Method for constructing beta-alanine producing strain

Abstract

The invention discloses a method for constructing a beta-alanine producing strain, which comprises the following steps: interrupting or down-regulating adhE and eda, and overexpressing or enhancing aspartate decarboxylase panD at the same time; gene ackA, ldhA and mgsA in a genome are interrupted or down-regulated, aspartate decarboxylase panD is overexpressed in a free plasmid form at the same time, anaerobic fermentation domestication is carried out on the strain B / C / D in a culture medium with glucose as a carbon source, and the strain with the beta-alanine production capacity improved, such as CGMCC No.28214, is obtained. The engineering bacteria constructed by the invention can be used for producing beta-alanine through anaerobic fermentation by taking glucose as a carbon source.

Description

Technical Field

[0001] The invention belongs to the field of metabolic engineering, and relates to a method for constructing beta-alanine producing bacteria, and a beta-alanine producing bacteria capable of producing beta-alanine through anaerobic fermentation using glucose as a carbon source. Background Art

[0002] β-Alanine is a natural β-amino acid that exists in nature. It participates in the synthesis of various functional substances such as carnosine and vitamin B5, and is widely used in the pharmaceutical, food, chemical and other industries. For example, it can be used to synthesize pantothenic acid, calcium pantothenate, carnosine, sodium pamidronate, octachloronitrate, etc. It can also be used as a dietary supplement to provide energy for muscles. Due to its wide range of uses, the market demand is increasing day by day.

[0003] There are three methods for preparing β-alanine: chemical method, enzymatic method and fermentation method. Among them, chemical method and enzymatic method are the main production methods at present. Chemical methods include acrylonitrile method, acrylic acid method, β-aminopropionitrile method, succinimide degradation method, etc., but these methods are high temperature and high pressure reactions, with harsh process conditions, and more by-products in the reaction process, complex extraction process, high cost, and unfriendly environment. These inevitable shortcomings have led to the increasingly weak competitiveness of chemical synthesis of β-alanine in the market.

[0004] The enzymatic method includes a variety of pathways, for example: L-aspartate α-decarboxylase catalyzes the decarboxylation of L-aspartate to produce β-alanine. The defect of this method is that the enzyme-catalyzed substrate aspartate is relatively expensive, resulting in high production costs and great restrictions on industrialization; the enzymatic method catalyzes the addition of ammonia to acrylic acid to synthesize β-alanine, but the post-processing steps are cumbersome and the production cost is high; the inventor reported in the previous study CN201911182183.2 that aspartate ammonia lyase catalyzes the reaction of acrylic acid or acrylonitrile to produce β-alanine, but it still needs to be further improved in terms of industrial production.

[0005] Fermentation has gradually become a new research direction in recent years. Patent document CN110272857A discloses that glucose is used as raw material to ferment and produce β-alanine in Escherichia coli engineering bacteria, with a yield of up to 50g / L and a sugar acid conversion rate of 40%, but the method is still only in the laboratory research stage. Patent documents CN112662609A, CN112625985A, and CN103898033A also disclose the use of Escherichia coli engineering bacteria to ferment and produce β-alanine, but they are also limited to the laboratory research stage and cannot be industrialized. In addition, patent documents CN107236752A and CN110016459A also disclose the use of glycerol as a carbon source in Escherichia coli engineering bacteria to produce β-alanine by fermentation, but the cost of glycerol is relatively high, and the high economic cost factor causes it to be unable to achieve industrial production. Summary of the invention

[0006] Escherichia coli is one of the fastest growing microorganisms. It is the most studied and widely used model strain in the field of genetic engineering. It has been widely researched and developed, and even industrially applied in the amino acid fermentation industry. Considering that glucose is the most commonly used fermentation carbon source and anaerobic fermentation is the most economical liquid deep culture method, the inventors interrupted the mixed acid fermentation pathway of Escherichia coli under anaerobic conditions through metabolic engineering. After strengthening the β-alanine fermentation pathway, the obtained strain is basically unable to grow using glucose under anaerobic conditions, but after anaerobic acclimation, the engineered bacteria grow again and produce high β-alanine. Based on this unexpected discovery, the present invention includes the following technical solutions:

[0007] A method for constructing a β-alanine producing bacterium comprises the following steps:

[0008] A. Using Escherichia coli as the base strain, overexpressing aspartate decarboxylase panD or enhancing the expression of aspartate decarboxylase panD, and interrupting or downregulating the expression of alcohol dehydrogenase gene adhE (NCBI accession number NC_000913.3) and / or 2-keto-3-deoxy-6-phosphogluconate aldolase gene eda (NCBI accession number NC_000913.3), to obtain strain A;

[0009] B. interrupting or down-regulating the expression of one or more genes selected from the following group of genes in the genome: acetate kinase gene ackA (NCBI accession number NC_000913.3), D-lactate dehydrogenase gene ldhA (NCBI accession number NC_000913.3), methylglyoxal synthase gene mgsA (NCBI accession number NC_000913.3), to obtain one of strains B, C and D;

[0010] C. In a culture medium with glucose as the carbon source, one of the strains B, C and D is subjected to anaerobic fermentation domestication to obtain a strain with improved β-alanine production capacity.

