CN110551669B

CN110551669B - Near-infrared light control dynamic regulation and control system and application thereof

Info

Publication number: CN110551669B
Application number: CN201910836782.5A
Authority: CN
Inventors: 陈修来; 丁强; 刘立明; 李洋; 刘佳; 罗秋玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2022-05-06
Anticipated expiration: 2039-09-05
Also published as: CN110551669A

Abstract

The invention discloses a near-infrared light controlled dynamic regulation and control system and application thereof, particularly discloses a near-infrared light combined control system and application thereof in acetoin production, and belongs to the technical field of metabolic engineering. The invention selects near infrared light photosensitive protein by means of molecular biology. The acetoin is produced in an inorganic salt culture medium by introducing a dynamic regulation gene line of the target Rpos into engineering escherichia coli by utilizing the light source characteristic that photosensitive protein specifically responds to a specific wavelength and the advantage of no need of exogenous addition of an inducer. The yield of acetoin produced by the method can reach 66.8g/L, and the conversion rate reaches 0.58g/g glucose.

Description

Near-infrared light control dynamic regulation and control system and application thereof

Technical Field

The invention relates to a near-infrared light controlled dynamic regulation and control system and application thereof, in particular to a near-infrared light controlled dynamic regulation and control system and application thereof in acetoin production, and belongs to the technical field of metabolic engineering.

Background

The dynamic regulation and control system is a new metabolic flow regulation and control means in the field of metabolic engineering, and is different from static regulation and control, and is mainly characterized in that in the fermentation process, an engineering strain can make corresponding enzyme activity regulation according to fermentation time, physiological state, intracellular metabolite concentration and extracellular environment change, so that the metabolic flow distribution is influenced, and the product production capacity is improved. The dynamic regulation and control system has the advantages of no need of artificial regulation and control in the fermentation process and no need of exogenous addition of an inducer, and has remarkable advantages in the production of high value-added compounds.

The current dynamic regulation system is mainly controlled by a quorum sensing system, namely a trigger of the system consists of a quorum sensing system of a local source or a heterogeneous source. When the concentration of the thalli reaches a certain concentration, the micromolecular substances released and accumulated by the quorum sensing system can act on a receptor of the regulation and control system, so that the regulation and control system can make corresponding actions. For example, in 2017, Apoorv Gupta et al, which is a dynamic system expressed in Nature biotechnology, is composed of an EsaI quorum sensing system and a target protein C-terminal degradation tag, when the concentration of bacteria reaches a certain threshold, the target protein is degraded, and the intracellular absolute concentration reaches 0, so that the aims and effects of enzyme activity shutdown and metabolic flux regulation are achieved. The system has the characteristic of path independence and is successfully applied to the production and application of inositol, glucaric acid and acetoin. In addition, in 2017, the AND logic gate dynamic regulation system published by Xinyuan He et al in ACS synthetic biology consisted of a quorum sensing system AND stationary phase sensory proteins. When the thallus concentration reaches a certain threshold value, the transcription expression of the target protein is started, thereby realizing the purpose and the effect of regulating and controlling the metabolic flux. When the system is applied to PHB production, the yield of PHB is improved by 1-2 times. In summary, both dynamic regulation systems require the introduction of heterologous quorum sensing systems, however, the introduction of heterologous quorum sensing systems, which often consist of multiple transcriptional regulatory proteins, undoubtedly affects the growth and fermentation performance of the strain, while limiting the amount of expression of pathway enzymes.

Acetoin is a precursor for the preparation of flavors and fragrances and is synthesized in escherichia coli by the pyruvate pathway. The traditional method for producing acetoin realizes the accumulation of the acetoin by over-expressing pathway enzyme. However, the production of acetoin is limited by the specific surface area, which causes the problem of uneven dissolved oxygen mass transfer, and directly influences the growth and accumulation of acetoin in escherichia coli. Therefore, the method for producing acetoin in a simple culture medium is provided, and the method has important significance for industrial production of acetoin.

Disclosure of Invention

The first purpose of the invention is to provide an engineering strain, which expresses near infrared light photosensitive pigment, near infrared light photoresponse component and target protein together, wherein the near infrared light photosensitive pigment comprises Bphs, Bphos, Yhjh and/or Mrkh; the near infrared light photoresponse component comprises a promoter P_mrkAThe amino acid sequence of the Bphs is shown as SEQ ID NO.14, the amino acid sequence of the Bpho is shown as SEQ ID NO.15, the amino acid sequence of the Yhjh is shown as SEQ ID NO.16, the amino acid sequence of the Mrkh is shown as SEQ ID NO.17, and the P is shown as_mrkAThe nucleotide sequence of (A) is shown in SEQ ID NO. 5.

In one embodiment of the invention, the nucleotide sequence encoding the Bphs is shown as SEQ ID NO.1, the nucleotide sequence encoding the Bpho is shown as SEQ ID NO.2, the nucleotide sequence encoding the Yhjh is shown as SEQ ID NO.3, and the nucleotide sequence encoding the Mrkh is shown as SEQ ID NO. 4.

In one embodiment of the invention, the target protein is RpoS, and the amino acid sequence of the RpoS is shown in SEQ ID No. 18.

In one embodiment of the invention, P is used_mrkA-Rpos and P_J23119-SO-P_J23119-Y₃₀M-P_J23119-BudAB-NOX as an expression vector.

