CN114517206B

CN114517206B - Recombinant Mediterranean salt-rich bacteria and application thereof in preparation of PHBV

Info

Publication number: CN114517206B
Application number: CN202011277922.9A
Authority: CN
Inventors: 韩静; 向华; 林琳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2024-05-24
Anticipated expiration: 2040-11-16
Also published as: CN114517206A

Abstract

The invention discloses recombinant Mediterranean salt-rich bacteria and application thereof in preparation of PHBV. The invention discloses a recombinant Mediterranean salt-rich bacterium, which comprises the following construction steps: 1) Knocking out cas3 genes in the salt-rich bacteria of the origin Zhonghai to obtain cas3 knocked-out bacteria; 2) Introducing a crRNA expression cassette for transcriptional inhibition of citrate synthase gene expression into cas3 knockout bacteria to obtain recombinant Mediterranean salt-rich bacteria. Experiments prove that the recombinant Mediterranean salt-rich bacteria have higher biomass and faster glucose consumption rate, and the cell dry weight and PHBV accumulation are obviously improved. The recombinant Mediterranean salt-rich bacteria has good application prospect.

Description

Recombinant Mediterranean salt-rich bacteria and application thereof in preparation of PHBV

Technical Field

The invention relates to the field of biotechnology, and discloses recombinant Mediterranean salt-rich bacteria and application thereof in preparation of PHBV.

Background

Polyhydroxyalkanoate (PHA) is an intracellular polymer produced by microorganisms in the presence of an excess of a carbon source and a limited amount of other elements such as nitrogen, phosphorus, sulfur, oxygen, etc., and is mainly used as a carbon source and a reservoir of energy in cells. PHA is mainly classified into short-chain PHA of 3 to 5 carbon atoms and medium-long-chain PHA of 6 carbon atoms or more, depending on the number of carbon atoms of the PHA monomer. In addition, copolymerized PHAs composed of short-chain PHA monomers and medium-long chain PHA monomers are also included. PHAs can be classified into homopolymers and copolymers according to whether the monomers contained in the PHA are single. Homopolymers contain only one monomer, such as poly-3-hydroxybutyrate (PHB), which is the most common and simplest PHA homopolymer in bacteria. The copolymer is composed of two or more monomers, such as Polyhydroxybutyrate Hydroxyvalerate (PHBV), comprising two monomers of 3HB and 3 HV. PHB and PHBV are two common PHAs that have been mass-produced industrially.

The variety and composition of PHA monomers can lead the material properties to have diversified characteristics and plasticity advantages, and can meet different application requirements. PHA has similar material properties to traditional petrochemical plastics, except that PHA can be completely degraded into the ecological cycle in the natural environment and is therefore considered to be a "green plastic". It can replace traditional plastics which are not degradable, so as to relieve the problem of increasingly serious white pollution. In addition, PHA has a certain application prospect in the field of biological medicine due to good biocompatibility, and can be used for tissue engineering materials, drug slow release carriers and the like.

The Mediterranean salt-rich bacteria (Haloferax mediterranei) is a strain with the potential of PHBV industrialized production. The PHBV production by using the Mediterranean salt-rich bacteria has the following advantages: PHBV can be synthesized by using cheap non-relevant carbon sources (such as starch, waste molasses, alcohol industrial waste, biodiesel industrial byproducts and the like), so that the raw material cost is reduced; seawater culture can be utilized to reduce the consumption of fresh water resources; the high-salt culture environment reduces the risk of bacteria contamination, reduces the sterilization requirement and can even realize continuous fermentation without sterilization; the cells can be cracked in clear water, so that the extraction cost of PHBV is reduced; the low-cost ceramic, plastic or cement fermentation tank can be used for fermentation, so that the equipment cost is saved; has no pathogenicity and high safety.

At present, the main factor limiting the application of PHBV is that the production cost is higher, and the PHBV yield can not meet the industrial requirement when the Mediterranean salt-rich bacteria is used as a strain with the industrial production potential of PHBV. Therefore, there is a need to increase PHBV yield of Salmonella rich in Mediterranean to reduce the production cost of PHBV.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the yield of Polyhydroxybutyrate Hydroxyvalerate (PHBV) of Mediterranean salt-rich bacteria.

In order to solve the technical problems, the invention firstly provides a method for constructing recombinant Mediterranean salt-rich bacteria, which comprises the following steps:

1) Knocking out cas3 genes in the salt-rich bacteria of the origin Zhonghai to obtain cas3 knocked-out bacteria;

2) Introducing a crRNA expression cassette for transcriptional inhibition of citrate synthase gene expression into the cas3 knockout bacterium to obtain recombinant Mediterranean salt-rich bacteria.

