Acid-resistant expression cassette and screening method thereof

Abstract

The present invention relates to the fields of genetic engineering and synthetic biology, and discloses an acid-resistant expression cassette and a screening method thereof. Specifically, the present invention relates to a step-by-step evaluation method for high-throughput screening of acid-resistant expression cassettes for Escherichia coli and industrial microorganisms, which can be used to improve the acid-resistant and fermentation production performance of industrial microorganisms under industrial conditions.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and synthetic biology. Specifically, the present invention relates to a step-by-step evaluation method for high-throughput screening of acid-resistant expression cassettes of Escherichia coli and industrial microorganisms, which can be used to improve the acid-resistant and fermentation production performance of industrial microorganisms under industrial conditions. Background Art

[0002] my country's organic acid fermentation industry is an important part of the bio-industry and has developed rapidly in recent years. However, there are still many problems that need to be solved in terms of production economy and environmental protection.

[0003] During the fermentation process, industrial microorganisms gradually accumulate large amounts of acidic products or byproducts, continuously acidifying the fermentation environment. In acidic environments, the growth and metabolism of industrial microorganisms are severely inhibited, resulting in a decrease in production capacity. Therefore, actual industrial fermentation processes (such as amino acid fermentation) use large amounts of base (such as liquid ammonia) to neutralize the acidic substances produced by fermentation and maintain neutral fermentation conditions. To obtain acidic products, large amounts of acid must be used after neutral fermentation. The extensive use of acid and base generates large amounts of industrial wastewater. If the acid resistance of industrial microorganisms could be improved and acid-resistant industrial microorganisms could be used to ferment under acidic conditions, the amount of acid and base used in the fermentation process could be effectively reduced, reducing wastewater discharge, while also inhibiting bacterial contamination and lowering related energy consumption, generating significant economic benefits and promoting energy conservation and emission reduction.

[0004] Domestic and international research on microbial stress resistance, such as acid resistance, high temperature resistance, and high osmotic pressure resistance, mainly involves traditional mutagenesis, domestication, simple gene cloning, and knockout, but the industrialization efficiency of these methods is relatively low. In recent years, some new gene-based methods have been developed to improve the stress resistance of microorganisms, such as the global transcription machinery gTME, artificial transcription factors, and global regulatory factor engineering, which have expanded the technical methods and ideas for microbial stress resistance modification [1].

[0005] In recent years, synthetic biology has made significant progress in conceptual theory, functional applications, and methodological techniques. Based on known stress-resistance-related structural and regulatory genes, synthetic biology methods can be used to artificially modify and integrate them, thereby designing standardized stress-resistance meta-expression cassettes, endowing microorganisms with highly effective stress-resistance properties for practical industrial microbial production.

[0006] Escherichia coli has three main types of acid resistance (AR) mechanisms[2]. Among them, the second type of acid resistance system (AR2) is a very important system with a relatively clear mechanism. The second type of acid resistance system (AR2) consists of glutaminase YbaS[3], glutamate decarboxylases GadA and GadB[4], and glutamate and γ-aminobutyric acid (GABA) antiporter GadC[5]. YbaS in this system converts glutamine into glutamate, releasing NH3 to neutralize intracellular protons; GadA and GadB cause glutamate to undergo decarboxylation to produce γ-aminobutyric acid and consume intracellular protons; GadC takes in glutamine and glutamate, and excretes glutamate and GABA.

[0007] EvgA is a response regulatory protein of the two-component signal transduction system EvgAS and an important middle-level regulatory factor in Escherichia coli. It can regulate the expression of downstream structural genes and regulatory genes. The genes regulated by it include a variety of acid-resistant related genes, such as gadB and gadC of the second type of acid-resistant system, and the local regulatory factors ydeO and gadX that regulate the expression of gadB and gadC[6]. Overexpression of the evgA gene in Escherichia coli can improve the survival rate of Escherichia coli against acid shock[7].

[0008] Although the mechanism of Escherichia coli acid-resistant system genes has been studied in detail, there is currently no research on using the above genes in combination with artificial promoters to develop standardized synthetic biology acid-resistant expression cassettes, and there is no method for high-throughput screening and step-by-step evaluation of the acid-resistant properties of acid-resistant expression cassettes and their impact on fermentation production performance. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention provides an expression cassette comprising one or more promoters, one or more acid-resistant genes selected from the group consisting of evgA, ybaS, gadB, and gadC. The expression cassette of the present invention, when introduced into a host cell, can enhance the acid resistance of the host cell.

[0010] In some embodiments, the evgA gene in the expression cassette of the present invention encodes the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the ybaS gene in the expression cassette of the present invention encodes the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the gadB gene in the expression cassette of the present invention encodes the amino acid sequence set forth in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. In some embodiments, the gadC gene in the expression cassette of the present invention encodes the amino acid sequence set forth in SEQ ID NO: 78.

[0011] In some embodiments, the promoter in the expression cassette of the present invention is selected from the group consisting of the promoters set forth in SEQ ID NOs: 1-10, the evgA gene promoter, the ybaS gene promoter, and the gadB gene promoter. In some embodiments, the nucleotide sequence of the evgA gene promoter is set forth in SEQ ID NO: 67. In some embodiments, the nucleotide sequence of the ybaS gene promoter is set forth in SEQ ID NO: 70. In some embodiments, the nucleotide sequence of the gadB gene promoter is set forth in SEQ ID NO: 72.

[0012] In some embodiments, the terminator in the expression cassette of the present invention is rrnBT terminator. In some embodiments, the nucleotide sequence of the rrnBT terminator is shown in SEQ ID NO: 69.

[0013] In some embodiments of the present invention, the expression cassette comprises, from 5' to 3', a promoter selected from SEQ ID NOs: 1-10 and the evgA gene promoter, the evgA gene, and the rrnBT terminator. In some specific embodiments, the nucleotide sequence of the expression cassette is set forth in any one of SEQ ID NOs: 26-36. In some preferred embodiments, the nucleotide sequence of the expression cassette is set forth in SEQ ID NOs: 34 or 35.

[0014] In other embodiments of the present invention, the expression cassette consists of two parts: the first part, from 5' to 3', comprises the ybaS gene promoter, the ybaS gene, and the rrnBT terminator; and the second part, from 5' to 3', comprises the gadB gene promoter, the gadB gene, the gadC gene, and the rrnBT terminator. In some specific embodiments, the nucleotide sequence of the expression cassette is set forth in any one of SEQ ID NOs: 55-59. In some preferred embodiments, the nucleotide sequence of the expression cassette is set forth in SEQ ID NOs: 58 or 59.

[0015] In other embodiments of the present invention, the expression cassette is composed of three parts: the first part comprises the ybaS gene promoter, the ybaS gene, and the rrnBT terminator from 5' to 3'; the second part comprises the gadB gene promoter, the gadB gene, the gadC gene, and the rrnBT terminator from 5' to 3'; and the third part comprises a promoter selected from SEQ ID NOs: 1-10 and the evgA gene promoter, the evgA gene, and the rrnBT terminator from 5' to 3'. In some specific embodiments, the nucleotide sequence of the expression cassette is shown in any one of SEQ ID NOs: 62-66. In some preferred embodiments, the nucleotide sequence of the expression cassette is SEQ ID NO: 62.

[0016] In a second aspect, the present invention provides an expression construct comprising an expression cassette of the present invention.

[0017] In a third aspect, the invention provides a recombinant host cell comprising an expression cassette of the present invention or an expression construct of the present invention. The recombinant host cell is preferably a prokaryotic cell, more preferably a bacterial cell, most preferably an Escherichia coli cell. The recombinant host cell of the present invention has an improved acid resistance compared to the corresponding cell that does not contain the expression cassette or expression construct. The acid resistance comprises a survival rate under acid shock and a growth rate under acid stress conditions.

[0018] In a fourth aspect, the present invention provides a method for producing an organic acid by microbial fermentation, the method comprising:

[0019] (a) introducing the expression cassette or expression construct of the present invention into an organic acid-producing microorganism;

[0020] (b) fermenting the microorganism; and

[0021] (c) harvesting the produced organic acids.

[0022] The organic acid producing microorganism used in the method for producing organic acids by microbial fermentation of the present invention is preferably a prokaryotic microorganism, more preferably a bacterium, most preferably Escherichia coli. The organic acid that can be produced by the method of the present invention includes amino acids (such as lysine, threonine, tryptophan, glutamic acid), succinic acid, citric acid and lactic acid.

[0023] In a fifth aspect, the present invention also provides a method for high-throughput screening of stress-resistant expression cassettes for microbial fermentation, the method comprising the following steps:

[0024] (a) introducing the expression cassette to be screened into the microorganism;

[0025] (b) high-throughput evaluation of the survival rate of the microorganism under stress shock;

[0026] (c) evaluating the growth rate of the microorganism under stress environment using a high-throughput growth tester such as BioscreenC;

[0027] (d) evaluating the fermentation performance of the microorganism under stress conditions using a high-throughput micro-fermentation instrument such as BioLector;

[0028] (e) identifying an expression cassette capable of improving the survival rate and / or the growth rate and / or the fermentation performance of the microorganism as a stress resistance expression cassette that can be used for microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. Shows the construction of the pNatevgA-rrnBT acid-resistant expression cassette expression plasmid pACYC184-pNatevgA-rrnBT.

[0030] Figure 2 . Shows the construction of the pQevgA-rrnBT acid-resistant expression cassette intermediate expression plasmid pBR322-pQevgA-rrnBT.

[0031] Figure 3 . Shows the construction of the pQevgA-rrnBT acid-resistant expression cassette expression plasmid pACYC184-pQevgA-rrnBT.

[0032] Figure 4 . Shows the construction of the control plasmid pACYC184-rrnBT.

[0033] Figure 5 . Shows the construction of the intermediate cloning vector pACYC184-HindIII-pNatevgA-Xho I-rrnBT-BamH I for the acid-resistant expression cassette of the ybaS-gadB-gadC triple gene.

[0034] Figure 6 . Shows the construction of the intermediate cloning plasmid pBR322-gadB-gadC470-rrnBT of the acid-resistant expression cassette of the ybaS-gadB-gadC triple gene.

[0035] Figure 7 . Shows the construction of the intermediate cloning plasmid pBR322-gadB(H465A / dHT / H465A&E89Q / dHT&E89Q)-gadC470-rrnBT of the ybaS-gadB-gadC triple gene acid-resistant expression cassette.

