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

Abstract

The invention discloses a near-infrared light-controlled dynamic regulation system and its application, and specifically discloses a near-infrared light combined-controlled dynamic regulation system and its application in the production of acetoin, belonging to the technical field of metabolic engineering. The present invention selects the near-infrared photosensitive protein by means of molecular biology. The production of acetoin in inorganic salt medium was achieved by introducing a dynamic regulatory gene circuit targeting RpoS in engineered E. The yield of acetoin produced by the method can reach 66.8 g/L, and the conversion rate can reach 0.58 g/g glucose.

Description

A near-infrared light-controlled dynamic control system and its application

technical field

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

Background technique

The dynamic regulation system is an emerging metabolic flow regulation method in the field of metabolic engineering. The main feature that is different from the static regulation is that during the fermentation process, the engineered strain will act according to the fermentation time, physiological state, intracellular metabolite concentration, and changes in the extracellular environment. The corresponding enzyme activity adjustment can be made, which in turn affects the distribution of metabolic flux and improves the production capacity of the product. The dynamic regulation system has the advantages that the fermentation process does not need manual regulation and does not need to add external inducers, and has significant advantages in the production of high value-added compounds.

The current dynamic regulation system is mainly controlled by the quorum sensing system, that is, the trigger of the system is composed of the original or heterologous quorum sensing system. When the concentration of bacteria reaches a certain concentration, the accumulated small molecules released by the quorum sensing system will act on the receptors of the regulatory system, so that the regulatory system will act accordingly. For example, in 2017, Apoorv Gupta et al. presented the dynamic system of Nature biotechnology, which is composed of the EsaI quorum sensing system and the addition of a degradation tag to the C-terminus of the target protein. When the bacterial concentration reaches a certain threshold, the target protein is degraded and the cell The internal absolute concentration reaches 0, so as to achieve the purpose and effect of closing the enzyme activity and regulating the metabolic flow. The system is path-independent and has been successfully applied in the production of inositol, glucaric acid and acetoin. In addition, in 2017, the AND logic gate dynamic regulation system published by Xinyuan He et al. in ACSsynthetic biology consists of a quorum sensing system and a stationary phase sensing protein. When the bacterial cell concentration reaches a certain threshold, the transcription and expression of the target protein is started, so as to achieve the purpose and effect of metabolic flux regulation. When the system is used in PHB production, the PHB output is increased by 1-2 times. In summary, both dynamic regulatory systems require the introduction of a heterologous quorum sensing system. However, the heterologous quorum sensing system is often composed of multiple transcriptional regulatory proteins, and its introduction will undoubtedly affect the growth and fermentation performance of the strain, while limiting the expression of the pathway enzymes.

Acetoin is a precursor for the preparation of flavors and fragrances, which is synthesized by the pyruvate pathway in Escherichia coli. The traditional method of producing acetoin achieves the accumulation of acetoin by overexpressing the pathway enzymes. However, the production of acetoin is limited by the specific surface area, causing the problem of uneven mass transfer of dissolved oxygen, which directly affects the growth and accumulation of acetoin in E. coli. Therefore, to provide a method for high-yield acetoin in a simple medium is of great significance for the industrial preparation of acetoin.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide an engineered strain that co-expresses a near-infrared photophytochrome, a near-infrared photosensitive component and a target protein, the near-infrared photophytochrome includes Bphs, Bpho, Yhjh and/or Mrkh The near-infrared light-responsive component comprises a promoter _PmrkA , the amino acid sequence of Bphs is shown in SEQ ID NO.14, the amino acid sequence of Bpho is shown in SEQ ID NO.15, and the amino acid sequence of Yhjh is shown in SEQ ID NO.15. The amino acid sequence is shown in SEQ ID NO.16, the amino acid sequence of Mrkh is shown in SEQ ID NO.17, and the nucleotide sequence of P _mrkA is shown in SEQ ID NO.5.

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

In one embodiment of the present 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 present invention, P _mrkA -RpoS and P _J23119 -SO-P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX are used as expression vectors.

