), the propionyl-CoA carboxylase gene (SpccB), and the methylmalonyl-CoA epimerase gene (Pmce). Ec-aceE-F / Ec-aceE-R Ec-aceE-YF / KAN-R GAla06

[0101] Example 2: Construction of an Escherichia coli strain that synthesizes alanine using methanol

[0102] (1) Construction of plasmids pY-GsLADH and pY-StDADH

[0103] DNA fragments containing the L-alanine dehydrogenase gene (sequence shown in SEQ ID NO.1) and the D-alanine dehydrogenase gene (sequence shown in SEQ ID NO.3) were synthesized by Kangwei Century Co., Ltd., and named pGsLADH and pStDADH, respectively. Using pGsLADH as a template and GsLADH-F1 and GsLADH-R1 as primers, fragment GsLADH-1 was amplified by PCR using Phanta Max Super-Fidelity DNA Polymerase and purified using a gel extraction kit. Fragment StDADH-1 was amplified by PCR using pStDADH as a template and StDADH-F1 and StDADH-R1 as primers and purified using a gel extraction kit.

[0104] Using pYB1k plasmid as a template and pY-F and pY-R as primers, the PCR product pYk_BB was obtained by PCR amplification. DpnI was added to the PCR product for digestion for 4 hours, and then the PCR product was recovered using a gel extraction kit to obtain the purified fragment pYk_BB.

[0105] The fragments pYk_BB and GsLADH-1 were ligated using 2× MultiF Seamless Assembly Mix via Gibson assembly, transformed into DH5α competent cells, screened for positive clones, and sequenced for verification, ultimately yielding the correct plasmid pY-GsLADH.

[0106] The fragments pYk_BB and StDADH-1 were ligated using Gibson assembly with 2× MultiF Seamless Assembly Mix, transformed into DH5α competent cells, screened for positive clones, and sequenced for verification, finally obtaining the correct plasmid pY-StDADH.

[0107] (2) Construction of recombinant Escherichia coli producing D-alanine and L-alanine

[0108] The pY-GsLADH and pY-StDADH plasmids were transformed into GL05, GAla05, and GAla06, respectively, using the calcium chloride transformation method. After overnight culture on LB plates containing kanamycin at 37°C, clones were selected and named GL05-Gs, GL05-St, GAla05-Gs, GAla05-St, GAla06-Gs, and GAla06-St, respectively, and stored at -80°C.

[0109] Example 3: Production of L-alanine and D-alanine using recombinant Escherichia coli strains with methanol as a carbon source.

[0110] (1) Using methanol as a raw material, L-alanine was synthesized by recombinant Escherichia coli strain.

[0111] Overnight-cultured recombinant Escherichia coli GAla05-Gs, GAla06-Gs, and control strain GL05-Gs were inoculated at a 1% inoculation rate into shake flasks containing 200 mL of inorganic salt medium with kanamycin and incubated at 37°C for 3-4 h until OD (dose retardation). 600 After the concentration of nm was 0.6-0.8, arabinose was added to a final concentration of 0.2 g / L, and the cells were cultured at 37°C for 12 h. The cells were then collected by centrifugation at 8000 rpm for 10 min.

[0112] The collected bacterial cells were converted to OD per liter of bacterial culture. 600 A bacterial culture of 20 μL was resuspended in shake flasks containing 10 mL of fermentation medium and incubated at 220 rpm and 37 °C for 24 h. The supernatant was collected by centrifugation and filtered. The L-alanine content was determined by HPLC. The experiment was repeated in three Erlenmeyer flasks.

[0113] Sample derivatization method: First, take 50 μl of the filtered supernatant and add 50 μl of 0.5 M sodium bicarbonate (Aladdin, catalog number: S112331) solution and 50 μl of 1% (v / v) 2,4-dinitrofluorobenzene (Maclean, catalog number: F830061) solution. After adding the solutions, mix well and heat in a 60℃ metal bath for 1 h. Finally, add 150 μl of deionized water, filter, and then detect. The L-alanine content was detected using a Shimadzu C18 column (Shim-pack GIST C18 5μm, 4.6×250mm) (mobile phase: 0.1% formic acid solution (65%), 100% acetonitrile (35%); flow rate: 1 mL / min; detection wavelength: 350 nm). L-alanine (China National Institutes for Food and Drug Control, product catalog number 140680) was used as a standard. The L-alanine content was qualitatively analyzed based on the retention time of the standard and quantitatively analyzed using the standard curve method (external standard method).

