CN117187275A - Expression system, construction method and application thereof - Google Patents

Abstract

The application relates to an expression system and a structure thereofMethods of construction and use, the expression system comprisingnadKHost bacteria with deleted genes and expression vectors comprisingnadKGenes of the order ofnadKThe gene deletion type host bacterium contains the expression vector. The system not only improves the passage stability of the vector in the genetic engineering strain under the condition of no addition of antibiotics; can also promote the synthesis of exogenous proteins or enzymes and promote the synthesis of various target metabolites.

Description

Expression system and its construction methods and applications

Technical field

This application relates to the fields of synthetic biotechnology and genetic engineering, specifically to an expression system and its construction method and application.

Background technique

Plasmids are a type of genetic elements that are independent of chromosomes and can replicate autonomously. They are widely used in metabolic engineering research for the overexpression of target genes. Compared with genome-integrated expression, the use of artificial plasmids to express target genes has the advantages of simple operation, high copy number, and high target gene expression. However, currently artificially developed plasmid vectors often suffer from instability problems, that is, the recombinant bacteria lose their plasmids and lose their original phenotypic characteristics during the culture process, thus causing unstable fermentation production. Most artificial plasmids carry antibiotic resistance genes. Adding corresponding antibiotics during laboratory fermentation can maintain the stability of the plasmid; however, in industrial fermentation, the addition of antibiotics not only greatly increases the cost, but also limits the scope of use of specific fermentation products. . Furthermore, the misuse of antibiotics poses ecological safety risks and may lead to the emergence of super-resistant bacteria. Therefore, the development of a stable plasmid expression system that does not rely on antibiotic selection pressure has important application value.

Researchers have developed methods to maintain stable passage of plasmids. For example, the use of toxin-antitoxin systems (such as parD and parE ), or the use of operator-repressor neutralization systems (ie, Operator-repressor titration, ORT). However, these methods increase the physiological burden on cells, resulting in slower growth and affecting the synthesis of target products. Some researchers have also tried to knock out infA , ispH , lpxA , etc. on the genome, and then use plasmids for complementary expression, which can make the growth of cells dependent on the presence of plasmids, thereby ensuring stable passage of plasmids. However, when these genes have been reported to be expressed using plasmids, multi-copy plasmids often lead to up-regulation of expression levels, which in turn leads to cellular metabolic imbalance, or negative feedback regulation reduces the copy number of the plasmid, thereby affecting the production efficiency of the target product of the recombinant strain.

Contents of the invention

Based on this, this application provides the nadK gene for constructing an expression system, which can be used to achieve stable inheritance of the vector without relying on antibiotic selective pressure. In addition, overexpression of the nadK gene in this expression system will not cause cellular metabolic burden. Aggravating or even promoting the synthesis of target metabolites is very important for using microorganisms as cell factories and using synthetic biotechnology to produce high-yield target metabolites.

The specific technical solutions are as follows:

An expression system, the expression system includes a nadK gene-deficient host cell and an expression vector, the nadK gene-deficient host cell contains the expression vector; the expression vector includes the nadK gene.

In one embodiment, the expression vector further includes a gene of interest.

A method for constructing an expression system, including the following steps:

Transform the above expression vector into host cells; and

Knock out the nadK gene in the host cell genome.

Application of nadK gene in constructing vector stable expression system.

The expression system is used in the expression of exogenous proteins, such as exogenous enzymes, or the synthesis of target metabolites.

Compared with traditional technology, this application has the following beneficial effects:

This application developed a vector stable expression system based on the nadK gene, which can not only improve the passage stability of the vector in host cells without adding antibiotics, but also promote the expression of exogenous proteins or exogenous enzymes, as well as target metabolites. Synthesis. The above-mentioned expression system is further used for the stable and high expression of exogenous proteins or exogenous enzymes, and the synthesis of various target metabolites.

Description of the drawings

Figure 1 is a schematic diagram of the construction of plasmid pET32a-PnhM-mcherry-nadK carrying the nadK gene;

Figure 2 is a schematic diagram of the construction of recombinant E. coli VG1△ nadK (pET32a-PnhM-mcherry-nadK);

Figure 3 shows the plasmid stability comparison of recombinant E. coli VG1△nadK (pET32a-PnhM-mcherry-nadK) and VG1 (pET32a-PnhM-mcherry);

Figure 4 is a comparison of the fluorescence intensity of recombinant E. coli VG1△nadK (pET32a-PnhM-mcherry-nadK) and VG1 (pET32a-PnhM-mcherry);

Figure 5 shows the plasmid stability comparison of recombinant E. coli VG1△nadK (pTU14-nadK) and VG1 (pTU14);

Figure 6 is a comparison of the plasmid stability of recombinant Bacillus subtilis THY7△nadK (pHT08-Pg3-lacZ-nadK) and THY7 (pHT08-Pg3-lacZ);

Figure 7 is a comparison of the plasmid stability of recombinant Rhodococcus rubrum THdAdN△nadK (pNVSm-SBMDB-nadK) and THdAdN (pNVSm-SBMDB);

Figure 8 is a comparison of the plasmid stability of recombinant Corynebacterium glutamicum ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK) and ATCC13032 (pXMJ19-Ptac-ssehasA-hasB).

Detailed ways

In order to make the above objects, features and advantages of the present application more obvious and understandable, the specific implementation modes of the present application are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be implemented in many other ways different from those described here. Those skilled in the art can make similar improvements without violating the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing specific embodiments only and is not intended to limit the application.

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

One embodiment of the present application provides an expression vector, which includes the nadK gene.

nadK gene (NAD Kinase) refers to the gene encoding NAD kinase. The nadK gene may be the entire nucleotide fragment or part of the nucleotide fragment of the nadK gene. Any nucleotide fragment that can encode NAD kinase falls within the protection scope of this application.

In a specific example, the expression vector also includes the gene of interest.

In a specific example, the expression vector also includes a promoter and a terminator.

In a specific example, the target gene and the nadK gene can be transcribed and expressed by the same promoter or different promoters.