P _J23119 -SO-P _J23119 -Y ₃₀ M is constructed as:

(1) Based on the commercial plasmid pTargetF (Addgene Plasmid#62226), the whole plasmid PCR method was used to insert the T7Te terminator sequence after the rrnB T1 terminator to reduce leaked expression; the whole plasmid PCR method was used to remove the sgRNA Expression cassette, obtain the engineering plasmid P _J23119 containing only Pj23119 constitutive promoter and double termination;

(2) Using the genome of Rhodobacter sphaeroides as a template, the Bphs, Bpho and Yhjh gene fragments were amplified respectively; the genome of Klebsiella pneumoniae was used as a template, and the mrkH gene fragment was obtained by amplification; homologous recombination was used In the same way, the Bphs and Bpho fragments of B0034RBS with B0034RBS (nucleotide sequence shown in SEQ ID NO. 7) were inserted into the P _J23119 expression frame to obtain the P _J23119- SO plasmid; The Yhjh of B0030RBS (nucleotide sequence shown in SEQ ID NO. 8) and the mrkH fragment of B0034RBS were inserted into the P _J23119 expression frame to obtain the P _J23119 -Y ₃₀ M plasmid; the P J23119-SO and P _J23119- SO and The two plasmids P _J23119 -Y ₃₀ M were integrated together to obtain the P _J23119 -SO-P _J23119 -Y ₃₀ M plasmid.

The construction method of P _mrkA- BFP plasmid is as follows: using bfp synthetic gene fragment (as shown in SEQ ID NO. 6) as a template, amplify the bfp gene containing B0034RBS, connect it to the P _J23119 plasmid by homologous recombination, and reverse it at the same time. The P _{mrkA plasmid was obtained by amplification, and the P mrkA-BFP plasmid} was obtained by homologous recombination.

The construction method of P _J23119 -BudAB-NOX plasmid is as follows: using Serratia marcescens H30 genome as a template, design primers with B0034RBS (nucleotide sequence shown in SEQ ID NO. 7), respectively. Amplify to obtain budA (nucleotide sequence as shown in SEQ ID NO. 9) and budB fragments (nucleotide sequence as shown in SEQ ID NO. 10) containing B0034RBS, and the two fragments of budA and budB adopt multi-fragment one-step synchronization. The mode of source recombination was inserted into the expression frame of P _J23119 (nucleotide sequence as shown in SEQ ID NO.11) to obtain the P _J23119 -BudAB plasmid; the Lactobacillus brevis genome was used as a template to amplify nox gene, the nox gene fragment (as shown in SEQ ID NO. 12) was inserted into the expression frame of P _J23119 -BudAB by means of multi-fragment one-step homologous recombination to obtain the P _J23119 -BudAB-NOX plasmid.

The construction method of the P _J23119 -SO-P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX plasmid is as follows: using the P _J23119 -BudAB-NOX plasmid as a template, the P _J23119 -BudAB-NOX fragment is amplified by homologous recombination, Then, using P _J23119 -SO-P _J23119 -Y ₃₀ M as a vector, enzyme digestion, homologous recombination, and inserting the P _J23119 -SO-P _J23119 -Y ₃₀ M expression cassette to obtain P _J23119 -SO-P _J23119 -Y ₃₀ MP _J23119 - BudAB-NOX plasmid.

The construction method of described _PmrkA- RpoS plasmid is: utilize the method of homologous recombination to replace the gene encoding BFP fluorescent protein in the _PmrkA -BFP plasmid with the gene encoding the endogenous RpoS protein of Escherichia coli (the nucleotide sequence is as shown in Fig. shown in SEQ ID NO. 14).

In one embodiment of the present invention, E. coli F0601 is used as the host cell.

The second object of the present invention is to provide a method for regulating the expression of a target protein gene, which is to supplement near-infrared light in the fermentation process of an engineered strain to control the expression of the target protein gene, and the engineered strains co-express near-infrared photophytochrome, A near-infrared light-responsive component and a target protein, the near-infrared photophytochrome includes Bphs, Bpho, Yhjh or Mrkh; the near-infrared light-responsive component includes a promoter P _mrkA , and the amino acid sequence of the Bphs is as shown in SEQ ID NO.14, the amino acid sequence of Bpho is shown in SEQ ID NO.15, the amino acid sequence of Yhjh is shown in SEQ ID NO.16, and the amino acid sequence of Mrkh is shown in SEQ ID NO.17 As shown, the nucleotide sequence of the P _mrkA is shown in SEQ ID NO.5.

In an embodiment of the present invention, the illumination intensity of the near-infrared light is 0.2W/cm ² -0.8W/cm ² , the wavelength is 600-650nm, and the distance from the strain medium is fixed at 5-15cm.

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

The third object of the present invention is to provide a method for producing acetoin, in which a single colony of the above-mentioned engineering strain is inoculated into an LB medium for activation, and then inserted into a fermentation medium for fermentation after activation.

In one embodiment of the present invention, the fermentation medium comprises NBS inorganic salt medium.