In one embodiment of the invention, the open-country sea salt-rich bacteria is Mediterranean salt-rich bacteria (Haloferax mediterranei) DF50- ΔEPS.

The sequence of the cas3 gene is SEQ ID No.3 in a sequence table.

In the above method, the citrate synthase gene may be an HFX_0432 gene and/or an HFX_6170 gene, the sequence of the HFX_0432 gene is shown as SEQ ID No.5, and the sequence of the HFX_6170 gene is shown as SEQ ID No. 6.

In the above method, the crRNA may target a coding region or a promoter region of the citrate synthase gene.

In the method, the expression cassette can be obtained by connecting a promoter, a repeat sequence, a spacer sequence and a terminator sequence; the repeat sequence is 55-84 th bit in SEQ ID No.4 in the sequence table, and the spacer sequence targets the coding region or the promoter region of the citrate synthase gene.

The spacer sequence can be the 85 th to 119 th and/or 150 th to 184 th positions in SEQ ID No.4 in the sequence table.

In the method, the promoter can be a promoter P _phaR shown in positions 1-54 of SEQ ID No.4 in a sequence table; the terminator may be a T8 terminator shown in positions 215-222 of SEQ ID No.4 of the sequence Listing.

In the above method, the expression cassette may be a DNA fragment shown in SEQ ID No.4 of the sequence Listing.

The expression cassette can be introduced into the Zhonghai salt-rich bacteria in the place of departure through a recombinant vector containing the expression cassette.

The recombinant vector may be pWL.about.502-crCS, and pWL.about.502-crCS is a recombinant vector obtained by replacing the DNA fragment between the BamH I and Kpn I recognition sequences of pWL.about.502 with the expression cassette.

The invention also provides another method for constructing recombinant Mediterranean salt-rich bacteria, which comprises inhibiting the expression of the citrate synthase gene in the origin Mediterranean salt-rich bacteria or reducing the content or activity of the citrate synthase in the origin Mediterranean salt-rich bacteria to obtain the recombinant Mediterranean salt-rich bacteria.

The recombinant Mediterranean salt-rich bacteria obtained by the method for constructing the recombinant Mediterranean salt-rich bacteria also belong to the protection scope of the invention.

The present invention also provides a method of producing PHBV, the method comprising: culturing the recombinant Mediterranean salt-rich bacteria in a culture medium containing glucose or starch to obtain PHBV.

In the above method, the medium may be MG medium.

The MG culture medium consists of a solvent and a solute, wherein the solvent is water, and the concentration of the solute in the MG culture medium are NaCl 110g/L、MgSO₄·7H₂O 29.52g/L、MgCl₂·6H₂O 20.51g/L、KCl 5g/L、NH₄Cl 2g/L、CaCl₂ 1g/L、KH₂PO₄ 37.5mg/L、FeSO₄·7H₂O 5mg/L、MnCl₂·4H₂O0.036mg/L、 piperazine-1, 4-diethyl sulfonic acid (PIPES) 15g/L, glucose (Glucose) 10g/L and pH7.0-7.2.

In the above method, the culturing may be performed at 37 ℃.

The invention also provides a kit of reagents, which consists of the cas3 knockout bacterium and the expression cassette, or consists of the cas3 knockout bacterium and the recombinant vector.

The kit can be used for constructing recombinant bacteria for preparing PHBV and can be used for preparing PHBV.

The method for constructing the recombinant Mediterranean salt-rich bacteria or the application of the recombinant Mediterranean salt-rich bacteria in preparing PHBV also belong to the protection scope of the invention.

Experiments prove that the recombinant Mediterranean salt-rich bacteria obtained by the method for constructing the recombinant Mediterranean salt-rich bacteria have higher biomass and faster glucose consumption rate, all glucose is consumed during 120h of culture, and the residual sugar is consumed 72h earlier than that of a control strain, so that the fermentation period is shortened. Compared with a control strain, the cell dry weight and the PHBV accumulation amount of the recombinant Mediterranean salt-rich bacteria obtained by the invention are obviously improved, the cell dry weight of the recombinant Mediterranean salt-rich bacteria is improved to 5.66g/L from 3.63g/L of the control strain, the PHBV yield is improved to 3.14g/L from 1.78g/L, and the PHBV yield is improved by 76.4% compared with the control strain. The method of the invention has good application prospect with the obtained recombinant Mediterranean salt-rich bacteria.

Drawings

FIG. 1 shows the change in the transcript levels of HFX_0432 in the Mediterranean salt-rich bacteria DF 50. DELTA. EPS. DELTA.cas 3-crCS after simultaneous inhibition of HFX_0432 and HFX_6170. no crRNA represents DF 50. DELTA. EPS. DELTA.cas 3-no crRNA, and crCS represents DF 50. DELTA. EPS. DELTA.cas 3-crCS.