In a specific example, the expression vector is a plasmid. Alternatively, plasmids include pUC series, pET series, pNV series and pXMJ19 and their derivative plasmids. Further optionally, plasmids include pUC19, pET32a, pNV18, pXMJ19 and derivative plasmids thereof.

In a specific example, the target genes include genes related to target metabolite synthesis. Optionally, the target metabolite synthesis-related genes include target metabolite synthesis-related enzyme genes. Alternatively, target metabolites include, but are not limited to, hyaluronic acid and PHA. Optionally, the PHA includes PHB.

In a specific example, the target genes include fluorescent reporter genes, nitrile hydratase genes, β-galactosidase genes, hyaluronic acid synthesis-related genes and PHA synthesis-related genes.

Optionally, the hyaluronic acid synthesis-related genes include one or more of the gene hasA encoding hyaluronic acid synthase and the gene hasB encoding UDP-glucose dehydrogenase.

In vivo, hyaluronic acid synthase and/or UDP-glucose dehydrogenase can catalyze the synthesis of hyaluronic acid.

Optionally, the PHA synthesis-related genes include PHB synthesis-related genes, and the PHB synthesis-related genes include the gene phaA encoding β-ketothiolase, the gene phaB encoding acetoacetyl-CoA reductase, and the gene phaC encoding PHA polymerase. one or more.

In vivo, β-ketothiolase, acetoacetyl-CoA reductase and/or PHA polymerase can catalyze the synthesis of PHB.

In a specific example, the source of the nadK gene includes Escherichia coli, Bacillus subtilis, Rhodococcus or Corynebacterium glutamicum.

In a specific example, the nucleotide fragment of the nadK gene is selected from any one of SEQ ID NO: 1 to 5.

One embodiment of the present application also provides the application of the nadK gene in constructing a vector-stable expression system.

The nadK gene is an essential gene for growth. The nicotinamide adenine dinucleotide (NAD) kinase it encodes is the only enzyme in the body that can catalyze the phosphorylation of NAD ⁺ to generate NADP ⁺ . Its overexpression can increase the concentration of NADP(H). Content, for products of NADPH-dependent pathways such as amino acids/proteins, oleic acid and polyhydroxyalkanoates (PHA; representative, Poly-β-hydroxybutyrate (PHB)) Synthesis has a promoting effect. At the same time, the activity of NAD kinase is regulated by negative feedback of NADPH. Excessive NADPH content will in turn inhibit the activity of NAD kinase, thus avoiding imbalance of cellular metabolic balance. That is, overexpression of NAD kinase will not significantly increase the metabolic burden of cells.

One embodiment of the present application also provides an expression system that uses an expression vector to compensate for the deletion of the nadK gene in the host bacterial genome, so that the expression vector can stably exist in the bacterial cell and perform its expression function without the addition of antibiotics. Specifically, the expression system includes a nadK gene-deficient host cell and any of the above expression vectors, and the nadK gene-deficient host cell contains the above-mentioned expression vector.

In a specific example, host cells include prokaryotic host cells and eukaryotic host cells.

Alternatively, the prokaryotic host cell includes one or more of E. coli, Bacillus subtilis, Rhodococcus, and Corynebacterium glutamicum.

This expression system has the following advantages: on the one hand, it improves the passage stability of the expression vector of host cells (such as genetically engineered strains). After 12 passages of culture without adding antibiotics, the plasmid retention rate was greater than 93%, while the control strain The plasmid retention rates are all less than 45%; on the other hand, it promotes the expression of various foreign proteins and the synthesis of target metabolites, such as the synthesis of important microbial synthetic products such as PHB, hyaluronic acid and nitrile hydratase, among which PHB The production of hyaluronic acid increased by 15.6%, the production of hyaluronic acid increased by 30.4%, and the fermentation enzyme activity of nitrile hydratase increased by 26%.

In a specific example, deletion of the nadK gene in the host cell includes complete or partial knockout of the nadK gene in the genome of the host cell.

In a specific example, the nadK gene knockout method includes using CRISPR gene editing method, Red recombination method or homologous recombination method. Optionally, the homologous recombination method is a homologous recombination double crossover method.

An embodiment of the present application also provides a method for constructing an expression system, including the following steps:

Transform the above expression vector into host cells; and

Knock out the nadK gene in the host cell genome.

It should be noted that the two steps in the expression system construction method may be performed in no particular order. As long as the expression system described in this application can be constructed. Those skilled in the art can perform operations as needed.

One embodiment of the present application also provides a method for maintaining stable passage of a vector (such as a plasmid) without relying on antibiotic selection pressure. The above expression vector is transformed into a host bacterium, and the nadK gene on the genome of the host bacterium is knocked out. Host bacteria lacking the nadK gene rely on expression vectors, allowing the expression vectors (such as plasmids) to stably exist in the bacteria during subsequent passages. At the same time, it can promote the expression of foreign genes (target genes) and the synthesis of target metabolites.

In order to achieve stable and high expression of foreign proteins by recombinant microorganisms and fermentation to synthesize target metabolites, one embodiment of the present application also provides a method for efficiently expressing foreign proteins and/or synthesizing target metabolites, including:

The nadK gene-deficient host cells containing the above-mentioned expression vector are cultured to express foreign proteins and/or ferment to synthesize target metabolites.

In a specific example, the medium for red fluorescent protein expression includes LB medium.

In a specific example, the culture medium for β-galactosidase expression includes LB medium containing 0.5 mM~0.6 mM isopropyl-β-D-thiogalactopyranoside.

In a specific example, the medium for PHB fermentation includes LB medium containing 20 g/L~30 g/L glucose.

In a specific example, the culture medium for nitrile hydratase fermentation includes glucose 25 g/L~28 g/L, urea 10g/L~15 g/L, yeast extract 1 g/L~2 g/L, and peptone 10 g /L~12 g/L, KH ₂ PO ₄ 3 g/L~5 g/L, K ₂ HPO ₄ 3 g/L~5 g/L, MgSO ₄ ·7H ₂ O 1 g/L~2 g/ L, MSG 1 g/L~2 g/L, CoCl ₂ 0.08 mM~0.1 mM, pH value 7.0~7.5.