[0036] Figure 8 . Shows the construction of the ybaS-gadB-gadC triple gene acid-resistant expression cassette expression plasmid pACYC184-ybaS-rrnBT-gadB-gadC470-rrnBT.

[0037] Figure 9 . Shows the construction of the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette expression plasmid pACYC184-ybaS-rrnBT-gadB-gadC470-rrnBT-pSevgA-rrnBT.

[0038] Figure 10 . Shows the survival results of the evgA acid-resistant expression cassette under acid stress shock.

[0039] Figure 11. Shows the survival results of the acid-resistant expression cassette of the ybaS-gadB-gadC three genes under acid stress shock.

[0040] Figure 12 . Shows the survival results of the acid-resistant four-gene expression cassette ybaS-gadB-gadC-evgA under acid stress shock.

[0041] Figure 13 . Shows the results of the acid stress growth test of the evgA acid-resistant expression cassette.

[0042] Figure 14 . Shows the results of the acid-resistant growth test of the ybaS-gadB-gadC three-gene expression cassette under acid stress.

[0043] Figure 15 . Shows the results of the acid-resistant growth test of the ybaS-gadB-gadC-evgA four-gene expression cassette under acid stress. Detailed Description of the Invention

[0044] The present invention provides an expression cassette composed of one or more promoters, one or more acid-resistant genes, and one or more terminators. The expression cassette is capable of expressing the acid-resistant genes in a host cell, thereby improving the acid resistance of the host cell, such as the acid shock survival rate, the growth rate in an acid stress environment, and the fermentation performance in an acid stress environment.

[0045] In the expression cassette of the present invention, the promoter, the acid-resistant gene and the terminator are operably linked to achieve the desired transcription of the acid-resistant gene and ultimately express the acid-resistant protein encoded by the acid-resistant gene in the host cell.

[0046] As used herein, "acid-resistant gene" refers to a gene encoding an acid-resistant protein of Escherichia coli or a functional variant thereof, such as genes encoding acid-resistant regulatory proteins such as evgA, ydeO and gadX, and genes encoding acid-resistant structural proteins, including the acid-resistant structural protein genes ybaS, gadB, gadC, etc. of the second type of acid-resistant system (AR2) of Escherichia coli [6]. Genes encoding other acid-resistant proteins of Escherichia coli can also be used in the expression cassette of the present invention. As used herein, a "functional variant" of an acid-resistant protein refers to a variant of a wild-type acid-resistant protein obtained by replacing, deleting, or adding one or more amino acids, provided that its activity is retained.

[0047] In some embodiments, the acid-resistant gene in the expression cassette of the present invention is the acid-resistant regulatory gene evgA of Escherichia coli. In a specific embodiment, the evgA gene encodes the wild-type EvgA shown in SEQ ID NO:68.

[0048] In some embodiments, the acid-resistant gene in the expression cassette of the present invention is the wild-type acid-resistant structural gene ybaS of Escherichia coli. In a specific embodiment, the ybaS gene encodes the wild-type YbaS shown in SEQ ID NO:71.

[0049] In some embodiments, the acid-resistant gene in the expression cassette of the present invention is the acid-resistant structural gene gadB of Escherichia coli. In one embodiment, the gadB gene encodes the wild-type GadB shown in SEQ ID NO: 73. In one embodiment, the gadB gene encodes the variant GadB (H465A) shown in SEQ ID NO: 74. In one embodiment, the gadB gene encodes the variant GadB (dHT) shown in SEQ ID NO: 75 (the last two amino acids of the C-terminus of GadB are removed). In one embodiment, the gadB gene encodes the variant GadB (H465A & E89Q) shown in SEQ ID NO: 76. In one embodiment, the gadB gene encodes the variant GadB (dHT & E89Q) shown in SEQ ID NO: 77

[10] .

[0050] In some embodiments, the acid-resistant gene in the expression cassette of the present invention is the acid-resistant structural gene gadC of Escherichia coli. In a specific embodiment, the gadC gene encodes the variant GadC470 (C-terminally truncated, retaining amino acids 1-470) shown in SEQ ID NO: 78

[11] .

[0051] The regulatory factor EvgA mentioned above can regulate a variety of acid-resistant genes, including gadB and gadC of the second type of acid-resistant system, as well as local regulatory factors ydeO and gadX that regulate the expression of gadB and gadC. The above-mentioned mutations of GadB and GadC have the characteristic of being more active than the wild type in a near-neutral acid environment. The above-mentioned GadB mutation has a higher activity at pH 6.0, while the activity of wild-type GadB at pH 6.0 is almost zero. The activity of wild-type GadB is higher only when the environmental pH is reduced to 5.3

[10] . The above-mentioned GadC mutation GadC470 has a significantly higher activity at pH 6.5 compared to the wild-type GadC

[11] .

[0052] The promoters of the expression cassette of the present invention include the natural promoter of the evgA gene shown in SEQ ID NO: 67, the natural promoter of the ybaS gene shown in SEQ ID NO: 70, the natural promoter of the gadB gene shown in SEQ ID NO: 72, and 10 artificial promoters pQ, pZ, pV, pW, pN, pS, pAA, pJJ, pII, and pOO, whose sequences are shown in SEQ ID NOs: 1-10, respectively. The above 10 artificial promoters are P of bacteriophage. L A series of constitutive promoters with strengths ranging from strong to weak were obtained after artificial mutation of the λ constitutive promoter [8]. Various promoters that can express target genes in host cells are known in the art, and such promoters are also included in the scope of the present invention.

[0053] The terminator of the expression cassette of the present invention includes the terminator rrnBT of the 16s ribosomal RNA rrnB operon [9] whose sequence is shown in SEQ ID NO: 69. Various terminators that can terminate the transcription of the target gene in the host cell are known in the art, and such terminators are also encompassed within the scope of the present invention.

[0054] In some embodiments, the present invention provides a single-gene acid-resistant expression cassette constructed using the Escherichia coli regulatory gene evgA, comprising a promoter, the regulatory gene evgA, and a terminator. In some specific embodiments, evgA expression is initiated using a promoter selected from the natural evgA promoter pNat, the artificial promoters pQ, pZ, pV, pW, pN, pS, pAA, pJJ, pII, and pOO, and evgA transcription is terminated using the terminator rrnBT. In some specific embodiments, the nucleotide sequence of the evgA single-gene expression cassette of the present invention is set forth in any one of SEQ ID NOs: 26-36.

[0055] In other embodiments, the present invention provides a ybaS-gadB-gadC three-gene acid-resistant expression cassette constructed using the ybaS, gadB, and gadC genes of the second acid-resistant system of Escherichia coli. The ybaS used is wild-type ybaS; the gadB used includes wild-type gadB and gadB mutants gadB(H465A), gadB(dHT) (truncated at the last two amino acids at the C-terminus), gadB(H465A&E89Q), and gadB(dHT&E89Q); and the gadC used is the gadC mutant gadC470 (truncated at the C-terminus, retaining the mutation at amino acids 1-470). In some specific embodiments, the acid-resistant triple gene expression cassette of ybaS, gadB, and gadC comprises two parts: the first part comprises the ybaS native promoter operably linked to ybaS, ybaS, and the rrnBT terminator; the second part comprises an operon containing gadB and gadC, with the gadB native promoter and the rrnBT terminator. In some specific embodiments, the nucleotide sequence of the acid-resistant triple gene expression cassette of the present invention is set forth in any one of SEQ ID NOs: 55-59.

[0056] In other embodiments, the present invention provides a four-gene acid-resistant expression cassette that combines the evgA regulatory gene with the three structural genes ybaS, gadB, and gadC. In some specific embodiments, the evgA used is wild-type evgA; the ybaS used is wild-type ybaS; the gadB used is selected from wild-type gadB, mutant gadB(H465A), gadB(dHT), gadB(H465A&E89Q), and gadB(dHT&E89Q); and the gadC used is the gadC mutant gadC470. In some specific embodiments, the four-gene acid-resistant expression cassette comprises three parts: the first part is a ybaS natural promoter operably linked to ybaS, ybaS, and an rrnBT terminator; the second part is an operator comprising gadB and gadC, whose promoter is the gadB natural promoter and whose terminator is rrnBT; the third part is a promoter selected from the evgA natural promoter pNat, artificial promoters pQ, pZ, pV, pW, pN, pS, pAA, pJJ, pII, and pOO, evgA, and an rrnBT terminator, which is operably linked to evgA. In some specific embodiments, the nucleotide sequence of the four-gene acid-resistant expression cassette of the present invention is shown in any one of SEQ ID NOs: 62-66.

[0057] In another aspect, the present invention provides an expression construct comprising an expression cassette of the present invention. The expression construct of the present invention can be based on any suitable vector. Vectors used in the expression construct of the present invention include those that autonomously replicate in a host cell, such as plasmid vectors, and also include vectors that can integrate into and replicate along with host cell DNA. Many vectors suitable for the present invention are commercially available. In one embodiment, the expression construct of the present invention is based on the commercial plasmid pACYC184 (NEB).

[0058] The present invention also provides recombinant host cells on the other hand, which comprise expression cassettes of the present invention or expression constructs of the present invention. It is known in the art that by means such as codon optimization, genes from a kind of organism can also work in other organisms. Therefore, expression cassettes of the present invention are not limited to use in Escherichia coli. Recombinant host cells of the present invention are preferably prokaryotic cells, more preferably bacterial cells, most preferably Escherichia coli cells such as MG1655 strains. Various methods of importing expression cassettes or expression constructs into host cells are well known in the art, such as CaCl2 method, electroporation, etc. As illustrated in Examples 5 and 6, recombinant host cells comprising expression cassettes of the present invention have improved acid resistance compared to corresponding control cells that do not contain the expression cassettes or expression constructs. As used herein, "acid resistance" includes survival rate under acid shock and growth rate under acid stress conditions.

[0059] In another aspect, the present invention provides a method for producing an organic acid by microbial fermentation, the method comprising:

[0060] (a) introducing the expression cassette or expression construct of the present invention into an organic acid-producing microorganism;

[0061] (b) fermenting the microorganism; and

[0062] (c) harvesting the produced organic acids.