[0011] The β-alanine producing bacteria constructed by the method can be used to produce β-alanine through anaerobic fermentation using glucose as a carbon source.

[0012] The interruption or down-regulation of gene expression in the above steps A and B can interrupt or reduce the mixed acid fermentation pathway of Escherichia coli under anaerobic conditions, thereby inhibiting the production of L-lactic acid, ethanol, L-formic acid and / or acetic acid.

[0013] The interruption method of the gene in the above steps A and B is selected from gene knockout, deletion, and inactivation.

[0014] Preferably, the aspartate decarboxylase panD in the above step A can be selected from the following group: aspartate decarboxylase panD derived from Bacillus subtilis (NCBI accession number NP_390122), aspartate decarboxylase panD derived from Corynebacterium glutamicum (NCBI accession number BAB97528), aspartate decarboxylase panD derived from Escherichia coli (NCBI accession number ACA79142), etc.

[0015] The method of overexpressing aspartate decarboxylase panD in step A above may be:

[0016] Integrate a single copy or multiple copies (i.e., two or more copies, three or more copies, four or more copies, five or more copies, or ten or more copies) of an aspartate decarboxylase panD gene into the host bacterial genome, wherein the aspartate decarboxylase panD may be a panD from different sources, such as a panD from Corynebacterium glutamicum (cgpanD) and a panD from Bacillus subtilis (BspanD); or

[0017] The aspartate decarboxylase panD gene is expressed in a host bacterium in the form of a plasmid. The plasmid vector is, for example, pKK233, but is not limited thereto, and may also be any vector suitable for expressing a target gene in Escherichia coli.

[0018] The method of enhancing the expression of aspartate decarboxylase panD in the above step A can be: using a strong promoter such as tac or trc promoter to regulate the expression of aspartate decarboxylase panD gene, or other related strong promoters capable of constitutive expression or inducible expression.

[0019] In one embodiment, the interruption of the gene in the above steps A and B can be the loss of the gene or a significant downregulation of its function, including knockout, deletion, and inactivation of the gene, which can generally be achieved by conventional gene manipulation methods such as knockout, insertion inactivation, frameshift mutation, introduction of stop codons to terminate translation in advance, etc. The gene inactivation can be selected from the following group of methods: complete deletion of nucleotide sequence, partial deletion of nucleotide sequence, gene mutation, and mutation of stop codon in the reading frame.

[0020] The down-regulated expression of genes in the above steps A and B can be selected from the following groups: protein ubiquitination modification, RNA interference (RNAi), structural changes in the expression system, negative regulation at the transcriptional level, negative regulation at the post-transcriptional level, reduced gene transcription, reduced gene translation, enhanced protein degradation, and a combination of two or more thereof.

[0021] In one embodiment, the interruption and down-regulation of gene expression in the above steps A and B, and the genomic integration of the aspartate decarboxylase panD gene in the above step A can be implemented by gene editing technology, and the gene editing technology is selected from the following group: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), and CRISPRi.

[0022] Preferably, the interruption and / or insertion of genes in the above steps A and B are implemented by gene editing technology.

[0023] The domestication of one of the strains B, C and D in the above step C comprises the following steps: obtaining an engineered bacterium having a β-alanine production capacity more than 10 times higher than that of the base bacterium by continuous subculture in a fermenter without oxygen.

[0024] Preferably, the above-mentioned base bacteria is Escherichia coli MG1655.

[0025] According to a second aspect of the present invention, a genetically engineered bacterium (or β-alanine producing bacterium, β-alanine engineering bacterium) is provided, which is constructed according to the above method.

[0026] In this case, the genotype of strain A can be MG1655△adhE::cgpanD△eda::cgpanD, that is, panD is overexpressed and adhE and eda are knocked out. The genotype of strain B can be MG1655△adhE::cgpanD△eda::cgpanD△ackA, that is, panD is overexpressed and adhE, eda and ackA are knocked out. The genotype of strain C can be MG1655△adhE::cgpanD△eda::cgpanD△ackA△ldhA, that is, panD is overexpressed and adhE, eda, ackA and ldhA are knocked out. The genotype of strain D can be MG1655△adhE::cgpanD△eda::cgpanD△ackA△ldhA△mgsA, that is, panD is overexpressed and adhE, eda, ackA, ldhA and mgsA are knocked out.

[0027] Preferably, the genetically engineered bacteria is Escherichia coli, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.28214.

[0028] According to a third aspect of the present invention, there is provided the use of the above-mentioned genetically engineered bacteria in producing β-alanine using glucose as a carbon source.

[0029] In one embodiment, the genetically engineered bacteria produce β-alanine by anaerobic fermentation using glucose as a carbon source.

[0030] The β-alanine engineering bacteria constructed by the present invention can use cheap and readily available glucose as a carbon source to produce β-alanine through submerged culture of anaerobic fermentation, which can significantly reduce the production cost of β-alanine and promote the industrial application of the Escherichia coli fermentation method to produce β-alanine.

