Escherichia coli probiotic with high yield of beta-alanine and application thereof

Abstract

The application discloses an Escherichia coli probiotic with high beta-alanine yield and application thereof, and the recombinant genetically engineered bacterium takes a wild-type Escherichia coli probiotic as a starting strain and is subjected to one or more gene edits, including Trc promoter strengthening of a panD gene, an aspC gene, an sdhC gene and a ppc gene, overexpression of a panD gene from Bacillus subtilis, an aspB gene from Corynebacterium glutamicum, an aspA gene and a pyc gene from Corynebacterium glutamicum on a plasmid pTrc99a, and knockout of a cycA gene. 600 Compared with an existing reported Escherichia coli probiotic beta-alanine production strain, the OD of the recombinant genetically engineered bacterium is 4.4 times that of the existing reported strain, and the beta-alanine yield is 6 times that of the existing reported strain, so that the recombinant genetically engineered bacterium has great industrial application potential.

Description

(I)TECHNICAL FIELD

[0001] The present application relates to a high-yield beta-alanine of escherichia coli probiotics and its application. (II)BACKGROUND

[0002] Beta-alanine, also known as 3-amino propionic acid, is the only beta-type amino acid and non-protein amino acid in nature. Although it does not participate in the synthesis of enzymes and proteins, it still has important physiological functions in the growth process of organisms. In cell metabolism, beta-alanine can be used as a precursor to synthesize pantothenic acid (vitamin B5) and coenzyme A (CoA), which is related to fatty acid metabolism, sugar metabolism and other metabolic activities. Plants and microorganisms can synthesize beta-alanine independently, while mammals need exogenous supplementation. According to the current research, beta-alanine has important application value in food, medicine, chemical and feed industries.

[0003] In the field of medicine, beta-alanine is a key precursor for the production of pantothenic acid, calcium pantothenate, coenzyme A (CoA), and acyl carrier protein (ACP), etc. It plays an important role in helping cell metabolism and the development of the central nervous system. At the same time, beta-alanine can also be used to synthesize drugs such as balsalazide and pamidronate disodium. In the food industry, beta-alanine can be used as a food additive, often used in food flavors to improve food flavor. At the same time, due to its antioxidant properties, it is also often used as a food preservative. According to relevant clinical studies, beta-alanine can also be used as a nutritional supplement for athletes. After supplementing beta-alanine, the human body can significantly improve fatigue caused by long-term high-intensity exercise and improve human tolerance. In the chemical industry, beta-alanine can not only be used as a precursor to produce pantothenic acid and calcium pantothenate, as well as poly-3-hydroxypropionic acid and poly-beta-alanine chemical products, but also be used as a precipitant in pharmaceuticals, electroplating corrosion inhibitor, and lead poisoning antidote, etc. In the field of feed, beta-alanine is increasingly used in animal feeding. Studies have found that adding beta-alanine to feed can improve the utilization of feed by livestock and poultry, reduce feed intake; at the same time, it can improve the antioxidant capacity of muscle and increase the content of muscle-derived active peptides, effectively improving meat quality. In addition, beta-alanine also plays an important role in the fields of environment and cosmetics.

[0004] Currently, there are three main methods for producing beta-alanine: (1) chemical synthesis, which uses nitrile substances such as acrylonitrile, acrylic acid, and beta-amino propionitrile to synthesize beta-alanine under high pressure and high temperature in strong acid and strong base conditions. However, chemical synthesis requires high energy consumption and high equipment requirements, and produces substances harmful to the environment and human body during production, as well as byproducts that make subsequent separation and purification difficult. (2) biological enzyme catalysis, which uses aspartate as a substrate and aspartate decarboxylase from strains such as Bacillus subtilis or Corynebacterium glutamicum to express and catalyze the production of beta-alanine in Escherichia coli. This method is more environmentally friendly, safe, and less polluting, and has attracted more attention from scholars. However, biological enzyme catalysis is unstable, costly, and susceptible to substrate inhibition. (3) microbial fermentation to produce beta-alanine, which uses synthetic biology, systems metabolic engineering, protein engineering, and transcriptome and metabolome technologies to modify the metabolic process of the target product beta-alanine, allowing more metabolic flow to flow towards the target product. As climate change and environmental problems become increasingly severe, the use of inexpensive glucose and other carbon sources to explore clean, environmentally friendly, and low-energy production methods has attracted the attention of many scholars.

[0005] With the development of biotechnology, the emergence of systems metabolic engineering technology has enabled humans to move beyond traditional strain screening techniques such as mutagenesis and directed evolution. By quantitatively analyzing the metabolic pathways and metabolic fluxes of strains, and purposefully modifying metabolic pathways, the production efficiency of target metabolites can be maximized, and the production performance of strains can be significantly improved. Unlike traditional breeding techniques, systems metabolic engineering is an effective method for improving the genetic traits of organisms by rationally designing cellular metabolic pathways and reconstructing metabolic networks.

[0006] In current research, Escherichia coli, yeast, Corynebacterium glutamicum, and Bacillus subtilis are commonly used as chassis strains to construct engineered strains. The metabolic genetic background and physiological characteristics of these chassis strains are relatively clear. Combined with mature gene editing technology and rational metabolic flux design, the modification process of strain metabolic pathways can be greatly accelerated, and the time to achieve optimal production performance can be shortened. Escherichia coli, as the most clear prokaryotic bacterium currently studied, has the characteristics of rapid reproduction, short fermentation period, high expression level of target genes, and mature and perfect expression system, and has been widely used in the industrial production of various biological products. Escherichia coli Nissle 1917, abbreviated as EcN, is a subspecies of Escherichia coli and a gram-negative bacterium. In addition to the advantages of Escherichia coli, EcN also has biological safety, but it is rarely used for the production of industrial metabolites. Therefore, EcN has good application prospects in the fields of metabolic engineering and synthetic biology.

[0007] The de novo synthesis pathway of beta-alanine in the E. coli probiotic bacteria includes glucose uptake, glycolysis pathway, TCA cycle, L-aspartate synthesis and its beta-alanine synthesis pathway. First, glucose is phosphorylated under the PTS or non-PTS system to synthesize glucose-6-phosphate, and the carbon flow enters the glycolysis pathway to synthesize the precursor of beta-alanine, phosphoenolpyruvate. Under the catalysis of phosphoenolpyruvate carboxylase, phosphoenolpyruvate generates oxaloacetate. Oxaloacetate is unstable and is easily decomposed to generate aspartic acid under the catalysis of aspartate transaminase, and then generates beta-alanine under the catalysis of aspartate decarboxylase. L-lysine, L-threonine, O-succinylhomoserine and homoserine belong to the aspartic acid family of amino acids, and they and beta-alanine have the common precursor L-aspartate, so weakening the competition branch is an effective construction strategy. However, it is still necessary to further explore the bottleneck affecting the yield of beta-alanine in the E. coli probiotic bacteria. The E. coli probiotic bacteria contains two highly stable cryptic plasmids, pMUT1 (NCBI number MW240712, 3.2kb) and pMUT2 (NCBI number CP023342, 5.6kb). Due to the presence of the two plasmids, the potential of the engineered bacteria is limited, not only the expression of heterologous genes is limited, but also the effect of protein expression is poor. Because of the incompatibility of the plasmids, it is difficult to transfer the exogenous plasmid into the bacteria and successfully express it. How to study and modify the cryptic plasmid is the key to improve the transformation efficiency of the exogenous plasmid. (III)SUMMARY

[0008] The purpose of the present application is to provide a high-yield beta-alanine E. coli probiotic bacteria and its application in the preparation of beta-alanine by microbial fermentation method, which lays a foundation for the industrial production of beta-alanine.

[0009] The technical scheme adopted by the present application is:

[0010] The present application provides a high-yield beta-alanine E. coli probiotic bacteria, which takes wild-type E. coli probiotic bacteria as the starting strain and performs one or more of the following gene editing: Trc promoter strengthening panD gene, aspC gene, sdhC gene, ppc gene, overexpression of panD gene from Bacillus subtilis, aspB gene from Corynebacterium glutamicum, aspA gene, pyc gene from Corynebacterium glutamicum on plasmid pTrc99a, and knock-out of cycA gene.