[0114] The results show (Table 4, Figure 1 The supernatant yields of L-alanine produced by GLA05-Gs, GAla06-Gs, and GL05-Gs were 1.53±0.05 g / L, 2.06±0.15 g / L, and 0 g / L, respectively. The constructed engineered strains can effectively utilize methanol to synthesize L-alanine.

[0115] (2) Using methanol as a raw material, D-alanine was synthesized by recombinant Escherichia coli strain.

[0116] Overnight-cultured recombinant Escherichia coli GLA05-St, GLA06-St, and control strain GL05-St were inoculated at a 1% inoculum into shake flasks containing 200 mL of inorganic salt medium with kanamycin and incubated at 37°C for 3-4 h until OD (digestive oxygen saturation) was reached. 600 After the concentration of nm was 0.6-0.8, arabinose was added to a final concentration of 0.2 g / L, and the cells were cultured at 37°C for 12 h. The cells were then collected by centrifugation at 8000 rpm for 10 min.

[0117] The collected bacterial cells were converted to OD per liter of bacterial culture. 600 A bacterial culture of 20 μL was resuspended in shake flasks containing 10 mL of fermentation medium and incubated at 220 rpm and 37 °C for 24 h. The supernatant was collected by centrifugation and filtered. The D-alanine content was determined by HPLC. The experiment was repeated in three Erlenmeyer flasks.

[0118] Sample derivatization method: First, take 50 μl of the filtered supernatant and add 50 μl of 0.5 M sodium bicarbonate (Aladdin, catalog number: S112331) solution and 50 μl of 1% (v / v) 2,4-dinitrofluorobenzene (Maclean, catalog number: F830061) solution. After adding the solutions, mix well and heat in a 60℃ metal bath for 1 h. Finally, add 150 μl of deionized water, filter, and then detect. The D-alanine content was detected using a Shimadzu C18 column (Shim-pack GIST C18 5μm, 4.6×250mm) (mobile phase: 0.1% formic acid solution (65%), 100% acetonitrile (35%); flow rate: 1 mL / min; detection wavelength: 350 nm). D-alanine (China National Institutes for Food and Drug Control, product catalog number 140680) was used as a standard to qualitatively analyze the D-alanine content based on the retention time of the standard and to quantitatively analyze the D-alanine content using the standard curve method (external standard method).

[0119] The results show (Table 4, Figure 1 The supernatant yields of D-alanine produced by GLA05-St, GLA06-St, and GL05-St were 1.31±0.04 g / L, 1.74±0.11 g / L, and 0 g / L, respectively. The constructed engineered strains can effectively utilize methanol to synthesize D-alanine.

[0120] Table 4 Comparison of yields of different strains

[0121]

Claims

1. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli can synthesize alanine from methanol; the alanine is L-alanine or D-alanine.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant Escherichia coli has the following characteristics (A1) or (A2): (A1) Contains the L-alanine dehydrogenase gene, and / or contains substances that enhance the expression of the L-alanine dehydrogenase gene, and / or contains substances that enhance the activity or content of L-alanine dehydrogenase. Furthermore, the alanine in question is L-alanine; (A2) Contains the D-alanine dehydrogenase gene, and / or contains substances that enhance the expression of the D-alanine dehydrogenase gene, and / or contains substances that enhance the activity or content of D-alanine dehydrogenase. Furthermore, the alanine in question is D-alanine.