[0031] The Latin name of the β-alanine engineering bacteria SH2356 constructed by the present invention is Escherichia coli, and the Chinese name is Escherichia coli, that is, Escherichia coli. It has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and the preservation date is August 23, 2023. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 28214. DETAILED DESCRIPTION

[0032] Escherichia coli is a facultative anaerobic bacterium. Under anaerobic conditions, its fermentation with glucose as a carbon source is a mixed acid fermentation, and the main metabolites include L-lactic acid, ethanol, L-formic acid and acetic acid. Studies have found that when these pathways are blocked, the anaerobic growth of Escherichia coli with glucose as a carbon source will be affected, and even manifest as being unable to grow under anaerobic conditions. However, through anaerobic growth domestication, and coupling the energy required for growth with certain metabolites, Escherichia coli may grow again and produce specific metabolites at a high yield. This strategy has been successfully applied to the anaerobic production of L-alanine by Escherichia coli (see Appl Microbiol Biotechnol (2007) 77: 355–366.), succinic acid (see Metabolic Engineering, Volume 24, July 2014, Pages 87-96.), D-lactic acid (see CN201310293659.6), etc., but this strategy has not yet been applied to the production of β-alanine.

[0033] As we all know, compared with liquid aerobic fermentation (aerobic fermentation), anaerobic fermentation has many advantages, such as no need for strict oxygen supply / dissolved oxygen control, smaller power / heat supply, a wider pH range, easy to achieve continuous fermentation production, etc., which makes anaerobic fermentation have outstanding advantages in economy, energy saving, environmental protection and carbon neutrality.

[0034] Glucose is a commonly used and readily available fermentation raw material, and is widely used in industrial-scale production of biotechnology. The inventors transformed Escherichia coli through metabolic engineering, interrupted the mixed acid fermentation pathway under anaerobic conditions, and strengthened the β-alanine fermentation pathway, but also found that the genetically engineered strains were basically unable to grow using glucose under anaerobic conditions. Therefore, after anaerobic domestication, the engineered bacteria were not only able to achieve anaerobic fermentation and proliferation, but also greatly improved the β-alanine production capacity, thereby constructing a relatively ideal glucose anaerobic fermentation engineered bacteria, which has the potential to be used in the de novo synthesis of β-alanine using glucose as a carbon source through anaerobic fermentation.

[0035] The experiment found that simply changing the metabolic pathway of E. coli through genetic engineering could not make the genetically engineered bacteria use glucose as a carbon source for anaerobic fermentation to express β-alanine, or the expression level was very low, which was far from or even the opposite of the theoretical expected results. In order to break through this technical bottleneck, the inventor introduced the domestication method commonly used in traditional microbial breeding. After multiple generations of domestication, the β-alanine production capacity of the strain was greatly improved.

[0036] Strain domestication is achieved by continuous subculture, applying anaerobic environmental pressure in glucose carbon source to the strain, forcing it to undergo adaptive evolution (domestication), so that the physiological characteristics of each generation of proliferating strains change, gradually adapting to the anaerobic fermentation conditions in glucose carbon source, and being able to gradually increase the expression of β-alanine under environmental pressure.

[0037] Although domestication can significantly improve the strain phenotype, the molecular targets behind it are not completely clear. Many mechanistic studies are comparative omics analysis of strains before and after domestication to obtain differential molecular targets, and then reverse the corresponding mutations in the strains after domestication or forward the strains before domestication to verify the effectiveness of the targets and obtain some molecular targets that are beneficial to β-alanine expression. Furthermore, based on these potential molecular targets, it is possible to achieve rational reconstruction of β-alanine-producing bacteria.

[0038] In some embodiments, the term "(β-alanine-producing capacity) increase" or "enhancement" may refer to an increase of at least about 1 fold, at least about 2 fold, or at least about 3 fold, or at least about 4 fold, or at least about 5 fold, or at least about 10 fold compared to a reference level.

[0039] The terms "β-alanine genetically engineered bacteria", "β-alanine producing bacteria", "genetically engineered bacteria", and "engineered (domesticated) bacteria" in this article have the same meaning, and all refer to strains of Escherichia coli that can produce β-alanine after genetic engineering modification or even further domestication.

[0040] The terms "anaerobic fermentation with glucose as the carbon source", "anaerobic fermentation in glucose carbon source", "anaerobic fermentation of glucose carbon source" and "anaerobic fermentation of glucose" have the same meaning in this article, especially referring to the liquid culture fermentation situation with glucose as the only or main carbon source in the absence of oxygen (air).

[0041] In this article, for the sake of simplicity of description, sometimes a certain protein such as aspartate decarboxylase (panD) and its encoding gene (DNA) are used interchangeably. Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings according to the context. For example, for panD, when used to describe the function or category of aspartate decarboxylase, it refers to the protein; when described as a gene, it refers to the gene encoding the aspartate decarboxylase.

[0042] As used herein, wild-type aspartate decarboxylase such as NCBI Accession No. ACA79142, NCBI Accession No. NP_390122, NCBI Accession No. BAB97528, and their mutants may be collectively referred to as aspartate decarboxylase (panD).