FIG. 2 shows the change in the transcript levels of HFX_0432 and HFX_6170 in the salt-rich Mediterranean bacteria DF 50. DELTA. EPS. DELTA.cas 3-crCS after simultaneous inhibition. no crRNA represents DF 50. DELTA. EPS. DELTA.cas 3-no crRNA, and crCS represents DF 50. DELTA. EPS. DELTA.cas 3-crCS.

FIG. 3 shows the growth of citrate synthase gene-inhibiting strains. no crRNA represents DF 50. DELTA. EPS. DELTA.cas 3-no crRNA, and crCS represents DF 50. DELTA. EPS. DELTA.cas 3-crCS.

FIG. 4 shows the glucose consumption of the citrate synthase gene-inhibiting strain. no crRNA represents DF 50. DELTA. EPS. DELTA.cas 3-no crRNA, and crCS represents DF 50. DELTA. EPS. DELTA.cas 3-crCS.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The Mediterranean salt-rich bacterium (Haloferax mediterranei) DF 50-delta EPS is a uracil auxotroph high-yield PHBV Mediterranean salt-rich bacterium with a knocked-out extracellular polysaccharide synthesis gene cluster, and is described in "Zhao D,Cai L,Wu J,et al.Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei[J].Applied Microbiology and Biotechnology,2013,97:3027-3036.", the biological material is available to the public from the applicant, and is only used for repeated experiments related to the invention, but can not be used for other purposes.

The following steps of plasmid transformation of Mediterranean salt-rich bacteria are as follows:

① Single colonies of the Mediterranean salt-rich bacteria are inoculated into AS-168 culture medium containing uracil (namely, uracil is added to the AS-168 culture medium, the concentration of the uracil in the culture medium is 50 mg/L), and the culture is carried out until the culture medium reaches a stable phase, and then the culture medium is used for culturing the Mediterranean salt-rich bacteria at the following condition of 1:50 was transferred to fresh medium, cultured at 37℃for about 24h for transformation;

② Taking 1mL of bacterial liquid into a sterilized 1.5mL centrifuge tube, centrifuging at 6,000rpm for 3min;

③ Discarding the supernatant, adding 200 mu L of BSS-LS solution, gently blowing and sucking suspended thalli by a pipetting gun, centrifuging at 6,000rpm for 3min;

④ Discarding the supernatant, adding 100 mu L of BSS-LS+ Glycerol solution, and lightly blowing uniformly by a pipetting gun;

⑤ Adding 10 mu L of 0.5M EDTA solution (pH 8.0) to the wall of the centrifuge tube, gently beating the wall of the centrifuge tube by hand, mixing the EDTA solution with the bacterial liquid, and standing at room temperature for 10min to form protoplast;

⑥ Sucking 10 mu L of plasmid to be converted, adding the plasmid to the tube wall, tapping the centrifuge tube, mixing the plasmid with bacterial liquid, and standing at room temperature for 5min;

⑦ 120 mu L of 60% (volume percent) PEG600 (solvent is UBSS-LS solution and solute is PEG 600) is added to the tube wall, the centrifuge tube is quickly turned upside down, the solution becomes uniform and transparent, and the solution is kept stand at room temperature for 25min;

⑧ Adding 1mL of recovery culture medium, mixing the mixture upside down, centrifuging the mixture at 6,000rpm for 3min, discarding the supernatant, adding 600 mu L of recovery culture medium, and carrying out shaking table recovery at 200rpm for 24h at 37 ℃;

⑨ The bacterial liquid is added to an AS-168SY culture medium flat plate, the flat plate is swayed to enable the bacterial liquid to be uniformly adsorbed on the flat plate, the flat plate is placed in a constant temperature incubator at 37 ℃, after the bacterial liquid on the flat plate is dried, the flat plate is placed in a sealed plastic bag, and the flat plate is cultured in an inverted mode at 42 ℃ for 4 days, so that light pink transformants can be seen.

BSS-LS solution:

sterilizing at 115deg.C for 30min, adding 5mL sterilized 1M Tris-HCl (pH 8.2), and packaging.

BSS-ls+ Glycerol solution:

UBSS-LS solution:

Regulating pH to 7.2 with NaOH, sterilizing at 115deg.C under high pressure for 30min, and packaging.

Resuscitating medium:

The pH was adjusted to 7.5 with Tris base or NaOH and autoclaved at 115℃for 30min.

