L-tryptophan genetically engineered bacteria under the influence of regulatory factors, construction method and application thereof

Abstract

The present application relates to the field of genetic engineering, in particular to L-tryptophan genetically engineered bacteria under the influence of regulatory factors and a construction method and application thereof, the Escherichia coli is reformed by using metabolic engineering means to improve the yield of L-tryptophan, mainly by inactivating the regulatory factor RpoS in the Escherichia coli, and simultaneously carrying out site-directed mutagenesis on FlhD and Crp, optimizing the metabolic flow of the Escherichia coli, and improving the activity of the regulatory factor capable of promoting L-tryptophan, further improving the ability of the Escherichia coli to produce L-tryptophan and the efficiency of the whole metabolic pathway, effectively improving the production of L-tryptophan, so that the yield of L-tryptophan is doubled compared with the original strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to L-tryptophan-engineered bacteria under the influence of regulatory factors, their construction methods, and applications. Background Technology

[0002] L-Tryptophan is widely used in the pharmaceutical, food, and feed industries and is an essential amino acid for the human body. In the medical field, L-Tryptophan can be used in amino acid injections, essential amino acid drugs, and additives for hydrolyzed proteins. L-Tryptophan can be used as a food additive to enhance the body's utilization efficiency of plant protein. Adding L-Tryptophan to feed can regulate the balance of amino acids in the feed and promote the growth of poultry and livestock.

[0003] L-tryptophan production methods include chemical synthesis, conversion, and microbial fermentation. Microbial fermentation was the earliest developed method for L-tryptophan production, using inexpensive raw materials such as glucose. Due to its advantages such as low-cost raw materials, high product purity, and easy extraction, microbial fermentation has received increasing attention. However, since the 1990s, with the rapid development of DNA recombination technology, especially the rise of metabolic engineering breeding, researchers have gradually bred a number of high-yield L-tryptophan-producing strains, significantly improving the efficiency of microbial fermentation for L-tryptophan production and making it the main industrial method for L-tryptophan production.

[0004] In early studies, traditional chemical or physical methods were typically used to select L-tryptophan-producing strains through single mutagenesis. However, after multiple rounds of chemical or physical mutagenesis, it became difficult to confirm the effects of multiple mutations on the strain during the mutagenesis process, and the unclear strain background hindered further improvement.

[0005] With the development of gene recombination technology, the use of genetic engineering to modify the L-tryptophan synthesis pathway has gradually developed. Without involving chemical mutagenesis, Zhao Zhijun et al. used the research strategy of metabolic engineering to increase the L-tryptophan production to 17.7 g / L based on E. coli W3110, which does not produce L-tryptophan, through a series of gene operations (Zhao Zhijun. Construction and metabolic regulation of L-tryptophan producing strains [D]. Wuxi: Jiangnan University, 2011.). In 2017, Chen et al. obtained a high-yield L-tryptophan strain S028 through the pure rational modification method of genetic engineering. After fermentation for 61 h, the L-tryptophan production reached 40.3 g / L (Chen L et al., Applied microbiology and biotechnology, 2017, 101(2): 559-568.).

[0006] In-depth research into genetic engineering technology has revealed that regulatory factors can directly increase L-tryptophan production by regulating metabolic pathways, activating or inhibiting the activity of key enzymes, relieving the repression of transcription factors, optimizing the tryptophan transport system, and precisely controlling fermentation conditions. Furthermore, regulatory factors can also influence L-tryptophan synthesis efficiency by regulating the supply of precursors and cofactors, altering the distribution of metabolic flux, and precisely controlling the expression of key genes through genetic engineering. The combined effect of these regulatory mechanisms allows for the optimization of regulatory factor application and the enhancement of target synthetic pathways, thereby significantly improving L-tryptophan production efficiency and yield.

[0007] In summary, given the current state of research, there is still a lack of a more efficient method to utilize regulatory factors to construct L-tryptophan-engineered bacteria, thereby further increasing L-tryptophan production. Summary of the Invention

[0008] The purpose of this invention is to provide an L-tryptophan-engineered bacterium under the influence of regulatory factors;

[0009] Another objective of this invention is to provide a method for constructing L-tryptophan-engineered bacteria under the influence of regulatory factors and its application, thereby increasing the yield of L-tryptophan.

[0010] The technical solution adopted to achieve the purpose of this invention is:

[0011] This application uses genetic engineering methods to modify the relevant regulatory factors in the L-tryptophan metabolic pathway of the starting strain, thereby obtaining a strain that expands L-tryptophan production.