[0063] The organic acid producing microorganism used in the method for producing organic acids by microbial fermentation of the present invention is preferably a prokaryotic microorganism, more preferably a bacterium, most preferably Escherichia coli. The organic acid that can be produced by the method of the present invention includes amino acids (such as lysine, threonine, tryptophan, glutamic acid), succinic acid, citric acid and lactic acid.

[0064] In one embodiment, the present invention provides a method for producing lysine by microbial fermentation, the method comprising:

[0065] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59 and SEQ ID NOs: 62-66 or an expression construct comprising the expression cassette into a lysine-producing microorganism;

[0066] (b) fermenting the microorganism; and

[0067] (c) harvesting the produced lysine.

[0068] Preferably, the expression cassette is selected from SEQ ID NO: 34, 35, 58, 59, 62. Preferably, the lysine-producing microorganism is lysine-producing Escherichia coli. More preferably, the lysine-producing microorganism is Escherichia coli SCEcL3 (pSLL1) strain.

[0069] In one embodiment, the present invention provides a method for producing threonine by microbial fermentation, the method comprising:

[0070] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59, and SEQ ID NOs: 62-66, or an expression construct comprising the expression cassette into a threonine-producing microorganism;

[0071] (b) fermenting the microorganism; and

[0072] (c) harvesting the produced threonine.

[0073] In one embodiment, the present invention provides a method for producing tryptophan by microbial fermentation, the method comprising:

[0074] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59, and SEQ ID NOs: 62-66, or an expression construct comprising the expression cassette into a tryptophan-producing microorganism;

[0075] (b) fermenting the microorganism; and

[0076] (c) harvesting the produced tryptophan.

[0077] In one embodiment, the present invention provides a method for producing glutamic acid by microbial fermentation, the method comprising:

[0078] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59, and SEQ ID NOs: 62-66, or an expression construct comprising the expression cassette into a glutamate-producing microorganism;

[0079] (b) fermenting the microorganism; and

[0080] (c) harvesting the produced glutamate.

[0081] In one embodiment, the present invention provides a method for producing succinic acid by microbial fermentation, the method comprising:

[0082] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59, and SEQ ID NOs: 62-66, or an expression construct comprising the expression cassette into a succinate-producing microorganism;

[0083] (b) fermenting the microorganism; and

[0084] (c) harvesting the produced succinic acid.

[0085] In one embodiment, the present invention provides a method for producing citric acid by microbial fermentation, the method comprising:

[0086] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59 and SEQ ID NOs: 62-66 or an expression construct comprising the expression cassette into a citric acid-producing microorganism;

[0087] (b) fermenting the microorganism; and

[0088] (c) harvesting the produced citric acid.

[0089] In one embodiment, the present invention provides a method for producing lactic acid by microbial fermentation, the method comprising:

[0090] (a) introducing an expression cassette shown in any one of SEQ ID NOs: 26-36, SEQ ID NOs: 55-59 and SEQ ID NOs: 62-66 or an expression construct comprising the expression cassette into a lactic acid-producing microorganism;

[0091] (b) fermenting the microorganism; and

[0092] (c) harvesting the lactic acid produced.

[0093] In a final aspect, the present invention provides a method for high-throughput screening of stress-resistant expression cassettes for microbial fermentation, the method comprising the following steps:

[0094] (a) introducing the expression cassette to be screened into the microorganism;

[0095] (b) high-throughput evaluation of the survival rate of the microorganism under stress shock;

[0096] (c) evaluating the growth rate of the microorganism under stress environment using a high-throughput growth tester such as BioscreenC;

[0097] (d) evaluating the fermentation performance of the microorganism under stress conditions using a high-throughput micro-fermentation instrument such as BioLector;

[0098] (e) identifying an expression cassette capable of improving the survival rate and / or the growth rate and / or the fermentation performance of the microorganism as a stress resistance expression cassette that can be used for microbial fermentation.

[0099] As used herein, "stress shock" refers to culturing a microorganism under extreme stress conditions that can affect the survival of the microorganism for a suitable period of time. Those skilled in the art can determine stress shock conditions for microorganisms. For example, for E. coli, acid shock refers to culturing at pH 2.0-pH 3.0, preferably pH 2.5, for example, 1, 2, 3, 4, 5 hours, preferably 2 hours. "Stress pressure" refers to stress conditions that primarily affect the growth of a microorganism. Those skilled in the art can determine stress pressure conditions for microorganisms. For example, for E. coli, acid stress refers to a pH below 7.0 but above 4.0, such as pH 6.0.

[0100] The high-throughput screening method for stress-resistant expression cassettes of the present invention is a comprehensive and integrated evaluation method that is carried out step by step. The method associates survival rate, growth rate and fermentation performance, avoids simply ignoring the impact of stress-resistant pressure on growth and fermentation performance through extreme stress impact survival evaluation, and also avoids simply ignoring the impact of the balance between growth and fermentation performance through growth evaluation. After two-step screening, high-throughput micro-fermentation is used to evaluate stress resistance and fermentation performance. Finally, a small number of the most promising stress-resistant expression cassettes obtained through screening are subsequently evaluated in industrial fermentation tanks, thereby avoiding direct large-scale fermentation evaluation of all acid-resistant expression cassettes and improving the screening effect and efficiency. Example

[0101] The present invention will be further described below by way of examples, but the present invention is not limited to the described examples.

[0102] Example 1: Construction of regulatory elements of promoters with different strengths

[0103] In this example, 10 artificial constitutive promoters [8] and RBS sequences with different strengths were selected and cloned into the commercial pUC57 vector by Genscript China (Nanjing GenScript). The sequence and strength information are shown in Table 1.

[0104] Table 1 Artificial promoter sequences and strengths

[0105]

[0106] Example 2: Construction of evgA acid-resistant expression cassette

[0107] 2.1 Construction of pNatevgA-rrnBT acid-resistant expression cassette

[0108] 2.1.1 Construction of the pBR322 intermediate expression vector for pNatevgA-rrnBT

[0109] The construction process of the pNatevgA-rrnBT intermediate expression vector pBR322-pNatevgA-rrnBT used in the examples of this application is as follows Figure 1 As shown:

[0110] First, the genome of Escherichia coli MG1655 (Novagen) was extracted using a kit (Promega, A1125) as a template, and the pNatevgA polynucleotide fragment containing the natural promoter was obtained by PCR amplification using the following forward primers and reverse primers according to conventional methods: upstream primer 5'-GAGATT CCATGG GACTAAACCGTGGCTTTTGCAAT-3' (evgA_U, SEQ ID NO: 11, the underlined bases are the restriction endonuclease Nco I recognition site), and the downstream primer 5'-TTCCTT C TCGAG TTAGCCGATTTTGTTACGTTGTG-3' (evgA_L, SEQ ID NO: 12; the underlined bases indicate the restriction endonuclease Xho I recognition site). PCR was performed using Transgen Fast pfu polymerase. The reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 62°C for 20 sec, and 72°C for 30 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The fragment was then separated and recovered using the Tiangen High-Purity DNA Miniprep Kit.

[0111] The resulting PCR product was double-digested with restriction endonucleases Nco I and Xho I, and then ligated with plasmid pBR322-temp1-rrnBT (this vector was constructed by the inventors, and the full-length sequence of the plasmid can be found in SEQ ID NO: 13) that had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced. The sequencing results showed that the cloned pBR322-pNatevgA-rrnBT sequence was correct.

[0112] 2.1.2 Construction of the pACYC184 expression vector for pNatevgA-rrnBT

[0113] The construction process of the pNatevgA-rrnBT expression vector pACYC184-pNatevgA-rrnBT used in the examples of this application is as follows Figure 1 As shown:

[0114] The pBR322-pNatevgA-rrnBT plasmid obtained in Example 2.1.1 was extracted using a high-purity plasmid extraction kit from Tiangen. The pNatevgA-rrnBT (Xba I, Sal I) polynucleotide fragment was obtained by PCR amplification using the following forward and reverse primers according to conventional methods: upstream primer 5'-GAGATT TCTAGA GACTAAACCGTGGCTTTTGCAATA-3' (pAE-F1, SEQ ID NO: 14, the underlined bases are the restriction endonuclease Xba I recognition site), and the downstream primer 5'-TAGATT GTCGAC GGATCCTAGATATGAC-3' (pAE-R, SEQ ID NO: 15; the underlined bases indicate the restriction endonuclease Sal I recognition site). PCR was performed using Transgen Fast Pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 60°C for 20 sec, and 72°C for 30 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis. The expected band was amplified and recovered by gel separation. The PCR product was double-digested with restriction endonucleases Xba I and Sal I, and then ligated with plasmid pACYC184 (NEB) double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. The plasmid was extracted and sequenced. The sequencing results showed that the cloned pACYC184-pNatevgA-rrnBT sequence was correct.

[0115] 2.2 Construction of pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT acid-resistant expression cassette

[0116] 2.2.1 Construction of pBR322 intermediate cloning vectors for pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT

[0117] The construction process of the intermediate cloning vector pBR322-tempevgA-rrnBT used in the examples of this application is as follows Figure 2 As shown:

[0118] The pBR322-pNatevgA-rrnBT plasmid was extracted using a high-purity plasmid extraction kit from Tiangen. The evgA (Kpn I, Xho I) polynucleotide fragment was obtained by PCR amplification using the following forward and reverse primers according to conventional methods: upstream primer 5'-TCCCTT GGTACC GCATGAACGCAATAATTATTGATGAC-3' (evgA-K, SEQ ID NO: 16, the underlined bases are the restriction endonuclease Kpn I recognition site), and the downstream primer 5'-TACAGA CTCGAG TTAGCCGATTTTGTTAC-3' (evgA-X, SEQ ID NO: 17; the underlined bases indicate the restriction endonuclease Xho I recognition site). PCR was performed using Transgen Fast pfu polymerase. The reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 60°C for 20 sec, and 72°C for 30 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then separated and recovered from the gel. The PCR product was double-digested with restriction endonucleases Kpn I and Xho I, and then ligated with plasmid pBR322-temp2-rrnBT (a vector constructed by the inventors, the full-length sequence of which can be found in SEQ ID NO: 18) that had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced, and the sequencing results indicated that the sequence of the cloned pBR322 intermediate cloning vector was correct.