[0043] In order to optimally express exogenous aspartate decarboxylase (panD) in the most commonly used Escherichia coli in genetic engineering, the expression genes of these enzymes and their mutants can be codon optimized. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms usually show a special preference for one of some codons encoding the same amino acid due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its respective genome tRNA library. Therefore, in fast-growing microorganisms, low-frequency codons of amino acids can be used for the same amino acids but high-frequency codon replacement. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0044] The aspartate decarboxylase (panD) in the present invention has a limited number of amino acids, wherein the aspartate decarboxylase from Escherichia coli (NCBI accession number ACA79142) has 126 amino acids, the aspartate decarboxylase from Bacillus subtilis (NCBI accession number NP_390122) has 127 amino acids, and the aspartate decarboxylase from Corynebacterium glutamicum (NCBI accession number BAB97528) has 136 amino acids, and has a clear structure, so those skilled in the art can easily obtain the encoding genes, expression cassettes and plasmids containing these genes.

[0045] It should be understood that in a specific genetic engineering implementation scheme, the order of step A and step B is not completely fixed from front to back according to the alphabetical order. They can be operated crosswise or reversely as long as each step can achieve its respective function and complete the directional change of the host cell genotype.

[0046] In the description of the technical solution of the present invention, the terms used, such as "A and / or B", "A and / or B", and the term "and / or" used in them are intended to include both A and B; A or B; A (alone); and B (alone).

[0047] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only for illustrative purposes and are not intended to limit the present invention. In addition, it should be understood that after reading the concept of the present invention, various changes or adjustments made by those skilled in the art should fall within the scope of protection of the present invention, and these equivalent forms also belong to the scope of the present invention.

[0048] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0049] Example

[0050] Materials and methods

[0051] The primer synthesis and sequencing in the examples were completed by Anhui General Biotechnology Co., Ltd.

[0052] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., mainly with reference to "Molecular Cloning Experiment Guide" (3rd edition), compiled by J. Sambrook, DW Russell (USA), translated by Huang Peitang, etc., Science Press, Beijing, 2002). For example, the competent cell transformation method and the competent preparation method are all carried out with reference to "Molecular Cloning Experiment Guide" (3rd edition), Chapter 1, page 96. If necessary, the specific experimental conditions can be determined by simple experiments.

[0053] Main culture medium:

[0054] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride. (Add 20g / L agar powder to solid medium.)

[0055] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K 2 HPO 4 .3H 2 O, 2.31g / LKH 2 PO 4, 5g / L glycerol, pH7.0-7.5. (TB solid medium plus 20g / L agar powder.)

[0056] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0057] The primer sequence information used in the examples is shown in Table 1.

[0058] Table 1. Primer sequences

[0059]

[0060]

[0061] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0062] The CRISPR-Cas9 system plasmids such as pEcgRNA and pEcCas for knocking out the genes ackA, ldhA, mgsA, adhE, and eda in Escherichia coli were constructed and donated by Yang Sheng's research group at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. Any unit or individual may obtain the plasmids for verification of the present invention, but may not be used for other purposes, including development and utilization, scientific research, and teaching, without the permission of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences and Zhejiang Huarui Biotechnology Co., Ltd.

[0063] β-Alanine liquid chromatography (HPLC) detection conditions:

[0064] The determination was carried out by HPLC, with a mixture of acetonitrile / water (50:50, v / v) and 50 mM sodium acetate as the mobile phase, a flow rate of 1 mL / min, and an ultraviolet detection wavelength of 360 nm.

[0065] Example 1: Construction of pTrc99a-cgpanD plasmid

[0066] The cgpanD fragment was amplified by PCR using the primer pair cgpanD-99F / cgpanD-99R and the genome of Corynebacterium glutamicum ATCC13032 as a template; the vector fragment was amplified by PCR using two synthetic oligosaccharide nucleic acids 99A-F / 99A-R as primers and plasmid pTrc99a as a template.

[0067] The PCR amplification system is as follows: KOD one 25 μl, upstream and downstream primers 0.3 μl each (50 μM), PCR template 0.5 μl, ddH 2 0 to make up to 50 μl.

[0068] The PCR amplification program was as follows: 98°C for 2 min, 98°C for 10 s, 58°C for 5 s, 68°C for 30 s (1-10 KB, 5 s / KB), for a total of 30 cycles, 68°C for 5 min, and insulation at 16°C.

[0069] The two PCR fragment products were purified and recovered, Gibson spliced, and transformed into DH5α competent cells (Shanghai Weidi Biotechnology). The transformant plates were cultured overnight at 37°C, and positive clones were obtained by colony PCR identification. Plasmids were extracted for use, and positive strains were stored in a final concentration of 30% glycerol solution and stored at -80°C. The nucleotide sequence of the extracted plasmid pTrc99a-cgpanD is SEQ ID NO: 1.

[0070] Example 2: Integration of L-aspartate-α-decarboxylase at the adhE and eda sites

[0071] The adhE gene encodes an alcohol dehydrogenase. Studies have found that this protein seriously affects the metabolic flow of alanine under anaerobic fermentation conditions. Therefore, knocking out this protein can increase the concentration of alanine during anaerobic fermentation of the strain and reduce the production of by-product ethanol; the eda gene encodes a kinase that can catalyze the conversion of pyruvate to lactate. Knocking out this gene can reduce the competition for pyruvate in the branch, thereby increasing the production of alanine; while knocking out the gene, the panD gene (cgpanD) from Corynebacterium glutamicum is integrated. This gene encodes L-aspartate-α-decarboxylase to increase the production of alanine.