[0012] Furthermore, the originating bacterium is Escherichia coli strain KW;

[0013] The regulatory factors include RpoS, FlhD, and Crp.

[0014] Specifically, the plasmid PH5a-aroG fbr -trpE fbr DCBA was transformed into the starting strain and overexpressed on the genome of the starting strain using genetic engineering technology. ppsA Genes, and knock them out trpR Genes and knockout genes tnaA Strain 1 was constructed, and the relevant regulatory factors in the L-tryptophan metabolic pathway of strain 1 were modified: the regulatory factor RpoS was inactivated, and / or FlhD and / or Crp were subjected to site-directed mutagenesis, thereby obtaining an L-tryptophan engineered bacterium under the influence of the regulatory factors, which is referred to as the L-tryptophan engineered bacterium in this application; thereby obtaining a strain for expanding L-tryptophan production.

[0015] Furthermore, the regulatory factor RpoS is inactivated by introducing a mutation Q33 into the regulatory factor RpoS. Inactivate the protein;

[0016] Further, the site-directed mutagenesis of the regulatory factor FlhD is performed by introducing a mutation site V84F into the regulatory factor FlhD to obtain a FlhD protein mutant, the amino acid sequence of which is shown in SEQ ID NO: 1; SEQ ID NO: 1 is:

[0017] MHTSELLKHIYDINLSYLLLAQRLIVQDKASAMFRLGINEEMATTLAALTLPQMVKLAETNQLVCHFRFDSHQTITQLTQDSRFDDLQQIHTGIMLSTRLLNDVNQPEEALRKKRA;

[0018] Furthermore, the site-directed mutagenesis of the regulatory factor Crp is performed by introducing a mutation site T29K into the regulatory factor Crp to obtain a Crp protein mutant, the amino acid sequence of which is shown in SEQ ID NO: 2; SEQ ID NO: 2 is:

[0019] MVLGKPQTDPTLEWFLSHCHIHKYPSKSKLIHQGEKAETLYYIVKGSVAVLIKDEEGKEMILSYLNQGDFIGELGLFEEGQERSAWVRAKTACEVAEISYKKFRQ LIQVNPDILMRLSAQMARRLQVTSEKVGNLAFLDVTGRIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR.

[0020] Furthermore, the regulatory factor RpoS in strain 1 was inactivated, and site-directed mutagenesis was performed on FlhD and / or Crp to obtain L-tryptophan engineered bacteria;

[0021] Furthermore, the regulatory factor RpoS in strain 1 was inactivated, and FlhD and Crp were subjected to site-directed mutagenesis to obtain L-tryptophan engineered bacteria.

[0022] This application also provides a method for constructing L-tryptophan-engineered bacteria under the influence of regulatory factors, comprising the following steps:

[0023] 1) In Escherichia coli strain KW (Chen, Y et al. Journal of Industrial Microbiol & Biotechnology, 2018, 45(5): 357-367.), a gene-containing... aroG fbr and trpE fbr DCBA Recombinant plasmid PH5a-aroG fbr -trpE fbr DCBA, overexpressed on the genome ppsA Gene knockout trpR Genes, gene knockout tnaA Strain 1 was constructed, which is referred to as BDX1 in this application;

[0024] 2) In the BDX1 strain, the Q33 mutant was introduced into the global regulator RpoS. The protein was inactivated, and strain 2 was constructed, referred to as BDX2 in this application;

[0025] 3) In strain BDX1, a mutant FlhD protein was obtained by introducing the mutation site V84F into the flagellar transcription regulator FlhD, and strain 3 was constructed, which is referred to as BDX3 in this application; the amino acid sequence of the FlhD protein mutant is shown in SEQ ID NO: 1;

[0026] 4) In strain BDX1, a mutation site T29K was introduced into the transcriptional regulator Crp to obtain a Crp protein mutant, which was used to construct strain 4, referred to as BDX4 in this application; the amino acid sequence of the Crp protein mutant is shown in SEQ ID NO: 2;

[0027] 5) Based on strain BDX2, the FlhD protein mutant was integrated into the genome to obtain strain 5, which is referred to as BDX5 in this application;

[0028] 6) Based on strain BDX5, the Crp protein mutant was integrated into the genome to obtain strain 6, which is referred to as BDX6 in this application, namely the L-tryptophan genetically engineered strain under the influence of regulatory factors.