[0119] 2.2.2 Construction of 10 pBR322 intermediate expression vectors of pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT (pBR322-pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT)

[0120] The construction process of the 10 pBR322 intermediate expression vectors used in the examples of this application is as follows: Figure 2 As shown:

[0121] Using the 10 pUC57 plasmids containing the artificial promoter sequence synthesized in Example 1 as templates, the following two sets of forward primers and reverse primers were used to perform PCR amplification according to conventional methods to obtain 10 polynucleotide fragments containing partial sequences of the artificial promoter:

[0122] The first set of primers: upstream primer 5'-CGAGCC AAGCTT AAGCTTCAATTC-3' (5N-U: SEQ ID NO: 19, the underlined bases are the restriction endonuclease Hind III recognition site), and the downstream primer 5'-TTTAAT GAATTC GGTCAGTGCGTC-3' (5N-L: SEQ ID NO: 20, the underlined bases are the restriction endonuclease EcoRI recognition site) was used to amplify 9 polynucleotide fragments containing partial sequences of artificial promoters (pQ / pZ / pV / pW / pN / pS / pAA / pJJ / pOO).

[0123] Second set of primers: Upstream primer 5'-CGAGCC AAGCTT AAGCTTCAATC-3' (9II-U: SEQ ID NO: 21, the underlined bases are the restriction endonuclease Hind III recognition site) and a downstream primer (5N-L: SEQ ID NO: 20) were used to amplify a polynucleotide fragment (pII).

[0124] The PCR reaction used Transgen Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 minutes, followed by 30 cycles of 95°C for 20 seconds, 63°C for 20 seconds, and 72°C for 20 seconds, and 72°C for 5 minutes. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, revealing the expected correct band. The resulting PCR product was then separated and recovered by gel separation. The resulting PCR product was double-digested with restriction endonucleases Hind III and EcoRI and ligated with the intermediate cloning vector pBR322-tempevgA-rrnBT, which had been double-digested with the same enzymes. The ligation product was then transformed into competent Escherichia coli MG1655 cells. The transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was then extracted and sequenced, confirming the correct sequence of the cloned pBR322 intermediate expression vector.

[0125] 2.2.3 Construction of 10 pACYC184 expression vectors containing pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT (pACYC184-pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT)

[0126] The construction process of the 10 pACYC184 expression vectors used in the examples of this application is as follows: Figure 3 As shown:

[0127] Ten pBR322 intermediate expression vectors (pBR322-pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT) constructed in Example 2.2.2 were extracted using Tiangen's high-purity plasmid extraction kit as templates. The following four sets of forward and reverse primers were used to perform PCR amplification according to conventional methods to obtain polynucleotide fragments of ten artificial promoters -evgA-rrnBT (pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT) (Xba I, Sal I):

[0128] The first set of primers: upstream primer 5'-GAGATT TCTAGA AAGCTTCAATTCCGACGTCTAAGA-3' (pAE-F2, SEQ ID NO: 22, the underlined bases are the restriction endonuclease Xba I recognition site) and a downstream primer (pAE-R, SEQ ID NO: 15) were used to amplify seven polynucleotide fragments (pQevgA / pZevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA-rrnBT).

[0129] Second set of primers: Upstream primer 5'-GAGATT TCTAGA AAGCTTCAATTCCGACGTCTAAGA-3' (pAE-F3, SEQ ID NO: 23, the underlined bases are the restriction endonuclease Xba I recognition site) and a downstream primer (pAE-R, SEQ ID NO: 15) were used to amplify a polynucleotide fragment (pVevgA-rrnBT).

[0130] The third set of primers: upstream primer 5'-GAGATT TCTAGA AAGCTTCAATCCGACGTCTGAGA-3' (pAE-F4, SEQ ID NO: 24, the underlined bases are the restriction endonuclease Xba I recognition site) and a downstream primer (pAE-R, SEQ ID NO: 15) were used to amplify a polynucleotide fragment (pIIevgA-rrnBT).

[0131] The fourth set of primers: upstream primer 5'-GAGATT TCTAGA AAGCTTCAATTCCGACGTCTAAG-3' (pAE-F5, SEQ ID NO: 25, the underlined bases are the restriction endonuclease Xba I recognition site) and a downstream primer (pAE-R, SEQ ID NO: 15) were used to amplify a polynucleotide fragment (pOOevgA-rrnBT).

[0132] The PCR reaction used Transgen Fast pfu polymerase under the following conditions: 95°C for 5 minutes, followed by 30 cycles of 95°C for 20 seconds, 62°C for 20 seconds, and 72°C for 30 seconds, and finally 72°C for 5 minutes. After completion of the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis, revealing the expected correct band. The resulting PCR product was then separated and recovered by gel separation. The resulting PCR product was double-digested with restriction endonucleases Xba I and Sal I and ligated with the intermediate cloning vector pACYC184, which had been double-digested with the same enzymes. The ligation product was then transformed into competent Escherichia coli MG1655 cells, which were plated on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. Plasmids were then extracted and sequenced, confirming the correct sequences of the 10 pACYC184 expression vectors expressing the artificial promoter evgA.

[0133] 2.3 Construction of control plasmid pACYC184-rrnBT

[0134] The construction process of the control plasmid pACYC184-rrnBT used in the examples of this application is as follows Figure 4 As shown:

[0135] The pACYC184-pNatevgA-rrnBT plasmid obtained in Example 2.2 was extracted using Tiangen's high-purity plasmid miniprep kit. After double digestion with the restriction endonucleases Xba I and Xho I, the vector backbone was blunt-ended with T4 DNA polymerase (NEB) at 12°C for 30 minutes, followed by the addition of 1.5 μl of 0.2 M EDTA at 75°C for 20 minutes. After purification, the plasmid was ligated with T4 DNA ligase (NEB) and transformed into competent Escherichia coli MG1655 cells. Transformed cells were plated on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. The plasmid was extracted and sequenced, confirming the correct sequence for the cloned pACYC184-rrnBT.

[0136] The acid-resistant evgA expression cassettes constructed in Example 2 include pNatevgA-rrnBT and pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT, and their sequences are shown in SEQ ID NOs: 26-36, respectively.

[0137] Example 3: Construction of a three-gene acid-resistant expression cassette of ybaS-gadB-gadC

[0138] The pACYC184 expression plasmid pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT containing the acid-resistant triple gene expression cassette of ybaS-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT was constructed, wherein the ybaS promoter was the ybaS native promoter (SEQ ID NO: 70), and the gadB-gadC470 operon promoter was the gadB native promoter (SEQ ID NO: 72).

[0139] 3.1 Construction of intermediate cloning plasmid pACYC184-Hind III-pNatevgA-Xho I-rrnBT-BamH I

[0140] The construction process of the intermediate cloning plasmid pACYC184-Hind III-pNatevgA-Xho I-rrnBT-BamH I used in the examples of this application is as follows Figure 5 As shown:

[0141] The pBR322-pNatevgA-rrnBT plasmid obtained in Example 2.1.1 was extracted using a high-purity plasmid extraction kit from Tiangen Company. The pNatevgA-rrnBT (Hind III, Xho I) polynucleotide fragment was obtained by PCR amplification according to conventional methods using the following forward and reverse primers: upstream primer 5'-AATGAT AAGCTT GACTAAACCGTGGCTTTTGCA-3' (evgA-Fn, SEQ ID NO: 37, the underlined bases are the restriction endonuclease Hind III recognition site), and the downstream primer 5'-TAGATT GGATCCTAGATATGACGACAG-3' (evgA-Rn, SEQ ID NO: 38, underlined bases indicate the restriction endonuclease Xho I recognition site). PCR was performed using Transgen Fastpfu polymerase. The PCR reaction conditions were: 95°C for 5 minutes; 95°C for 20 seconds, 58°C for 20 seconds, and 72°C for 1 minute, for a total of 30 cycles; and 72°C for 5 minutes. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then separated and recovered from the gel. The PCR product was double-digested with restriction endonucleases Hind III and Xho I, and then ligated with plasmid pACYC184, which had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. The plasmid was extracted and sequenced. Sequencing results showed that the cloned pACYC184-Hind III-pNatevgA-Xho I-rrnBT-BamH I sequence was correct.

[0142] 3.2 Amplification of the ybaS-rrnBT-gadB polynucleotide fragment

[0143] The amplification of the ybaS-rrnBT-gadB polynucleotide fragment used in the examples of this application is as follows Figure 8 As shown:

[0144] PCR amplification of the ybaS polynucleotide fragment: Using the E. coli MG1655 genome as a template, PCR amplification was performed according to conventional methods using the following forward and reverse primers: upstream primer 5'-AATGAT AAGCTT TCGATTTGCATCAGGATTAGACATT-3' (yS-F1-A, SEQ ID NO: 39, the underlined bases are the restriction endonuclease Hind III recognition site), and the downstream primer 5'-GCCT TCTAGA TCAGCCCTTAAACAC-3' (yS-R, SEQ ID NO: 40; the underlined bases indicate the recognition site for the restriction endonuclease Xba I). ​​PCR was performed using Transgen Fast Pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 60°C for 20 sec, and 72°C for 1 min, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then separated and recovered from the gel.

[0145] PCR amplification of the rrnBT polynucleotide fragment: Using the pBR322-pNatevgA-rrnBT plasmid obtained in Example 2.1.1 as a template, PCR amplification was performed according to conventional methods using the following forward and reverse primers: upstream primer 5'-GTTTAAGGGCTGA TCTAGA The primers were AGGCATCAAATAAAACGAAAGGCTCGTCGAA-3' (rB-F, SEQ ID NO: 41; the underlined bases indicate the restriction endonuclease Xba I recognition site) and the downstream primer 5'-TATTGTTTAACGGCAGTCCCGGGTAGATATGACGACAGGAAGAGTTTGTAGAA-3' (rB-R, SEQ ID NO: 42; the underlined bases indicate the restriction endonuclease Xma I recognition site). PCR was performed using Transgen Fast Pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 60°C for 20 sec, and 72°C for 30 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis, revealing the expected correct band, which was then recovered by gel separation.

[0146] PCR amplification of gadB polynucleotide fragment: Using the E. coli MG1655 genome as a template, PCR amplification was performed according to conventional methods using the following forward primers and reverse primers: upstream primer 5'- CCCGGG The PCR reaction used the following primers: ACTGCCGTTAAACAATATCTTTCTC-3' (gBC-F, SEQ ID NO: 43; the underlined bases indicate the restriction endonuclease Xma I recognition site) and the downstream primer 5'-TGTTAATGGATAAATCCATCAATTTGT-3' (gBC-R, SEQ ID NO: 44). The PCR reaction used Transgen Fast PFU polymerase. The PCR reaction conditions were: 95°C for 5 minutes; 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds, for a total of 30 cycles; and 72°C for 5 minutes. After completion of the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis, which revealed the expected correct band. The product was then separated and recovered from the gel.