In a specific example, the culture medium for hyaluronic acid fermentation includes glucose 35 g/L~40 g/L, ammonium sulfate 25g/L~30 g/L, corn steep liquor 15 g/L~20 g/L, phosphoric acid Potassium dihydrogen 1 g/L~2 g/L, dipotassium hydrogen phosphate 1 g/L~2 g/L, magnesium sulfate heptahydrate 2 g/L~3 g/L, ferrous sulfate heptahydrate 0.05 g/L ~0.08 g/L, manganese sulfate heptahydrate 0.05 g/L~0.08 g/L, pH value 7.0~7.2.

One embodiment of the present application also provides the application of the above-mentioned expression system in the expression of exogenous proteins or the synthesis of target metabolites.

Optionally, the exogenous protein includes exogenous enzymes.

Optionally, the target metabolite includes a target metabolite synthesized by exogenous enzyme catalysis.

In a specific example, foreign proteins include, but are not limited to, fluorescent proteins, β-galactosidase, nitrile hydratase, nitrilase, epoxide hydrolase, hyaluronic acid synthase, UDP-glucose dehydrogenase, At least one of β-ketothiolase, acetoacetyl-CoA reductase and PHA polymerase.

In a specific example, target metabolites include PHA and/or hyaluronic acid.

The embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. For experimental methods that do not indicate specific conditions in the following examples, priority is given to the guidelines given in this application. You can also follow the experimental manuals or conventional conditions in this field. You can also follow the conditions recommended by the manufacturer, or refer to the experimental methods in this field. Known experimental methods.

In the following specific examples, the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.

Example 1 Construction of genomic nadK- deficient recombinant E. coli VG1△nadK (pET32a-PnhM-mcherry-nadK) and VG1△nadK (pTU14-nadK)

An Escherichia coli plasmid stable expression system was constructed based on nadK gene for the expression of red fluorescent reporter gene and the synthesis of PHB.

1. Construct plasmids pET32a-PnhM-mcherry-nadK and pTU14-nadK carrying nadK. The method includes the following steps:

The construction method of the expression vector pET32a-PnhM-mcherry of the red fluorescent protein gene mcherry is as follows: pET32a (Novagen commercial plasmid vector) was digested by Xba I and Eco RI to obtain the backbone; the promoter PnhM and mcherry genes were obtained by Jinweizhi Biotechnology Co., Ltd. Synthesize (SEQ ID NO: 1), use primers PnhM-F and mcherry-R to amplify the PnhM-mcherry fragment; the two are connected by Gibson, transformed into E. coli VG1 (CN1086203C), resuscitated and cultured, and spread with 100 μg/mL LB plate of ampicillin was used to obtain VG1 (pET32a-PnhM-mcherry).

The construction method of the plasmid vector pET32a-PnhM-mcherry-nadK is shown in Figure 1. The details are as follows: pET32a-PnhM-mcherry is digested with Eco RI to obtain the plasmid backbone; pXMJ19 (HonorGene commercial plasmid vector) is used as the template, and the primer rrnB is used -F, rrnB-R amplify the terminator rrnB; use E. coli VG1 as a template, use primers nadK-F, nadK-R to amplify the nadK gene and its promoter (SEQ ID NO: 2); the three fragments are connected by Gibson , transform E. coli VG1, resuscitate and culture, and then spread on LB plates containing 100 μg/mL ampicillin to obtain VG1 (pET32a-PnhM-mcherry-nadK).

The construction method of the plasmid vector pTU14-nadK used for PHB synthesis is as follows: the pTU14 plasmid (CN1086203C) is digested by Afl II to obtain the plasmid backbone; pET32a-PnhM-mcherry-nadK is used as the template, and primer rrnB-F2 and primer nadK-R2 are used The terminator and nadK fragment were amplified; the two were connected using Gibson, and transformed into E. coli VG1. After recovery and culture, they were spread on LB plates containing 100 μg/mL ampicillin to obtain VG1 (pTU14-nadK).

PnhM-mcherry sequence (SEQ ID NO:1):

acatctacacattgacatccgttccgatgtgatgtaaaaattgtcacgcgcatgcttaattaagaaggagatatacatatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacga gggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgacc gtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaaaaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggt caagaccaccctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtga.

Escherichia coli nadK gene sequence (SEQ ID NO:2):

tgctgatccatgataatttcttccggggcttgcccctcaggcgttttctgttctttactactcatgaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcggaaaaatga ataatcatttcaagtgtattggcattgtgggacacccacggcaccccactgcactgacaacacatgaaatgctctaccgctggctgtgcacaaaaggttacgaggtcatcgttgagcaacaaatcgctcacgaactgcaactgaagaatgtgaaaactggcacgctcgcggagatgggcaactagctgatctcgcggtagtcgttgg tggcgacggtaatatgctgggcgcggcacgcacactcgcccgttacgatattaaagttattggaatcaaccgtggcaacctgggtttcctgactgaccttgaccccgataacgcccagcaacagttagccgatgtgctggaaggccactacatcagcgagaaacgttttttgctggaagcgcaagtctgtcagca agattgccagaaacgcatcagcaccgcgataaatgaagtggtgcttcatccaggcaaagtggcgcatatgattgagttcgaagtgtatatcgacgagatctttgcgttttctcagcgatctgatggactaattatttcgacgccaacaggctccaccgcctattccctctctgcaggcggtcctattctgaccccctctctgg atgcgattaccctggtgcccatgttcccgcatacgttgtcagcacgaccactggtcataaacagcagcagcacgatccgtctgcgtttttcgcatcgccgtaacgacctggaaatcagttgcgacagccagatagcactgccgattcaggaaggtgaagatgtcctgattcgtcgctgtgattaccatctgaatct gattcatccgaaagattacagttatttcaacacattaagcaccaagctcggctggtcaaaaaaattattctaa.

The primers used in the above process are shown in Table 1.