[0011] Further, the nucleotide sequence of the panD gene is shown as SEQ ID NO. 3; the nucleotide sequence of the aspC gene is shown as SEQ ID NO. 7; the nucleotide sequence of the aspB gene is shown as SEQ ID NO. 8; the nucleotide sequence of the aspA gene is shown as SEQ ID NO. 9; the nucleotide sequence of the sdhC gene is shown as SEQ ID NO. 10; the nucleotide sequence of the ppc gene is shown as SEQ ID NO. 11; the nucleotide sequence of the pyc gene is shown as SEQ ID NO. 12; the nucleotide sequence of the cycA gene is shown as SEQ ID NO. 13; and the nucleotide sequence of the Trc promoter is shown as SEQ ID NO. 14.

[0012] Further, the wild-type E. coli probiotic is E. coli Nissle 1917.

[0013] Further, the E. coli probiotic takes the wild-type E. coli probiotic as a starting strain, and the following gene editing is sequentially performed: the Trc promoter strengthens the panD gene, the panD gene derived from Bacillus subtilis is overexpressed on the plasmid pTrc99a, the Trc promoter strengthens the aspC gene, the aspB gene derived from Corynebacterium glutamicum is overexpressed on the plasmid pTrc99a, the aspA gene is overexpressed on the plasmid pTrc99a, the Trc promoter strengthens the sdhC gene, the Trc promoter strengthens the ppc gene, the exogenous PC catalytic reaction is introduced, and the cycA gene is knocked out.

[0014] The E. coli probiotic according to the present application is constructed by the following method:

[0015] (1) The Trc promoter strengthens the panD gene: taking the genome of E. coli Nissle 1917 as a template, the Trc promoter is designed as a homologous arm on the primer, panD-FF / panD-FR and panD-RF / panD-RR are used as primers to amplify the upstream and downstream homologous arms of the original promoter of the panD gene, and donor DNA is obtained by fusion; the pTarget plasmid is taken as a template, pTarget-panD-F and pTarget-panD-R are used as primers to amplify the plasmid and linearize to obtain linearized pTarget-panD plasmid, the donor DNA and the linearized pTarget-panD plasmid are connected, and then transformed into the competent cells of the wild-type E. coli probiotic E. coli Nissle 1917 (denoted as starting strain E0) into which the pCas9 plasmid has been introduced, to eliminate the pTarget and pCas9 plasmids, and construct strain E1 (Trc-panD).

[0016] (2) Overexpression of codon-optimized Bacillus subtilis panD gene on plasmid pTrc99a: linearized plasmid pTrc99a-line was obtained using pTrc99a as template and pTrc99a-line-F / pTrc99a-line-R as primers; fragment panD was obtained using pTrc99a plasmid containing codon-optimized heterologous panD gene as template and panD-F / panD-R as primers; linearized plasmid pTrc99a-line was ligated with panD gene fragment to construct plasmid pTrc99a-panD, which was transformed into strain E1 (Trc-panD) competent cells to construct strain E4 (Trc-panD / pTrc99a-panD). BS BS BS BS BS BS BS

[0017] (3) Overexpression of codon-optimized Corynebacterium glutamicum aspB gene on plasmid pTrc99a-panD: linearized plasmid pTrc99a-panD-line was obtained using pTrc99a-panD as template and pTrc99a-D-line-F / pTrc99a-D-line-R as primers; fragment aspB was obtained using pTrc99a plasmid containing codon-optimized heterologous aspB gene as template and aspB-F / aspB-R as primers; linearized plasmid pTrc99a-panD-line was ligated with aspB gene fragment to construct plasmid pTrc99a-panD-aspB, which was transformed into strain E8 (Trc-panD Trc-aspC / pTrc99a-panD) competent cells to construct strain E10 (Trc-panD Trc-aspC / pTrc99a-panD-aspB). BS BS BS BS BS CG CG CG CG BS CG BS CG BS BS CG

[0018] ​​​​​​​​​​​​​​​​​​​​​​​(4) Strengthening the panD gene with a plasmid pTrc99a-panD BS -aspB CG Overexpressing the aspA gene: using the plasmid pTrc99a-panD BS -aspB CG as the template, and pTrc99a-D BS B CG -line-F / pTrc99a-D BS B CG as the primers to obtain the linearized plasmid pTrc99a-panD BS -aspB CG -line; using the wild-type E. coli probiotic as the template, and aspA-F / aspA-R as the primers to obtain the fragment aspA, and ligating the linearized plasmid pTrc99a-panD BS -aspB CG -line with the aspA gene fragment to construct the plasmid pTrc99a-panD BS -aspB CG -aspA, and transforming it into the strain E10 (Trc-panD Trc-aspC / pTrc99a-panD BS -aspB CG ) competent cells to construct the strain E11 (Trc-panD Trc-aspC / pTrc99a-panD BS -aspB CG -aspA).

[0019] (5) Strengthening the sdhC gene with a Trc promoter: using the genome of E. coli Nissle 1917 as the template, and designing the Trc promoter as the homologous arm on the primers, and using sdhC-FF / sdhC-FR and sdhC-RF / sdhC-RR as the primers to amplify the upstream and downstream homologous arms of the original promoter of the sdhC gene, and fusing to obtain the donor DNA; using the pTarget plasmid as the template, and pTarget-sdhC-F and pTarget-sdhC-R as the primers to amplify the plasmid and linearize to obtain the linearized pTarget-sdhC plasmid, and ligating the donor DNA and the linearized pTarget-sdhC plasmid, and transforming it into the strain E11 (Trc-panD Trc-aspC / pTrc99a-panD BS -aspB CGIn competent cells (-aspA), pTarget and pCas9 plasmids were eliminated to construct strain E12(Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD) BS -aspB CG -aspA).

[0020] (6) Trc promoter enhancement of the ppc gene: Using the genome of E. coli Nissle 1917 as a template, the Trc promoter was designed as a homologous arm on primers. Using ppc-FF / ppc-FR and ppc-RF / ppc-RR primers, the upstream and downstream homologous arms of the original ppc gene promoter were amplified and fused to obtain donor DNA. Using pTarget plasmid as a template, the plasmid was amplified and linearized using pTarget-ppc-F and pTarget-ppc-R primers to obtain linearized pTarget-ppc plasmid. The donor DNA and linearized pTarget-ppc plasmid were ligated and transformed into strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD) which had been transformed into pCas9 plasmid. BS -aspB CG In competent cells (-aspA), pTarget and pCas9 plasmids were eliminated to construct strain E13(Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD) strain. BS -aspB CG -aspA).