P_J23119-SO-P_J23119-Y₃₀The construction method of M comprises the following steps:

(1) based on a commercial Plasmid pTargetF (Addgene Plasmid #62226), a T7Te terminator sequence is inserted after an rrnB T1 terminator in a full-Plasmid PCR mode so as to reduce leakage expression; removing the sgRNA expression frame by adopting a full-plasmid PCR (polymerase chain reaction) mode to obtain an engineering plasmid P only containing a Pj23119 constitutive promoter and double termination_J23119；

(2) Taking Rhodobacter sphaeroides (Rhodobacter sphaeroides) genomes as templates, and respectively amplifying to obtain gene fragments of Bphs, Bphos and Yhjh; taking a pneumonia bacillus genome (Klebsiella pneumoniae) as a template, and amplifying to obtain an mrkH gene fragment; by means of homologous recombination, the Bphos fragment of Bphs and B0034RBS with B0034RBS (nucleotide sequence shown in SEQ ID NO. 7) is inserted into P_J23119In the expression cassette, obtaining P_J23119-a SO plasmid; in the same manner, the mrkH fragment of Yhjh and B0034RBS carrying B0030RBS (nucleotide sequence shown in SEQ ID NO. 8) was inserted into P_J23119In the expression cassette, obtaining P_J23119-Y₃₀M plasmid; method for transforming P by homologous recombination_J23119-SO and P_J23119-Y₃₀M two plasmids were integrated to obtain P_J23119-SO-P_J23119-Y₃₀M plasmid.

P_mrkAThe construction method of the BFP plasmid comprises the following steps: amplifying the bfp gene containing B0034RBS by using bfp synthetic gene fragment (shown in SEQ ID NO. 6) as template, and connecting to P by homologous recombination_J23119On plasmid, simultaneous reverse amplification to give P_mrkAPlasmid, homologous recombinationBy obtaining P_mrkA-a BFP plasmid.

P_J23119The construction method of the-BudAB-NOX plasmid comprises the following steps: using Serratia marcescens (Serratia marcescens) H30 genome as a template, designing a primer with B0034RBS (nucleotide sequence is shown as SEQ ID NO. 7), respectively amplifying to obtain budA (nucleotide sequence is shown as SEQ ID NO. 9) and budB fragment (nucleotide sequence is shown as SEQ ID NO. 10) containing B0034RBS, and inserting the budA and the budB into P by adopting a multi-fragment one-step homologous recombination mode_J23119(the nucleotide sequence is shown as SEQ ID NO. 11) to obtain P_J23119-a BudAB plasmid; amplifying the nox gene by using Lactobacillus brevis genome as a template, and inserting the nox gene segment (shown as SEQ ID NO. 12) into the P gene by adopting a multi-segment one-step homologous recombination mode_J23119In the expression cassette of BudAB, P is obtained_J23119-a BudAB-NOX plasmid.

The P is_J23119-SO-P_J23119-Y₃₀M-P_J23119The construction method of the-BudAB-NOX plasmid comprises the following steps: with P_J23119-BudAB-NOX plasmid as template, amplifying P by homologous recombination_J23119-BudAB-NOX fragment, followed by P_J23119-SO-P_J23119-Y₃₀M is a vector, enzyme digestion, homologous recombination and P insertion are carried out_J23119-SO-P_J23119-Y₃₀M expression cassette to obtain P_J23119-SO-P_J23119-Y₃₀M-P_J23119-a BudAB-NOX plasmid.

The P is_mrkAThe construction method of the-Rpos plasmid comprises the following steps: method for transforming P by homologous recombination_mrkA-the gene encoding the BFP fluorescent protein in the BFP plasmid is replaced by a gene encoding the endogenous RpoS protein of escherichia coli (nucleotide sequence shown in SEQ ID No. 14).

In one embodiment of the invention, e.

The second purpose of the invention is to provide a method for regulating and controlling the expression of target protein genes, which is to supplement near infrared light in the fermentation process of engineering strains to control the expression of the target protein genes, wherein the engineering strains jointly express near infrared light photosensitive pigment and near infrared light photosensitive pigmentA response component and a target protein, the near infrared light photosensitizer pigment comprising Bphs, Bpho, Yhjh, or Mrkh; the near infrared light photoresponse component comprises a promoter P_mrkAThe amino acid sequence of the Bphs is shown as SEQ ID NO.14, the amino acid sequence of the Bpho is shown as SEQ ID NO.15, the amino acid sequence of the Yhjh is shown as SEQ ID NO.16, the amino acid sequence of the Mrkh is shown as SEQ ID NO.17, and the P is shown as_mrkAThe nucleotide sequence of (A) is shown in SEQ ID NO. 5.

In one embodiment of the present invention, the illumination intensity of the near infrared light is 0.2W/cm²-0.8W/cm²The wavelength is 600-650nm, and the distance from the strain culture medium is fixed to be 5-15 cm.

In one embodiment of the invention, the wavelength is 650nm and the distance from the culture medium of the strain is fixed at 10 cm.

The third purpose of the invention is to provide a method for producing acetoin, wherein the engineering strain single colony is inoculated into an LB culture medium for activation, and is inoculated into a fermentation culture medium for fermentation after activation.

In one embodiment of the invention, the medium of the fermentation comprises NBS mineral salts medium.

In one embodiment of the present invention, the fermentation conditions are 35-38 deg.C, 200-220rpm, initial OD after activation₆₀₀Fermenting for 70-75h at 0.04-0.1; or, the fermentation conditions are 35-38 ℃, 480-₆₀₀0.04-0.1 percent, 5-10 percent of inoculation amount, 40-60 percent of liquid loading amount, 6.0-7.0 percent of initial pH value and 1-2vvm of ventilation amount, and fermenting for 70-80 hours.

The fourth purpose of the invention is to provide the application of the engineering strain in preparing target protein or in the fields of biology, pharmacy, food or chemical industry.