Table 1 Primers and sequences

2. Use the Red recombination method to construct genomic nadK- deficient recombinant E. coli. The construction process is shown in Figure 2. The specific steps are as follows:

Introduce the plasmid pEcCas (Addgene No. #73227) containing Red recombinase into the above-mentioned recombinant E. coli VG1 (pET32a-PnhM-mcherry-nadK) and VG1 (pTU14-nadK). After recovery and culture, apply 100 μg/mL Ampicillin and 50 μg/mL kanamycin were added to the LB plate to obtain VG1 (pEcCas+pET32a-PnhM-mcherry-nadK) and VG1 (pEcCas+pTU14-nadK), which were prepared into electrotransformation competent cells.

Use E. coli S17-1 (commercial strain, ATCC47055) as the template, use primers Sm-F and Sm-R to amplify the spectinomycin resistance gene expression cassette; use E. coli VG1 as the template, use the primer nad-up -F and nad-up-R amplify the upstream homology arm of the nadK gene; use primers nadK-down-F and nadK-down-R to amplify the downstream homology arm of the nadK gene; put the above three PCR fragments through Overlap PCR fusion resulted in the linear fragment nadK-arm-Sm for knockout.

NadK-arm-Sm was electroporated into (pEcCas+pET32a-PnhM-mcherry-nadK) and VG1 (pEcCas+pTU14-nadK) respectively. After recovery and culture, it was spread on cards containing 100 μg/mL ampicillin and 50 μg/mL. On the LB plate of namycin and 50 μg/mL spectinomycin, incubate at 37°C for 24 hours, and select single colonies for PCR verification to ensure that the nadK gene is deleted. The colonies were further streaked onto LB plates containing 100 μg/mL ampicillin and 10% sucrose, and the pEcCas plasmid was lost using sucrose reverse screening to obtain the final genetically engineered strains VG1△nadK (pET32a-PnhM-mcherry-nadK) and VG1ΔnadK (pTU14-nadK), used for subsequent evaluation.

The primers used in the above process are shown in Table 2.

Table 2 Primers and sequences

Example 2 Construction of genomic nadK- deficient recombinant Bacillus subtilis THY-7△nadK (pHT08-Pg3-lacZ-nadK)

This example uses the CRISPR/Cas9 method to construct a genomic nadK- deficient recombinant Bacillus subtilis and use it to express β-galactosidase. The specific steps are as follows:

First, the plasmid vector pHT08-Pg3-lacZ-nadK carrying the nadK gene was constructed as follows: Plasmid pTH08-Pg3-lacZ (Jiao, et al., Biotechnol. Bioeng., 2017, 114: 832-842.), generated by Sal I The plasmid skeleton was obtained by enzyme digestion; using pET32a-PnhM-mcherry-nadK as a template, rrnB-F3 and nadK-R3 were used to amplify the nadK gene expression cassette; the two were connected using Gibson, and Top10 competent cells were transformed to construct pHT08-Pg3- lacZ-nadK.

Then the CRISPR plasmid for nadK gene knockout was constructed as follows: the plasmid pJOE8999 (Altenbuchner, Applied and Environmental Microbiology, 2016, 82, 5421–5427) was digested with Bsa I to obtain the plasmid backbone; primers nadK-sgRNA1-F, nadK -sgRNA1-R has a 20 bp complementary pairing region, and the end 4 bp is the sticky end of BsaI enzyme, which can be complementary to the plasmid backbone. Dilute the two primers to 100 μmol/L, add 1 μL of each and dilute to 10 μL with water, and mix the system Denature at 95°C for 5 minutes, then cool to 25°C at 0.1°C/s, and anneal to form dsDNA. Use T4 DNA ligase to connect the annealing product and the plasmid backbone to obtain pJOE-sgRNA1. Further, pJOE-sgRNA1 was digested with Sfi I to obtain the plasmid skeleton; using Bacillus subtilis THY-7 (Jiao, et al., Biotechnol. Bioeng., 2017, 114: 832-842.) as the template, nadK-up- F2 and nadK-up-R2 were used to amplify the upstream homology arm of the nadK gene, and primers nadK-down-F2 and nadK-down-R2 were used to amplify the downstream homology arm of the nadK gene; the three were connected by Gibson to obtain pJOE-sgRNA1- nadKarm.

The genome nadK (SEQ ID NO:3) of Bacillus subtilis THY-7 was knocked out using CRISPR technology (Altenbuchner, Applied and Environmental Microbiology, 2016, 82, 5421–5427). The details are as follows: pHT08-Pg3-lacZ-nadK was introduced THY-7 gives THY-7(pHT08-Pg3-lacZ-nadK). Plasmid pJOE-sgRNA1-nadKarm was transformed into THY-7 (pHT08-Pg3-lacZ-nadK) by electroporation, and after recovery and culture, it was spread on a solution containing 15ug/mL kanamycin, 5ug/mL chloramphenicol and 0.2 % mannose on the LB plate, cultured at 30°C for 24 hours to grow a single colony, and colony PCR was used to confirm that the nadK gene was completely knocked out. Further inoculate the colonies into antibiotic-free LB medium and culture at 37°C for 12 hours. After dilution and coating on the plate, colonies will grow. Pick multiple colonies and streak them onto a plate containing kanamycin to verify whether they grow or not. Indicates that the colony has eliminated the pJOE-sgRNA1-nadKarm plasmid and can be used for subsequent evaluation.

Bacillus subtilis nadK sequence (SEQ ID NO:3):

atgaaatttgccgtatcatcaaaaggagatcaagtttctgatacgctgaaaagcaaaaatacaggcgtatttattggattttgatatggaactggatgaaaatgaaccggaaatcgttatttcagtcggaggcgacggaacgcttttgtatgcttttcacagatacagcgaccgtttggacaaaacagcatt tgtcggcgttcacacaggtcatctgggtttttatgccgactgggttcctcatgaaattgaaaaacttgttcttgccatcgcgaaaacgccgtatcatacagtcgaatatccgcttctcgaagtcattgtgacctatcacgaaaacgagcgggaagaaagatacttagctttgaatgaatgtacgattaaaagcatcga aggaagccttgttgctgatgtggaaatcaaagggcagctctttgaaaccttccggggcgacggcctctgcctgtcaactccatcaggcagcacagcctacaataaggcgctgggcggagcgattatccatccgtctatcagggcgatccagcttgcggaaatggcttcaattaataaccgcgtgttccgtacggtc gggtcacctctgcttctcccatcccatcatgattgcatgattaagccgagaaacgaagttgactttcaagtgacgattgaccatttaacccttctccataaggatgtaaagtcgatccgctgccaagttgcatccgaaaaagtgcggtttgcgagattccgtccgtttccattttggaaaagagtccaggattcgtttat ggaaaaggtgaatag.