[0021] (7) In plasmid pTrc99a-panD BS -aspB CG -aspA overexpression of the codon-optimized pyc gene from Corynebacterium glutamicum: using plasmid pTrc99a-panD BS -aspB CG -aspA is the template, and pTrc99a-D is used. BS B CG A-line-F / pTrc99a-D BS B CG A-line-R was used as primers to obtain the linearized plasmid pTrc99a-panD. BS -aspB CG -aspA-line; Uses heterogeneous pyc files with codon optimizations. CG Using the pTrc99a plasmid as a template, and with pyc CG -F / pycCG -R is the primer obtained fragment pyc CG The linearized plasmid pTrc99a-panD BS -aspB CG -aspA-line and pyc CG The gene fragments were ligated to construct plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG and transformed into strain E13 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc / pTrc99a-panD BS -aspB CG -aspA) competent cells, to construct strain E14 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0022] (8) Knockout of cycA gene: Taking the genome of E. coli Nissle 1917 as a template, cycA-FF / cycA-FR and cycA-RF / cycA-RR as primers, the upstream and downstream homologous arms of the cycA gene were amplified, and a donor DNA was obtained by fusion; taking the pTarget plasmid as a template, pTarget-cycA-F and pTarget-cycA-R as primers, the plasmid was amplified and linearized to obtain a linearized pTarget-cycA plasmid; after the donor DNA and the linearized pTarget-cycA plasmid were connected, the pTarget and pCas9 plasmids were eliminated in the strain E14 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ) competent cells into which the pCas9 plasmid had been introduced, to construct strain E15 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc AcycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0023] The beta-alanine synthesis network of EcN is reformed, the key enzyme for the biosynthesis of beta-alanine is aspartate-alpha-decarboxylase (ADC, encoded by panD gene), because the catalytic activity of the endogenous ADC in EcN is not high, only the needs of its normal growth can be met, which limits the accumulation of beta-alanine, so it is not suitable for constructing beta-alanine production strain, meanwhile, in EcN, ADC catalyzes aspartate to transform into beta-alanine, and aspartate is the precursor of threonine, lysine and homoserine, therefore, in the process of constructing beta-alanine production strain, the panD gene from Bacillus subtilis with excellent performance is screened, which can provide sufficient metabolic pull for the synthesis of beta-alanine, thereby enhancing the competition for the precursor aspartate. L-aspartate as the direct precursor of beta-alanine synthesis can be synthesized by two routes in EcN, oxaloacetate transamination route, i.e. TCA reduction branch pathway and fumaric acid amination route, i.e. TCA oxidation branch pathway. In the oxaloacetate transamination route, the intermediate oxaloacetate of TCA cycle can be converted into L-aspartate with L-glutamate as the amino donor under the catalysis of aspartate transaminase (encoded by aspC gene), and in the fumaric acid amination route, aspartate aminase (encoded by aspA gene) catalyzes the reversible conversion of the intermediate fumaric acid of TCA cycle and aspartate, which is one of the effective means to extract carbon flux from TCA cycle. The catalytic transamination reaction of aspC gene is affected by the substrates, products and cofactors in the reaction system, and the enzyme activity is not high, and by overexpressing the aspB gene from Corynebacterium glutamicum on the plasmid, oxaloacetate can be efficiently catalyzed to convert into aspartate, and by overexpressing the aspA gene on the plasmid, the fumaric acid amination route is strengthened, the carbon flux in TCA cycle is reduced, and the accumulation of intermediates in TCA cycle is prevented. By strengthening succinate dehydrogenase (encoded by sdhC gene), the carbon flux in TCA cycle can be extracted to enrich the aspartate substrate pool, and by strengthening phosphoenolpyruvate carboxylase (encoded by ppc gene), phosphoenolpyruvate (PEP) is directly converted into oxaloacetate, thereby reducing the carbon flux entering the oxidation branch of TCA cycle. In the process of glucose uptake, part of PEP is consumed to form pyruvate (PYR) in PTS with PTS as the phosphate donor, but with ATP instead of PEP as the phosphate donor, the metabolic burden in cells is increased. Meanwhile, the accumulation of pyruvate is one of the main reasons for the carbon loss in the process of beta-alanine synthesis.Therefore, in order to reduce the carbon loss in the synthesis of beta-alanine, make pyruvic acid for the synthesis of beta-alanine, effectively distribute the carbon flux of PEP and PYR nodes, which is the key to promote the synthesis of precursor oxaloacetate, by introducing exogenous PC to catalyze the reaction, redistributing the carbon flux of PEP / PYR nodes, not only can directly enrich the carbon flux of beta-Ala synthesis, reduce carbon loss, but also can avoid the metabolic burden and the increase of organic acid caused by replacing PTS with non-PTS, and further increase the yield of beta-alanine by knocking out the beta-alanine transporter (encoded by cycA gene).

[0024] The application also relates to the application of the E. coli probiotic bacteria in the microbial fermentation method for preparing beta-alanine, which comprises the following steps: inoculating the E. coli probiotic bacteria into a fermentation medium containing 50 mg / L kanamycin, culturing the bacteria under the condition of 30 DEG C and 180 rpm until the OD 600 =0.5, adding IPTG to a final concentration of 0.25 mM to induce gene expression, continuing to culture for 48 h until the fermentation is completed, and obtaining the fermentation liquor containing beta-alanine by separating and purifying the fermentation liquor.

[0025] The fermentation medium formula is as follows: glucose 18-22 g / L, (NH4)2SO4 14-18 g / L, yeast extract 2-3 g / L, KH2PO4 1-2 g / L, MgSO4 0.2-1 g / L, CaCO3 13-17 g / L, VB1 0.3-0.5 mg / L, VB 12 0.3-0.5 mg / L, trace element solution 0.5-1.5 mL / L, solvent is water, and the pH value does not need to be adjusted; further optimization is as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, CaCO3 15 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 , 1 mL / L trace element solution, solvent is water, and the pH value does not need to be adjusted; the components of the trace element solution are as follows: 8-12 g / L CaCl2, 8-12 g / L FeSO4.7H2O, 0.8-1.2 g / L ZnSO4.7H2O, 0.1-0.3 g / L CuSO4, 0.01-0.04 g / L NiCl2.7H2O, solvent is deionized water; further optimization is as follows: 10 g / L CaCl2, 10 g / L FeSO4.7H2O, 1 g / L ZnSO4.7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2.7H2O, solvent is deionized water.

[0026] The E. coli probiotic is inoculated into LB medium containing 50 mg / L kanamycin before fermentation, and seed liquid is prepared by culturing at 37°C and 180 rpm overnight. The seed liquid is inoculated into fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%.

[0027] The fermentation is carried out in a fermentation tank: the E. coli probiotic is inoculated into LB medium containing 50 mg / L kanamycin, and seed liquid is prepared by culturing at 37°C and 180 rpm overnight. The seed liquid is inoculated into LB medium containing 50 mg / L kanamycin at a volume concentration of 2%.

[0028] The fermentation medium formula in a 5-L fermentation tank: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 12 0.2 mg / L, 1 mL / L trace element solution, solvent is deionized water; composition of trace element solution: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water;

[0029] The composition of the feed medium: glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 , solvent is water, and the pH is adjusted to 6.8 with 14% ammonia water.

[0030] The feed rate is 25 mL / h, and the total amount of feed medium added is 1700 mL / 2L.

[0031] The fermentation liquid separation and purification method is as follows: firstly, centrifuging at 12000 rpm and 4 DEG C for 10 min to remove the bacteria and other solid particles in the fermentation liquid, then removing the macromolecular protein in the fermentation liquid by microfiltration, then removing the inorganic salt in the fermentation liquid by deionization technology, adding activated carbon for decolorization, separating beta-alanine by ion exchange resin, vacuum concentration, adding ethanol for cooling crystallization, and obtaining beta-alanine crystal.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The present application screens the panD gene from Bacillus subtilis with excellent performance, enhances the competition for the precursor aspartate, provides sufficient metabolic pull for the synthesis of beta-alanine, overexpresses the aspB gene from Corynebacterium glutamicum on a plasmid to efficiently catalyze the conversion of oxaloacetate into aspartate, overexpresses the aspA gene on a plasmid to strengthen the amination route of fumaric acid, reduces the carbon flux in the TCA cycle, prevents the accumulation of intermediates in the TCA cycle, strengthens the sdhC gene to extract the carbon flux from the TCA cycle to enrich the aspartate substrate pool, strengthens the ppc gene to promote the direct conversion of phosphoenolpyruvate (PEP) into oxaloacetate, thereby reducing the carbon flux into the oxidative branch of the TCA cycle and increasing the carbon flow of the key metabolic precursor oxaloacetate, introduces the exogenous PC to catalyze the reaction and directly enrich the carbon flux for the synthesis of beta-Ala, reduces the loss of central metabolic carbon flow, avoids the metabolic burden and the increase of organic acid caused by the replacement of PTS with non-PTS, and knocks out the cycA gene to further increase the yield of beta-alanine. 600 In the fed-batch fermentation, the beta-alanine yield of the strain E15 reaches 70.4 g / L, and the sugar acid conversion rate reaches 37%. Compared with the reported E. coli probiotic beta-alanine production strain, the OD 600 of the E. coli probiotic of the present application is 4.4 times that of the reported strain, and the beta-alanine yield is 6 times that of the reported strain, which has great industrial application potential. (IV) DESCRIPTION OF DRAWINGS

[0034] Figure 1 The column chart of the biomass OD 600 and the beta-alanine concentration of the strains E4, E8, E9, E10 and E11.

[0035] Figure 2 The column chart of the biomass OD 600 and the beta-alanine concentration of the strains E4, E8, E9, E10 and E11.

[0036] Figure 3 The column chart of the biomass OD 600Bar chart of biomass OD and beta-alanine concentration.

[0037] Figure 4 Biomass OD for strains E12 and E13 600 Bar chart of biomass OD and beta-alanine concentration.

[0038] Figure 5 Biomass OD for strains E13 and E14 600 Bar chart of biomass OD and beta-alanine concentration.

[0039] Figure 6 Biomass OD for strains E14 and E15 600 Bar chart of biomass OD and beta-alanine concentration.

[0040] Figure 7 Biomass OD for strain E15 in 5-L fermenter fed-batch fermentation 600 Bar chart of biomass OD and beta-alanine concentration. (V) DETAILED DESCRIPTION

[0041] The application will be further described below in conjunction with specific examples, but the scope of protection of the application is not limited to this:

[0042] Wild-type Escherichia coli probiotic strain Escherichia coli Nissle 1917, i.e. wild strain E0, from Hangzhou BioSci Biotech Preservation Center, preservation date August 5, 2020, preservation number BSBCC T0023.