The fifth purpose of the invention is to provide the application of the method for regulating and controlling the gene expression of the target protein or the method for producing the acetoin in preparation of the target protein or in the fields of biology, pharmacy, food or chemical industry.

In one embodiment of the invention, the target protein comprises an enzymatic protein or a non-enzymatic protein.

The invention has the beneficial effects that:

the invention provides a method for constructing a dynamic regulation gene circuit, which has the advantage of exogenously adding an inducer in the fermentation process and space-time specificity. In addition, the method has simple design, few system elements and low strain growth load. By constructing an acetoin production engineering strain and introducing a dynamic regulation gene circuit, acetoin with the concentration of up to 66.8g/L can be accumulated in an inorganic salt culture medium, the conversion rate of the acetoin reaches 0.58g/g glucose, and the method has a good application prospect.

Drawings

FIG. 1: the fluorescent process curve of the near infrared light response under different illumination intensity and illumination period A is the feasibility of the near infrared light activation element; the dependence of the illumination intensity of near-infrared light activation; c, a fluorescence curve of a near infrared light activation process; the photoperiod dependence of near-infrared light activation.

FIG. 2: plasmid map, A: P_J23119-Y₃₀M-P_J23119-BudAB-NOX；B:P_mrkA-RpoS。

FIG. 3: a genetically engineered target of an acetoin-producing strain.

FIG. 4: acetoin shake flask fermentation parameters.

FIG. 5: acetoin 5L fermentor parameters.

Detailed Description

Materials and methods

The plasmid construction is carried out by classical molecular biology means.

Fluorescence process curve measurements were performed using a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, Calif.) with a controlled ambient temperature of 30 ℃.

Seed culture medium: LB culture medium, the ingredients include peptone 10g/L, yeast powder 5g/L, sodium chloride 10 g/L.

Fermentation medium: NBS inorganic salt culture medium with the components of 50g/L, CaCl glucose₂·2H₂O15 mg/L, 0.667mL/L of microelement liquid, sterilizing and supplementing MgSO₄·7H₂O 0.25g/L，V_B10.5mg/L, betaine hydrochloride 1 mM. Preparation method of trace element liquidIs FeCl₃·6H₂O 2.4g/L、CoCl₂·6H₂O 0.3g/L、CuCl₂ 0.15g/L、ZnCl₂·4H₂O 0.3g/L、NaMnO₄ 0.3g/L、H₃BO₃ 0.075g/L、MnCl₂·4H₂O0.5 g/L, dissolved in 0.1M HCl.

Preparation of a fermentation sample: taking a fermentation liquid sample, centrifuging at 12000rpm for 5min, taking supernatant liquid, diluting, filtering by a 0.22 mu m water system membrane, and taking filtrate as a sample for liquid chromatography analysis.

And (3) determining the acetoin content: the DEAN high performance liquid chromatograph (equipped with ultraviolet visible detector) adopts BerleAminex HPX-87H (300 × 7.8mm, 9 μ M) chromatographic column, and the mobile phase is H with concentration of 0.005M₂SO₄Filtering the mobile phase with 0.22 μm filter membrane, ultrasonic degassing at flow rate of 0.6mL/min and column temperature of 35 deg.C, and detecting at ultraviolet detection wavelength of 210 nm.

The nucleotide sequences of the promoters, genes or related elements involved, etc. are shown in Table 1.

TABLE 1 nucleotide sequence

Example 1 evaluation of near-infrared light-activated elements at different light intensities and light pulse periods

Will P_J23119-Y₃₀M-P_J23119-BudAB-NOX and P_mrkATransformation of the BFP plasmid into E.coli F0601 (see Dong X, Chen X, Qian Y, et al. Metabolic engineering of Escherichia coli W3110 to product L-plate [ J. ]].Biotechnology&Bioengineering,2016,114, (3):656-664.) engineered strains were obtained, cultured in LB medium and subjected to continuous fluorescence measurement using a SpectraMax M3 microplate reader. When the strain is under the near infrared light condition of 650nm, near infrared light photosensitive pigment (including Bphs, Bpho, Yhjh and/or Mrkh) is combined in front of the PmrkA promoter, so as to realize the expression of fluorescent protein BFP.

The experiment is carried out under dark or 650nm near infrared light conditions, the result is shown in figure 1, graph A and graph B show the dose dependence of the near infrared light system, when the near infrared light intensity is increased to 0.8W/cm²When the expression level of the BFP fluorescent protein is increased from 142.62OD (a.u.) to 1410.68OD (a.u.); the C diagram shows that the time (0-14h) and the illumination intensity (0.2-0.8W/cm)²) The expression quantity of BFP is gradually increased; graph D shows that when the pulse period of the near infrared light is increased to 100%, the expression level of the mKate fluorescent protein is increased from 152.69OD (a.u.) to 1437.9OD (a.u.).

Example 2 Shake flask fermentation Performance of acetoin-producing strains introduced by dynamic Gene lines

As shown in FIG. 3, with P_J23119-Y₃₀M-P_J23119-BudAB-NOX and P_mrkAIntroducing different dynamic gene circuits into E.coli F0601 strain to obtain Q1-Q77 strain engineering strain, wherein Q1 strain expresses that only P is expressed_J23119-Y₃₀M-P_J23119Engineered Strain obtained when BudAB-NOX, strain Q2 indicated expression of P alone_mrkAThe engineering strains obtained in the time of-Rpos, Q3-Q7 respectively represent that the illumination intensity of near infrared light is 0.2W/cm²、0.25W/cm²、0.3W/cm²、0.4W/cm²And 0.8W/cm.