The primers used in the above process are shown in Table 3.

Table 3 Primers and sequences

Example 3 Construction of genome nadK- deficient recombinant Rhodococcus rubra THdAdN△nadK (pNVSm-SBMDB-nadK)

The purpose of this example is to use the homologous recombination double-exchange method to construct a genomic nadK- deficient recombinant Rhodococcus rubrum and use it for the expression of nitrile hydratase. The specific steps are as follows:

First, construct the plasmid vector pNVSm-SBMDB-nadK carrying the nadK gene. The method is as follows: The plasmid vector pNVSm-SBMDB (CN107177581A) is digested with Eco RI to obtain the plasmid backbone; using pET32a-PnhM-mcherry-nadK as the template, use rrnB-F4, nadK-R4 amplifies the nadK gene expression cassette; the two are connected using Gibson, transformed into Top10 competent cells, and spread onto a plate containing 50 μg/mL spectinomycin to construct pNVSm-SBMDB-nadK.

The suicide plasmid pK18mobsacB-nadK-Rr for knocking out the nadK gene of Rhodococcus rubrum was constructed as follows: the commercial plasmid pK18mobsacB was digested with Hin dIII and Xba I to obtain the skeleton; using Rhodococcus rubrum THdAdN (CN105420154A) as a template, nadK was used -up-F3 and nadK-up-R3 amplified the upstream homology arm of nadK gene, and used primers nadK-down-F3 and nadK-down-R3 to amplify the downstream homology arm of nadK gene; the three were connected by Gibson to obtain pK18mobsacB -nadK-Rr.

The homologous recombination double exchange method was further used to knock out the nadK gene (SEQID NO: 4) of the Rhodococcus rubrum THdAN genome, as follows: pNVSm-SBMDB-nadK was introduced into Rhodococcus rubrum THdAdN to obtain THdAdN (pNVSm-SBMDB-nadK). ; The suicide plasmid pK18mobsacB-nadK-Rr was transformed by electroporation, and after recovery and culture, it was spread on an LB plate containing 25 μg/mL kanamycin and 50 μg/mL spectinomycin, and integrated into the genome through single exchange; single The exchanged colonies were further spread onto LB plates containing 50 μg/mL spectinomycin and 100 g/L sucrose, and cultured at 28°C for 3 days to grow colonies. Colony PCR was used to verify whether the nadK gene was successfully knocked out. The successfully knocked-out colony was named THdAdN△nadK (pNVSm-SBMDB-nadK).

Rhodococcus rubrum nadK sequence (SEQ ID NO:4):

gtgaccacagatagtgcagcgcccggcccggagcgcgcgacccgcgaggtcctgctcgtgtcccactcgggacgggtcgagatcgccgcgacggcgcagcgcacggcgaagatcttcggcgaggccggcatcgggctgcgggtgctcgacgacgaggtcgccagcaccggtctgggtcccctgg ccggacccgagggccgggtccgcatcgtcgaacccggaccggacgccgcccggggttgcgagatggtgatcgtgctcggcggcgacggcagtttcctgcgcgccgccgaactggcccagtgcgcgacggtgcccgtgctcggaatcaacctgggacgcatcggtttcctcgccgaggccgagaccga gcatctcgaggaggcgctggcacaggtggtgcgccgcgagtaccgggtcgaggagcggatgacgctggacgtcgcgatccgggtcgacgacgcgatcgtcgaccggggatgggcgctcaacgaggccagcctcgagaacaagtcccgcctgggcgtgctcgaggtggtgctcgaggtcgacggtc ggccggtgtcggcgttcggatgcgacggagtgctgatcgccaccccgaccggctcgaccgcctacgcgttctccgccggcggcccgatcgtctggccggaactcgaagcgttgctggtgattcccagcaacgcccacgctctgttcgcccggcccatggtgaccagccgggaatcgctgatcgccgt ggagaccctcgccggcagccacgacgggctggtgttctgcgacggccgccgcaccctcgacctgcccgcgggtgggcggctcgaggtggtccgcggcaagcatcccgtgcggtgggtgcgcctggactcggcgccgttcgccgaccgaatggtgcgcaagttcgatcttccggtgaagggctggcgggg aaggagactgtga.

The primers used in the above process are shown in Table 4.

Table 4 Primers and sequences

Example 4 Construction of genomic nadK-deleted recombinant Corynebacterium glutamicum ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK)

In this example, the homologous recombination double-exchange method is used to construct the genomic nadK- deficient Corynebacterium glutamicum and use it for the synthesis of hyaluronic acid. The specific steps are as follows:

First, plasmid pXMJ19-Ptac-ssehasA-hasB-nadK was constructed as follows: Plasmid pXMJ19-Ptac-ssehasA-hasB (Cheng, et al., Biotechnology Journal, 2016, 11, 574–584) was digested with Eco RI to obtain the plasmid backbone. ; Using pET32a-PnhM-mcherry-nadK as a template, use rrnB-F5 and nadK-R5 to amplify the nadK gene expression cassette; the two are connected by Gibson, transform Top10 competent cells, and spread to contain 10 μg/mL chloramphenicol. The plate was constructed to obtain pXMJ19-Ptac-ssehasA-hasB-nadK.

The suicide plasmid pK18mobsacB-nadK-Cg for knocking out the nadK gene of Corynebacterium glutamicum was constructed as follows: the plasmid pK18mobsacB (Cheng Fangyu literature) was digested with Hin dIII and Xba I to obtain the skeleton; the skeleton was obtained from Corynebacterium glutamicum ATCC13032 As a template, use nadK-up-F4 and nadK-up-R4 to amplify the upstream homology arm of the nadK gene, and use the primers nadK-down-F4 and nadK-down-R4 to amplify the downstream homology arm of the nadK gene; the three pK18mobsacB-nadK-Cg was obtained via Gibson ligation.