[0043] LB plate composition: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar powder, solvent is water, pH value is natural.

[0044] LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0045] The fermentation medium formula is as follows: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, CaCO3 15 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 , 1 mL / L trace element solution, solvent is water, no need to adjust pH value; the components of the trace element solution are as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water.

[0046] Table 1 Genes involved in the modification of the strains and the corresponding pathways

[0047]

[0048] Table 2 Primers used in the gene editing process

[0049]

[0050]

[0051]

[0052] Example 1, Trc promoter strengthens the panD gene in strain E0, to construct strain E1 (pTarget-Trc panD).

[0053] Using CRISPR / Cas9-mediated gene editing technology, the Trc promoter (nucleotide sequence as shown in SEQ ID No. 14) derived from pTrc99a is used to replace the original promoter of the aspartate-alpha-decarboxylase panD gene (nucleotide sequence as shown in SEQ ID No. 1) on the genome of the wild strain E0, to enhance the accumulation of beta-alanine. The specific operation is as follows:

[0054] (1) Construct pTarget-panD-line plasmid: use plasmid pTarget as template, and use pTarget-panD-F / pTarget-panD-R as primers to amplify N20 mutant product. After adding DpnI to the PCR product, digest at 37°C for 1 h to digest the methylated plasmid template. After purification with a purification kit, it is transformed into E. coli DH5α competent cells, spread on LB solid plates containing 50 mg / L spectinomycin, and incubated at 37°C for 12 h. Colony PCR is used for preliminary verification, and sequencing is used to verify the correctness of the pTarget-panD-PAM plasmid. The plasmid pTarget-panD-PAM is obtained. Then use pTarget-line-F / pTarget-line-R as primers, and use plasmid pTarget-panD-PAM as template to amplify plasmid linearization fragment. After adding DpnI to the PCR product, digest at 37°C for 3 h, and recover the DNA fragment with a purification kit to obtain the linearized plasmid pTarget-panD-line.

[0055] (2) Constructing plasmid pTarget-panD containing Donor: using the genome of E. coli Nissle 1917 as a template, and using panD-FF / panD-FR as primers to amplify the upstream homologous arm F1 (F1 primer contains Trc promoter sequence); using panD-RF / panD-RR as primers to amplify the downstream homologous arm R1 (R1 primer contains Trc promoter sequence). The PCR product is recovered by using a purification kit, which is the Trc-panD upstream and downstream fragments. Using the upstream and downstream homologous arms F1 and R1 as templates, and using panD-FF / panD-RR as primers to obtain the DNA fragment containing the upstream and downstream homologous arms of Trc-panD, and the DNA fragment is recovered by a purification kit to obtain the fused fragment. Then the fused fragment and the linearized plasmid pTarget-panD-line of step (1) are used to construct the plasmid pTarget-Trc panD by one-step cloning method, and the E. coli DH5α competent cells are transformed, and then coated on LB solid plates containing 50 mg / L spectinomycin, and incubated at 37°C for 12 h. The correct strains are preliminarily screened by colony PCR using primers pTarget-YZ-F / pTarget-YZ-R, and finally the correctness of the plasmid pTarget-Trc panD is verified by sequencing.

[0056] (3) The plasmid pCas9 is transformed into the competent cells E0, and then coated on LB solid plates containing 50 mg / L kanamycin, and incubated at 30°C overnight. Single colonies are picked into LB test tube medium containing 50 mg / L kanamycin resistance, and incubated at 30°C overnight. Then 100 mL of LB medium is inoculated with the competent cells at a volume concentration of 1%, and 50 mg / L of kanamycin resistance and 10 mM of L-arabinose are added, and then incubated at 180 rpm and 30°C until the OD 600 = 0.5, and centrifuged at 4°C and 4000 rpm. The cells are washed once with 0.1M calcium chloride aqueous solution at 4°C, and then washed once with 0.1M calcium chloride solution containing 15% glycerol, and finally resuspended with 15% glycerol calcium chloride, and then stored in aliquots, to obtain the competent cells containing plasmid pCas9, which is ready for use.

[0057] (4) Take 2 μL of pTarget-Trc panD plasmid constructed in step (2) and mix with 100 μL of competent cells prepared in step (3), and incubate on ice for 30 min. Then heat shock in a constant temperature water bath (HH-4) at 42 °C. Immediately after heat shock, add 600 μL of 4 °C pre-cooled LB medium, mix well and then transfer to a 30 °C shaker for 3 h at 150 rpm. Then spread on LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin, and incubate at 30 °C for 20 h. Then use panD-YZ-F / panD-YZ-R as primers for colony PCR verification, and sequencing verification to confirm the correctness of strain construction.

[0058] (5) Elimination of pTarget and pCas9 plasmids: pick a single colony of the positive strain in step (4) and inoculate into an LB liquid medium test tube containing 2 mM IPTG and 50 mg / L kanamycin, and incubate at 30 °C overnight. Then use the bacterial liquid to streak on LB solid medium containing 50 mg / L kanamycin, and incubate at 30 °C for 20 h until single colonies appear. Then pick a single colony and inoculate on LB solid medium containing 50 mg / L spectinomycin, and incubate at 30 °C for 20 h. If no single colony appears, it indicates that the pTarget plasmid has been eliminated from the strain. Then pick the strain with the pTarget plasmid eliminated and inoculate into an LB liquid medium test tube without antibiotics, and incubate at 42 °C for 10 h. Then use the bacterial liquid to streak on LB solid medium containing 50 mg / L kanamycin, and incubate at 37 °C for 10 h. If no single colony appears, it indicates that the pCas9 plasmid has been successfully eliminated, and the strain is used as a plasmid-free competent strain. Finally, the plasmid-free strain E1 (pTarget-Trc panD) is obtained.

[0059] (6) Take the wild-type strain E0 as a control, and pick a single colony of the strain E1 and inoculate into an LB liquid medium test tube without antibiotics, and incubate at 37 °C at 180 rpm overnight to obtain a seed liquid. Then inoculate 1 mL of the seed liquid into a 500 mL shake flask containing 50 mL of fermentation medium without antibiotics, and incubate at 30 °C at 180 rpm for 48 h. After fermentation, centrifuge 1 mL of the fermentation liquid at 12,000 rpm for 3 min, discard all the supernatant, and resuspend the bacterial cells and calcium carbonate in 1 mL of distilled water. Centrifuge at 12,000 rpm for 3 min, discard the supernatant, and resuspend the bacterial cells and calcium carbonate in 1 mL of distilled water again. Centrifuge at 12,000 rpm for 3 min, discard the supernatant, and finally resuspend the bacterial cells and calcium carbonate in 800 μL of distilled water. Then add 200 μL of 20% acetic acid aqueous solution, and let stand at room temperature for 5 min to dissolve the calcium carbonate. Take 100 μL of the bacterial liquid with dissolved calcium carbonate, dilute in 1900 μL of distilled water, and finally determine the biomass OD 600Take 1 mL of the fermentation broth 12000 rpm centrifugal 3 min, take the supernatant as sample for detecting the concentration of β-alanine, standby. Biomass OD 600 and the content of β-alanine as shown in Figure 1 , the biomass OD 600 of the control strain E0 was 12.02, and the content of β-alanine was 0 g / L; the biomass OD 600 of the strain E1 was 11.95, and the content of β-alanine was 0.01 g / L, and the content of β-alanine was not significantly improved.

[0060] (7) Determination of the concentration of β-alanine:

[0061] 1% 2,4-dinitrofluorobenzene configuration: take 1 mL of 2,4-dinitrofluorobenzene dissolved in 99 mL of acetonitrile.

[0062] 0.5M NaHCO3 solution configuration: take 21g NaHCO3 dissolved in 500 mL of deionized water.

[0063] 0.2M PB buffer: weigh 8.74g Na2HPO4·12H2O and 2.43g Na2HPO4·2H2O dissolved in 200 mL of deionized water, and store for use.

[0064] Sample treatment: dilute the sample with ultrapure water to 0.1-1 g / L.

[0065] Reaction conditions: take 100 μL of sample, 100 μL of 0.5M NaHCO3 solution and 100 μL of 1% 2,4-dinitrofluorobenzene, respectively, incubate at 60°C for 75 min, finally add 700 μL of 0.2M PB buffer and mix well, filter (polyvinylidene fluoride, organic membrane, 0.22 μm) standby.