[0029] 7) Shake-flask fermentation was performed to verify the L-tryptophan producing strains KW and BDX1-BDX6.

[0030] This application also includes the application of L-tryptophan genetically engineered bacteria in the fermentation production of L-tryptophan.

[0031] This application also includes the application of the method for constructing L-tryptophan genetically engineered bacteria in the fermentation production of L-tryptophan.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] Advanced metabolic engineering techniques were used to precisely and ingeniously modify Escherichia coli, significantly increasing L-tryptophan production. By studying the regulatory factors of E. coli, especially modifying the regulatory factors RpoS, FlhD, and Crp, the potential inhibitory effects of regulatory factors on the L-tryptophan synthesis pathway were weakened or eliminated, while the activity of regulatory factors that can promote L-tryptophan production was enhanced, thereby further improving the ability of E. coli to produce L-tryptophan and the efficiency of the entire metabolic pathway.

[0034] This design enabled the modified genetically engineered strain to increase its yield by 5 times compared to the original starting strain, which not only allowed for the effective accumulation of L-tryptophan but also laid the foundation for the industrial production of L-tryptophan. Attached Figure Description

[0035] Figure 1 PH5a-aroG in this invention fbr -trpE fbr DCBA plasmid map;

[0036] Figure 2 The figure shows the L-tryptophan fermentation yield of the BDX1-BDX6 strains of this invention;

[0037] Figure 3 This is a diagram showing the biomass test of the fermentation broth of the BDX1 and BDX2 strains of this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0040] The following examples are provided to help better understand the present invention, but are not intended to limit the invention.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0042] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0043] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0044] The amino acid sequence of the mutant FlhD (V84F) is shown in SEQ ID NO: 1. SEQ ID NO: 1 is:

[0045] MHTSELLKHIYDINLSYLLLAQRLIVQDKASAMFRLGINEEMATTLAALTLPQMVKLAETNQLVCHFRFDSHQTITQLTQDSRFDDLQQIHTGIMLSTRLLNDVNQPEEALRKKRA.

[0046] The amino acid sequence of the mutant Crp(T29K) is shown in SEQ ID NO: 2. SEQ ID NO: 2 is:

[0047] MVLGKPQTDPTLEWFLSHCHIHKYPSKSKLIHQGEKAETLYYIVKGSVAVLIKDEEGKEMILSYLNQGDFIGELGLFEEGQERSAWVRAKTACEVAEISYKKFRQ LIQVNPDILMRLSAQMARRLQVTSEKVGNLAFLDVTGRIAQTLLNLAKQPDAMTHPDGMQIKITRQEIGQIVGCSRETVGRILKMLEDQNLISAHGKTIVVYGTR.

[0048] The KW strain is described in the following literature: Chen, Y. et al. Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production. Journal of Industrial Microbiol & Biotechnology, 45(5), 357-367 (2018).

[0049] Example 1: Construction of L-tryptophan-producing strain BDX1

[0050] PH5a-aroG fbr -trpE fbr DCBA plasmid construction: Using wild-type Escherichia coli MG1655 as a template, the aroG fragment with adapter was amplified using primers aroG-F and aroG-R.

[0051] Using PH5a plasmid as a template, PH5a-MF and PH5a-MR amplifications were performed on the PH5a-M fragment with adapter.

[0052] Using aroG and PH5a-M fragments as templates, the aroG-M-Gibson fragment for Gibson assembly was amplified using aroG-F and PH5a-MR primers. Using MG1655 as a template, the adapter-containing trpEDCBA-Gibson fragment was amplified using primers trpEDCBA-F and trpEDCBA-R. The adapter-containing plasmid backbone was amplified using PH5a-ver-F and PH5a-ver-R, and then assembled with the aroG-M-Gibson and trpEDCBA-Gibson fragments to obtain the PH5a-aroG-trpEDCBA plasmid. Using the PH5a-aroG-trpEDCBA plasmid as a template, the aroG... fbr -F and trpE fbr -R primers amplify the PH5a-1-Gibson fragment with the adapter, using aroG fbr -R and trpE fbr -F primers amplify the PH5a-2-Gibson fragment with the adapter. The PH5a-1-Gibson fragment and the PH5a-2-Gibson fragment are then assembled with Gibson to obtain PH5a-aroGibson. fbr -trpE fbr DCBA plasmid, plasmid map as shown Figure 1 As shown.