[0147] Overlapping PCR amplification of the ybaS-rrnBT-gadB polynucleotide fragment: Overlapping PCR reactions were performed using the previously obtained ybaS, rrnBT, and gadB as templates. Initially, without primers, PCR was performed using Transgen's Fastpfu polymerase. The reaction conditions were: 95°C for 5 min, 95°C for 20 sec, 65°C for 20 sec, and 72°C for 1 min 30 sec, for a total of 10 cycles. Then, an upstream primer (yS-F1-A, SEQ ID NO: 39) and a downstream primer (gBC-R, SEQ ID NO: 44) were added. PCR was performed using Transgen's Fastpfu polymerase. The reaction conditions were: 95°C for 5 min, 95°C for 20 sec, 65°C for 20 sec, and 72°C for 1 min 30 sec, for a total of 25 cycles. After completion of the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis. The expected bands were amplified and recovered by gel separation.

[0148] 3.3 Construction of intermediate cloning plasmid pBR322-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT

[0149] 3.3.1 Construction of the intermediate cloning plasmid pBR322-gadB-gadC470-rrnBT

[0150] The construction process of the intermediate cloning plasmid pBR322-gadB-gadC470-rrnBT used in the examples of this application is as follows Figure 6 As shown:

[0151] Using the E. coli MG1655 genome as a template, the gadB-gadC470 polynucleotide fragment was amplified by PCR using the following forward and reverse primers according to conventional methods: upstream primer 5'-AATGAA CCATGG ACTGCCGTTAAACAATATCTTTCTC-3' (Bp-For, SEQ ID NO: 45, the underlined bases are the restriction endonuclease Nco I recognition site), and the downstream primer 5'-TAGATT CTCGAGTTATTTACGATCATGAACAGCA-3' (BCp-Rev, SEQ ID NO: 46; the underlined bases indicate the restriction endonuclease Xho I recognition site). PCR was performed using Transgen Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 62°C for 20 sec, and 72°C for 2 min 20 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band, which was then recovered by gel separation. The PCR product was double-digested with restriction endonucleases Nco I and Xho I, and then ligated with plasmid pBR322-pNatevgA-rrnBT, which had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced, which showed that the cloned pBR322-gadB-gadC470-rrnBT sequence was correct.

[0152] 3.3.2 Construction of intermediate cloning plasmid pBR322-gadB(H465A)-gadC470-rrnBT

[0153] The construction process of the intermediate clone plasmid pBR322-gadB(H465A)-gadC470-rrnBT used in the examples of this application is as follows: Figure 7 As shown:

[0154] Using the E. coli MG1655 genome as a template, two polynucleotide fragments were amplified by PCR using the following two sets of forward primers and reverse primers according to conventional methods:

[0155] The first set of primers: an upstream primer (Bp-For, SEQ ID NO: 45), and a downstream primer 5'-CGTTAAACGTTATCAGGTAGCTTTAAAGCTGTTCTG-3' (H465A(C470)-rev, SEQ ID NO: 47).

[0156] The second set of primers: upstream primer 5'-AAAGCTACCTGATAACGTTTAACGGTAAC-3' (C470(H465A)-for, SEQ ID NO: 48), and downstream primer (BCp-Rev, SEQ ID NO: 46).

[0157] The PCR reaction used Transgen's Fast pfu polymerase, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 62°C 20 sec, 72°C 1 min 30 sec, for a total of 30 cycles; 72°C 5 min. After the reaction was completed, the PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the PCR amplified correct bands were consistent with the expectations and were recovered by gel separation. The two fragments obtained were used as templates for overlapping PCR reactions. First, without adding primers, Transgen's Fast pfu polymerase was used, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 65°C 20 sec, 72°C 2 min 30 sec, for a total of 10 cycles. An upstream primer (Bp-For, SEQ ID NO: 45) and a downstream primer (BCp-Rev, SEQ ID NO: 46) were then added, and PCR was performed using Transgen Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 minutes, followed by 25 cycles of 95°C for 20 seconds, 62°C for 20 seconds, and 72°C for 2 minutes and 30 seconds. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then recovered by gel separation.

[0158] The PCR product was double-digested with restriction endonucleases Nco I and Xho I, and then ligated with plasmid pBR322-pNatevgA-rrnBT, which had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced, which showed that the cloned pBR322-gadB-gadC470-rrnBT sequence was correct.

[0159] 3.3.3 Construction of the intermediate cloning plasmid pBR322-gadB(dHT)-gadC470-rrnBT

[0160] The construction process of the intermediate cloning plasmid pBR322-gadB(dHT)-gadC470-rrnBT used in the examples of this application is as follows: Figure 7 As shown:

[0161] The first set of primers: an upstream primer (Bp-For, SEQ ID NO: 45), and a downstream primer 5'-GTTACCGTTAAACGTTATCATTTAAAGCTGTTCTGTTGGG-3' (dHT(C470)-rev, SEQ ID NO: 49).

[0162] The second set of primers: upstream primer 5'-GAACAGCTTTAAATGATAACGTTTAACGGTAAC-3' (C470(dHT)-for, SEQ ID NO: 50), and downstream primer (BCp-Rev, SEQ ID NO: 46).

[0163] The PCR reaction used Transgen's Fast pfu polymerase, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 62°C 20 sec, 72°C 1 min 30 sec, for a total of 30 cycles; 72°C 5 min. After the reaction was completed, the PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the PCR amplified correct bands were consistent with the expectations and were recovered by gel separation. The two fragments obtained were used as templates for overlapping PCR reactions. First, without adding primers, Transgen's Fast pfu polymerase was used, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 65°C 20 sec, 72°C 2 min 30 sec, for a total of 10 cycles. An upstream primer (Bp-For, SEQ ID NO: 45) and a downstream primer (BCp-Rev, SEQ ID NO: 46) were then added, and PCR was performed using Transgen Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 minutes, followed by 25 cycles of 95°C for 20 seconds, 62°C for 20 seconds, and 72°C for 2 minutes and 30 seconds. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then recovered by gel separation.

[0164] The PCR product was double-digested with restriction endonucleases Nco I and Xho I, and then ligated with plasmid pBR322-pNatevgA-rrnBT, which had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced, which showed that the cloned pBR322-gadB-gadC470-rrnBT sequence was correct.

[0165] 3.3.4 Construction of intermediate cloning plasmid pBR322-gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT

[0166] The construction process of the intermediate cloning plasmid pBR322-gadB (H465A & E89Q) / gadB (dHT & E89Q) - gadC470-rrnBT used in the examples of this application is as follows Figure 7 As shown:

[0167] The pBR322-gadB(H465A)-gadC470-rrnBT plasmid obtained in Example 3.3.2 was extracted using Tiangen's high-purity plasmid mini-preparation kit as a template. Two polynucleotide fragments were amplified by PCR using the following two sets of forward and reverse primers, respectively, according to conventional methods:

[0168] The first set of primers: an upstream primer (Bp-For, SEQ ID NO: 45), and a downstream primer 5'-GGATTGCGGATACTGTTCTTTGTC-3' (E89Q-rev, SEQ ID NO: 51).

[0169] The second set of primers: upstream primer 5'-GACAAAGAACAGTATCCGCAATCC-3' (E89Q-for, SEQ ID NO: 52), and downstream primer (BCp-Rev, SEQ ID NO: 46).

[0170] The PCR reaction used Transgen's Fast pfu polymerase, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 63°C 20 sec, 72°C 1 min 30 sec, for a total of 30 cycles; 72°C 5 min. After the reaction was completed, the PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that the PCR amplified correct bands were consistent with the expected ones, and the bands were separated and recovered by gel separation. The two fragments obtained were used as templates for overlapping PCR reactions. First, without adding primers, Transgen's Fast pfu polymerase was used, and the PCR reaction conditions were: 95°C, 5 min; 95°C 20 sec, 65°C 20 sec, 72°C 2 min 30 sec, for a total of 10 cycles. An upstream primer (Bp-For, SEQ ID NO: 45) and a downstream primer (BCp-Rev, SEQ ID NO: 46) were then added, and PCR was performed using Transgen Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 minutes, followed by 25 cycles of 95°C for 20 seconds, 62°C for 20 seconds, and 72°C for 2 minutes and 30 seconds. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then recovered by gel separation.

[0171] The resulting PCR product was double-digested with restriction endonucleases Nco I and Xho I, and then ligated with plasmid pBR322-pNatevgA-rrnBT, which had been double-digested with the same enzymes. The ligation product was transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 50 μg / mL ampicillin to screen for positive clones. The plasmid was extracted and sequenced, which indicated that the cloned pBR322-gadB(H465A&E89Q)-gadC470-rrnBT sequence was correct.

[0172] Using the pBR322-gadB(dHT)-gadC470-rrnBT plasmid obtained in Example 3.3.3 as a template, pBR322-gadB(dHT&E89Q)-gadC470-rrnBT was cloned through the same steps. The sequencing results showed that the sequence was correct.

[0173] 3.4 Amplification of the gadB-gadC470 / gadB(H465A)-gadC470 / gadB(dHT)-gadC470 / gadB(H465A&E89Q)-gadC470 / gadB(dHT&E89Q)-gadC470 polynucleotide fragment

[0174] The amplification of the polynucleotide fragments used in the examples of this application is as follows Figure 8 As shown:

[0175] The five intermediate clone plasmids obtained in Example 3.3 were extracted using the Tiangen high-purity plasmid extraction kit as templates, and the five polynucleotide fragments were amplified by PCR according to conventional methods using the following forward primers and reverse primers: upstream primer 5'-ACAAATTGATGGATTTATCCATTAACA-3' (gadC470-F, SEQ ID NO: 53), and downstream primer 5'-TAGATT CTCGAG TTATTTACGATCATGAAC-3' (gC-R1, SEQ ID NO: 54; the underlined bases indicate the restriction endonuclease Xho I recognition site). PCR was performed using Transgen Fast pfu polymerase. The reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 62°C for 20 sec, and 72°C for 1 min 45 sec, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then separated and recovered from the gel.