[0066] Detection condition: HPLC model: Thermo Scientific Utimate 3000, HPLC detection wavelength is 360 nm. Gradient elution program is used to separate beta-alanine, wherein the mobile phase A component is methanol: acetonitrile: ultrapure water = 45:45:10 (v:v:v); the mobile phase B component is 10 mM potassium dihydrogen phosphate, and the pH value is adjusted to 7.0 by KOH. The elution program is 0-2.5 min 10% A, 90% B; 2.5-2.6 min A 10%→14%, B 90%→86%; 2.6-13 min A 14%→34%, B 86%→66%; 13-13.1 min A 34%→38% B 66%→62%; 13.1-28 min A 38%→100% B 62%→0; 28-28.1 min A 100%→10% B 0→90%; 28.1-32 min A 10%→14% B 90%→86%.

[0067] Example 2: Overexpression of endogenous panD in strain E0 on pTrc99a plasmid ECN Gene (nucleotide sequence as shown in SEQ ID No. 1), construct strain E2 (Trc-panD / pTrc99a-panD ECN ).

[0068] (1) Construct pTrc99a-line plasmid: use plasmid pTrc99a as template, use pTrc99a-line-F / pTrc99a-line-R as primers to amplify plasmid linearization fragment, add DpnI to the PCR product after digestion of the methylated vector at 37°C, purify and recover the purified product by purification kit to obtain linearized plasmid pTrc99a-line.

[0069] (2) Construct pTrc99a-panD ECN plasmid containing Donor: use wild type strain E0 as template, use panD ECN -F / panD ECN -R as primers to amplify panD ECN fragment, purify and then clone and ligate to the linearized vector pTrc99a-line in step (1), transform into E. coli DH5α competent cells, plate to LB plate containing 50 mg / L kanamycin, 37°C culture for 12 h, use pTrc99a-YZ-F / pTrc99a-YZ-R as primers for colony PCR verification, and sequencing verification of strain construction correctness, successfully construct pTrc99a-panD ECN plasmid.

[0070] (3) Transformation plasmid: Extract the constructed pTrc99a-panD ECN The plasmid was transformed into competent cells of the E1 plasmid-free strain, plated onto LB agar plates containing 50 mg / L kanamycin, and incubated at 37°C for 12 h to construct strain E2 (Trc-panD / pTrc99a-panD). ECN ).

[0071] (4) Using strain E1 as a control, strain E1 was subjected to shake-flask fermentation test according to the method in Example 1. A single colony of strain E2 was picked and cultured overnight at 37°C and 180 rpm in LB broth, and the resulting seed culture was obtained. 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium and 50 mg / L kanamycin, and the cells were cultured at 30°C and 180 rpm until the OD value was reached. 600 =0.5, add 0.25mM IPTG to a final concentration, and incubate at 30℃ and 180rpm for 48h with shaking. The β-alanine content was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 1 As shown, the biomass OD of control strain E1 600 The OD value of strain E2 was 11.95, and the β-alanine content was 0.01 g / L; 600 The concentration was 11.84, and the β-alanine content was 0.09 g / L, showing a slight increase.

[0072] Example 3: The panD gene (nucleotide sequence shown in SEQ ID No. 2) from Bacillus tequilensis was overexpressed on the pTrc99a plasmid to construct strain E3 (Trc-panD / pTrc99a-panD). BT ).

[0073] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method in step (1) of Example 2.

[0074] (2) Construct pTrc99a-panD containing Donor BT Plasmid: heterologous panD with codon optimization BT Using pTrc99a plasmid as a template, panD BT -F / panD BT -R indicates primers that amplify panD BT The fragment, using the method of step (2) of Example 2, is made by panD BTFragment and Step (1) Linearization of plasmid pTrc99a-line was performed for one-step cloning and verification, and pTrc99a-panD was constructed. BT Plasmid.

[0075] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD BT Transformed into competent cells of E1 plasmid-free strain, strain E3 (Trc-panD / pTrc99a-panD) was constructed. BT ).

[0076] (4) Using strain E2 as a control, strains E2 and E3 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 1 As shown, the biomass OD of control strain E2 600 The OD value of strain E3 was 11.84, and the β-alanine content was 0.09 g / L; the biomass OD of strain E3 was... 600 The value was 11.37, and the β-alanine content was 0.23 g / L.

[0077] Example 4: Overexpression of the panD gene (nucleotide sequence shown in SEQ ID No. 3) from Bacillus subtilis on the pTrc99a plasmid to construct strain E4 (Trc-panD / pTrc99a-panD) BS ).

[0078] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method in step (1) of Example 2.

[0079] (2) Construct pTrc99a-panD containing Donor BS Plasmid: heterologous panD with codon optimization BS Using pTrc99a plasmid as a template, panD BS -F / panD BS -R indicates primers that amplify panD BS The fragment, using the method of step (2) of Example 2, is made by panD BS Fragment and Step (1) Linearization of plasmid pTrc99a-line was performed for one-step cloning and verification, and pTrc99a-panD was constructed. BS Plasmid.

[0080] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD BSTransformed into competent cells of E1 plasmid-free strain, strain E4 (Trc-panD / pTrc99a-panD) was constructed. BS ).

[0081] (4) Using strain E2 as a control, strains E2 and E4 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 1 As shown, the biomass OD of control strain E2 600 The OD value of strain E4 was 11.84, and the β-alanine content was 0.09 g / L; the biomass OD of strain E4 was... 600 The value was 11.63, and the content of β-alanine was 0.4 g / L.

[0082] Example 5: Overexpression of the panD gene (nucleotide sequence shown in SEQ ID No. 4) from Caulobacter crescentus on the pTrc99a plasmid to construct strain E5 (Trc-panD / pTrc99a-panD) CC ).

[0083] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method in step (1) of Example 2.

[0084] (2) Construct pTrc99a-panD containing Donor CC Plasmid: heterologous panD with codon optimization CC Using pTrc99a plasmid as a template, panD CC -F / panD CC -R is the primer that amplifies panD CC The fragment, using the method of step (2) of Example 2, is made by panD CC Fragment and Step (1) Linearization of plasmid pTrc99a-line for one-step cloning and verification, constructing pTrc99a-panD CC Plasmid.

[0085] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD CC Transformed into competent cells of E1 plasmid-free strain, strain E5(Trc-panD / pTrc99a-panD) was constructed. CC ).

[0086] (4) Using strain E2 as a control, strains E2 and E5 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD600 and the content of beta-alanine is as shown in Table 1. Figure 1 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L. 600 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L. 600 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L.

[0087] Example 6: Overexpression of the panD gene (nucleotide sequence as shown in SEQ ID No. 5) from Mycobacterium tuberculosis H37RV on the pTrc99a plasmid to construct strain E6 (Trc-panD / pTrc99a-panD MB ).

[0088] (1) Construction of the pTrc99a-line plasmid: the linearized plasmid pTrc99a-line was obtained by the method of step (1) in Example 2.

[0089] (2) Construction of the pTrc99a-panD MB plasmid containing the Donor: the panD MB fragment was amplified by using the pTrc99a plasmid containing the heterologous codon-optimized panD MB as a template and panD MB -F / panD MB -R as primers, and the panD MB fragment was cloned into the linearized plasmid pTrc99a-line of step (1) and verified to construct the pTrc99a-panD MB plasmid.

[0090] (3) The pTrc99a-panD MB plasmid was transformed into the E1 plasmid-free strain competent cells by the method of step (3) in Example 2 to construct strain E6 (Trc-panD / pTrc99a-panD MB ).

[0091] (4) The strain E2 was used as a control strain, and the strains E2 and E6 were subjected to shake flask fermentation test by the method of Example 2. The content of beta-alanine was detected according to the method of Example 1. The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E6 was 11.73, and the content of beta-alanine was 0.24 g / L. 600 and the content of beta-alanine is as shown in Table 1. Figure 1 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L. 600 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L. 600 The OD of the biomass of the control strain E2 was 11.84, and the content of beta-alanine was 0.09 g / L; the OD of the biomass of strain E5 was 10.33, and the content of beta-alanine was 0.19 g / L.

[0092] Example 7: Overexpression of the panD gene (nucleotide sequence shown in SEQ ID No. 6) from Helicobacter pylori Puno135 on the pTrc99a plasmid to construct strain E7 (Trc-panD / pTrc99a-panD) HP ).