[0053] Construction of the Cas9-PPSA plasmid: Using wild-type Escherichia coli MG1655 as a template, the ppsA-UP fragment with the adapter was amplified using primers ppsA-up-F and ppsA-up-R, and the ppsA-Down fragment with the adapter was amplified using primers ppsA-down-F and ppsA-down-R. The ppsA-UP and ppsA-Down fragments were then assembled into the ppsA-UD fragment.

[0054] Using the Cas9 plasmid as a template, the plasmid backbone ppsA-ver1 fragment was amplified using primers ppsA-N20-F and ppsA-ver-R, and the plasmid backbone tnaA-ver2 fragment was amplified using primers ppsA-ver-F and ppsA-N20-R. The plasmid backbones ppsA-ver1 and ppsA-ver2, along with the above fragment ppsA-UD, were then subjected to Gibson assembly (the Gibson assembly method, invented by Gibson et al., involves the intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the Cas9-ppsA plasmid.

[0055] Construction of the cas9-trpR plasmid: Using wild-type Escherichia coli MG1655 as a template, the trpR-UP fragment with the adapter was amplified using primers trpR-up-F and trpR-up-R, and the trpR-Down fragment with the adapter was amplified using primers trpR-down-F and trpR-down-R. The trpR-UP and trpR-Down fragments were then assembled into the trpR-UD fragment.

[0056] Using the Cas9 plasmid as a template, the plasmid backbone trpR-ver1 fragment was amplified using primers trpR-N20-F and trpR-ver-R, and the plasmid backbone trpR-ver2 fragment was amplified using primers trpR-ver-F and trpR-N20-R. The plasmid backbones trpR-ver1 and trpR-ver2, along with the above fragment trpR-UD, were then subjected to Gibson assembly (the Gibson assembly method, invented by Gibson et al., involves the intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the Cas9-trpR plasmid.

[0057] Construction of the Cas9-TNAA plasmid: Using MG1655 as a template, the tanA-UP fragment with the adapter was amplified using primers tnaA-up-F and tnaA-up-R, and the tnaA-Down fragment with the adapter was amplified using primers tnaA-down-F and tnaA-down-R. The tnaA-UP and tnaA-Down fragments were then assembled into the tnaA-UD fragment.

[0058] Using the Cas9 plasmid as a template, the plasmid backbone tnaA-ver1 fragment was amplified using primers tnaA-N20-F and tnaA-ver-R, and the plasmid backbone tnaA-ver2 fragment was amplified using primers tnaA-ver-F and tnaA-N20-R. The plasmid backbones tnaA-ver1 and tnaA-ver2, along with the above fragment tnaA-UD, were then subjected to Gibson assembly (the Gibson assembly method, invented by Gibson et al., involves the intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the Cas9-tnaA plasmid.

[0059] The primers used in this section are as follows:

[0060] Table 1 Primers used to construct L-tryptophan-producing strain BDX1

[0061] Primer Name Nucleotide Sequence (5'-3') Primer aroG-F (SEQ ID NO: 3) CGCATCCGACAATTAAACCTTACCCGCGACGCGCTTTTA Primer aroG-R (SEQ ID NO: 4) TGGCAACACTGGAACAGACATGAATTATCAGAACGACGA Primer PH5a-M-F (SEQ ID NO: 5) CGTCGTTCTGATAATTCATGTCTGTTCCAGTGTTGCCAT Primer PH5a-M-R (SEQ ID NO: 6) AGCGGCGACGCGCAGTTAATCCCACAGCCGCCAGTTCCG Primer trpEDCBA-F (SEQ ID NO: 7) GGAACTGGCGGCTGTGGGATTAACTGCGCGTCGCCGCTT Primer trpEDCBA-R (SEQ ID NO: 8) ACAAAATTAGAGAATAACAATGCAAACACAAAAACCGAC Primer PH5a-ver-F (SEQ ID NO: 9) TCGGTTTTTGTGTTTGCATTGTTATTCTCTAATTTTGTT Primer PH5a-ver-R (SEQ ID NO: 10) AAAAGCGCGTCGCGGGTAAGGTTTAATTGTCGGATGCGC <![CDATA[Primer aroG fbr -F (SEQ ID NO: 11)]]> CATCAGGGCTTTTTTGTCCGGTCGGCTTCAAAAATG <![CDATA[Primer aroG fbr -R (SEQ ID NO: 12)]]> AAGCCGACCGGACAAAAAAGCCCTGATGCCAGTTCG <![CDATA[Primer trpE fbr -F (SEQ ID NO: 13)]]> GCTGCTGCTGGAATTCGCAGATATCGACAGCAAAG <![CDATA[Primer trpE fbr -R (SEQ ID NO: 14)]]> TGTCGATATCTGCGAATTCCAGCAGCAGCGTTGCC Primer ppsA-up-F (SEQ ID NO: 15) GAATCCATGGGCCTGTTGAAAGCATAAATTAAAAACG Primer ppsA-up-R (SEQ ID NO: 16) TTAAACAAAATTATTGGGGAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACGAACAATCCTTTTGTGATA Primer ppsA-down-F (SEQ ID NO: 17) ATTCCCCAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCCAACAATGGCTCGTCACCGCTCGTGCTTTGGTATAACCAAC Primer ppsA-down-R (SEQ ID NO: 18) TCCAAGCTTCCATTCAGAAGGGAGTGTCGATAATCC Primer ppsA-ver-F (SEQ ID NO: 19) TCGACACTCCCTTCTGAATGGAAGCTTGGATTCTC Primer ppsA-ver-R (SEQ ID NO: 20) AATTTATGCTTTCAACAGGCCCATGGATTCTTC Primer ppsA-N20-F (SEQ ID NO: 21) TAGCCAACAATGGCTCGTCACCGCGTTTTAGAGCTAGAAATAGC Primer ppsA-N20-R (SEQ ID NO: 22) CGCGGTGACGAGCCATTGTTGGCTAAGATCTGACTCCATAA Primer trpR-up-F (SEQ ID NO: 23) AATCCATGGGCCTGTAGCAGCTTATAACGCCGGA Primer trpR-up-R (SEQ ID NO: 24) ATCAGGCCTACAAAAAATATGTCGCCATTGTTAGC Primer trpR-down-F (SEQ ID NO: 25) CAATGGCGACATATTTTTTGTAGGCCTGATAAGAC Primer trpR-down-R (SEQ ID NO: 26) CCAAGCTTCCATTCATGGTCCCGTGATGTCGCGT Primer trpR-ver-F (SEQ ID NO: 27) ACATCACGGGACCATGAATGGAAGCTTGGATTCTC Primer trpR-ver-R (SEQ ID NO: 28) GCGTTATAAGCTGCTACAGGCCCATGGATTCTTC Primer trpR-N20-F (SEQ ID NO: 29) GCCAGATGAGCGCGAAGCGTGTTTTAGAGCTAGAAATAGC Primer trpR-N20-R (SEQ ID NO: 30) ACGCTTCGCGCTCATCTGGCGCTAAGATCTGACTCCATAA Primer tnaA-up-F (SEQ ID NO: 31) ACGAAGAATCCATGGGCCTGTGCATTATGAATATCTTACATA Primer tnaA-up-R (SEQ ID NO: 32) ATAGCCACTCTGTAGTATTAATACATAATCCTTCATTTATTT Primer tnaA-down-F (SEQ ID NO: 33) AAATAAATGAAGGATTATGTATTAATACTACAGAGTGGCTAT Primer tnaA-down-R (SEQ ID NO: 34) TGAGAATCCAAGCTTCCATTCAGCCGACGGGATAATTTAAATTTG Primer tnaA-vec-F (SEQ ID NO: 35) CAAATTTAAATTATCCCGTCGGCTGAATGGAAGCTTGGATTCTCA Primer tnaA-vec-R (SEQ ID NO: 36) TATGTAAGATATTCATAATGCACAGGCCCATGGATTCTTCGT Primer tnaA-N20-F (SEQ ID NO: 37) TAACTCTGCAGGTGGTCAGCGTTTTAGAGCTAGAAATAGCAAG Primer tnaA-N20-R (SEQ ID NO: 38) GCTGACCACCTGCAGAGTTAGCTAAGATCTGACTCCATAACAG

[0062] Plasmid PH5a-aroG fbr-trpE fbr DCBA-transformed strain KW was introduced and overexpressed in its genome by introducing the tac promoter. ppsA Genes, knockout regulatory factors trpR and L-tryptophan degradation gene tnaA Subsequently, the tryptophan-producing strain BDX1 was constructed.