As shown in FIGS. 4 and 5, the growth of the strain and the synthesis of the product were examined in NBS mineral salts medium. Culturing single bacterial colony of engineering strain in LB culture medium at 37 deg.C and 200rpm to OD₆₀₀The initial inoculation amount is 0.05, the initial inoculation amount is 1%, the fermentation is carried out for 70 hours, and the results of shake flask fermentation show that the strains Q1-Q2 respectively express only an acetoin production path and express accelerated division genes, the acetoin yield of the strains Q1-Q2 is improved by 18.5g/L from 13.5g/L, and the relative survival rate of cells is improved to 74.6% from 70.5%. The fermentation performance of the Q7 strain is 31.8g/L of acetoin accumulation.

Example 3 fermentation Performance of fermenter with introduction of acetoin-producing Strain by dynamic Gene line

The fermentation performance of the Q7 strain was tested in a 5L fermentor. Culturing single bacterial colony of engineering strain in LB culture medium at constant temperature of 37 ℃ and 500rpm to OD₆₀₀0.05, initial inoculum size 5%, aeration 1vvm, fermentation period 72h, liquid loading 3L, initial pH 6.8 with 4M sodium hydroxide and 2M hydrochloric acid. At the end of fermentation, the accumulated amount of acetoin reaches 66.8g/L, and the conversion rate reaches 0.58g/g glucose.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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<400> 9