[0093] (1) Construction of pTrc99a-line plasmid: The linearized plasmid pTrc99a-line was obtained by using the method in step (1) of Example 2.

[0094] (2) Construct pTrc99a-panD containing Donor HP Plasmid: heterologous panD with codon optimization HP Using pTrc99a plasmid as a template, panD HP -F / panD HP -R is the primer that amplifies panD HP The fragment, using the method of step (2) of Example 2, is made by panD HP Fragment and Step (1) Linearization of plasmid pTrc99a-line for one-step cloning and verification, constructing pTrc99a-panD HP Plasmid.

[0095] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD HP Transformed into competent cells of E1 plasmid-free strain, strain E7 (Trc-panD / pTrc99a-panD) was constructed. HP ).

[0096] (4) Using strain E2 as a control, strains E2 and E7 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 1 As shown, the biomass OD of control strain E2 600 The OD value of strain E7 was 11.84, and the β-alanine content was 0.09 g / L; the biomass OD of strain E7 was... 600 The value was 10.92, and the content of β-alanine was 0.17 g / L.

[0097] Example 8: The aspartate transaminase aspC gene (nucleotide sequence shown in SEQ ID No. 7) in strain E4, reinforced with the Trc promoter, was used to construct strain E8 (Trc-panDTrc-aspC / pTrc99a-panD). BS ).

[0098] (1) Construction of pTarget-aspC-line plasmid: The N20 mutant product was amplified using plasmid pTarget as the template and pTarget-aspC-F / pTarget-aspC-R as the primers, purified and transformed using the method of step (1) of Example 1, and then the correctness of the pTarget-aspC-PAM plasmid was verified by sequencing to obtain the plasmid pTarget-aspC-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain the linearized plasmid pTarget-aspC-line.

[0099] (2) Construction of pTarget-aspC plasmid containing Donor: The upstream homologous arm F1 (F1 primer containing Trc promoter sequence) was amplified using the genome of E. coli Nissle 1917 as the template and aspC-FF / aspC-FR as the primers; the downstream homologous arm R1 (R1 primer containing Trc promoter sequence) was amplified using the genome of E. coli Nissle 1917 as the template and aspC-RF / aspC-RR as the primers. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-aspC-line of step (1) using the method of step (1) of Example 1, and then transformed and verified to construct the pTarget-aspC plasmid.

[0100] (3) The plasmid pCas9 was transformed into the pTrc99a-panD BS competent cells of step (2) using the method of step (3) of Example 1, and then the competent cells were prepared.

[0101] (4) The plasmid pTarget-aspC was transformed into the competent cells of step (3) using the method of step (4) of Example 1, and then verified to successfully construct the Trc-aspC strain.

[0102] (5) Elimination of pTarget and pCas9 plasmids: The plasmid-free strain was obtained using the method of step (5) of Example 1.

[0103] (6) Transformation of pTrc99a-panD BS plasmid: The plasmid pTrc99a-panD BS was extracted from the strain E4, and then transformed into the competent cells of the plasmid-free strain to construct the strain E8 (Trc-panD BS Trc-aspC / pTrc99a-panD 600 ).

[0104] (7) The strain E4 was used as the control strain, and the strains E4 and E8 were subjected to shake flask fermentation test using the method of Example 2. The content of β-alanine was detected according to the method of Example 1. The biomass OD 600 and the content of β-alanine were as follows: Figure 2The biomass OD of the control strain E4 was 11.63, and the content of β-alanine was 0.4 g / L. 600 The biomass OD of the strain E8 was 11.1, and the content of β-alanine was 0.4 g / L, and the content of β-alanine was not improved. 600 The biomass OD of the strain E8 was 11.1, and the content of β-alanine was 0.4 g / L, and the content of β-alanine was not improved.

[0105] Example 9: pTrc99a-panD BS overexpressing the endogenous aspC gene on the plasmid in the strain E0 ECN The nucleotide sequence is shown as SEQ ID No. 7), and the strain E9 (Trc-panD Trc-aspC / pTrc99a-panD BS -aspC ECN ) was constructed.

[0106] (1) Construction of pTrc99a-D BS -line plasmid: The plasmid pTrc99a-panD BS -line was used as the template, and pTrc99a-D BS -line-F / pTrc99a-D BS -line-R were used as primers to amplify the plasmid linearization fragment, and the linearized plasmid pTrc99a-panD BS -line was obtained by using the method of step (1) in Example 2.

[0107] (2) Construction of pTrc99a-panD BS -aspC ECN plasmid: The wild-type strain E0 was used as the template, and aspC ECN -F / aspC ECN -R were used as primers to amplify the aspC ECN fragment, and the aspC ECN fragment was cloned into the linearized plasmid pTrc99a-panD BS -line of step (1) by using the method of step (2) in Example 2, and the pTrc99a-panD BS -aspC ECN plasmid was constructed and verified.

[0108] (3) The plasmid pTrc99a-panD BS -aspC ECN was transformed into the E8 plasmid-free strain competent cells by using the method of step (3) in Example 2, and the strain E9 (Trc-panD Trc-aspC / pTrc99a-panD BS -aspC ECN ) was constructed.

[0109] (4) Using strain E8 as a control, strains E8 and E9 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 2 As shown, the biomass OD of control strain E8 600 The OD value of strain E9 was 11.1, with a β-alanine content of 0.4 g / L; the biomass OD of strain E9 was... 600 The concentration was 10.6, and the β-alanine content was 0.35 g / L, showing a slight decrease.

[0110] Example 10: pTrc99a-panD BS The aspB gene (nucleotide sequence shown in SEQ ID No. 8) from Corynebacterium glutamicum was overexpressed on the plasmid, and strain E10 (Trc-panDTrc-aspC / pTrc99a-panD) was constructed. BS -aspB CG ).

[0111] (1) Constructing pTrc99a-D BS -line plasmid: The linearized plasmid pTrc99a-D was obtained by using the method in step (1) of Example 9. BS -line.

[0112] (2) Construct pTrc99a-panD containing Donor BS -aspB CG Plasmid: heterogeneous aspB with codon optimization CG Using pTrc99a plasmid as a template, aspB CG -F / aspB CG -R indicates primers that amplify aspB CG The fragment, using the method of step (2) of Example 2, is aspB CG Fragments and steps (1) Linearization of plasmid pTrc99a-panD BS -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG Plasmid.

[0113] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD BS -aspB CG Transformed into competent cells of E9 plasmid-free strain, strain E10 (Trc-panDTrc-aspC / pTrc99a-panD) was constructed. BS -aspBCG ).

[0114] (4) Using strain E9 as a control, strains E9 and E10 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 2 As shown, the biomass OD of control strain E9 600 The OD value of strain E10 was 10.6, and the β-alanine content was 0.35 g / L; the biomass OD of strain E10 was... 600 The value was 10.3, and the content of β-alanine was 0.7 g / L.

[0115] Example 11: pTrc99a-panD BS -aspB CG The endogenous aspA gene (nucleotide sequence shown in SEQ ID No. 9) from strain E0 was overexpressed on the plasmid, and strain E11 (Trc-panDTrc-aspC / pTrc99a-panD) was constructed. BS -aspB CG -aspA).

[0116] (1) Construct pTrc99a-panD BS -aspB CG -line plasmid: using plasmid pTrc99a-panD BS -aspB CG Using pTrc99a-D as a template BS B CG -line-F / pTrc99a-D BS B CG -line-R is the primer amplification of the linearized plasmid fragment. The linearized plasmid pTrc99a-D was obtained by using the method in step (1) of Example 2. BS B CG -line.

[0117] (2) Construct pTrc99a-panD containing Donor BS -aspB CG -aspA plasmid: Using wild-type strain E0 as a template, the aspA fragment was amplified using aspA-F / aspA-R primers. The aspA fragment was then amplified using the method in step (2) of Example 2, and the aspA fragment was combined with the linearized plasmid pTrc99a-D from step (1). BS B CG -line performs a one-step cloning and verification, constructing pTrc99a-panD BS -aspB CG -aspA plasmid.

[0118] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD BS -aspB CG -aspA was transformed into competent cells of E10 plasmid-free strain to construct strain E11(Trc-panDTrc-aspC / pTrc99a-panD) BS -aspB CG -aspA).

[0119] (4) Using strain E10 as a control, strains E10 and E11 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 2 As shown, the biomass OD of control strain E10 600 The OD value of strain E11 was 10.3, and the β-alanine content was 0.7 g / L; the biomass OD of strain E11 was... 600 The value was 11.62, and the content of β-alanine was 1.04 g / L.