[0176] 3.5 Amplification of the ybaS-rrnBT-gadB-gadC470 / ybaS-gadB(H465A)-gadC470 / ybaS-gadB(dHT)-gadC470 / ybaS-gadB(H465A&E89Q)-gadC470 / ybaS-gadB(dHT&E89Q)-gadC470 polynucleotide fragment

[0177] The amplification of the polynucleotide fragments used in the examples of this application is as follows Figure 8 As shown:

[0178] Overlapping PCR amplification of the five polynucleotide fragments described above: Overlapping PCR reactions were performed using the five polynucleotide fragments obtained in Example 3.4 and ybaS-rrnBT-gadB obtained in Example 3.2 as templates. Initially, without adding primers, PCR was performed using Transgen's Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 61°C for 20 sec, and 72°C for 3 min, for a total of 10 cycles. Then, an upstream primer (yS-F1-A, SEQ ID NO: 39) and a downstream primer (gC-R1, SEQ ID NO: 54) were added, and PCR was performed using Transgen's Fast pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 61°C for 20 sec, and 72°C for 3 min, for a total of 25 cycles. After completion of the reaction, the PCR amplification products were analyzed by 1% agarose gel electrophoresis. The expected bands were amplified and recovered by gel separation.

[0179] 3.6 Construction of the ybaS-gadB-gadC triple gene acid-resistant expression cassette expression plasmid pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT

[0180] The construction process of the three-gene acid-resistant expression cassette expression plasmid pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT used in the examples of this application is as follows: Figure 8 As shown:

[0181] The five PCR products obtained in Example 3.5 were double-digested with restriction endonucleases Hind III and Xho I, and then ligated with the plasmid pACYC184-Hind III-pNatevgA-Xho I-rrnBT-BamH I obtained in Example 3.1, which had been double-digested with the same enzymes. The ligation products were transformed into Escherichia coli MG1655 competent cells, and the transformed cells were plated on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. The plasmids were extracted and sequenced. The sequencing results indicated that the sequences of the five cloned ybaS-gadB-gadC triple gene acid-resistant expression cassette expression plasmids were correct.

[0182] The five ybaS-gadB-gadC triple-gene acid-resistant expression cassettes constructed in this Example 3 include ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT, and their sequences are shown in SEQ ID NOs: 55-59, respectively.

[0183] Example 4: Construction of the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette

[0184] The construction process of the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette expression plasmid pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT-pSevgA-rrnBT used in the examples of the present application is as follows: Figure 9 As shown:

[0185] The pBR322-pSevgA-rrnBT plasmid obtained in Example 2.2 was extracted using a high-purity plasmid extraction kit from Tiangen Company. The pSevgA-rrnBT polynucleotide fragment was amplified by PCR using the following forward and reverse primers according to conventional methods: upstream primer 5'-AATGAA GGATCC CAATTCCGACGTCTAAGAAACCATTATT-3' (pAYE-2, SEQ ID NO: 60, the underlined bases are the restriction endonuclease BamHI recognition site), and the downstream primer 5'-TAGATT CCTGCATTAGGGACGTCTAGATATGACGACAGGAAGAGTTTGTAGAA-3' (pAYE-R, SEQ ID NO: 61; the underlined bases indicate the EcoNI restriction endonuclease recognition site). PCR was performed using Transgen Fast Pfu polymerase. The PCR reaction conditions were: 95°C for 5 min; 95°C for 20 sec, 62°C for 20 sec, and 72°C for 1 min, for a total of 30 cycles; and 72°C for 5 min. After completion of the reaction, the PCR amplification product was analyzed by 1% agarose gel electrophoresis, which revealed the expected band. The product was then separated and recovered from the gel. The obtained PCR product was double-digested with restriction endonucleases BamH I and EcoN I, and then ligated with the five ybaS-gadB-gadC three-gene acid-resistant expression cassette expression plasmids obtained in Example 3.6, which had been double-digested with the same enzymes. The ligation products were transformed into Escherichia coli MG1655 competent cells, and the transformed cells were spread on LB plates supplemented with 34 μg / mL chloramphenicol to screen for positive clones. The plasmids were extracted and sequenced. The sequencing results showed that the sequences of the five cloned ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette plasmids were correct.

[0186] The five ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassettes constructed in this Example 4 include ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT-pSevgA-rrnBT, and their sequences are shown in SEQ ID NOs: 62-66, respectively.

[0187] Example 5: Effect of the Acid Resistance Expression Cassette on Extreme Acid Shock Survival of Escherichia coli MG1655

[0188] The evgA acid-resistant expression cassette expression plasmid obtained in Example 2 (pACYC184-pNatevgA / pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT) and the ybaS-gadB-gadC three-gene acid-resistant expression cassette expression plasmid obtained in Example 3 (pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(d The ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette expression plasmid (pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT-pSevgA-rrnBT) obtained in Example 4, and the control plasmid (pACYC184-rrnBT) were respectively transformed into Escherichia coli MG1655 competent cells, and positive clones were obtained by colony PCR and plasmid sequencing. The bacteria were respectively inoculated into LB medium and cultured overnight at 37°C and 250rpm, mixed at a ratio of 0.8mL of bacterial solution and 0.2mL of 60% glycerol, and frozen in a -80°C refrigerator. The corresponding strains were named as: control blank strain MG, control plasmid strain RB; strains containing evgA acid-resistant expression cassette En / E1 / E2 / E3 / E4 / E5 / E6 / E7 / E8 / E9 / E10; strains containing ybaS-gadB-gadC three-gene acid-resistant expression cassette YBC1 / YBC2 / YBC3 / YBC4 / YBC5; strains containing ybaS-gadB-gadC four-gene acid-resistant expression cassette YBC1E6 / YBC2E6 / YBC3E6 / YBC4E6 / YBC5E6.

[0189] The above-mentioned frozen strains containing the acid-fast expression cassette plasmid, the control plasmid strain and E. coli MG1655 were restored to LB solid medium plates (for E. coli MG1655, no antibiotics were added; for the strains containing the acid-fast expression cassette plasmid and the control plasmid, 34 μg / mL chloramphenicol was added; the same below), and cultured at 37°C overnight. The bacteria were inoculated into LB medium (LBG) supplemented with 2% glucose at 37°C and 250 rpm overnight, and then transferred to fresh LBG medium at a ratio of 1:100 and cultured at 37°C and 250 rpm for about 1.5 hours. When the bacterial solution OD 600When the OD value reaches 0.5-0.6, transfer to fresh LBG-pH 2.5 medium (LBG medium is adjusted to pH 2.5 by hydrochloric acid) and perform acid shock for 2 hours. The initial OD value is 600 The bacterial suspension after shock was diluted with fresh non-resistant LBG medium at a dilution ratio of 1:10, 1:100, 1:1000, and 1:10000, and 4 μL was spotted on a non-resistant LB solid medium plate. The plates were cultured at 37°C overnight and the growth of colonies on the plates was observed.

[0190] result:

[0191] 1. For the evgA acid-resistant expression cassette, survival after shock is as follows Figure 10 shown.

[0192] (1) The survival of the control strains MG and RB after acid shock was almost zero.

[0193] (2) Strain containing the acid-resistant expression cassette of evgA from the natural promoter (En): The survival rate after acid shock was about 10 higher than that of the control strains MG and RB. 4 times.

[0194] (3) Strains containing evgA acid-resistant expression cassettes with 10 constitutive artificial promoters of different strengths (E1 / E2 / E3 / E4 / E5 / E6 / E7 / E8 / E9 / E10): The strains with stronger promoter expression strength (E1 / E2 / E3 / E4 / E5 / E6) showed a 10% improvement in survival after acid shock compared with the control strains MG and RB. 4 The strains with weaker promoter expression (E7 / E8 / E9) showed a 10-fold increase in survival after acid shock compared to the control strains MG and RB. 2 ~10 3 The strain with the weakest promoter expression strength (E10) had a survival rate similar to that of the control strains MG and RB after acid shock.

[0195] 2. For the acid-resistant expression cassette of the ybaS-gadB-gadC triple gene, survival after shock is as follows: Figure 11 shown.

[0196] (1) The strains containing the three-gene acid-resistant expression cassette (YBC1 / YBC2 / YBC4) showed a 2- to 10-fold increase in survival after acid shock compared to the control strains MG and RB.

[0197] (2) The strain containing the three-gene acid-resistant expression cassette (YBC5) had a survival rate of about 10% higher after acid shock than the control strains MG and RB. 2 times.

[0198] (3) The strain containing the three-gene acid-resistant expression cassette (YBC3) had a survival rate of about 10% higher after acid shock than the control strains MG and RB.3 times.

[0199] 3. For the acid-resistant expression cassette of the ybaS-gadB-gadC-evgA four-gene gene, survival after shock is as follows: Figure 12 shown.

[0200] (1) The strains containing the four-gene acid-resistant expression cassette (YBC1E6 / YBC2E6 / YBC3E6 / YBC4E6 / YBC5E6) showed a 10% improvement in survival after acid shock compared to the control strains MG and RB. 2 ~10 3 times, and about 10 times lower than that of the strain containing the evgA acid-resistant expression cassette (E6).

[0201] (2) The strains containing four acid-resistant gene expression cassettes (YBC1E6 / YBC2E6 / YBC4E6 / YBC5E6) showed a survival rate of approximately 10% after acid shock compared to the corresponding strains containing three acid-resistant gene expression cassettes (YBC1 / YBC2 / YBC4 / YBC5). 2 The strain containing four acid-resistant gene expression cassettes (YBC3E6) had a survival rate about 10 times higher after acid shock than the corresponding strain containing three acid-resistant gene expression cassettes (YBC3).

[0202] This Example 5 illustrates that: evgA acid-resistant expression cassettes of different expression intensities bring about different degrees of improvement in the strain's survival under acid shock, with the stronger the expression, the more significant the improvement in survival; the ybaS-gadB-gadC three-gene acid-resistant expression cassette brings about an improvement in the strain's survival under acid shock; and the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette can bring about improved strain acid shock survival compared to the corresponding ybaS-gadB-gadC three-gene acid-resistant expression cassette.