In one embodiment of the present invention, the fermentation conditions are 35-38°C, 200-220rpm, the initial OD ₆₀₀ after activation is 0.04-0.1, and the fermentation conditions are 70-75h; or, the fermentation conditions are 35-38°C , 480-530rpm, the initial OD ₆₀₀ after activation is 0.04-0.1, the inoculum volume is 5-10%, the liquid filling volume is 40%-60%, the initial pH is 6.0-7.0, the aeration volume is 1-2vvm, fermentation 70 -80h.

The fourth object of the present invention is to provide the application of the above-mentioned engineered strain in the preparation of target protein or in the fields of biology, pharmacy, food or chemical industry.

The fifth object of the present invention is to provide the application of the above-mentioned method for regulating the expression of target protein gene or the above-mentioned method for producing acetoin in the preparation of target protein or in the fields of biology, pharmacy, food or chemical industry.

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

Beneficial effects of the present invention:

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

Description of drawings

Figure 1: Fluorescence process curves of NIR photoresponse under different illumination intensities and photoperiods, A: Feasibility of NIR photoactivated elements; B: Illumination intensity dependence of NIR photoactivation; C: NIR photoactivation process Fluorescence curves; D: photoperiod dependence of near-infrared light activation.

Figure 2: Plasmid map, A: P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX; B: P _mrkA -RpoS.

Figure 3: Genetically engineered targets of acetoin-producing strains.

Figure 4: Acetoin shake flask fermentation parameters.

Figure 5: Acetoin 5L fermenter parameters.

Detailed ways

Materials and Methods

Plasmid construction was carried out by classical molecular biology methods.

The fluorescence process curve was measured using a SpectraMax M3 microplate microplate reader (Molecular Devices, Sunnyvale, CA), and the ambient temperature was controlled at 30 degrees.

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

Fermentation medium: NBS inorganic salt medium, the ingredients include glucose 50g/L, CaCl ₂ ·2H ₂ O 15mg/L, trace element liquid 0.667mL/L, and MgSO ₄ ·7H ₂ O 0.25g/L after sterilization , V _B1 0.5mg/L, betaine hydrochloride 1mM. The configuration method of the trace element liquid is FeCl ₃ ·6H ₂ O 2.4g/L, CoCl ₂ ·6H ₂ O 0.3g/L, CuCl ₂ 0.15g/L, ZnCl ₂ ·4H ₂ O 0.3g/L, NaMnO ₄ 0.3 g/L, H ₃ BO ₃ 0.075g/L, MnCl ₂ ·4H ₂ O 0.5g/L, dissolved in 0.1M HCl to prepare.

Preparation of fermentation samples: take fermentation broth samples, centrifuge at 12,000 rpm for 5 min, take the supernatant and dilute it, filter it through a 0.22 μm aqueous membrane, and use the filtrate for liquid chromatography analysis.

Determination of acetoin content: Dionex high performance liquid chromatograph (with UV-visible detector), using Biole AminexHPX-87H (300×7.8mm, 9μm) chromatographic column, the mobile phase is H ₂ SO ₄ with a concentration of 0.005M, The mobile phase was filtered through a 0.22 μm filter membrane, degassed by ultrasonic, the flow rate was 0.6 mL/min, the column temperature was 35 °C, and the detection was performed at an ultraviolet detection wavelength of 210 nm.

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

Table 1 Nucleotide sequences

Example 1 Evaluation of near-infrared light-activated elements under different light intensities and light pulse periods

The P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX and P _mrkA -BFP plasmids were transferred into E. coli F0601 (see Dong X, Chen X, Qian Y, et al. Metabolic engineering of Escherichia coli W3110 to produce L- malate[J].Biotechnology&Bioengineering,2016,114(3):656-664.), obtained the engineered strain, placed it in LB medium for cultivation, and used the SpectraMax M3 microplate microplate reader to continuously carry out the above engineered strains. Fluorescence assay. When the strain is exposed to near-infrared light at 650 nm, near-infrared photophytochromes (including Bphs, Bpho, Yhjh and/or Mrkh) bind to the front of the PmrkA promoter to achieve the expression of the fluorescent protein BFP.

Experiments were carried out under dark or 650nm near-infrared light illumination conditions. The results are shown in Figure 1. Figures A and B show the dose dependence of the illumination intensity of the near-infrared light system. When the illumination intensity of the near-infrared light increases to 0.8W At /cm ² , the expression of BFP fluorescent protein increased from 142.62 OD (au) to 1410.68 OD (au); Figure C shows that with time (0-14h) and light intensity (0.2-0.8W/cm ² ) Increase, the expression of BFP gradually increased; D figure shows that when the near-infrared light pulse period increased to 100%, the mKate fluorescent protein expression increased from 152.69OD(au) to 1437.9OD(au).