The homologous recombination double exchange method was further used to knock out the nadK gene (SEQ ID NO: 5) in the genome of Corynebacterium glutamicum ATCC13032, as follows: pXMJ19-Ptac-ssehasA-hasB-nadK was introduced into Corynebacterium glutamicum ATCC13032, ATCC13032 (pNVSm-SBMDB-nadK) was obtained; the suicide plasmid pK18mobsacB-nadK-Cg was transformed by electroporation. After recovery and culture, it was spread onto an LB plate containing 25 μg/mL kanamycin and 5 μg/mL chloramphenicol. Exchange to integrate it into the genome; the single-exchange colony was further spread on an LB plate containing 5 μg/mL chloramphenicol and 100 g/L sucrose, and cultured at 30°C for 2 days to grow colonies, and colony PCR was used to verify whether the nadK gene was successfully knocked out. remove. The successfully knocked-out colony was named ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK).

Corynebacterium glutamicum nadK sequence (SEQ ID NO:5):

atgactgcacccacgaacgctggggaactcaggcgagttttgctggttccacacaccgggcgttcttccaatattgaatccgccatcttggcagccaagctgctcgacgatgctggaatcgatgtgagggtgctgatcaatgatgcagatgatccaatgcagagcactccgttttaggccgtttcacccatgt caggcacgctgcagacgccgctgacggcgcagaactagttctggtgctgggtggagatggcaccttcctccgcgcagcagatatggcccacgctgttgatttgcctgttctgggcatcaacctaggccatgtgggattcttggctgaatgggagtctgactcacttgaagaggcactcaaacgtgtgatcgaccgcg attaccgtattgaagatcgcatgaccttaactgtcgttgtcctagacggcggtggagaagaaatcggccgaggctgggctctcaatgaggtcagtattgaaaacttaaaccgcaggggagtgctcgatgcaaccctcgaggtagatgcacgaccagttgcttcctttggttgcgatggcgtgctgatttccaccccaaccggctcc accgcttatgcattttccgccggtggtcctgtactgtggccagaactcgatgccatcttggtggttcctaataacgcccacgcgctgtttacccaaaccgctggttgtgagcccaaaatccaccgtagctgtggaatccaattcagatacttcagcagcgatggccgtcatggatggtttccgtcccattcctatg cctccaggatcccgtgttgaggtcaccaggggtgagcgtcccgtgcgttgggtgaggcttgattcttcaccgtttaccgaccgacttgtgagcaaattaaggctccccgttaccggttggcggggtccgcaaaaacaggcggaaaaataaagatcccaggtcagcggggtaa.

The primers used in the above process are shown in Table 5.

Table 5 Primers and sequences

Example 5 Evaluation of plasmid stability and fluorescence intensity of recombinant strain VG1△nadK (pET32a-PnhM-mcherry-nadK) and control strain VG1 (pET32a-PnhM-mcherry)

This example evaluates the impact of a plasmid stable expression system constructed based on the nadK gene on the expression intensity and stability of red fluorescent protein in recombinant E. coli.

The recombinant strain VG1△nadK (pET32a-PnhM-mcherry-nadK) and the control strain VG1 (pET32a-PnhM-mcherry) were respectively inoculated into LB medium without antibiotics, and subcultured at 37°C for a total of 12 times. Each transfer The ratio is 1% and the culture time is 12 hours. After each culture, the mcherry fluorescence intensity is measured. The bacterial solution is appropriately diluted and spread onto an LB plate without or containing 100 μg/mL ampicillin, and the colonies are counted. Calculate plasmid retention (i.e., the ratio of the number of colonies on antibiotic plates to antibiotic-free plates).

The plasmid retention rate during passage is shown in Figure 3. After 12 transfers, the plasmid retention rate of the control strain VG1 (pET32a-PnhM-mcherry) was only 25%, while the plasmid retention rate of VG1△nadK (pET32a-PnhM-mcherry-nadK ), the plasmid retention rate was 95%, indicating that the method of the present application can significantly improve the passage stability of the plasmid. The changes in fluorescence intensity during the passage process are shown in Figure 4. Overexpression of the nadK gene on the plasmid has almost no effect on the initial fluorescence intensity, but significantly improves the stability of the fluorescence intensity during the passage process.

Example 6 Evaluation of plasmid stability and PHB production of recombinant strain VG1△nadK (pTU14-nadK) and control strain VG1 (pTU14)

This example is to evaluate the impact of a plasmid stable expression system constructed based on the nadK gene on the PHB yield and plasmid passage stability of recombinant E. coli.

The recombinant strain VG1△nadK (pTU14-nadK) and the control strain VG1 (pTU14) were inoculated into LB medium without antibiotics and containing 20 g/L glucose, and subcultured at 37°C for a total of 12 times. The transfer ratio of each time is 1%, and the culture time is 12 hours. After each culture, the bacterial solution is appropriately diluted and spread on an LB plate without or containing 100 μg/mL ampicillin, and the colonies are counted to calculate the plasmid retention rate (i.e. The ratio of the number of colonies on antibiotic plates to those on antibiotic-free plates). The culture fluid of the 1st and 12th passage was inoculated into LB medium containing 30g/L glucose at a ratio of 1%, cultured at 37°C and 200 rpm for 48 hours, and the PHB content was measured (refer to CN1086203C for the measurement method).

The plasmid retention rate during passage is shown in Figure 5. After 12 transfers, the plasmid retention rate of the control strain VG1 (pTU14) was 45%, while the plasmid retention rate of VG1△nadK (pTU14-nadK) was 96%. The PHB production of the 1st and 12th transfer bacterial solution is shown in Table 6. The initial PHB production of VG1△nadK (pTU14-nadK) was 15.6% higher than that of the control bacteria; after 12 passages, the PHB production of VG1△nadK (pTU14- The production of nadK) was basically unchanged (94.2% of the initial level), while the production of the control strain remained only 39.2%. The results show that the plasmid stable expression system based on nadK of this application can not only improve the stability of plasmid passage in E. coli, but also promote the synthesis of PHB.