[0120] Example 12: The succinate dehydrogenase sdhC gene (nucleotide sequence shown in SEQ ID No. 10) in strain E11, reinforced with the Trc promoter, was used to construct strain E12 (Trc-panDTrc-aspCTrc-sdhC / pTrc99a-panD...). BS -aspB CG -aspA).

[0121] (1) Construction of pTarget-sdhC-line plasmid: Using plasmid pTarget as a template, the N20 mutant product was amplified using pTarget-sdhC-F / pTarget-sdhC-R primers. The product was purified and transformed using the method in step (1) of Example 1, and then the correctness of pTarget-sdhC-PAM plasmid was verified by sequencing to obtain plasmid pTarget-sdhC-PAM. Linearization was performed using the method in step (1) of Example 1 to obtain linearized plasmid pTarget-sdhC-line.

[0122] (2) Constructing plasmid pTarget-sdhC containing Donor: using the genome of E. coli Nissle 1917 as a template, the upstream homology arm F1 (F1 primer contains Trc promoter sequence) is amplified with primers sdhC-FF / sdhC-FR; the downstream homology arm R1 (R1 primer contains Trc promoter sequence) is amplified with primers sdhC-RF / sdhC-RR. The upstream and downstream homology arms are cloned into the linearized plasmid pTarget-sdhC-line of step (1) by the method of step (1) of Example 1, and transformation and verification are performed to construct the pTarget-sdhC plasmid.

[0123] (3) The plasmid pCas9 is transformed into the pTrc99a-panD BS -aspB CG -aspA competent cells E11 by the method of step (3) of Example 1.

[0124] (4) The plasmid pTarget-sdhC is transformed into the competent cells of step (3) by the method of step (4) of Example 1, and verification is performed, successfully constructing the Trc-sdhC strain.

[0125] (5) Elimination of pTarget and pCas9 plasmids: the plasmid-free strain is obtained by the method of step (5) of Example 1.

[0126] (6) Transforming the pTrc99a-panD BS -aspB CG -aspA plasmid: the pTrc99a-panD BS -aspB CG -aspA plasmid is extracted from the strain E11, and the plasmid is transformed into the competent cells of the plasmid-free strain to construct the strain E12 (Trc-panD Trc-aspC Trc-sdhC / pTrc99a-panD BS -aspB CG -aspA).

[0127] (7) Using the strain E11 as a control strain, the strains E11 and E12 are subjected to shake flask fermentation test by the method of Example 2. The content of β-alanine is detected according to the method of Example 1. The biomass OD 600 and the content of β-alanine are shown in Table 1. Figure 3 The biomass OD 600 of the control strain E11 is 11.62, and the content of β-alanine is 1.04 g / L; the biomass OD 600 of the strain E12 is 12.51, and the content of β-alanine is 1.36 g / L.

[0128] Example 13: Trc promoter strengthens the phosphoenolpyruvate carboxylase ppc gene (nucleotide sequence as shown in SEQ ID No. 11) in strain E12, and constructs strain E13 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc / pTrc99a-panD BS -aspB CG -aspA).

[0129] (1) Construction of pTarget-ppc-line plasmid: N20 mutant product was amplified with plasmid pTarget as template and pTarget-ppc-F / pTarget-ppc-R as primers, purified and transformed using the method of step (1) of Example 1, and then the correctness of pTarget-ppc-PAM plasmid was verified by sequencing to obtain plasmid pTarget-ppc-PAM. Linearization was performed using the method of step (1) of Example 1 to obtain linearized plasmid pTarget-ppc-line.

[0130] (2) Construction of pTarget-ppc plasmid containing Donor: the upstream homologous arm F1 (F1 primer contains Trc promoter sequence) was amplified with the genome of E. coli Nissle 1917 as template and ppc-FF / ppc-FR as primers; the downstream homologous arm R1 (R1 primer contains Trc promoter sequence) was amplified with ppc-RF / ppc-RR as primers. The upstream and downstream homologous arms were cloned into the linearized plasmid pTarget-ppc-line of step (1) using the method of step (1) of Example 1, and transformed and verified to construct pTarget-ppc plasmid.

[0131] (3) The plasmid pCas9 was transformed into the pTrc99a-panD BS -aspB CG -aspA competent cells E12 using the method of step (3) of Example 1, and competent cells were prepared.

[0132] (4) The plasmid pTarget-ppc was transformed into the competent cells of step (3) using the method of step (4) of Example 1, and verified, and the Trc-ppc strain was successfully constructed.

[0133] (5) Elimination of pTarget and pCas9 plasmids: the plasmid-free strain was obtained using the method of step (5) of Example 1.

[0134] (6) Transformation of pTrc99a-panD BS -aspB CG -aspA plasmid: the plasmid pTrc99a-panDBS -aspB CG -aspA, the plasmid was transformed into competent cells of a plasmid-free strain to construct strain E13(Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA).

[0135] (7) Using strain E12 as a control, strains E12 and E13 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 4 As shown, the biomass OD of control strain E12 600 The OD value of strain E13 was 12.51, and the β-alanine content was 1.36 g / L; the biomass OD of strain E13 was... 600 The value was 11.66, and the content of β-alanine was 1.57 g / L.

[0136] Example 14: pTrc99a-panD BS -aspB CG Overexpression of the pyc gene derived from Corynebacterium glutamicum (nucleotide sequence shown in SEQ ID No. 12) on the -aspA plasmid was performed on strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0137] (1) Construct pTrc99a-panD BS -aspB CG -aspA-line plasmid: pTrc99a-panD BS -aspB CG -aspA is the template, and pTrc99a-D is used. BS B CG A-line-F / pTrc99a-D BS B CG A-line-R is the primer that amplifies the linearized fragment of the plasmid. The linearized plasmid pTrc99a-panD was obtained by using the method in step (1) of Example 2. BS -aspB CG -aspA-line.

[0138] (2) Construct pTrc99a-panD containing DonorBS -aspB CG -aspA-pyc CG Plasmid: heterogeneous pyc containing codons optimized CG Using the pTrc99a plasmid as a template, and pyc CG -F / pyc CG -R indicates primers that amplify pyc CG The fragment, using the method in step (2) of Example 2, is made into pyc CG Fragments and steps (1) Linearization of plasmid pTrc99a-panD BS -aspB CG -aspA-line performs a one-step cloning and verification, and builds pTrc99a-panD. BS -aspB CG -aspA-pyc CG Plasmid.

[0139] (3) Using the method in step (3) of Example 2, the plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG Transformed into competent cells of plasmid-free strain E13, strain E14 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc / pTrc99a-panD) was constructed. BS -aspB CG -aspA-pyc CG ).

[0140] (4) Using strain E13 as a control, strains E13 and E14 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 5 As shown, the biomass OD of control strain E13 600 The OD value of strain E14 was 11.66, and the β-alanine content was 1.57 g / L; the biomass OD of strain E14 was... 600 The value was 10.67, and the content of β-alanine was 1.87 g / L.

[0141] Example 15: Knock out the cycA transporter gene (nucleotide sequence shown in SEQ ID No. 13) in strain E14 to construct strain E15 (Trc-panDTrc-aspCTrc-sdhCTrc-ppc△cycA / pTrc99a-panD BS -aspB CG -aspA-pyc CG ).

[0142] (1) Construct pTarget-cycA-line plasmid: Amplify N20 mutant product with plasmid pTarget as template and pTarget-cycA-F / pTarget-cycA-R as primers, purify and transform by the method of step (1) of Example 1, and then verify the correctness of pTarget-cycA-PAM plasmid by sequencing to obtain plasmid pTarget-cycA-PAM. Linearize by the method of step (1) of Example 1 to obtain linearized plasmid pTarget-cycA-line.

[0143] (2) Construct pTarget-cycA plasmid containing Donor: Amplify upstream homologous arm with the genome of E. coli Nissle 1917 as template and cycA-FF / cycA-FR as primers; amplify downstream homologous arm with cycA-RF / cycA-RR as primers. Clone the upstream and downstream homologous arms into linearized plasmid pTarget-cycA-line of step (1) by the method of step (1) of Example 1, and then transform and verify to construct pTarget-cycA plasmid.

[0144] (3) Transform plasmid pCas9 into pTrc99a-panD BS -aspB CG -aspA-pyc CG in competent cells E14 and prepare competent cells.