Example 2 The shake flask fermentation performance of acetoin production strain introduced by dynamic gene line

As shown in Figure 3, using P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX and P _mrkA -RpoS (see Figure 2 for the plasmid map) as expression vectors, different dynamic gene lines were introduced into E.coli F0601 strain, a total of Seven engineering strains Q1-Q7 were obtained. The Q1 strain represented the engineered strain obtained when only P _J23119 -Y ₃₀ MP _J23119 -BudAB-NOX was expressed, and the Q2 strain represented the engineered strain obtained when only P _mrkA -RpoS was expressed. Indicates the engineering strains when the near-infrared light intensity is 0.2W/cm ² , 0.25W/cm ² , 0.3W/cm ² , 0.4W/cm ² , and 0.8W/cm , respectively.

As shown in Figures 4 and 5, strain growth and product synthesis were detected in NBS inorganic salt medium. 37°C, 200rpm, single colony of engineered strain was cultured in LB medium to OD ₆₀₀ of 0.05, the initial inoculum was controlled to 1%, and fermented for 70h. The results of shake flask fermentation showed that strains Q1-Q2 expressed only acetoin Production path, expression of accelerated division gene, the acetoin yield of Q1-Q2 strain increased from 13.5g/L to 18.5g/L, and the relative cell survival rate increased from 70.5% to 74.6%. The fermentation performance of strain Q7 was 31.8g/L of acetoin accumulation.

Example 3 The fermentor fermentation performance of the acetoin-producing strain introduced into the dynamic gene line

The fermentation performance of the Q7 strain was tested in a 5L fermenter. The temperature was constant at 37°C, 500rpm, and the single colony of the engineered strain was cultured in LB medium to OD ₆₀₀ of 0.05, the initial inoculum was controlled to 5%, the ventilation rate was 1vvm, the fermentation period was 72h, the liquid volume was 3L, and 4M hydrogen was used. Sodium oxide and 2M hydrochloric acid control the initial pH to 6.8. At the end of fermentation, the accumulation of acetoin reached 66.8 g/L, and the conversion rate reached 0.58 g/g glucose.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

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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> Synthetic

<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 ccccgttatct 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> Synthetic

<400> 11

ttgacagcta gctcagtcct aggtataat 29

<210> 12

<211> 1392

<212> DNA

<213> Synthetic

<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> Synthetic

<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> Synthetic

<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> Synthetic

<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> Synthetic

<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> Synthetic

<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> Synthetic

<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 (5)

1. an engineering strain, is characterized in that, described engineering bacteria jointly expresses near-infrared phytochrome, acetoin synthesis pathway related gene, near-infrared photoresponse component and target protein, and described near-infrared phytochrome comprises Bphs, Bpho, Yhjh and Mrkh; the near-infrared light-responsive component includes a promoter P _mrkA ; the amino acid sequence of the Bphs is shown in SEQ ID NO.14, and the amino acid sequence of the Bpho is shown in SEQ ID NO.15 shown, the amino acid sequence of the Yhjh is shown in SEQ ID NO.16, the amino acid sequence of the Mrkh is shown in SEQ ID NO.17, and the nucleotide sequence of the P _mrkA is shown in SEQ ID NO.5; the Acetoin synthesis pathway related genes budA and budB, the budA nucleotide sequence is shown in SEQ ID NO.9, and the budB nucleotide sequence is shown in SEQ ID NO.10; The gene encoding the target protein is located downstream of the promoter P _mrkA ; The target protein is RpoS; the amino acid sequence of the RpoS is shown in SEQ ID NO.18; E.coli F0601 was used as the host cell. 2. The engineering strain of claim 1, wherein the nucleotide sequence encoding the Bphs is shown in SEQ ID NO.1, and the nucleotide sequence encoding the Bpho is shown in SEQ ID NO.2 , the nucleotide sequence encoding the Yhjh is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Mrkh is shown in SEQ ID NO.4. 3. a method for producing acetoin, is characterized in that, the single colony of engineering strain described in claim 1 or 2 is inoculated in LB medium to carry out activation, and after activation, access fermentation medium to carry out fermentation. 4. The method of claim 3, wherein the fermentation conditions are 35-38°C, 200-220rpm, the initial OD ₆₀₀ after activation is 0.04-0.1, and the fermentation conditions are 70-75h; or, the fermentation conditions 35-38°C, 480-530rpm, initial OD ₆₀₀ after activation is 0.04-0.1, inoculum volume is 5-10%, filling volume is 40%-60%, initial pH is 6.0-7.0, aeration volume is 1 -2vvm, fermentation 70-80h. 5. the application of the engineering strain described in claim 1 or 2 in the preparation of acetoin.

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