30% Artificial brine:

Reagents other than CaCl ₂ were dissolved first and then CaCl ₂ solution was slowly added.

0.5M EDTA solution (pH 8.0): EDTA was dissolved in water to a concentration of 0.5M and pH was adjusted to 8.0 with NaOH.

The formula of AS-168SY medium is AS follows:

Adjusting pH to 7.0-7.2 with NaOH.

The formula of AS-168 medium is AS follows:

Adjusting pH to 7.0-7.2 with NaOH.

Wherein Casamino acids is BD company in the united states (Becton, dickinson and company), product number 7241547; trisodium citrate dihydrate is a product of national pharmaceutical group chemical reagent limited company, cat# 10019418; the L-sodium glutamate is a product of national pharmaceutical group chemical reagent limited company, and the product number is 62010638; yeast extract is an OXOID product with a product number of 2831838-02; peptone (soya) is a Beijing bispin microbiological media manufacturing factory product, under the product number CM:02-19.

The MG medium formulation was as follows:

adjusting pH to 7.0-7.2 with NaOH, and sterilizing at 115deg.C under high pressure for 30min.

Wherein PIPES is a product of Beijing Ding Guo prosperous biotechnology Limited liability company, and the product number is BP248-250G.

Example 1,

1. Knockout cas3 Gene

Taking Mediterranean salt-rich bacteria (Haloferax mediterranei) DF 50-delta EPS with high yield of PHBV as an initial strain, and knocking out cas3 genes in the Mediterranean salt-rich bacteria DF 50-delta EPS to construct the Mediterranean salt-rich bacteria DF50 delta EPS delta cas3. The method comprises the following steps:

(1) PCR amplifying the upper and lower homologous arms of Cas3 gene by using the primer Cas3-U-F/Cas3-U-R and Cas3-D-F/Cas3-D-R as templates, respectively, then performing overlap PCR by using the amplified PCR product containing the upper and lower homologous arms as templates, performing enzyme digestion by using the primer Cas3-U-F and Cas3-D-R, recovering the product, performing enzyme digestion by using BamH I and Kpn I, recovering large fragments, connecting the obtained large fragments with a carrier skeleton obtained by performing double enzyme digestion on the carrier pHFX(Liu H,Han J,Liu X,et al.Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica[J].Journal of Genetics and Genomics,2011,38:261-269.) by using BamH I and Kpn I, and recording the obtained recombinant carrier with correct sequence as pHFX-Cas3.

The primer sequences used were as follows:

Cas3-U-F:5'-ATAGGATCCGCTCGTTGACCCGGCGTA-3', underlined, represents the recognition sequence of BamH I;

Cas3-U-R：5′ the bolded part is a partial sequence in the upstream homology arm, and the square part is a partial sequence in the downstream homology arm;

Cas3-D-F：5′ The bolded part is a partial sequence in the downstream homology arm, and the square part is a partial sequence in the upstream homology arm;

the Cas3-U-R is reverse complementary to the Cas 3-D-F;

Cas3-D-R:5'-ATAGGTACCCAGTCAATTCTACGTCTT-3', underlined, indicates the recognition sequence of Kpn I.

The PCR product amplified by the Cas3-U-F and the Cas3-U-R contains an upstream homology arm shown as SEQ ID No.1 in a sequence table, and the PCR product amplified by the Cas3-D-F and the Cas3-D-R contains a downstream homology arm shown as SEQ ID No.2 in the sequence table.

(2) Introducing pHFX-cas3 obtained in the step (1) into escherichia coli E.coli JM110 (product of the department of Optimus and the product of the name of the engineering, TSC 08) for demethylation, extracting recombinant vector pHFX-cas3, and introducing into Mediterranean salt-rich bacteria (Haloferax mediterranei) DF 50-delta EPS to obtain recombinant bacteria; the obtained recombinant strain was cultured on an AS-168SY medium plate for about 4 days, and the grown transformant was streaked on the AS-168SY medium plate, and cultured for about 2 days, and the strain having undergone single crossover (strain having undergone first homologous recombination in the upstream or downstream homology arm) was identified by PCR amplification of the grown monoclonal genomic DNA.

The primers used were Cas3-U-F and Cas3-D-R.

Can be amplified into single-exchange strains with the size of 3419bp and 828bp, or non-exchange strains.

(3) The single-crossover strain was inoculated into AS-168+FU liquid medium (the medium is a medium with uracil concentration of 50mg/L and 5-fluoroorotic acid concentration of 250mg/L obtained by adding uracil and 5-fluoroorotic acid to AS-168 medium), cultured at 37℃for 2-3 days at 200rpm, the obtained culture solution was diluted 10 ⁵ times with AS-168 medium, and then 200. Mu.L was applied to AS-168+FU medium plates to continue culturing at 37 ℃.