[0145] (4) Transform plasmid pTarget-cycA into the competent cells of step (3) by the method of step (4) of Example 1 and verify to successfully construct cycA knockout strain.

[0146] (5) Eliminate pTarget and pCas9 plasmids: Obtain plasmid-free strain by the method of step (5) of Example 1.

[0147] (6) Transform pTrc99a-panD BS -aspB CG -aspA-pyc CG plasmid: Extract plasmid pTrc99a-panD BS -aspB CG -aspA-pyc CG from strain E14, transform the plasmid into the competent cells of plasmid-free strain, and construct strain E15 (Trc-panD Trc-aspC Trc-sdhC Trc-ppc ΔcycA / pTrc99a-panD BS -aspBCG -aspA-pyc CG ).

[0148] (7) Using strain E14 as a control, strains E14 and E15 were subjected to shake-flask fermentation tests according to the method in Example 2. The content of β-alanine was determined according to the method in Example 1. Biomass OD 600 and the content of β-alanine, such as Figure 6 As shown, the biomass OD of control strain E14 600 The OD value of strain E15 was 10.67, and the β-alanine content was 1.87 g / L; the biomass OD of strain E15 was... 600 The value was 11.1, and the content of β-alanine was 2.17 g / L.

[0149] Example 16: Fed-feed fermentation in a 5-L fermenter

[0150] Strawberry strain E15 from Example 15 was streaked onto an LB agar plate containing 50 mg / L kanamycin resistance and incubated overnight at 37°C. Single colonies were picked and transferred to LB tubes containing 50 mg / L kanamycin resistance, and incubated overnight at 37°C and 150 rpm to prepare the primary seed culture. The primary seed culture was inoculated at a volume concentration of 5% into 100 mL of LB medium containing 50 mg / L kanamycin resistance and incubated overnight at 37°C and 150 rpm to obtain the secondary seed culture. The secondary seed culture was inoculated at a volume concentration of 15% into a 5-L fermenter containing 2 L of fermentation medium, and IPTG was added to a final concentration of 0.2 mM. Fermentation was carried out at 30℃, 300 rpm, and an aeration rate of 4 V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), automatic feeding was activated, and feed medium was added at a rate of 25 mL / h until the pH value was lower than 6.80. Feeding was then stopped, and the residual sugar in the fermenter was maintained at a low level, with a sugar concentration below 1 g / L. The total amount of feed medium added to strain E15 was 1700 mL, and the culture was carried out for 81 h. The yield of β-alanine in the fermentation broth was detected using the method in Example 1, and the biomass OD was detected by spectrophotometry. 600 Sugar concentration was detected using the DNS method; Figure 7 As shown, β-alanine production reached 70.4 g / L at 81 h, and the final biomass OD 600 The concentration was maintained at 74.3. Fermentation results indicate that the metabolically engineered Escherichia coli probiotics exhibit excellent performance in producing β-alanine, laying the foundation for the industrial production of β-alanine.

[0151] Fermentation medium formulation for a 5-L fermenter: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB 120.2 mg / L, 1 mL / L trace element solution, solvent is deionized water; the composition of the trace element solution: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water.

[0152] Feed medium: glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 , solvent is deionized water, and the pH is adjusted to 6.8 by 14% ammonia water.

Claims

1. A high-yield β-alanine-producing Escherichia coli probiotic bacterium, characterized by, The E. coli probiotic is a wild-type E. coli probiotic E. coli Nissle 1917 as a starting strain, successively carrying out the following gene editing: overexpression of the endogenous panD gene on plasmid pTrc99a, overexpression of the gene from Bacillus subtilis panD gene, Trc promoter enhancement aspC gene on plasmid pTrc99a, overexpression of the gene from Corynebacterium glutamicum aspB gene on plasmid pTrc99a, overexpression of the gene from Bacillus subtilis aspA gene, Trc promoter enhancement sdhC gene, Trc promoter enhancement ppc gene on plasmid pTrc99a, overexpression of the gene from Corynebacterium glutamicum pyc gene, knockout cycA gene; the E. coli probiotic contains the plasmid pTrc99a; The substance derived from Bacillus subtilis panD The nucleotide sequence of the gene is shown in SEQ ID NO.3; aspC The nucleotide sequence of the gene is shown in SEQ ID NO.7; aspB The nucleotide sequence of the gene is shown in SEQ ID NO. 8; aspA The nucleotide sequence of the gene is shown in SEQ ID NO. 9; sdhC The nucleotide sequence of the gene is shown in SEQ ID NO. 10; ppc The nucleotide sequence of the gene is shown in SEQ ID NO.11; pyc The nucleotide sequence of the gene is shown in SEQ ID NO.12; cycA The nucleotide sequence of the gene is shown in SEQ ID NO.13; the nucleotide sequence of the Trc promoter is shown in SEQ ID NO.

14.

2. The E. coli probiotic bacteria of claim 1 for use in the microbial fermentation process for preparing beta-alanine.

3. Use according to claim 2, wherein the compound is ###0002### The application is: inoculating the E. coli probiotic bacteria into a fermentation medium containing 50 mg / L kanamycin, culturing the bacteria at 30 DEG C and 180 rpm to OD 600 =0.5, adding IPTG to a final concentration of 0.25 mM to induce gene expression, continuing to culture for 48 h to the end of fermentation, obtaining a fermentation liquor containing beta-alanine, and separating and purifying the fermentation liquor to obtain beta-alanine. The fermentation medium formula is as follows: glucose 18~22 g / L, (NH4)2SO4 14~18 g / L, yeast extract 2~3 g / L, KH2PO4 1~2 g / L, MgSO4 0.2~1 g / L, CaCO3 13~17 g / L, VB10.3~0.5 mg / L, VB 12 0.3~0.5 mg / L, trace element solution 0.5~1.5 mL / L, solvent is water, and pH value is not required to be adjusted; components of the trace element solution are as follows: 8~12 g / L CaCl2, 8~12 g / L FeSO4·7H2O, 0.8~1.2 g / L ZnSO4·7H2O, 0.1~0.3 g / L CuSO4, 0.01~0.04 g / L NiCl2·7H2O, and solvent is deionized water.

4. Use according to claim 3, wherein the compound is ###0002### Before fermentation, the E. coli probiotic bacteria is inoculated into LB medium containing 50 mg / L kanamycin, 37℃, 180 rpm overnight to prepare seed liquid, and the seed liquid is inoculated into fermentation medium containing 50 mg / L kanamycin at a volume concentration of 2%.

5. The use according to claim 3, wherein the compound is ###0002### The fermentation is carried out in a fermentation tank: the E. coli probiotic bacteria is inoculated into LB plate containing 50 mg / L kanamycin resistance, 37℃, overnight culture, single colony is picked to LB test tube containing 50 mg / L kanamycin resistance, 37℃, 150-180 rpm overnight culture to prepare primary seed liquid; the primary seed liquid is inoculated into LB medium containing 50 mg / L kanamycin at a volume concentration of 5%, 37℃, 150-180 rpm overnight culture as secondary seed liquid; The secondary seed liquid is inoculated into the fermentation tank containing fermentation medium containing 50 mg / L kanamycin at a volume concentration of 15%, and 0.2 mM IPTG is added, and the fermentation culture is carried out at 30℃, 300-500 rpm, and aeration amount of 4 V / V·min, when the pH value is higher than 6.80, the automatic feeding is started, and the feeding medium is added until the pH value is lower than 6.80, and the culture is stopped, and the culture is carried out for 70-81h to obtain fermentation liquid containing beta-alanine; The fermentation medium formula in the fermenter: glucose 20 g / L, (NH4)2SO4 16 g / L, yeast extract 2 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, VB1 0.5 mg / L, VB6 0.2 mg / L, 1 mL / L trace element solution, solvent is deionized water; the composition of the trace element solution: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water; 12 0.2 mg / L, 1 mL / L trace element solution, solvent is deionized water; the composition of the trace element solution: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water; The feed medium consisted of glucose 500 g / L, (NH4)2SO4 10 g / L, yeast extract 2 g / L, KH2PO4 14 g / L, MgSO4 8 g / L, 0.5 mg / L VB1, 0.2 mg / L VB 12 , with water as solvent and pH adjusted to 6.8 with 14% ammonia.

6. The use according to claim 5, wherein the compound is ###0002### The feeding speed is 25 mL / h, and the total amount of feeding medium added is 1700 mL / 2L.

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