Table 6 PHB production comparison of recombinant E. coli

Further, VG1△nadK (pTU14-nadK) and VG1 (pTU14) were fermented and cultured in a 5 L fermentor. Pick a single colony and inoculate it into LB medium containing 20 g/L glucose. After culturing at 37°C for 12 hours, inoculate it into a 5 L fermenter at a ratio of 10% (LB medium containing 40 g/L glucose, filling volume 2 L), the culture temperature is 37°C, the initial stirring speed is 200-400 rpm, and the pH is controlled at 6.5-7.5. During the fermentation process, yeast extract, glucose and ammonium sulfate are added as needed to maintain the glucose concentration in the early and middle stages of fermentation. 20~40 g/L, the ammonium sulfate concentration is maintained at 1~2 g/L, and the carbon-nitrogen ratio is maintained between 10~20; when the dissolved oxygen decreases, gradually adjust the stirring speed to keep the dissolved oxygen above 5%. Fermentation is completed after 48~65 hours. Without the addition of antibiotics, the PHB production was as high as 152 g/L after 48 hours of fermentation culture; while the PHB production of the comparison strain VG1 (pTU14) without the addition of antibiotics was only 88 g/L, and under the conditions of the addition of antibiotics, the PHB production was 102 g/L. The plasmid stable expression system based on the nadK gene significantly improved the plasmid stability of the recombinant strain and the production of the target product PHB.

Example 7 Evaluation of plasmid stability and β-galactosidase activity of recombinant Bacillus subtilis THY-7△nadK (pHT08-Pg3-lacZ-nadK) and control strain THY-7 (pHT08-Pg3-lacZ)

This example is to evaluate the impact of a plasmid stable expression system constructed based on the nadK gene on the β-galactosidase activity and plasmid passage stability of recombinant Bacillus subtilis.

The recombinant strain THY-7△nadK (pHT08-Pg3-lacZ-nadK) and the control strain THY-7 (pHT08-Pg3-lacZ) were inoculated into antibiotic-free and containing 0.5mM isopropyl-β-D-thio Galactoside's LB medium was subcultured at 37°C for a total of 12 times, with a transfer ratio of 1% each time and a culture time of 12 hours. After each culture, the bacterial solution was appropriately diluted and spread to a level that did not contain or contain on an LB plate with 5ug/mL chloramphenicol, and count the colonies to calculate the plasmid retention rate (i.e., the ratio of the number of colonies on the antibiotic plate and the antibiotic-free plate). In the culture medium of the 1st and 12th passage, β-galactosidase activity was measured (for the measurement method, please refer to Jiao, et al., Biotechnol. Bioeng., 2017, 114: 832-842.).

The plasmid retention rate during passage is shown in Figure 6. After 12 transfers, the plasmid retention rate of the control strain THY-7 (pHT08-Pg3-lacZ) was only 12%, while that of THY-7△nadK (pHT08-Pg3 -lacZ-nadK) plasmid retention rate reached 97%. The β-galactosidase activity of the 1st and 12th transfer bacterial solution is shown in Table 7. The initial β-galactosidase activity of THY-7△nadK (pHT08-Pg3-lacZ-nadK) and the control There was almost no difference between the bacteria, but after 12 passages, it was significantly higher than the control bacteria. The results show that the nadK -based plasmid stable expression system can improve the plasmid passage stability of recombinant Bacillus subtilis spores.

Table 7 Comparison of β-galactosidase activities of recombinant Bacillus subtilis

Example 8 Evaluation of plasmid stability and nitrile hydratase activity of recombinant Rhodococcus rubrum THdAdN△nadK (pNVSm-SBMDB-nadK) and control strain THdAdN (pNVSm-SBMDB)

This example is to evaluate the impact of a plasmid stable expression system constructed based on the nadK gene on the nitrile hydratase activity and plasmid passage stability of recombinant Rhodococcus rubrum.

The recombinant strain THdAdN△nadK (pNVSm-SBMDB-nadK) and the control strain THdAdN (pNVSm-SBMDB) were inoculated into Rhodococcus seed culture medium without antibiotics (glucose 10-50 g/L, yeast extract 1-4 g/ L, peptone 1-10g/L, KH ₂ PO ₄ 0.2-3 g/L, K ₂ HPO ₄ 0.2-3 g/L, MgSO ₄ ·7H ₂ O 0.2-3 g/L, MSG 1 g/L, pH value is 7.5), subcultured at 28°C for a total of 12 times, the transfer ratio each time was 1%, and the culture time was 36 hours. After each culture, the bacterial solution was appropriately diluted and spread to a level that did not contain or contained 50ug/ mL of spectinomycin on the LB plate, and the colonies were counted to calculate the plasmid retention rate (i.e., the ratio of the number of colonies on the antibiotic plate and the antibiotic-free plate). The culture broth of the 1st and 12th passages was inoculated into Rhodococcus fermentation medium (the medium for nitrile hydratase fermentation includes glucose 25 g/L, urea 10 g/L, yeast extract 1 g/L, and peptone 10g/L). L, KH ₂ PO ₄ 3 g/L, K ₂ HPO ₄ 3 g/L, MgSO ₄ ·7H ₂ O 1 g/L, MSG 1 g/L, CoCl ₂ 0.08mM, pH value 7.5), 28℃ , ferment and culture at 200 rpm for 48 hours, and measure nitrile hydratase activity (method reference CN107177581A).