[0203] Example 6: Effect of the acid-resistant expression cassette on acid-stress growth

[0204] The strain containing the acid-resistant expression cassette plasmid obtained in Example 5, the strain containing the control plasmid, and Escherichia coli MG1655 were inoculated into LBG medium and cultured overnight at 37°C and 250 rpm. At an initial OD6000 of 0.05, the culture was transferred to 300 μL of fresh lysine fermentation medium with an initial pH of 7.0 (or supplemented with 10 mM glutamine and 10 mM sodium glutamate). High-throughput growth assays were performed using a fully automated growth curve analyzer, Bioscreen C (Oy Growth Curves Ab Ltd, Finland) and 100-well plates (Honeycompb Plate, two plates can be used simultaneously).

[0205] The composition of the lysine fermentation medium used in the growth test is shown in Table 2. The medium pH was adjusted to 7.0 with KOH. Glucose and magnesium sulfate were sterilized separately and then added to the medium. As the growth test progressed, the medium pH gradually decreased to approximately 4.8, meeting the acid stress assessment for the growth test.

[0206] Table 2 Composition of lysine fermentation medium in growth test

[0207] Culture medium components concentration

[0208] glucose 75g / L corn steep liquor 100g / L <![CDATA[(NH4)2SO4]]> 40g / L <![CDATA[KH2PO4]]> 1g / L <![CDATA[MgSO4·7H2O]]> 0.4g / L <![CDATA[FeSO4·7H2O]]> 0.01g / L <![CDATA[MnSO4·H2O]]> 6mg / L D-Biotin 0.3mg / L Thiamine hydrochloride 0.5mg / L D-Pantothenic acid 0.01g / L

[0209] result:

[0210] 1. For the evgA acid-resistant expression cassette, the acid stress growth test results are as follows: Figure 13 shown.

[0211] After 24 hours of growth in fermentation medium without glutamine and sodium glutamate, the growth of each strain (ΔOD at the initial time and 24 hours measured by Bioscreen C) 600 ) as shown in Table 3.

[0212] Table 3 Acid stress growth test of strains containing evgA acid-resistant expression cassette

[0213]

[0214] (1) The strain (En) containing the pNatevgA-rrnBT acid-resistant expression cassette had a 24-hour growth rate higher than that of the control strains MG and RB, with the growth rate being 8.6% higher than that of the control strain MG.

[0215] (2) Strains containing evgA acid-resistant expression cassettes under artificial promoters of different strengths, among which: strains E1 / E2 / E3 / E4 / E5 / E6 / E7, 24-hour growth rates were higher than those of the control strains MG and RB, with increases of 23.3% / 28.0% / 42.3% / 25.1% / 17.9% / 11.6% / 2.7% respectively compared with the control strain MG; strain E8, 24-hour growth rate was 0.4% higher than that of the control strain MG, and 2.0% lower than that of the control strain RB; strains E9 / E10, 24-hour growth rates were lower than those of the control strains MG and RB, with decreases of 3.8% / 5.4% respectively compared with the control strain MG.

[0216] (3) The evgA acid-resistant expression cassettes with different expression intensities brought about different degrees of improvement in the strains' tolerance to acid stress growth. Among them, the acid-resistant expression cassette pVevgA-rrnBT was able to improve the acid stress growth tolerance of the control strain Escherichia coli MG1655 in the above culture medium, with a 42.3% increase in growth at 24 hours.

[0217] 2. For the acid-resistant expression cassette of the ybaS-gadB-gadC triple gene, the results of the acid stress growth test are as follows: Figure 14 shown.

[0218] Without adding ( Figure 14 A) or add ( Figure 14 B) Growth of each strain after 24 hours of growth in fermentation medium containing 10 mM glutamine and 10 mM sodium glutamate (ΔOD between the initial time and the 24-hour time measured by Bioscreen C) 600 ) as shown in Table 4.

[0219] Table 4 Acid stress growth test of strains containing the acid-resistant expression cassette of the ybaS-gadB-gadC gene

[0220]

[0221] (1) When no additives were added, the strains containing the acid-resistant expression cassette of the ybaS-gadB-gadC gene (YBC1 / YBC2 / YBC3 / YBC4 / YBC5) had lower 24-hour growth rates than the control strains MG and RB.

[0222] (2) When added, the strains containing the acid-resistant expression cassette of the ybaS-gadB-gadC gene (YBC1 / YBC2 / YBC3 / YBC4 / YBC5) showed higher 24-hour growth than the control strains MG and RB, with increases of 40.6% / 36.2% / 64.6% / 28.9% / 54.8% respectively compared with the control strain MG.

[0223] (3) The acid-resistant expression cassette of the ybaS-gadB-gadC triple gene can improve the acid stress tolerance of the control strain Escherichia coli MG1655 in the above culture medium when glutamine and sodium glutamate are added.

[0224] 3. For the acid-resistant expression cassette of the ybaS-gadB-gadC-evgA four-gene, the results of the acid stress growth test are as follows: Figure 15 shown.

[0225] Without adding ( Figure 15 A) or add ( Figure 15 B) Growth of each strain after 24 hours of growth in fermentation medium containing 10 mM glutamine and 10 mM sodium glutamate (ΔOD between the initial time and the 24-hour time measured by Bioscreen C) 600 ) as shown in Table 5.

[0226] Table 5 Acid stress growth test of strains containing the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette

[0227]

[0228] (1) When no additives were added, the strains containing the acid-resistant expression cassette of the four genes ybaS-gadB-gadC-evgA (YBC1E6 / YBC2E6 / YBC3E6 / YBC4E6 / YBC5E6) showed higher 24-hour growth than the control strains MG and RB, with increases of 57.6% / 54.3% / 68.7% / 42.4% / 57.6% compared with the control strain MG, respectively.

[0229] (2) When added, the strains containing the acid-resistant expression cassette of the four genes ybaS-gadB-gadC-evgA (YBC1E6 / YBC2E6 / YBC3E6 / YBC4E6 / YBC5E6) showed higher 24-hour growth than the control strains MG and RB, with increases of 72.5% / 68.1% / 86.1% / 71.7% / 70.6% respectively compared with the control strain MG.

[0230] (3) The acid-resistant expression cassette of the four genes ybaS-gadB-gadC-evgA can improve the acid stress tolerance of the control strain Escherichia coli MG1655 in the above culture medium when glutamine or sodium glutamate is not added or added.

[0231] Example 7: Effect of acid-resistant expression cassette on lysine fermentation

[0232] The evgA acid-resistant expression cassette expression plasmid obtained in Example 2 (pACYC184-pNatevgA / pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pJJevgA / pIIevgA / pOOevgA-rrnBT), the ybaS-gadB-gadC three-gene acid-resistant expression cassette expression plasmid obtained in Example 3 (pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT), and the ybaS-gadB-gadC three-gene acid-resistant expression cassette expression plasmid obtained in Example 4 were transformed by electroporation. The ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette expression plasmid (pACYC184-ybaS-rrnBT-gadB / gadB(H465A) / gadB(dHT) / gadB(H465A&E89Q) / gadB(dHT&E89Q)-gadC470-rrnBT-pSevgA-rrnBT) was transformed into a modified lysine-producing Escherichia coli MG1655 strain (the strain is based on the MG1655 strain and is modified according to the method disclosed in Chinese patent application CN103773745A to obtain the lysine-producing strain SCEcL3(pSLL1)) and electroporated competent cells, and positive clones were obtained by colony PCR and plasmid sequencing. The bacteria were inoculated into LB medium and cultured overnight at 37°C and 250 rpm. 0.8 mL of the bacterial suspension was mixed with 0.2 mL of 60% glycerol and stored at -80°C. The corresponding strains were designated as follows: parental lysine-producing strain P; strains containing the evgA acid-resistant expression cassette PEn / PE1 / PE2 / PE3 / PE4 / PE5 / PE6 / PE7 / PE8 / PE9 / PE10; strains containing the ybaS-gadB-gadC triple-gene acid-resistant expression cassette PYBC1 / PYBC2 / PYBC3 / PYBC4 / PYBC5; and strains containing the ybaS-gadB-gadC quadruple-gene acid-resistant expression cassette PYBC1E6 / PYBC2E6 / PYBC3E6 / PYBC4E6 / PYBC5E6.

[0233] The frozen lysine-producing strain containing the acid-resistant expression cassette plasmid and the parent lysine-producing strain were restored to LB solid medium plates (for the parent lysine-producing strain, 50 μg / mL ampicillin mL was added; for the lysine-producing strain containing the acid-resistant expression cassette plasmid, 50 μg / mL ampicillin mL and 34 μg / mL chloramphenicol were added) and cultured overnight at 37°C. Each strain was inoculated into 2 mL of LB medium and cultured overnight at 37°C and 200 rpm. The strains were transferred to 1.2 mL of fermentation medium at a ratio of 1:10, and high-throughput acid pressure fermentation tests were performed using a microbioreactor BioLector (m2p-labs GmbH, Germany) and a 48-well plum blossom plate (MTP-48-Flowerplate, LOT 1401-hc-Temp37).

[0234] The fermentation medium was as follows: glucose 40 g / L, ammonium sulfate 10 g / L, phosphoric acid 0.6 mL / L, potassium chloride 0.8 g / L, betaine 0.4 g / L, magnesium sulfate 1.2 g / L, manganese sulfate 0.03 g / L, ferrous sulfate 0.03 g / L, corn steep liquor organic nitrogen 0.4 g / L, 5% defoamer 0.5 mL / L, and threonine 0.2 g / L. The pH of the medium was adjusted to 7.0 with aqueous ammonia. Glucose and magnesium sulfate were sterilized separately and then added to the medium. In the fermentation test, the initial pH of the fermentation medium was 7.0. After about 6 hours of fermentation, the pH dropped to 6.0. After that, the pH was maintained at 6.0 by adding aqueous ammonia. The fermentation was carried out for a total of 48 hours in a BioLector. The lysine·HCl content in the fermentation broth was analyzed using a biosensor analyzer SBA-40E (Institute of Biology, Shandong Academy of Sciences).

[0235] result:

[0236] For the ybaS-gadB-gadC three-gene acid-resistant expression cassette and the ybaS-gadB-gadC-evgA four-gene acid-resistant expression cassette, the results after 48 hours of fermentation (the lysine·HCl production of the parent lysine-producing strain P was taken as 100%) are shown in Table 6.