(4) After single colony is grown, AS-168+FU culture medium plate is streaked, and the culture is carried out for about 2 days, and whether the single colony is double-exchanged strain is identified by PCR amplification of the grown single genome DNA.

The primers used were Cas3-U-F and Cas3-D-R.

The strain can be amplified to a single band with the size of 828bp and is a double-exchange strain, and the strain can be amplified to a single band with the size of 3419bp and is a wild strain. The obtained double-exchange strain is Mediterranean salt-rich bacteria DF 50-delta EPS with cas3 gene knocked out, and is marked as Mediterranean salt-rich bacteria DF50 delta EPS delta cas3. The sequence of cas3 gene in the Mediterranean salt-rich bacteria DF 50-delta EPS is SEQ ID No.3 in a sequence table.

2. Construction of crRNA expression plasmid pWL-crCS targeting citrate synthase candidate Gene

The mini-CRISPR structure is synthesized artificially, and the sequence is as follows (5 '-3'):

(SEQ ID No.4 of the sequence Listing).

The mini-CRISPR structure can transcribe crRNA of two promoter regions of the respectively targeted citrate synthase genes HFX_0432 (the sequence of which is SEQ ID No.5 and HFX_6170 (the sequence of which is SEQ ID No. 6). In the above sequence, the underlined part is the strong constitutive promoter P _phaR sequence, the bolded part is the repeat sequence, the square is the spacer sequence targeting HFX_0432, the double underlined part is the spacer sequence targeting HFX_6170, and the hatched part is the T8 terminator sequence.

The synthesized mini-CRISPR structure is amplified by using the primers crRNA-F and crRNA-R, then the obtained PCR product is subjected to double digestion by using BamH I and Kpn I, the obtained large fragment is connected with a vector skeleton obtained by carrying out BamH I and Kpn I double digestion on pWL plasmid (Cai S,Cai L,Liu H,et al.Identification of the haloarchaeal phasin(PhaP)that functions in polyhydroxyalkanoate accumulation and granule formation in Haloferax mediterranei[J].Applied and Environmental Microbiology,2012,78:1946-1952.), the crRNA expression plasmid is obtained, and the recombinant plasmid with the correct sequence is marked as pWL502-crCS.

PWL502-crCS are recombinant plasmids obtained by replacing the DNA fragment between BamHI and KpnI recognition sequences of pWL502 with the mini-CRISPR structure described above.

The primer sequences used were as follows:

crRNA-F：5′-ACAACAACCCCCCATGGATCCAATGGTGTCGAAGGGAAC-3′；

crRNA-R：5′-CGCACACAAGAAAACGGTACCAAAAAAAAGCTTCAACCC-3′。

3. transformation pWL of 502-crCS plasmid into Mediterranean salt-rich bacteria

The obtained crRNA expression plasmid pWL-crCS is introduced into E.coli JM110 for demethylation, then the crRNA expression plasmid is extracted and then introduced into Mediterranean salt-rich bacteria DF50 delta EPS delta cas3 to obtain recombinant bacteria, and the recombinant bacteria containing pWL-crCS are marked as Mediterranean salt-rich bacteria DF50 delta EPS delta cas3-crCS.

PWL502,502 was demethylated by E.coli JM110 and introduced into the Mediterranean salt-rich bacterium D50ΔEPS Δcas3 as an empty vector control strain, and the obtained strain was designated as D50ΔEPS Δcas3-no crRNA.

RT-QPCR detection of the Gene expression of the citrate synthase of the salt-rich Mediterranean bacterium DF50 DeltaEPS Deltacas 3-crCS

Culturing the Mediterranean salt-rich bacteria DF50 delta EPS delta cas3-crCS obtained in the step 3 in an MG culture medium for 72 hours, extracting RNA, transcribing the RNA into cDNA, detecting the expression condition of two citrate synthase genes HFX_0432 and HFX_6170 by REAL TIME PCR, taking 7S rRNA as an internal reference, and taking the DF50 delta EPS delta cas3-no crRNA as a control.

The primers used were 0432-RT-F/0432-RT-R and 6170-RT-F/6170-RT-R for detecting the mRNA levels of HFX_0432 and HFX_6170, respectively. The primer sequences were as follows:

0432-RT-F：5′-ATGCGGATGCTCAAGGATGT-3′；

0432-RT-R：5′-AGACCCTTCTCCTCGCTGAT-3′；

6170-RT-F：5′-CCGACTCCCAACCGATAGAG-3′；

6170-RT-R：5′-AGTACGCGGCTACAATCGTC-3′。

the RT-QPCR results show that the transcription of HFX_0432 and HFX_6170 are obviously inhibited. Wherein, the transcription level of HFX_0432 is inhibited to 7% (see FIG. 1), and the transcription level of HFX_6170 is inhibited to 21% (see FIG. 2).