The plasmid retention rate during passage is shown in Figure 7. After 12 transfers, the plasmid retention rate of the control strain THdAdN (pNVSm-SBMDB) was only 34%, while the plasmid retention rate of THdAdN△nadK (pNVSm-SBMDB-nadK) The rate reaches 93%. The 1st and 12th transfer strains were used for fermentation culture. The nitrile hydratase activity is shown in Table 8. The initial nitrile hydratase activity of THdAdN△nadK (pNVSm-SBMDB-nadK) was 25% higher than that of the control strain, and There was almost no decrease in enzyme activity after 12 passages; while the enzyme activity of the control bacteria decreased to 26% of the original level after 12 passages. The results show that the nadK-based plasmid stable expression system can not only improve the plasmid passage stability of recombinant Rhodococcus rubrum, but also significantly promote the expression of nitrile hydratase.

Table 8 Comparison of nitrile hydratase activities of recombinant Rhodococcus rubrum

Example 9 Evaluation of plasmid stability and hyaluronic acid production of recombinant Corynebacterium glutamicum ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK) and control strain ATCC13032 (pXMJ19-Ptac-ssehasA-hasB)

This example is to evaluate the impact of a plasmid stable expression system constructed based on the nadK gene on the hyaluronic acid production and plasmid passage stability of recombinant Corynebacterium glutamicum.

Recombinant Corynebacterium glutamicum ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK) and control strain ATCC13032 (pXMJ19-Ptac-ssehasA-hasB) were inoculated into LB medium without antibiotics and containing 20g/L glucose. , a total of 12 times of subculture at 30°C, the transfer ratio of each time is 1%, and the culture time is 24 hours. After each culture, the bacterial solution is appropriately diluted and spread into a medium without or containing 5ug/mL chloramphenicol. on the LB plate, and count the colonies to calculate the plasmid retention rate (i.e., the ratio of the number of colonies on the antibiotic plate and the antibiotic-free plate). The culture broth of the 1st and 12th passages was inoculated into the fermentation medium (glucose 40 g/L, ammonium sulfate 30 g/L, corn steep liquor 20 g/L, potassium dihydrogen phosphate 1g/L, dihydrogen phosphate Potassium 1 g/L, magnesium sulfate heptahydrate 2 g/L, ferrous sulfate heptahydrate 0.05 g/L, manganese sulfate heptahydrate 0.05g/L, pH value 7.2), add 1mM isopropyl after incubation at 28°C for 3 hours -β-D-thiogalactopyranoside, continue culturing for 48 hours, and measure the hyaluronic acid concentration (refer to Cheng, et al., Biotechnology Journal, 2016, 11, 574–584 for the measurement method).

The plasmid retention rate during passage is shown in Figure 8. After 12 transfers, the plasmid of the control strain ATCC13032 (pXMJ19-Ptac-ssehasA-hasB) was almost completely lost, while the plasmid of ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB- nadK) plasmid retention rate reached 94%. The first and twelfth transfer strains were used for fermentation culture. The hyaluronic acid production is shown in Table 9. The initial hyaluronic acid production of ATCC13032△nadK (pXMJ19-Ptac-ssehasA-hasB-nadK) was higher than that of the control bacteria. 30.4%, and the yield after 12 passages was 92% of the original level; while the yield of the control strain was zero after 12 passages. The results show that the nadK-based plasmid stable expression system can not only improve the plasmid passage stability of Corynebacterium glutamicum, but also significantly promote the synthesis of hyaluronic acid.

Table 9 Comparison of hyaluronic acid production by recombinant Corynebacterium glutamicum

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual.

The above-described embodiments only express several implementation modes of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. Therefore, the scope of protection of the patent of this application shall be determined by the appended claims, and the description may be used to interpret the content of the claims.

Claims (17)

1. An expression system, comprisingnadKGene-deficient host cells and expression vectors, said host cellsnadKThe gene deletion type host cell contains the expression vector;

the expression vector comprisesnadKAnd (3) a gene.

2. The expression system of claim 1, wherein the expression vector further comprises a gene of interest.

3. The expression system of claim 1 or 2, wherein the expression vector is a plasmid.

4. The expression system of claim 3, wherein the plasmids include pUC series, pET series, pNV series and pXMJ19 and its derivative plasmids.

5. The expression system of claim 4, wherein the plasmid comprises pUC19, pET32a, pNV18, pXMJ19 and derived plasmids thereof.

6. The expression system of any one of claims 1-2 and 4-5, wherein the gene of interest comprises a gene related to synthesis of a target metabolite.

7. The expression system of any one of claims 1-2 and 4-5, wherein the gene of interest comprises a fluorescent reporter gene, a nitrile hydratase gene, a β -galactosidase gene, a gene involved in hyaluronic acid synthesis, or a gene involved in PHA synthesis.

8. The expression system of any one of claims 1-2 and 4-5, wherein thenadKSources of genes include E.coli, B.subtilis, rhodococcus or C.glutamicum.

9. The expression system of claim 8, wherein thenadKThe nucleotide fragment of the gene is selected from any one of fragments shown in SEQ ID NO. 1-5.

10. The expression system of any one of claims 1-2, 4-5, and 9, wherein the host cell comprises a prokaryotic host cell and a eukaryotic host cell.

11. The expression system of claim 10, wherein the prokaryotic host cell comprises one or more of escherichia coli, bacillus subtilis, rhodococcus, and corynebacterium glutamicum.

12. The expression system of any one of claims 1-2, 4-5, 9, and 11, wherein the host cellnadKGene deletions include genomic expression in a host cellnadKKnocking out all or part of the gene.

13. According to claim 12The expression system of (2), characterized in thatnadKThe gene knockout method comprises CRISPR gene editing, red recombination or homologous recombination.

14. A method of constructing an expression system, comprising the steps of:

transforming the expression vector of any one of claims 1-13 into a host cell; and

Knocking out the host cell genomenadKAnd (3) a gene.

15.nadKThe application of the gene in constructing a vector stable expression system.

16. The use of the expression system of any one of claims 1-13 for expression of a foreign protein, or synthesis of a target metabolite.

17. The use according to claim 16, wherein the exogenous protein comprises at least one of fluorescent protein, β -galactosidase, nitrile hydratase, nitrilase, epoxide hydrolase, hyaluronate synthase, UDP-glucose dehydrogenase, β -ketothiolase, acetoacetyl-coa reductase, and PHA polymerase; and/or

The target metabolite includes at least one of PHA and hyaluronic acid.