[0237] Table 6 contains the acid-resistant expression cassette of the three genes ybaS-gadB-gadC, ybaS-gadB-gadC-evgA

[0238] Acid-pressure fermentation test of lysine-producing strains with four-gene acid-resistant expression cassette

[0239]

[0240] (1) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, the 48-h lysine·HCl production of the lysine-producing strain PE6 containing the evgA acid-resistant expression cassette pSevgA-rrnBT was 55.0% of that of the parental lysine-producing strain P, which was lower than that of the parental lysine-producing strain P.

[0241] (2) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, for the lysine-producing strain PYBC1 containing the acid-resistant expression cassette ybaS-gadB-gadC three genes ybaS-rrnBT-gadB-gadC470-rrnBT, the 48-hour lysine·HCl production was 66.3% of that of the parent lysine-producing strain P, which was lower than that of the parent lysine-producing strain P; for the lysine-producing strain PYBC1E6 containing the acid-resistant expression cassette ybaS-gadB-gadC-evgA four genes ybaS-rrnBT-gadB-gadC470-rrnBT-pSevgA-rrnBT, the 48-hour lysine·HCl production was 116% of that of the parent lysine-producing strain P, which was higher than that of the parent lysine-producing strain P.

[0242] (3) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, for the lysine-producing strains PYBC2 / PYBC3 containing the acid-resistant expression cassette ybaS-gadB-gadC three genes ybaS-rrnBT-gadB(H465A / dHT)-gadC470-rrnBT, the 48-hour lysine·HCl production was 68.7% and 87.4% of that of the parent lysine-producing strain P, respectively, which was lower than that of the parent lysine-producing strain P; for the lysine-producing strains PYBC2E6 / PYBC3E6 containing the acid-resistant expression cassette ybaS-gadB-gadC-evgA four genes ybaS-rrnBT-gadB(H465A / dHT)-gadC470-rrnBT-pSevgA-rrnBT, the 48-hour lysine·HCl production was 72.4% and 70.0% of that of the parent lysine-producing strain P, respectively, which was lower than that of the parent lysine-producing strain P.

[0243] (4) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, for the lysine-producing strains PYBC4 / PYBC5 containing the acid-resistant expression cassette of the ybaS-gadB-gadC triple gene ybaS-rrnBT-gadB(H465A&E89Q / dHT&E89Q)-gadC470-rrnBT, the 48-hour lysine·HCl production was 165% and 215% of that of the parental lysine-producing strain P, respectively, which was higher than that of the parental lysine-producing strain P; for the strain containing ybaS The lysine-producing strains PYBC4E6 / PYBC5E6, which expressed the acid-resistant four-gene expression cassette aS-gadB-gadC-evgA ybaS-rrnBT-gadB(H465A&E89Q / dHT&E89Q)-gadC470-rrnBT-pSevgA-rrnBT, had lysine·HCl production of 43.8% and 68.7% of that of the parental lysine-producing strain P in 48 hours, respectively, which was lower than that of the parental lysine-producing strain P.

[0244] For the evgA acid-resistant expression cassette, the results after 48 hours of fermentation (the lysine·HCl production of the parent lysine-producing strain was taken as 100%) are shown in Table 7.

[0245] Table 7 Acid pressure fermentation test of lysine-producing strains containing evgA acid-resistant expression cassette

[0246]

[0247] (1) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, the lysine-producing strain PEn containing the evgA acid-resistant expression cassette pNatevgA-rrnBT had a 48-hour lysine·HCl production of 58.5% of that of the parental lysine-producing strain P, which was lower than that of the parental lysine-producing strain P.

[0248] (2) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, the lysine-producing strains PE8 / PE9 containing the evgA acid-resistant expression cassette pJJevgA / pIIevgA-rrnBT had lysine·HCl production of 312% and 171% of that of the parental lysine-producing strain P in 48 h, respectively, which were higher than that of the parental lysine-producing strain P.

[0249] (3) Under the above fermentation medium and acid pressure fermentation conditions of pH 6.0, the lysine·HCl production of the lysine-producing strains PE1 / PE2 / PE3 / PE4PE5 / PE6 / PE7 / PE10 containing the evgA acid-resistant expression cassette pQevgA / pZevgA / pVevgA / pWevgA / pNevgA / pSevgA / pAAevgA / pOOevgA-rrnBT within 48 h was 88.1%, 89.6%, 93.3%, 77.7%, 79.3%, 45.1%, 68.9% and 53.4% ​​of that of the parental lysine-producing strain P, respectively, which was lower than that of the parental lysine-producing strain P.

[0250] Conclusion: Although all acid-resistant expression cassettes constructed in the examples improved acid resistance in E. coli after transformation, surprisingly, different combinations of promoters, acid-resistant genes, and terminators had significantly different effects on E. coli fermentation. For lysine fermentation production, expression cassettes PYBC1E6, PYBC4, PYBC5, PE8, and PE9 achieved unexpectedly excellent results.

[0251] References

[0252] 1. Lin, Z., Y. Zhang, and J. Wang, Engineering of transcriptional regulators microbial stress tolerance. Biotechnol Adv, 2013.31(6): p.986-91.

[0253] 2.Lin,J.,et al.,Comparative analysis of extreme acid survival inSalmonella typhimurium,Shigella flexneri,and Escherichia coli.J Bacteriol,1995.177(14):p.4097-104.

[0254] 3.Lu, PL, et al., L-glutamine provides acid resistance for Escherichiacoli through enzymatic release of ammonia. Cell Research, 2013.23(5):p.635-644.

[0255] 4.Capitani,G.,et al.,Crystal structure and functional analysis ofEscherichia coli glutamate decarboxylase.EMBO J,2003.22(16):p.4027-37.

[0256] 5.Hersh,B.M.,et al.,A glutamate-dependent acid resistance gene inEscherichia coli.J Bacteriol,1996.178(13):p.3978-81.

[0257] 6.Masuda,N.and G.M.Church,Regulatory network of acid resistance genesin Escherichia coli.Mol Microbiol,2003.48(3):p.699-712.

[0258] 7.Masuda,N.and G.M.Church,Escherichia coli gene expression responsiveto levels of the response regulator EvgA.Journal of Bacteriology,2002.184(22):p.6225-6234.

[0259] 8.Alper,H.,et al.,Tuning genetic control through promoterengineering.Proceedings of the National Academy of Sciences of the UnitedStates of America,2005.102(36):p.12678-12683.

[0260] 9.Orosz,A.,I.Boros,and P.Venetianer,Analysis of the ComplexTranscription Termination Region of the Escherichia-Coli Rrnb Gene.EuropeanJournal of Biochemistry,1991.201(3):p.653-659.

[0261] 10.Ho,N.A.T.,et al.,Expanding the active pH range of Escherichiacoliglutamate decarboxylase by breaking the cooperativeness.Journal ofBioscience and Bioengineering,2013.115(2):p.154-158.

[0262] 11.Ma,D.,et al.,Structure and mechanism of a glutamate-GABAantiporter.Nature,2012.483(7391):p.632-U161.

Claims

1. An expression cassette, which consists of one or more promoters, one or more acid-resistant genes and one or more terminators, wherein the acid-resistant gene is selected from the group consisting of evgA gene, ybaS gene, gadB gene and gadC gene, wherein the expression cassette i) consisting of the promoter of SEQ ID NO: 8 or 9, the evgA gene, and the rrnBT terminator from 5' to 3'; or ii) consisting of two parts, the first part comprising, from 5' to 3', the ybaS gene promoter, the ybaS gene, and the rrnBT terminator, and the second part comprising, from 5' to 3', the gadB gene promoter, the gadB gene, the gadC gene, and the rrnBT terminator, wherein the gadB gene encodes the amino acid sequence shown in SEQ ID NO: 76 or 77; or iii) consisting of three parts, the first part comprising the ybaS gene promoter, the ybaS gene, and the rrnBT terminator from 5' to 3', the second part comprising the gadB gene promoter, the gadB gene, the gadC gene, and the rrnBT terminator from 5' to 3', and the third part comprising the promoter of SEQ ID NO: 6, the evgA gene, and the rrnBT terminator from 5' to 3', in, The expression cassette is used for producing lysine by microbial fermentation. 2 . The expression cassette according to claim 1 , which is capable of improving the acid resistance of a host cell after being introduced into the host cell.

3. The expression cassette of claim 1, wherein the evgA gene encodes the amino acid sequence shown in SEQ ID NO:

68.

4. The expression cassette of claim 1, wherein the ybaS gene encodes the amino acid sequence shown in SEQ ID NO:

71.

5. The expression cassette of claim 1, wherein the gadB gene in iii) encodes the amino acid sequence shown in SEQ ID NO:

73. The expression cassette of claim 1 , wherein the gadC gene encodes the amino acid sequence shown in SEQ ID NO:

78.

7. The expression cassette of claim 1, wherein the nucleotide sequence of the ybaS gene promoter is shown in SEQ ID NO: 70, and the nucleotide sequence of the gadB gene promoter is shown in SEQ ID NO:

72.

8. The expression cassette of claim 1, wherein the nucleotide sequence of the rrnBT terminator is shown in SEQ ID NO:

69.

9. The expression cassette of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO: 34 or 35.

10. The expression cassette of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO: 58 or 59. The expression cassette of claim 1 , wherein the nucleotide sequence is shown in SEQ ID NO:

62.

12. An expression construct comprising the expression cassette of any one of claims 1 to 11.

13. A recombinant host cell comprising the expression cassette of any one of claims 1 to 11 or the expression construct of claim 12, wherein the recombinant host cell is an Escherichia coli SCEcL3 (pSLL1) strain cell.

14. The recombinant host cell of claim 13, which has increased acid resistance compared to a corresponding cell not containing the expression cassette or expression construct.

15. The recombinant host cell of claim 14, wherein the acid resistance comprises survival rate under acid shock and growth rate under acid stress conditions.

16. A method for producing lysine by microbial fermentation, the method comprising: (a) introducing the expression cassette according to any one of claims 1 to 11 or the expression construct according to claim 12 into a lysine-producing microorganism; (b) fermenting the microorganism; and (c) harvesting the produced lysine, The microorganism is the Escherichia coli SCEcL3 (pSLL1) strain.

17. The method of claim 16, wherein the nucleotide sequence of the expression cassette is shown in any one of SEQ ID NOs: 34, 35, 58, 59, and 62.

18. Use of the expression cassette according to any one of claims 1 to 11, the expression construct according to claim 12 and the recombinant host cell according to any one of claims 13 to 15 for producing lysine by microbial fermentation.

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