[0072] 2.1 Construction of pEcgRNA-adhE and pEcgRNA-eda plasmids

[0073] The synthesized oligosaccharide nucleic acids sgRNA-adhE-F / sgRNA-adhE-R and sg-eda-F / sg-eda-R were annealed to form double-stranded sequences, connected with the linearized plasmid pEcgRNA, and transformed into DH5α competent cells (Shanghai Weidi Biotechnology). The transformants were cultured on plates at 37°C overnight, and the transformants were identified by PCR to obtain positive clones, thereby obtaining pEcgRNA-adhE and pEcgRNA-eda plasmids.

[0074] Experimental process: pEcgRNA plasmid was cut with restriction endonuclease Bsa I, and the pEcgRNA linearized vector fragment was purified and recovered; oligosaccharide nucleic acids sgRNA-adhE-F / sgRNA-adhE-R, sg-eda-F / sg-eda-R were annealed to form a double-stranded sequence; then 1 μl of the pEcgRNA linearized vector fragment was taken, annealed to form 1 μl of the double-stranded fragment, connected with T4 ligase, and transformed into DH5α competent cells. After recovery at 37°C for 1 hour, 150 μl of bacterial solution was spread on LB+Spec100 plates, and cultured overnight in a constant temperature incubator at 37°C to obtain the target plasmid.

[0075] Annealing self-built system

[0076]

[0077] Annealing self-PCR program

[0078]

[0079] 2.2 Preparation of donor fragments

[0080] Using the primer pairs AdhE-UP-F / AdhE-UP-R and AdhE-DN-F / AdhE-DN-R, 165bp and 179bp fragments were PCR amplified respectively using the Escherichia coli MG1655 genome as a template; using the primer pair TRC-UP / CgpanD-AE-R, 906bp fragment was PCR amplified using the plasmid pTrc99a-cgpanD constructed in Example 1 as a template; using the primer pair AdhE-UP-F / CgpanD-AE-R, OE-PCR was performed using the above three fragments as templates to obtain the adhE-cgpanD donor fragment.

[0081] Using the primer pairs eda-up-F / eda-up-R and Eda-DN-FN / Eda-DN-R, 213bp and 226bp fragments were PCR amplified respectively using the MG1655 genome as template; using the oligonucleotide Trc-panD-F / CgpanD-eda-R as primers and the Ptrc99a-cgpanD plasmid as template, 906bp fragment was PCR amplified; using the primer pair eda-up-F / CgpanD-eda-R, OE-PCR was performed with the above three fragments as templates to obtain the eda-cgpanD donor fragment.

[0082] 2.3 Construction of MG1655ΔadhE::cgpanDΔeda::cgpanD / pEcCa strain

[0083] The pEcCas (Addgene plasmid #73227 donated by Yang Sheng's research group at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, see Li Q, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021Apr 15; 53(5): 620-627.) plasmid was transformed into Escherichia coli Mg1655 chemically competent cells, and screened on LB solid plates containing kanamycin (50 μg / mL) to obtain Mg1655 / pEcCas transformants (the preparation method of chemically transformed competent cells refers to the Molecular Cloning Experiment Guide (3rd Edition)). A single colony of Mg1655 / pEcCas was picked and inoculated into a 4 mL LB test tube containing kanamycin (50 μg / mL), and cultured at 37°C and 220 rpm. When the bacterial concentration OD 600 When the p-value was 0.4, arabinose was added at a final concentration of 10 mM for induction, and the culture was continued for 1 hour to prepare electrocompetent cells (for the preparation method of electrocompetent cells, refer to Li Q, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta BiochimBiophys Sin (Shanghai). 2021 Apr 15; 53(5): 620-627.).

[0084] The adhE-cgpanD fragment and pEcgRNA-adhE plasmid were electroporated into Mg1655 / pEcCas competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin (50 μg / ml) and kanamycin (50 μg / ml), and cultured at 37°C overnight; single colonies were grown and identified by colony PCR using the AdhE-VF / AdhE-VR primer pair, and the positive clone obtained was the MG1655△adhE::cgpanD / pEcCas+pEcgRNA-adhE strain.

[0085] A single colony that was positive by PCR was picked and inoculated into an LB test tube containing kanamycin (final concentration of 50 μg / ml), and rhamnose with a final concentration of 10 mM was added at the same time, and cultured at 37°C overnight; the next day, the bacterial liquid in the test tube was directly streaked onto an LB plate containing kanamycin (final concentration of 50 μg / ml) and cultured at 37°C overnight; the next day, a single colony was picked and transferred to an LB plate containing spectinomycin (final concentration of 50 μg / ml). If it could not grow, it indicated that the pEcgRNA-adhE plasmid had been lost, and the MG1655△adhE::cgpanD / pEcCas strain was obtained.

[0086] The same operation steps were performed as above, and the MG1655△adhE::cgpanD / pEcCas strain was used to prepare electroporation competent cells, and then the adhE-cgpanD fragment and the pEcgRNA-adhE plasmid were electroporated into the above competent cells, and the MG1655△adhE::cgpanD△eda::cgpanD / pEcCas strain was obtained by colony PCR identification and pEcgRNA-eda elimination steps, and named MG1655△adhE::cgpanD△eda::cgpanD, which can be referred to as strain A.