5. Inhibition of citrate synthase gene by CRISPRi improves PHBV yield of salt-rich Mediterranean bacteria

The strain to be tested is: and D, obtaining the Mediterranean salt-rich bacteria DF50 delta EPS delta cas3-crCS and DF50 delta EPS delta cas3-no crRNA in the step 3.

An equal amount (initial OD 0.1) of the strain to be tested was cultured in a 250mL shake flask with 50mL of MG medium until glucose was consumed, at 37℃and 200rpm shaking culture for 192h total. And detecting the growth condition, the glucose consumption condition and the PHBV accumulation condition of the strain to be detected after the culture is finished.

And (3) detecting the growth condition: samples were taken at intervals of 24 hours, and absorbance of the bacterial liquid was measured at a wavelength of 600 nm.

Glucose consumption detection: sampling every 24h, centrifuging at 12,000rpm for 3min, collecting supernatant, sucking 50 μl of supernatant, diluting with 950 μl of distilled water, taking 25 μl, detecting with a glucose meter (SBA-40D, china), and multiplying the glucose concentration by dilution factor (20 times) to obtain the glucose concentration in the culture medium. The standard solution is glucose solution with the concentration of 1 g/L.

PHBV accumulation condition detection: PHBV detection is carried out by taking bacterial liquid cultured for 120 hours, and the specific method is as follows:

(1) Preparing an esterification liquid: 1g/L benzoic acid (internal standard) is dissolved in a mixed solution of methanol and concentrated sulfuric acid (volume ratio is 97:3), and esterified solution is obtained;

(2) Centrifuging to collect 20-50mL of bacterial liquid, freeze-drying, weighing, recording accurate values, and taking about 50mg of dry bacterial in an esterification pipe;

(3) After the step (2) is completed, adding 2mL of an esterification liquid and 2mL of chloroform into an esterification pipe, and esterifying for 4 hours at 100 ℃;

(4) After the step (3) is completed, naturally cooling, adding 1mL of distilled water, shaking and uniformly mixing, standing and layering;

(5) After the step (4) is completed, the lower chloroform phase is taken for gas chromatography analysis, and benzoic acid (product of national medicine group chemical reagent Co., ltd., product number 30018617) is used as an internal standard. Using AGILENT GC to 6820 gas chromatograph, the sample injection amount is 1 μl, the column temperature is set to 200 ℃, the temperature of the sample injector is set to 200 ℃, the temperature of the detector is set to 220 ℃, the column length is set to 30m, and the column head pressure is set to 0.25Mpa. The temperature programming conditions are as follows: after maintaining at 80℃for 1.5min, the temperature was raised to 140℃at a rate of 30℃per minute, and then to 220℃at a rate of 40℃per minute, and maintained at this temperature for 0.5min.

The fermentation result shows that compared with the control strain DF50 delta EPS delta cas3-no crRNA, the strain DF50 delta EPS delta cas3-crCS for inhibiting the citrate synthase gene has higher biomass and faster glucose consumption rate (see figures 3 and 4), and all glucose is consumed when the strain is cultured for 120 hours, and the residual glucose is consumed 72 hours earlier than the control strain DF50 delta EPS delta cas3-no crRNA, so that the fermentation period is shortened.

In addition, compared with the control strain DF50 DeltaEPS Deltacas 3-no crRNA, the dry cell weight and the PHBV accumulation amount of the DF50 DeltaEPS Deltacas 3-crCS at the end of fermentation are both remarkably improved (see table 1), the dry cell weight is improved to 5.66g/L from 3.63g/L of the control strain DF50 DeltaEPS Deltacas 3-no crRNA, the PHBV yield is improved to 3.14g/L from 1.78g/L, and the PHBV yield is improved by 76.4% compared with the control strain. Compared with the control strain DF50 delta EPS delta cas3-no crRNA, the 3HV monomer proportion of PHBV synthesized by DF50 delta EPS delta cas3-crCS is changed from 10.57mol percent to 6.07 percent at the end of fermentation, which shows that the method has certain application potential in synthesizing PHBV with different monomer proportions.

TABLE 1 accumulation of PHBV in citrate synthase candidate Gene-inhibiting Strain

^a CDW, CELL DRY WEIGHT, dry cell weight.

PHBV content (%), i.e., PHBV content (%), means the percentage of PHBV mass to dry cell weight.