atgaacgaaa aacacgggtg ttcctgtgcg cgccatttgg cgcagggttt tgccaggcag 60

tcgatcaacg ccggggaggg cgaaatctat caaatctcgc tgatgagcgc gctcatcgac 120

ggggtctacg agggggaaac caccattgcc gagttgctca aacacggcaa ctttggcctc 180

ggtaccttca atcatctgga cggtgaactg atcgctttcg accaggaaat acaccagctg 240

cgcgccgacg gcagcgcccg cccggccggc ctgcaacaga aaaccccctt cgccgtcgtc 300

acctttttcc agcccagcgt cagccagcaa ttcgatcggc cgatcaccaa ggcgcagctg 360

caccaatgca tcgatgaaca ggtcgcctcg ccgaacctgt tctgtgcggt gcgggtcgac 420

ggcgagttca gccacgtgga aacccgcacc gtgccgcgcc aggagcggcc ttaccgcccg 480

atgctggaag cgatagaaga gcagccgacc ttctcgttcc atcagcggcg cggcacgctg 540

gtcggcttcc gctcgccgga ttacatgcag ggcatcggcg tggccggcta tcacgaacac 600

ttcgttaccg acgaccgcag cggcggcggc cacgtgctgg actaccagct cgatcatggc 660

cgtctgcagt tcggcgtcat cacgcgtctc aatcttcagt taccgcatga tgcggatttc 720

ttgcgcgcca acctctgccc agaggatttg gaccgcgcga ttcgttccgc cgagggctaa 780

<210> 10

<211> 1686

<212> DNA

<213> Artificial Synthesis

<400> 10

atggcacagg aaaaaacagg caatgactgg caatgcggcg ccgatttggt ggtgaaaaac 60

ctggaagcgc agggcgtcaa acacgttttc ggtattccgg gcgccaagat cgaccgggtg 120

ttcgactcgc tggaggacgc gccgtcgatc gaaacggtgg tggtgcgcca tgaggccaac 180

gcggccttta tggcggcggc ggtcggccgc ttgaccggca aggccggggt ggcgctggtc 240

acctccgggc cgggcagctc caacctgatc accgggctgg ctaccgccac ctcggaaggg 300

gacgcggtgg tggccttcgg cggggcggtg aagcgcgccg acagcctgaa gcagacgcac 360

cagagcatgg ataccgtcag catgttccgg ccggtcacca agtattgcgc cgaagtgcat 420

gccggttcgg cgatttccga ggtgatcgcc aacgccttcc gccgcgccga gttcggccgg 480

ccgggggcat cgttcgtcag cctgccgatg gatatcgtca atgaaccggt cagcgcgccg 540

gtactggcgg gctgccgctt gccgcgcatg ggggccgccg cggcagacga cattcaggcg 600

gcggtgaaac tgatccgcca ggccaaatgc ccggtgctgc tgctgggctt gcaggccagc 660

cggccggaga acagcgaggc ggtgcgccat ctgctgtacc gcacccatat gccggtggtc 720

ggcacctatc aggcggcggg ggtgatcgac gtcaaccact tcgcccgttt cgccggccgc 780

gtcggcctgt tcaacaacca gccggccgat cagctgctgc agaaggccga tctggtggtg 840

agcgtcggct atgatccgat cgaatacgat ccctgcatgt ggaacagcca cgggcgactg 900

aagctggtgc atctcgacgt gctgccggcg gatatcgata cctgctatcg gccggacgtc 960

gagctggtgg gcaatatcag cgccacgctg aacatgatga ccgaagcatt cagtgaggcg 1020

gtctgcgtgc cgccggaggt ggagctgatc ctctccgatc tcggccgcca gcgcactgag 1080

ctggcggagc gcgccgcccg ccgcggcggc atgccgatcc acccgctgcg catcgtcaag 1140

gagctgcagg acatcgtcag cgacgacgtg acgctgtgtg tggacatggg cagtttccac 1200

atctggatcg cccgttatct ttacagcttc cgtgcccgtc agctgttgat ctccaacggc 1260

cagcaaacca tgggggtggc attgccgtgg gccatcggtg cggcgctggt gcgccccggc 1320

gacaaggtgg tgtctatatc cggtgacggc ggcttcatgc agtcgagcat ggagctggag 1380

accgcggtgc ggctgaaaaa caacatcgtg cacgtgatct gggtcgataa cgcctacaac 1440

atggtggaaa tgcaggaagt gaacaaatac cagcgcaagt ccggcgtgga gttcgggccg 1500

attgacttca aggcctacgc ggaatcctgc ggcgcggtcg gtttcgccgt gcagtcggtg 1560

gaagatctgc ggccgatgct gcgcaaggcg atggcgatcc aggggccggt agtggtggcc 1620

atcccggtcg actatgccga caactataag ctgatggcgc agatgaactt cagccagatg 1680

atttaa 1686

<210> 11

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 11

ttgacagcta gctcagtcct aggtataat 29

<210> 12

<211> 1392

<212> DNA

<213> Artificial Synthesis

<400> 12

atgaaaatca ttagcattaa attcgtgctc ggcggcaaca tcatgaaggt gaccgtggtt 60

ggctgtaccc atgccggcac cttcgcgatc aagcagattc tggcggaaca cccagacgcc 120

gaggtgacgg tttacgagcg caacgacgtg atcagctttc tcagctgtgg catcgcgctg 180

tatctgggtg gtaaagtggc cgacccacaa ggtctgtttt acagcagccc ggaagaactg 240

caaaagctgg gcgcgaacgt gcagatgaac cataacgttc tggccatcga tccggaccag 300

aagaccgtga ccgtggagga tctgaccagc catgcgcaga ccacggagag ctacgacaag 360

ctcgttatga ccagcggtag ctggccaatc gtgccaaaga tcccgggcat tgacagcgac 420

cgcgttaaac tgtgcaagaa ctgggcccat gcgcaagcgc tgatcgagga tgccaaggaa 480

gcgaaacgca tcacggtgat cggtgcgggt tacattggcg ccgaactggc cgaggcctat 540

agcaccaccg gccatgacgt gaccctcatc gacgccatgg atcgtgtgat gccgaagtac 600

ttcgacgccg acttcaccga cgttatcgaa caagattatc gcgaccatgg tgtgcagctc 660

gcgctgagcg aaaccgtgga aagctttacg gacagcgcca ccggcctcac cattaagacc 720

gataagaaca gctatgagac cgatctggcg attctgtgca ttggcttccg tccgaatacc 780

gatctgctga aaggcaaagt ggatatggcg ccaaacggcg cgatcatcac cgatgactac 840

atgcgcagca gcaacccgga catctttgcc gccggcgata gcgccgccgt gcattacaac 900