[0087] Example 3: Knockout of genes related to mixed acid fermentation pathway

[0088] In order to inhibit the production of acetic acid, L-formic acid and L-lactic acid, the following related genes of the mixed acid fermentation pathway of Escherichia coli under anaerobic conditions were knocked out: acetate kinase gene ackA, D-lactate dehydrogenase gene ldhA, methylglyoxal synthase gene mgsA, and the operation steps are as follows.

[0089] 3.1 Construction of pEcgRNA-ackA, pEcgRNA-ldhA and pEcgRNA-mgsA plasmids

[0090] The pEcgRNA-ackA, pEcgRNA-ldhA and pEcgRNA-mgsA plasmids were constructed using synthetic oligosaccharide nucleic acids sg-ackA-F / sg-ackA-R, sg-ldhA-F / sg-ldhA-R and sg-mgsA-F / sg-mgsA-R as primer pairs, respectively, according to the construction scheme of the relevant plasmids in step 2.1 of Example 2, and the positive strains were extracted and plasmids were obtained for use.

[0091] 3.2 Preparation of homologous recombination fragments

[0092] Preparation of AckA-donor fragment: The two synthesized primers AckA-homo-F / AckA-homo-R were annealed (same as the annealing procedure in Example 2) to form a double-stranded fragment, which was stored at -20° C. LdhA-donor and mgsA-donor fragments were prepared in the same way.

[0093] 3.3 Construction of ackA knockout strain

[0094] The MG1655△adhE::cgpanD△eda::cgpanD / pEcCas strain was prepared into an electroporation competent cell, and then the pEcgRNA-ackA and AckA-donor fragments were simultaneously electroporated into the MG1655△adhE::cgpanD△eda::cgpanD / pEcCas competent cells, coated with LB+Kan50+Spec50 plates, and the plates were kept at 37°C for static culture. The positive clones were identified by colony PCR, and the positive clones were adjusted to single colonies for pEcgRNA-ackA resistance elimination (the detailed operation steps are the same as in Example 2). The MG1655△adhE::cgpanD△eda::cgpanD△ackA / pEcCas strain was obtained, which can be referred to as strain B, as the starting strain for ldhA gene knockout.

[0095] 3.4 Knockout of ldhA gene

[0096] The steps for knocking out the ldhA gene in strain B are the same as those in step 3.3 above, and the MG1655ΔadhE::cgpanDΔeda::cgpanDΔackAΔldhA / pEcCas strain is obtained, which may be referred to as strain C and serves as the mgsA gene knockout starting strain.

[0097] 3.5 Knockout of mgsA gene

[0098] The steps for knocking out the mgsA gene in strain C are the same as those in step 3.3 above, and the MG1655ΔadhE::cgpanDΔeda::cgpanDΔackAΔldhAΔmgsA / pEcCas strain is obtained, which can be referred to as strain D.

[0099] 3.6 The pEcCas-carrying strains B / C / D above lost the pEcCas plasmid

[0100] The positive clones of strains A / B / C / D carrying plasmid pEcCas were picked and directly cultured in LB liquid medium without antibiotics at 37°C overnight on a shaking table. A small amount of bacterial liquid was drawn and streaked on a 10 g / L sucrose LB solid plate to separate single bacteria. Single colonies were identified on LB plates containing kanamycin (50 μg / mL) resistance to verify the pEcCas elimination strain. The MG1655△adhE::cgpanD△eda::cgpanD strain (also referred to as strain A), MG1655△adhE::cgpanD△eda::cgpanD△ackA strain (also referred to as strain B), MG1655△adhE::cgpanD△eda::cgpanD△ackA△ldhA strain (also referred to as strain C) and MG1655△adhE::cgpanD△eda::cgpanD△ackA△ldhA△mgsA strain (also referred to as strain D) were obtained.

[0101] Example 4: Overexpression of panD from Bacillus subtilis

[0102] 4.1 Construction of pKK233-BspanD plasmid:

[0103] Using the pKK223-3 plasmid (preserved in the laboratory of Zhejiang Huarui Biotechnology Co., Ltd.) as a template and the pKK223-F / pKK233-R primer pair, PCR amplification was performed to obtain the pKK223 vector backbone fragment with a size of 4572 bp.

[0104] The PCR system was as follows: 25 μL of 2×TOROBlue Flash KOD Dye Mix, 0.3 μL of each of the upstream and downstream primers (50 μM), and 0.5 μL of the pKK223-3 plasmid template. 2 Add HO to make up to 50 μL.

[0105] PCR reaction program: 98°C for 2 min; 98°C for 10 s, 58°C for 5 s, 68°C for 5 s, a total of 32 cycles; 68°C for 2 min; 16°C.

[0106] The PCR product was digested with DpnI (1ul DpnI was added to the PCR system and digested at 37°C for 2h), and then recovered from agarose gel (according to the kit operating procedures). The genome of Bacillus subtilis 168 was used as a template, and PtacBspanD-F / PtacBspanD-R were used as primers to perform PCR amplification to obtain a 469bp BspanD fragment. The product was identified and purified and recovered after the size was consistent. The pKK223 vector backbone fragment was connected to the BspanD fragment using the DNA assembly method (DNA assembly Kit purchased from Quanshijin) to obtain the pKK233-BspanD plasmid, and the nucleotide sequence was SEQ ID NO: 2.