[0063] The strains and plasmids used in this section are as follows:

[0064] Table 2. Strains and plasmids used to construct L-tryptophan-producing strain BDX1

[0065]

[0066] Example 2: Construction of L-tryptophan-producing strain BDX2

[0067] RpoS encodes σs, a subunit of RNA polymerase, and acts as a major regulator of many stationary-phase genes in *E. coli* to adapt to nutrient deficiency and other stresses. Genome-wide analysis of RpoS-dependent gene expression revealed that up to 10% of genes in *E. coli* are directly or indirectly regulated by RpoS. Although the RpoS regulatory system is a large conserved system crucial for adaptation to various stresses, its regulatory mechanisms in specific metabolic pathways, such as L-tryptophan synthesis, remain incompletely understood. Therefore, we introduced the mutation site Q33. RpoS was inactivated to determine its effect on the L-tryptophan synthesis pathway.

[0068] Construction of the Cas9-RpoS plasmid: Using MG1655 as a template, the rpoS-up fragment with adapter was amplified using primers rpoS-up-F and rpoS-up-R, and the rpoS-down fragment with adapter was amplified using primers rpoS-down-F and rpoS-down-R. Using the rpoS-up and rpoS-down fragments as templates, the rpoS-UD fragment for Gibson assembly was amplified using primers rpoS-up-F and rpoS-down-R. Using the Cas9 plasmid as a template, the plasmid backbone rpoS-ver1 with adapter was amplified using primers rpoS-ver-F and rpoS-N20-R, and the plasmid backbone rpoS-ver2 with adapter was amplified using primers rpoS-N20-F and rpoS-ver-R. The plasmid backbones rpoS-ver1 and rpoS-ver2, along with the above rpoS-UD fragment, were assembled using Gibson to obtain the plasmid cas9-rpoS.

[0069] The primers used in this section are as follows:

[0070] Table 3 Primers used to construct L-tryptophan-producing strain BDX2

[0071]

[0072] The plasmid cas9-rpoS was introduced into strain BDX1, and induction was performed by adding arabinose to the culture medium, ultimately constructing a nonsense mutant RpoS (Q33). The strain is BDX2.

[0073] The strains and plasmids used in this section are as follows:

[0074] Table 4. Strains and plasmids used to construct L-tryptophan-producing strain BDX2

[0075]

[0076] Example 3 Construction of L-tryptophan-producing strain BDX3

[0077] FlhD, which encodes a flagellated transcriptional regulator, is involved in the transcriptional regulation of multiple flagellated operons and the regulation of cell motility. It is closely related to the physiological changes of cells. By introducing the mutation site F at the V84 residue of FlhD, a mutant of the regulator FlhD (V84F) was obtained to investigate whether FlhD has an effect on L-tryptophan production in cell physiology.

[0078] Construction of the Cas9-FlhD plasmid: Using MG1655 as a template, the flhD-up fragment with adapter was amplified using primers flhD-up-F and flhD-up-R, and the flhD-down fragment with adapter was amplified using primers flhD-down-F and flhD-down-R. Using the flhD-up and flhD-down fragments as templates, the flhD-UD fragment for Gibsonassembly was amplified using primers flhD-up-F and flhD-down-R. Using the Cas9 plasmid as a template, the plasmid backbone flhD-ver1 with adapter was amplified using primers flhD-ver-F and flhD-N20-R, and the plasmid backbone flhD-ver2 with adapter was amplified using primers flhD-N20-F and flhD-ver-R. The plasmid skeletons flhD-ver1 and flhD-ver2, along with the above flhD-UD fragment, were assembled using Gibson to obtain the plasmid cas9-flhD.

[0079] The primers used in this section are as follows:

[0080] Table 5 Primers used to construct L-tryptophan-producing strain BDX3

[0081]

[0082] The plasmid cas9-flhD was introduced into strain BDX1. By adding arabinose to the culture medium for induction, strain BDX3, with the V84 amino acid mutation of FlhD, was finally constructed.

[0083] The strains and plasmids used in this section are as follows:

[0084] Table 6. Strains and plasmids used to construct L-tryptophan-producing strain BDX3

[0085]

[0086] Example 4: Construction of L-tryptophan-producing strain BDX4

[0087] crp CRP, encoded by the gene, is a transcription factor that binds to intracellular 3',5'-cyclic adenosine monophosphate (cAMP) to form a complex that participates in the regulation of transcription of multiple genes. The CRP-cAMP complex can bind to specific sites downstream of the transcription start site of the crp gene, negatively regulating its own transcription. The CRP-cAMP complex can also activate the transcription of other genes, especially in low-glucose environments, by binding to its regulatory regions and enhancing RNA polymerase binding and transcriptional activity. CRP senses cAMP levels in the cell and regulates genes related to glucose and amino acid metabolism, helping cells adapt to different environmental conditions and optimize energy utilization.