ccgacgcatc agaacgccta tatcccactg gccaccaatg ccgttcgcca aggcatcctc 960

gtgggcaaaa atctggttaa gccgacggtg aagtacatgg gcacgcagag cagcagtggt 1020

ctggcgctct acgatcgtac catcgttagt accggtctga cgctggcggc ggcgaaacag 1080

caaggcgtga atgcggaaca agttatcgtg gaagacaact accgcccgga gttcatgcca 1140

agcacggaac cagtgctgat gagtctggtg ttcgatccag acacccatcg cattctgggc 1200

ggtgcgctga tgagtaaata cgacgtgagc cagagcgcga atacgctgag tgtgtgcatc 1260

cagaacgaga atacgattga cgatctggcc atggtggata tgctgttcca gccgaacttc 1320

gaccgcccgt tcaactatct gaacattctg gcgcaagccg cgcaagccaa agttgcgcag 1380

agcgtgaatg cg 1392

<210> 13

<211> 1029

<212> DNA

<213> Artificial Synthesis

<400> 13

atgttccgtc aagggatcac gggtaggagc caccttatga gtcagaatac gctgaaagtt 60

catgatttaa atgaagatgc ggaatttgat gagaacggag ttgaggtttt tgacgaaaag 120

gccttagtag aagaggaacc cagtgataac gatttggccg aagaggaact gttatcgcag 180

ggagccacac agcgtgtgtt ggacgcgact cagctttacc ttggtgagat tggttattca 240

ccactgttaa cggccgaaga agaagtttat tttgcgcgtc gcgcactgcg tggagatgtc 300

gcctctcgcc gccggatgat cgagagtaac ttgcgtctgg tggtaaaaat tgcccgccgt 360

tatggcaatc gtggtctggc gttgctggac cttatcgaag agggcaacct ggggctgatc 420

cgcgcggtag agaagtttga cccggaacgt ggtttccgct tctcaacata cgcaacctgg 480

tggattcgcc agacgattga acgggcgatt atgaaccaaa cccgtactat tcgtttgccg 540

attcacatcg taaaggagct gaacgtttac ctgcgaaccg cacgtgagtt gtcccataag 600

ctggaccatg aaccaagtgc ggaagagatc gcagagcaac tggataagcc agttgatgac 660

gtcagccgta tgcttcgtct taacgagcgc attacctcgg tagacacccc gctgggtggt 720

gattccgaaa aagcgttgct ggacatcctg gccgatgaaa aagagaacgg tccggaagat 780

accacgcaag atgacgatat gaagcagagc atcgtcaaat ggctgttcga gctgaacgcc 840

aaacagcgtg aagtgctggc acgtcgattc ggtttgctgg ggtacgaagc ggcaacactg 900

gaagatgtag gtcgtgaaat tggcctcacc cgtgaacgtg ttcgccagat tcaggttgaa 960

ggcctgcgcc gtttgcgtga aatcctgcaa acgcaggggc tgaatatcga agcgctgttc 1020

cgcgagtaa 1029

<210> 14

<211> 687

<212> PRT

<213> Artificial Synthesis

<400> 14

Met Ala Arg Gly Cys Leu Met Thr Ile Ser Gly Gly Thr Phe Asp Pro

1 5 10 15

Ser Ile Cys Glu Met Glu Pro Ile Ala Thr Pro Gly Ala Ile Gln Pro

20 25 30

His Gly Ala Leu Met Thr Ala Arg Ala Asp Ser Gly Arg Val Ala His

35 40 45

Ala Ser Val Asn Leu Gly Glu Ile Leu Gly Leu Pro Ala Ala Ser Val

50 55 60

Leu Gly Ala Pro Ile Gly Glu Val Ile Gly Arg Val Asn Glu Ile Leu

65 70 75 80

Leu Arg Glu Ala Arg Arg Ser Gly Ser Glu Thr Pro Glu Thr Ile Gly

85 90 95

Ser Phe Arg Arg Ser Asp Gly Gln Leu Leu His Leu His Ala Phe Gln

100 105 110

Ser Gly Asp Tyr Met Cys Leu Asp Ile Glu Pro Val Arg Asp Glu Asp

115 120 125

Gly Arg Leu Pro Pro Gly Ala Arg Gln Ser Val Ile Glu Thr Phe Ser

130 135 140

Ser Ala Met Thr Gln Val Glu Leu Cys Glu Leu Ala Val His Gly Leu

145 150 155 160

Gln Leu Val Leu Gly Tyr Asp Arg Val Met Ala Tyr Arg Phe Gly Ala

165 170 175

Asp Gly His Gly Glu Val Ile Ala Glu Arg Arg Arg Gln Asp Leu Glu

180 185 190

Pro Tyr Leu Gly Leu His Tyr Pro Ala Ser Asp Ile Pro Gln Ile Ala

195 200 205

Arg Ala Leu Tyr Leu Arg Gln Arg Val Gly Ala Ile Ala Asp Ala Cys

210 215 220

Tyr Arg Pro Val Pro Leu Leu Gly His Pro Glu Leu Asp Asp Gly Lys

225 230 235 240

Pro Leu Asp Leu Thr His Ser Ser Leu Arg Ser Val Ser Pro Val His

245 250 255

Leu Asp Tyr Met Gln Asn Met Asn Thr Ala Ala Ser Leu Thr Ile Gly

260 265 270

Leu Ala Asp Gly Asp Arg Leu Trp Gly Met Leu Val Cys His Asn Thr

275 280 285

Thr Pro Arg Ile Ala Gly Pro Glu Trp Arg Ala Ala Ala Gly Met Ile

290 295 300

Gly Gln Val Val Ser Leu Leu Leu Ser Arg Leu Gly Glu Val Glu Asn

305 310 315 320

Ala Ala Glu Thr Leu Ala Arg Gln Ser Thr Leu Ser Thr Leu Val Glu

325 330 335

Arg Leu Ser Thr Gly Asp Thr Leu Ala Ala Ala Phe Val Ala Ala Asp

340 345 350

Gln Leu Ile Leu Asp Leu Val Gly Ala Ser Ala Ala Val Val Arg Leu

355 360 365

Ala Gly Gln Glu Leu His Phe Gly Arg Thr Pro Pro Val Asp Ala Met

370 375 380

Gln Lys Val Leu Asp Ser Leu Gly Arg Pro Ser Pro Leu Glu Val Leu

385 390 395 400

Ser Leu Asp Asp Val Thr Leu Arg His Pro Glu Leu Pro Glu Leu Leu

405 410 415

Ala Ala Gly Ser Gly Ile Leu Leu Leu Pro Leu Thr Ser Gly Asp Gly

420 425 430

Asp Leu Ile Ala Trp Phe Arg Pro Glu His Val Gln Thr Ile Thr Trp

435 440 445

Gly Gly Asn Pro Ala Glu His Gly Thr Trp Asn Pro Ala Thr Gln Arg

450 455 460