3HV fraction (mol%) is the 3HV monomer ratio, which represents the mole percentage of 3HV monomer in PHBV.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

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ctcctcttcg aacacctccg agatgtccga gaccgtgtcg atatggtcat ccctgagggg 120

gcgacgacac cagaaggaaa gccactttgt ggtgtcattc ggcgtctcgc acttgtccac 180

gacttcggga aggcaacgac attttttcag cagtatatcg gtgcacaact agggcagccg 240

acgcatgaca agttacggta tcacgctccg cttggctcat tagctgcata ctatgttctc 300

cgtgaaacgg ggcactcgac agcgacctgt ctcgcaggtt tcgtcgccgt tgcaaaacac 360

cacgggcgac tcccgaatgt cgttgaatac gtgttcaaac ggatggcaag tcctgacccg 420

gaaaagtgga tggcggataa gaagcaggtc gagaacatcc ataagaacgc gccgaggctt 480

gcaacagcaa tattcgagga agcaaccggg gacgatgacg cgtggctgga ttttgcacag 540

tcctgtgtca acgacgaatc gctgttcacc gaaattgctg atcacgtcac gagaaacggt 600

gagcgtccga taactgaacc aacgttcctc actgacgaat tctacggctt gatgttggag 660

tgttggggga cactagttct cgctgataag acgagtgccg ctggtgcacc ccaagcaagc 720

tccgtctacg acgccacgaa cccgaggaca gccgacctaa cgcagtatat cgacaacctt 780

ggggatggaa acaccgaccc ggacggctct cggaccgagc agctcaacta ttaccgctca 840

cgcgctcgcc aagatgtact tgactcagtc acggagtttg tcgagagtga atcggacgtt 900

gcgacgatta cgttgccgac cggaatggga aagactctca ccggtctgaa tgccgcacta 960

gaaatccgcg accagacggg tggcgaccgc attgtctacg cgttgccgtt tacgagtatt 1020

atcgatcagg tcggtgcaga agtacaggac atctttgata cggatgggtc cgatggaatc 1080

gttgctcttc accatcacct ttctgacacc cgctttgggt acagtgacgg agacgacgat 1140

gcatctgacc tgaacgacga cattgcaggg atgcttgggg agagttggcg ggcaggactc 1200

acagtaacta cattcgtcca actgttcgag agtttggctg gaccgcggaa tacgcagtcg 1260

atgaagattc cggcacttcg tgggaatgtc atcgtcctcg atgaacctca gtcactgccc 1320

cttgactggt ggaagctcgt tcctcggctt gtagacgttc ttactgaaca gtacggagca 1380

actgtcatct cgatgacggc gacgcaaccc gagctattcc ctgctccgat gtcgcttgtt 1440

tcagatgccg aacggtattt cacggtcgca gaacgcgtcc agtaccatct ccatgactcc 1500

gtcgaacggt tccttcgtgg tgaagagcag ccgctcgaat acaatgatgc cgcaaacgaa 1560

ctcgtcgaag tggcccaatc cggtgattca ctcctcgcaa tctgcaatac gattgatagc 1620

gcccgggtac tggctaacgc tgtaactgaa cgaattcagg cagttaacct cgcagaacag 1680

tattttgagt cactcaggaa tgggtcttcg gacccagttg cggagactgt tcaactggtc 1740

cgccaatcgt ctaaacaagc gttcgtacat ctctcgacgc gactgagacc gaccgatagg 1800

ctcgcactga tacgaattat caagcagctc cgtgcttctg gatctccggt cattgcagtt 1860

acgacacagc tcgtcgaagc aggtgttgac atcagcttcg agaacgtcta tcgagacctt 1920

gcgccggttg atagtatcgt tcaggcggcc ggtcgttgta atcgttcttt cgagcgtgaa 1980

ctcggtgctg ttacggtttg gtggctcaca cagccagcag aacaaaggca cacacccgca 2040

gttgcggtct acgatactca aggaccttcg cttactcctg taactgcctc cgcactcgat 2100

agcgttcgag atggtcagac gaaactagct ggccaatcag ttgcccgtgc agcggtgcaa 2160

gaatattacg gaacacttca taaagagaag aatgtcggga gagaggaata ccacgagtac 2220

gtcgatgacg ccgatgcaga gtcactgggc cgtctctcac tgataacaca gacgaaaacc 2280

gtcgatgttc ttatctgtgt caccgaagca gatcagacac ttgtagagtc tctggagggt 2340

gcctacgaga actacgattt tcaggaggtc aagcgactcc tcaacgcgac caagccgctt 2400