[0088] Construction of the Cas9-CrP plasmid: Using MG1655 as a template, the CRP-UP fragment with the adapter was amplified using primers CRP-UP-F and CRP-UP-R, and the CRP-Down fragment with the adapter was amplified using primers CRP-Down-F and CRP-Down-R. Using the CRP-UP and CRP-Down fragments as templates, the CRP-UD fragment for Gibson assembly was amplified using primers CRP-UP-F and CRP-Down-R. Using the Cas9 plasmid as a template, the plasmid backbone CRP-Ver1 with the adapter was amplified using primers CRP-Ver-F and CRP-N20-R, and the plasmid backbone CRP-Ver2 with the adapter was amplified using primers CRP-N20-F and CRP-Ver-R. The plasmid backbones CRP-Ver1 and CRP-Ver2, along with the CRP-UD fragment above, were assembled using Gibson assembly to obtain the plasmid Cas9-CrP.

[0089] The primers used in this section are as follows:

[0090] Table 7 Primers used to construct L-tryptophan-producing strain BDX3

[0091]

[0092] The plasmid cas9-crp was introduced into strain BDX1. By adding arabinose to the culture medium for induction, strain BDX4, in which the T29 amino acid of Crp was mutated to K, was finally constructed, which is the Crp (T29K) mutant.

[0093] The strains and plasmids used in this section are as follows:

[0094] Table 8. Strains and plasmids used to construct L-tryptophan-producing strain BDX4

[0095]

[0096] Example 5: Construction of L-tryptophan-producing strain BDX6

[0097] The results above show that, based on strain BDX1, when RpoS (Q33) is introduced... The L-tryptophan production capacity of strains BDX2, BDX3, and BDX4 was improved to some extent by using the FlhD (V84F) mutant and the Crp (T29K) mutant.

[0098] Therefore, in order to further increase the production of L-tryptophan, we superimposed three sites, specifically by introducing the regulatory factor FlhD (V84F) mutant and the regulatory factor Crp (T29K) mutant into the strain BDX2, thereby constructing strains BDX5 and BDX6.

[0099] The plasmid cas9-flhD was introduced into strain BDX2, and induced by adding arabinose to the culture medium, and finally strain BDX5, which mutated FlhD at V84 amino acid to F, was constructed.

[0100] The plasmid cas9-crp was introduced into strain BDX6, and knockout was induced by adding arabinose to the culture medium, finally constructing strain BDX6 with the T29 amino acid mutation of Crp to K.

[0101] The strains and plasmids used in this section are as follows:

[0102] Table 9. Strains and plasmids used to construct L-tryptophan-producing strain BDX6

[0103]

[0104] Example 6 Fermentation of L-tryptophan-producing strains

[0105] The shake-flask fermentation process of Escherichia coli L-tryptophan producing strains BDX1-BDX6 is as follows:

[0106] (1) Slant activation culture: Take out the preserved strain from the -80℃ freezer and streak it on a solid medium containing tetracycline resistance, and incubate at 37℃ for 12-18h.

[0107] (2) Seed culture: Use an inoculation loop to pick a single colony from the fresh activated slant and place it in seed basal medium (50 mL LB medium in a 500 mL Erlenmeyer flask, sealed with sealing film), and incubate at 37℃ and 220 r / min for 6-8 h until OD. 600 Approximately 2-3.

[0108] (3) Shaking flask batch fermentation culture: Inoculate the seed culture solution into a tetracycline-resistant fermentation basic medium (500mL Erlenmeyer flask, 50mL liquid volume, sealed with sealing film) at 10% inoculation amount, and carry out L-tryptophan batch fermentation at 37℃ and 220r / min for 36-42h.

[0109] The formulation of the shake flask fermentation medium is shown in Table 10:

[0110] Table 10 L-Tryptophan Fermentation Medium Formulation

[0111]

[0112] Example 7: Detection of fermentation strains by high performance liquid chromatography (HPLC)

[0113] The fermentation broth was centrifuged at 5500 rpm / min for 15-20 min in a refrigerated centrifuge, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0114] The HPLC conditions were as follows: ZORBAX Eclipse AAA (amino acid analysis) column; mobile phase A: 40 mM Na₂HPO₄, pH 7.8; mobile phase B: methanol:acetonitrile:water = 45:45:10, v / v / v. The elution gradient was: 0-1 min, 100% A; 9.8 min: 43% A + 57% B; 10 min: 100% B; 12 min: 100% B; 12.5 min: 100% A. The flow rate was 2.0 mL / min. A RID and VWD detector were connected in series. The detection cell temperature was controlled at 40℃. The injection volume was 10 μL. The analysis time was 26 min. The UV detection wavelength was 338 nm.