Met Arg Pro Arg Ala Ser Phe Asp Ala Trp Lys Glu Thr Val Thr Gly

465 470 475 480

Arg Ser Leu Pro Trp Thr Ser Ala Glu Arg Asn Cys Ala Arg Glu Leu

485 490 495

Gly Glu Ala Ile Ala Ala Glu Met Ala Gln Arg Thr Arg Ala Glu Glu

500 505 510

Leu Glu Arg Val Ala Met Val Asp Ser Leu Thr Arg Leu Trp Asn Arg

515 520 525

Leu Gly Ile Glu Thr Leu Leu Lys Arg Glu Trp Glu Tyr Ala Thr Arg

530 535 540

Lys Asn Ser Pro Ile Ser Ile Val Met Ile Asp Phe Asp Asn Phe Lys

545 550 555 560

Gln Ile Asn Asp Gln His Gly His Leu Val Gly Asp Glu Val Leu Gln

565 570 575

Gly Ser Ala Arg Leu Ile Ile Ser Val Leu Ala Ser Tyr Asp Ile Leu

580 585 590

Gly Arg Trp Gly Gly Asp Glu Phe Met Leu Ile Leu Pro Gly Ser Gly

595 600 605

Arg Glu Gln Thr Ala Val Leu Leu Glu Arg Ile Gln Ala Thr Ile Ala

610 615 620

Gln Asn Pro Val Pro Thr Ser Ala Gly Pro Met Ala Ile Ser Leu Ser

625 630 635 640

Met Gly Gly Val Ser Val Phe Thr Asn Gln Gly Glu Ala Leu Gln Tyr

645 650 655

Trp Val Glu Gln Ala Asp Asn Gln Leu Met Lys Val Lys Arg Leu Gly

660 665 670

Lys Gly Asn Phe Gln Leu Ala Glu Tyr His His His His His His

675 680 685

<210> 15

<211> 197

<212> PRT

<213> Artificial Synthesis

<400> 15

Met Pro Leu Ser Arg Asp Leu Arg Glu Lys Thr Gly Met Leu His Asn

1 5 10 15

Arg Ala Glu Thr Leu Leu Gly Leu Pro Ser Gly Ile Met Gly Trp Ala

20 25 30

Asp Tyr Val Asp Trp Leu Arg His Phe Leu Ala Leu Tyr Asp Pro Ile

35 40 45

Glu Arg Arg Ile Val Ala Phe Gly Gly Trp Ser Gly Leu Ala Ser Phe

50 55 60

Asp Pro Asp Pro Gly His Ser Arg Arg Leu Ile Gln Asp Leu His Ala

65 70 75 80

Leu Gly Ile Asp Thr Asp Arg Ile Pro Arg Ala Pro Ala Glu Tyr Cys

85 90 95

Pro Pro Leu Thr Asn Phe Ala Arg Ala Leu Gly Ala Arg Tyr Val Leu

100 105 110

Glu Gly Ser Ala Leu Gly Gly Arg Val Ile Leu His His Leu Lys Lys

115 120 125

Arg Ile Gly Asp Glu Ile Gly Asn Ala Thr Ala Phe Phe Gly Gly Pro

130 135 140

Ser His Gly Thr Ala Thr His Trp Arg Ala Phe Gln Ala Ala Leu Asp

145 150 155 160

Arg Phe Gly Ala Ala His Pro Asp Lys Arg Ala Asp Val Leu Ala Gly

165 170 175

Ala Ala Ala Thr Phe Thr Ala Leu Leu Glu Trp Phe Thr Pro Phe Val

180 185 190

Ala Ala Arg Arg Val

195

<210> 16

<211> 255

<212> PRT

<213> Artificial Synthesis

<400> 16

Met Ile Arg Gln Val Ile Gln Arg Ile Ser Asn Pro Glu Ala Ser Ile

1 5 10 15

Glu Ser Leu Gln Glu Arg Arg Phe Trp Leu Gln Cys Glu Arg Ala Tyr

20 25 30

Thr Trp Gln Pro Ile Tyr Gln Thr Cys Gly Arg Leu Met Ala Val Glu

35 40 45

Leu Leu Thr Val Val Thr His Pro Leu Asn Pro Ser Gln Arg Leu Pro

50 55 60

Pro Asp Arg Tyr Phe Thr Glu Ile Thr Val Ser His Arg Met Glu Val

65 70 75 80

Val Lys Glu Gln Ile Asp Leu Leu Ala Gln Lys Ala Asp Phe Phe Ile

85 90 95

Glu His Gly Leu Leu Ala Ser Val Asn Ile Asp Gly Pro Thr Leu Ile

100 105 110

Ala Leu Arg Gln Gln Pro Lys Ile Leu Arg Gln Ile Glu Arg Leu Pro

115 120 125

Trp Leu Arg Phe Glu Leu Val Glu His Ile Arg Leu Pro Lys Asp Ser

130 135 140

Thr Phe Ala Ser Met Cys Glu Phe Gly Pro Leu Trp Leu Asp Asp Phe

145 150 155 160

Gly Thr Gly Met Ala Asn Phe Ser Ala Leu Ser Glu Val Arg Tyr Asp

165 170 175

Tyr Ile Lys Ile Ala Arg Glu Leu Phe Val Met Leu Arg Gln Ser Pro

180 185 190

Glu Gly Arg Thr Leu Phe Ser Gln Leu Leu His Leu Met Asn Arg Tyr

195 200 205

Cys Arg Gly Val Ile Val Glu Gly Val Glu Thr Pro Glu Glu Trp Arg

210 215 220

Asp Val Gln Asn Ser Pro Ala Phe Ala Ala Gln Gly Trp Phe Leu Ser

225 230 235 240

Arg Pro Ala Pro Ile Glu Thr Leu Asn Thr Ala Val Leu Ala Leu

245 250 255

<210> 17

<211> 234

<212> PRT

<213> Artificial Synthesis

<400> 17

Met Thr Glu Gly Thr Ile Lys Thr Ser Lys Tyr Glu Ile Ile Ala Ile

1 5 10 15

Phe Arg Glu Glu Leu Arg Lys Arg Thr Glu Ile Glu Ile Phe Phe Asn

20 25 30

Asn Thr Ser Ile Ile Thr Gln Leu Thr Arg Val Asp Phe Ala Glu Phe

35 40 45

His Ile Gln Thr His Arg Lys Ile Pro Ser Gly His Lys Ile Arg Phe

50 55 60

Leu Leu His Ser Asp Ser Gly Lys Ile Glu Phe Asn Ala Ala Leu Thr

65 70 75 80

Lys His Asp Asn Ser Gly Val Asp Lys Gly Ile Arg Tyr Ala Phe Ser

85 90 95

Leu Pro Glu Cys Leu Gln Val Val Gln Arg Arg Arg Asp Pro Arg Phe

100 105 110

Arg Leu Arg His Glu His Asp Phe Tyr Cys Arg Gly Arg His Lys Asn

115 120 125

Gly Glu Asn Tyr Leu Phe Asp Ile Lys Asp Ile Ser Asp Gly Gly Cys

130 135 140

Ala Leu Met Thr Lys Thr Pro Asn Leu Lys Phe Leu Ser His Asn Ala