[0107] 4.2 Electroporation:

[0108] The method for preparing electroporation competent cells was as follows: refer to step 2.3, and the pKK233-BspanD plasmid was electroporated into the competent cells of strains B / C / D obtained in step 3.6 of Example 3. The transformant bacterial liquid was spread on an LB plate containing ampicillin (100 μg / ml) and cultured overnight at 37°C to obtain genetically engineered bacteria that simultaneously overexpressed panD (cgpanD) from Corynebacterium glutamicum and panD (BspanD) from Bacillus subtilis, which were still named strains B / C / D.

[0109] Example 5: Evaluation of the performance of anaerobic fermentation of glucose carbon source by engineered bacteria

[0110] The anaerobic fermentation performance of glucose carbon source between wild strain MG1655 (abbreviated as WT) and engineered bacteria BD was compared. The operation steps are as follows.

[0111] Each strain was picked from its LB plate into a seed medium, cultured with shaking at 37°C for 16 hours, and then inoculated into a fermentation medium at 20 v / v%, and cultured in a sealed manner for 48 hours to monitor the product.

[0112] The seed culture medium includes component A and component B:

[0113] Component A: Glucose 20-80g / L, MgSO 4 7H 2 O 0.5-2.5g / L, sterilize with moist heat at 121℃ for 25 minutes.

[0114] Component B: Yeast powder (Angel) 5-10g / L, peptone (Oxoid) 5-10g / L, (NH 4 ) 2 SO 4 2.0-13.2g / L, KH2 PO 4 1.0-3.5g / L, K 2 HPO 4 ·3H 2 O 1.0-5.0g / L, potassium bicarbonate 100-200mM, betaine 1mM, adjust pH to 7.2-7.4 with 5mol / LKOH before sterilization, and sterilize with wet heat at 121℃ for 25 minutes.

[0115] Mix component A and component B under sterile conditions to obtain the seed culture medium.

[0116] The fermenter medium also includes component A and component B:

[0117] Component A: Glucose 20-50g / L, MgSO 4 7H 2 O 0.5-2.5g / L, sterilize with moist heat at 121℃ for 25 minutes.

[0118] Component B: Yeast powder (Angel) 0.5-2.0g / L, (NH 4 ) 2 SO 4 2.0-5.0g / L, KH 2 PO 4 1.0-3.5g / L, betaine hydrochloride 0.5-2g / L, FeSO 4 7H 2 O 1.0-2.5mg / L, CoCl 2 6H 2 O 0.1-0.5mg / L, CuSO 4 0.1-0.5mg / L, ZnSO 4 7H 2 O 0.1-1.0mg / L, Na 2 MoO 4 .2H 2 O 0.1-0.5mg / L, H 3 BO 3 0.01-0.1mg / L, MnSO 4 ·H 2 O 0.1-1.0 mg / L, potassium bicarbonate 100-200 mM, sterilize at 121°C for 25 minutes.

[0119] Mix component A and component B under sterile conditions to obtain the fermentation medium.

[0120] The pH of the fermentation was maintained at 7.0 using 2.4M potassium carbonate + 1.2M ammonia water, and no aeration was used during the whole process. The results of anaerobic fermentation of eight strains with glucose as carbon source for 48h and the detection results of β-alanine content in the fermentation broth are listed in Table 2.

[0121] Table 2. Comparison of the results of anaerobic fermentation of glucose to produce β-alanine by four strains

[0122]

[0123] The strain suffixes -1 and -2 represent two parallel experiments.

[0124] As can be seen from Table 2, compared with each genetically engineered bacterium BD, the wild strain MG1655 has the best growth phenotype, but it does not produce β-alanine during anaerobic fermentation in glucose carbon source. Each genetically engineered bacterium BD is obviously affected by the physiological defects brought about by genetic engineering, has a poor growth state, and has a significantly weakened proliferation ability. Therefore, it is necessary to domesticate the genetically engineered bacterium BD in order to obtain a domesticated strain with a good growth phenotype and improved β-alanine expression ability in the anaerobic fermentation environment of glucose carbon source.

[0125] Example 6: Anaerobic domestication of engineered bacteria

[0126] In a glucose carbon source medium and under anaerobic conditions, the genetically engineered bacteria BD are domesticated so that the fermentation strain is adapted to anaerobic fermentation. The method is as follows.

[0127] Seed shake flask and seed tank culture medium: glucose 20-100g / L, (NH 4 ) 2 SO 4 13.2g / L, KH 2 PO 4 3.5g / L, K 2 HPO 4 5.0g / L, (NH 4 ) 2 HPO 4 3.5g / L, MgSO 4 7H 2 O 1mM, CaCl 2 ·2H 2 O 0.1mM, VB150μg / L, betaine 1mM, potassium bicarbonate 100-200mM, trace inorganic salts 1ml / L culture medium, pH 7.0.

[0128] Trace inorganic salt mother solution: FeCl 3 6H 2 O 1.6 g, CoCl 2 6H 2O 0.2g, CuCl 2 ·2H 2 O 0.1g, ZnCl 2 0.2g, Na 2 MoO 4 ·2H 2 O 0.2g, H 3 BO 3 0.05g, distilled water to 1L, filter sterilize.