[0115] Example 8: Fermentation results and analysis of L-tryptophan producing strains BDX1-BDX6

[0116] After fermentation culture of strains KW and BDX1-BDX6 for 38-42 hours, the supernatant of the fermentation broth was analyzed by HPLC. The results are as follows: Figure 2As shown in the figure. The fermentation results indicate that, based on strain BDX1, the global regulatory factor RpoS(Q33) was introduced. After that, tryptophan production increased from 0.74 g / L to 1.12 g / L, and as... Figure 3 As shown, the biomass of strain BDX2 was also increased. Based on strain KW, the introduction of the flagella-related regulatory factor FlhD (V84F) mutant increased tryptophan production to 0.85 g / L. Introducing the regulatory factor Crp (T29K) mutant into strain KW relieved its inhibitory or activating effect on the regulated gene, increasing tryptophan production to 0.9 g / L. Therefore, these three regulatory factors are closely related to L-tryptophan production.

[0117] Based on strain BDX2, the introduction of the regulatory factor FlhD(V84F) mutant increased tryptophan production to 1.23 g / L; further, the introduction of the Crp(T29K) mutant further increased L-tryptophan production to 1.42 g / L. Compared to the control strain KW, strain BDX6 showed an approximately 5-fold increase in L-tryptophan production. This demonstrates that regulatory factors have a significant impact on metabolic pathways, and that modifying corresponding regulatory factors can, to some extent, increase the yield of the target product and improve strain growth.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An L-tryptophan-engineered bacterium under the influence of regulatory factors, characterized in that: In Escherichia coli strain KW, a gene containing... aroG fbr and trpE fbr DCBA Recombinant plasmids, overexpression ppsA Gene knockout trpR Gene knockout tnaA Genes were used to construct strain BDX1; the L-tryptophan-producing genetically engineered bacteria were obtained by inactivating the regulatory factor RpoS and performing site-directed mutagenesis on FlhD in strain BDX1; or the L-tryptophan-producing genetically engineered bacteria were obtained by inactivating the regulatory factor RpoS and performing site-directed mutagenesis on both FlhD and Crp in strain BDX1; site-directed mutagenesis of FlhD was achieved by introducing the mutation site V84F into the regulatory factor FlhD to obtain the FlhD protein mutant, the amino acid sequence of which is SEQ ID NO: 1; site-directed mutagenesis of Crp was achieved by introducing the mutation site T29K into the regulatory factor Crp to obtain the Crp protein mutant, the amino acid sequence of which is SEQ ID NO:

2.

2. The L-tryptophan-engineered bacterium under the influence of regulatory factors as described in claim 1, characterized in that: Inactivation of the regulatory factor RpoS is achieved by introducing a mutation Q33 into the regulatory factor RpoS. The protein is inactivated.

3. A method for constructing L-tryptophan-engineered bacteria under the influence of regulatory factors, comprising the following steps: Step 1. In E. coli strain KW, a gene containing... aroG fbr and trpE fbr DCBA Recombinant plasmids, overexpression ppsA Gene knockout trpR Gene knockout tnaA Genes were used to construct strain BDX1; Step 2. In strain BDX1, the mutant Q33 was introduced into the global regulator RpoS. The protein was inactivated, and strain BDX2 was constructed. Step 3. In strain BDX2, a mutation site V84F was introduced into the flagellar transcription regulator FlhD to obtain the FlhD protein mutant, and strain BDX5 was constructed. The amino acid sequence of the FlhD protein mutant is shown in SEQ ID NO:

1. In strain BDX5, a mutation site T29K was introduced into the transcription regulator Crp to obtain the Crp protein mutant, and strain BDX6 was constructed. The amino acid sequence of the Crp protein mutant is shown in SEQ ID NO:

2. The strain BDX5 or strain BDX6 is an L-tryptophan genetically engineered bacterium.

4. The application of L-tryptophan genetically engineered bacteria under the influence of regulatory factors as described in claim 1 in the fermentation production of L-tryptophan.

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