145 150 155 160

Leu Leu Lys Asn Ala Val Leu Met Leu Ala Glu Tyr Gly Glu Ile Thr

165 170 175

Ile Asp Leu Val Val Lys Asn Val Ile Val Ile Thr Leu Asp Asn Ala

180 185 190

Asn Glu Glu Ser Glu Ser Tyr Tyr Gln Ile Ser Cys Gln Phe Lys Phe

195 200 205

Arg His Leu Asp Asp Gln Arg Arg Ile Glu Lys Ile Leu Leu Asp Leu

210 215 220

Ile Leu Glu Ala Lys Arg Lys Lys Arg Ile

225 230

<210> 18

<211> 342

<212> PRT

<213> Artificial Synthesis

<400> 18

Met Phe Arg Gln Gly Ile Thr Gly Arg Ser His Leu Met Ser Gln Asn

1 5 10 15

Thr Leu Lys Val His Asp Leu Asn Glu Asp Ala Glu Phe Asp Glu Asn

20 25 30

Gly Val Glu Val Phe Asp Glu Lys Ala Leu Val Glu Glu Glu Pro Ser

35 40 45

Asp Asn Asp Leu Ala Glu Glu Glu Leu Leu Ser Gln Gly Ala Thr Gln

50 55 60

Arg Val Leu Asp Ala Thr Gln Leu Tyr Leu Gly Glu Ile Gly Tyr Ser

65 70 75 80

Pro Leu Leu Thr Ala Glu Glu Glu Val Tyr Phe Ala Arg Arg Ala Leu

85 90 95

Arg Gly Asp Val Ala Ser Arg Arg Arg Met Ile Glu Ser Asn Leu Arg

100 105 110

Leu Val Val Lys Ile Ala Arg Arg Tyr Gly Asn Arg Gly Leu Ala Leu

115 120 125

Leu Asp Leu Ile Glu Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu

130 135 140

Lys Phe Asp Pro Glu Arg Gly Phe Arg Phe Ser Thr Tyr Ala Thr Trp

145 150 155 160

Trp Ile Arg Gln Thr Ile Glu Arg Ala Ile Met Asn Gln Thr Arg Thr

165 170 175

Ile Arg Leu Pro Ile His Ile Val Lys Glu Leu Asn Val Tyr Leu Arg

180 185 190

Thr Ala Arg Glu Leu Ser His Lys Leu Asp His Glu Pro Ser Ala Glu

195 200 205

Glu Ile Ala Glu Gln Leu Asp Lys Pro Val Asp Asp Val Ser Arg Met

210 215 220

Leu Arg Leu Asn Glu Arg Ile Thr Ser Val Asp Thr Pro Leu Gly Gly

225 230 235 240

Asp Ser Glu Lys Ala Leu Leu Asp Ile Leu Ala Asp Glu Lys Glu Asn

245 250 255

Gly Pro Glu Asp Thr Thr Gln Asp Asp Asp Met Lys Gln Ser Ile Val

260 265 270

Lys Trp Leu Phe Glu Leu Asn Ala Lys Gln Arg Glu Val Leu Ala Arg

275 280 285

Arg Phe Gly Leu Leu Gly Tyr Glu Ala Ala Thr Leu Glu Asp Val Gly

290 295 300

Arg Glu Ile Gly Leu Thr Arg Glu Arg Val Arg Gln Ile Gln Val Glu

305 310 315 320

Gly Leu Arg Arg Leu Arg Glu Ile Leu Gln Thr Gln Gly Leu Asn Ile

325 330 335

Glu Ala Leu Phe Arg Glu

340

Claims

1. An engineering strain is characterized in thatThe engineering bacteria jointly express near-infrared light photosensitive pigments, acetoin synthesis pathway related genes, near-infrared light response components and target proteins, wherein the near-infrared light photosensitive pigments comprise Bphs, Bphos, Yhjh and Mrkh; the near infrared light photoresponse component comprises a promoter P_mrkA(ii) a The amino acid sequence of the Bphs is shown as SEQ ID NO.14, the amino acid sequence of the Bpho is shown as SEQ ID NO.15, the amino acid sequence of the Yhjh is shown as SEQ ID NO.16, the amino acid sequence of the Mrkh is shown as SEQ ID NO.17, and the P is shown as_mrkAThe nucleotide sequence of (A) is shown as SEQ ID NO. 5; the nucleotide sequence of the budA is shown as SEQ ID NO.9, and the nucleotide sequence of the budB is shown as SEQ ID NO. 10;

the gene coding for the target protein is located in the promoter P_mrkADownstream;

the target protein is Rpos; the amino acid sequence of the Rpos is shown as SEQ ID NO. 18;

coli F0601 as host cells.

2. The engineered strain of claim 1, wherein the nucleotide sequence encoding the Bphs is set forth in SEQ ID No.1, the nucleotide sequence encoding the Bpho is set forth in SEQ ID No.2, the nucleotide sequence encoding the Yhjh is set forth in SEQ ID No.3, and the nucleotide sequence encoding the Mrkh is set forth in SEQ ID No. 4.

3. A method for producing acetoin is characterized in that a single colony of the engineering strain of claim 1 or 2 is inoculated into an LB culture medium for activation, and is inoculated into a fermentation culture medium for fermentation after activation.

4. The method as claimed in claim 3, wherein the fermentation conditions are 35-38 ℃, 200-₆₀₀Fermenting for 70-75h at 0.04-0.1; or, the fermentation conditions are 35-38 ℃, 480-₆₀₀0.04-0.1, 5-10% of inoculation amount, 40-60% of liquid loading amount, 6.0-7.0 of initial pH, 1-2vvm of ventilation amount, 70% of fermentation-80h。

5. Use of the engineered strain of claim 1 or 2 in the preparation of acetoin.