[0129] The 2.4L fermenter is filled with 1.5L of liquid, sterilized at 121℃ for 15min, and inoculated with BD or its successive bacteria after cooling, with an inoculation amount of 1-20v / v%, a fermentation temperature of 37℃, and a stirring speed of 100rpm. During the domestication process, 2.4M potassium carbonate + 1.2M ammonia water is used to maintain the pH at 7.0. The engineered bacteria are continuously subcultured in the fermenter, and the bacterial liquid in the fermenter is transferred to a new fermenter every 24-48h. After more than 400 transfers, a domesticated strain with significantly improved acid production is obtained. These domesticated strains are still referred to as B / C / D.

[0130] Example 7: Fermentation evaluation of domesticated strains

[0131] According to the method of Example 5, the domesticated strains of the engineering bacteria BD were streaked to isolate single clones, and two strains were selected for each test of growth phenotype and β-alanine production capacity of anaerobic fermentation of glucose carbon source. The results are listed in Table 3.

[0132] Table 3. Comparison of β-alanine production by fermentation of three engineered strains BD after acclimation

[0133] Bacteria β-Alanine after acclimation g / L <![CDATA[OD after domestication 600 > Sugar conversion rate % B-1 1.6 3.2 8.5 B-2 1.5 3.6 7.5 C-1 2.0 3.5 10 C-2 2.2 4.2 11 D-1 2.8 4.8 13.5 D-2 2.9 4.5 15.5

[0134] As can be seen from the table, after the strain BD was domesticated by continuous subculture, the growth phenotype of the domesticated strain for anaerobic fermentation of glucose carbon source, that is, the cell density, was greatly improved, generally increased by about 10 times, indicating that the environmental adaptability was significantly improved. With the improvement of growth and proliferation ability, the β-alanine production capacity of the domesticated strain was reflected, and the β-alanine production of anaerobic fermentation of glucose carbon source could reach up to 3g / L or more. Similar to the comparison results before domestication, the engineered strain D, which integrated aspartate decarboxylase panD in the genome and overexpressed aspartate decarboxylase panD through plasmid transformation, had the highest β-alanine expression ability. The domesticated strain of engineered strain D with the best growth phenotype was named SH2356.

[0135] The engineered domesticated bacteria SH2356 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.28214.

[0136] It should also be noted that the listing and discussion of previously published documents in this specification should not be regarded as an admission that the document is prior art or common knowledge.

Claims

1. A method for constructing a β-alanine producing bacterium, characterized in that: The following steps are involved: A. Using Escherichia coli as the base strain, overexpressing aspartate decarboxylase panD or enhancing the expression of aspartate decarboxylase panD, and interrupting or downregulating the expression of alcohol dehydrogenase gene adhE and / or 2-keto-3-deoxy-6-phosphogluconate aldolase gene eda, to obtain strain A; B. interrupting or down-regulating the expression of one or more genes selected from the following group in the genome: acetate kinase gene ackA, D-lactate dehydrogenase gene ldhA, methylglyoxal synthase gene mgsA, to obtain one of strains B, C and D; C. In a culture medium with glucose as the carbon source, one of the strains B, C and D is subjected to anaerobic fermentation domestication to obtain a strain with improved β-alanine production capacity.

2. The method according to claim 1, characterized in that The interruption mode of the gene in step A and step B is selected from gene knockout, deletion, and inactivation.

3. The method according to claim 1, characterized in that In step A, the expression of genes adhE and eda are simultaneously interrupted or downregulated, and / or In step B, the expression of genes ackA, ldhA and mgsA is simultaneously interrupted or downregulated.

4. The method according to claim 1, characterized in that The aspartate decarboxylase panD in step A is selected from the following group: aspartate decarboxylase panD derived from Bacillus subtilis (NCBI accession number NP_390122), aspartate decarboxylase panD derived from Corynebacterium glutamicum (NCBI accession number BAB97528), aspartate decarboxylase panD derived from Escherichia coli (NCBI accession number ACA79142).

5. The method according to claim 1, characterized in that The method of overexpressing aspartate decarboxylase panD in step A is: Integrate a single copy or multiple copies of the aspartate decarboxylase panD gene into the host bacterial genome, and / or The aspartate decarboxylase panD gene is expressed in the host bacteria in the form of a plasmid; The method for enhancing the expression of aspartate decarboxylase panD in step A is: The expression of aspartate decarboxylase panD gene was regulated by the strong promoter tac or trc promoter.

6. The method according to claim 1, characterized in that The domestication of one of the strains B, C and D in step C comprises the following steps: obtaining an engineered bacterium with a β-alanine production capacity more than 10 times higher than that of the base bacterium by continuous subculture in a fermenter without oxygen.

7. The method according to claim 1, characterized in that The base bacteria is Escherichia coli MG1655.

8. A genetically engineered bacterium, characterized in that: The method is constructed according to any one of claims 1 to 7.

9. The genetically engineered bacterium according to claim 8, characterized in that It is Escherichia coli, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.28214.

10. Use of the genetically engineered bacteria according to claim 8 or 9 in producing β-alanine using glucose as a carbon source.

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