3. The recombinant Escherichia coli according to any one of claims 1 or 2, characterized in that, The recombinant Escherichia coli also has the following characteristics: (A3) Contains a methanol dehydrogenase gene, and / or contains substances that enhance the expression of the methanol dehydrogenase gene, and / or contains substances that enhance the activity or content of methanol dehydrogenase; (A4) Contains the 3-hexylose-6-phosphate synthase gene, and / or contains substances that enhance the expression of the 3-hexylose-6-phosphate synthase gene, and / or contains substances that enhance the activity or content of 3-hexylose-6-phosphate synthase; (A5) Contains a gene for 3-phosphate-3-hexose isomerase, and / or contains substances that enhance the expression of the gene for 3-phosphate-3-hexose isomerase, and / or contains substances that enhance the activity or content of 3-phosphate-3-hexose isomerase; (A6) Does not contain the phosphorylacetyltransferase gene, and / or does not contain substances with phosphorylacetyltransferase activity; (A7) Does not contain the pyruvate formate lyase gene, and / or does not contain substances with pyruvate formate lyase activity; (A8) Does not contain the aldosterone dehydrogenase gene, and / or does not contain substances with aldosterone dehydrogenase activity; (A9) Does not contain the alanine racemase gene, and / or does not contain any substance with alanine racemase activity; (A10) Contains an alanine transporter gene, and / or contains substances that enhance the expression of the alanine transporter gene, and / or contains substances that enhance the activity or content of the alanine transporter.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that, The recombinant Escherichia coli also has the following characteristics: (A11) Does not contain the pyruvate dehydrogenase gene, and / or does not contain substances with pyruvate dehydrogenase activity; (A12) Contains the propionyl-CoA carboxylase gene, and / or contains substances that enhance the expression of the propionyl-CoA carboxylase gene, and / or contains substances that have propionyl-CoA carboxylase activity. (A13) Contains a methylmalonyl-CoA epimerase gene, and / or contains a substance that enhances the expression of the methylmalonyl-CoA epimerase gene, and / or contains a substance with methylmalonyl-CoA epimerase activity. (A14) Contains a methylmalonyl-CoA mutase gene, and / or contains a substance that enhances the expression of the methylmalonyl-CoA mutase gene, and / or contains a substance with methylmalonyl-CoA mutase activity.

5. The recombinant Escherichia coli according to claim 3, characterized in that, The recombinant Escherichia coli was prepared by performing the following steps on Escherichia coli, which served as the recipient bacterium: (M1) Introduce an alanine dehydrogenase gene, specifically an L-alanine dehydrogenase gene or a D-alanine dehydrogenase gene, which are used to synthesize L-alanine or D-alanine, respectively. (M2) Knockout of the phosphoacetyltransferase gene; (M3) Knock out the pyruvate formate lyase gene; (M4) Knockout of the aldol dehydrogenase gene; (M5) Knockout of the alanine racemase gene; (M6) enhances the expression of alanine transporter genes; The Escherichia coli used as the recipient bacterium is an Escherichia coli that can efficiently utilize methanol, preferably Escherichia coli GL05, whose preservation number is CGMCC No. 35139.

6. The recombinant Escherichia coli according to claim 4, characterized in that, The recombinant Escherichia coli was prepared by performing the following steps on Escherichia coli, which served as the recipient bacterium: (M1) Introduce an alanine dehydrogenase gene, specifically an L-alanine dehydrogenase gene or a D-alanine dehydrogenase gene, which are used to synthesize L-alanine or D-alanine, respectively. (M2) Knockout of the phosphoacetyltransferase gene; (M3) Knock out the pyruvate formate lyase gene; (M4) Knockout of the aldol dehydrogenase gene; (M5) Knockout of the alanine racemase gene; (M6) enhances the expression of alanine transporter genes; (M7) Knock out the pyruvate dehydrogenase gene; specifically, the pyruvate dehydrogenase E1 subunit gene; (M8) Introducing the propionyl-CoA carboxylase gene; (M9) Introducing the methylmalonyl-CoA epimerase gene; (M10) Introduce the methylmalonyl-CoA mutase gene; the methylmalonyl-CoA mutase gene specifically comprises the methylmalonyl-CoA mutase large subunit gene and the methylmalonyl-CoA mutase small subunit gene; The Escherichia coli used as the recipient bacterium is an Escherichia coli that can efficiently utilize methanol, preferably Escherichia coli GL05, whose preservation number is CGMCC No. 35139.

7. The use of the recombinant Escherichia coli according to any one of claims 1 to 6 in the production of alanine.

8. A method for producing alanine, characterized in that, The recombinant Escherichia coli according to any one of claims 1 to 6 is prepared by cell culture; the method includes: culturing the recombinant Escherichia coli in a fermentation medium, and collecting alanine from the fermentation product; wherein the fermentation medium uses methanol as a carbon source.

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