cgggtctcca tcccaatata ccgcgatgac tcaccagagg ctaatgtagt caccgagtta 2460

cggcctcttg cgggacgtaa ggatgagagt tcgattcgcg tactcgatgc gggaacgcga 2520

gactttgaaa aatacttcga ccacaccacg ggattcgtcg tagcagattc tacagttgag 2580

gaccgattcc tatga 2595

<210> 4

<211> 222

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

aatggtgtcg aagggaacat atatgttact gcaggtacaa caccgagtta ggaggttaca 60

gacgaaccct agttgggttg aagcatatgg taagcaaccc tttgggtatg tcaggcgaag 120

ttacagacga accctagttg ggttgaagcg tgttctcaga aagaatatgg ttcgaggcca 180

tctagttaca gacgaaccct agttgggttg aagctttttt tt 222

<210> 5

<211> 1140

<212> DNA

<213> Mediterranean salt-rich bacterium (Haloferax mediterranei)

<400> 5

atgtcaggcg aactgaagcg ggggctggaa ggtgtgctgg tcaccgaatc ggaactgagc 60

ttcatcgacg gcgacgcggg acaacttatc tatcggggct acgatatcga agacctcgct 120

cgcgatgcga gctacgagga agtgttgtac ctcctgtggc acggcgaact tccgaatcgt 180

acgcaactcg acgagttctc cgacgaactg gcagcccacc gcgacatcgg cgacggaatc 240

ctcgacgtgg cacgcgaact cgccgaacag gacgaatcgc cgatggctgc gcttcgaacg 300

ctcgtttccg cgatgtcggc gtacgacgaa aacgccgact tcgaggacgt gaccgaccgc 360

gaggtcaacc tcgagaaagc aaagcgcatc acggcgaaga tgccgtcggt gctcgcggcc 420

tacgctcggt tccggcgcgg tgacgactac gtcgcgccga acgacgacct gaatcacgcc 480

gcgaatttcc tctacatgct caacggcgaa gagccgaacg aggtgctggc ggagacgttc 540

gacatggcgc tcgtgctaca cgccgaccac ggactaaacg cgtcgacgtt ctccgcgatg 600

gtcacgtcgt ccaccctctc ggacatgtac agcgcggtca cgtccgcaat cgggacgctc 660

tccgggtcgc ttcacggcgg cgcgaacgcg aacgtcatgc ggatgctcaa ggatgtcgac 720

gacagcgaca tggaccccgt cgactgggtc gaagacgcac tcgaccgcgg cgagcgcgtc 780

gccggattcg gccaccgcgt ctacaacgtc aaggaccccc gcgcgaagat tctcggccag 840

aagtccgagg cactcggcga ggccgcaggc gacatgaagt ggtacgagat gtcggtcgca 900

atcgaggagt acatcagcga ggagaagggt ctcgcaccga acgtggactt ctactccgca 960

tcgacgtact accagatgga tatccccatc gacctttaca cacccatctt cgccgtctcg 1020

cgtagcggcg gatggattgc gcacatcctc gaacagtacg acgacaaccg gctcatccgc 1080

ccgcgcgctc ggtacaccgg ggacaaagac ctcgacttcc cgacgctcga cgagcggtaa 1140

<210> 6

<211> 1134

<212> DNA

<213> Mediterranean salt-rich bacterium (Haloferax mediterranei)

<400> 6

atgagcgata acaccacgaa ccaaggacta gaggggatca cagttgcaga gactcgaatt 60

agcgacatcg atggcgaaag tggcgaactt gtgattggtg ggtaccccat cgaagagctt 120

gcaacaaacg cgacctacga ggaaagtatc ttcctcctct tgaacggccg actcccaacc 180

gatagagaac tcgccaacct ccgagccaaa ctcgcggccc gccgggagat tagcgacgaa 240

gtacgggcgg tgctccggcg tgctgccgaa gaagaaaaac ccgcgatgga tgcccttcgg 300

atgggcgctg cgacagcaac catcgggacc gaagatggca ccccacagga cgatgcgcag 360

cgagtaatcg ccgccttccc gacgattgta gccgcgtact ggcgatatcg gcaggggaag 420

aagccggtcg caccgcgcga agatttggga cacgccgcaa attatctcta catgctgagc 480

ggtgaggagc cgaccgacgc cgccatccgt gggttagaaa cgtacctcaa cacggttgta 540

gaccacgggg tgaacgcctc gacatttact gcgagagtcg tggtttcgac ggagtcagat 600

ctcgtatcgg cagccaccgc cgccgtcggg acactcaagg ggccactcca cggcggcgct 660

ccgggacctg tcctcgacat gttcaaagaa gtccacgaat ccggggaacc agaggggtac 720

gtccgcgaga cgctggagcg tggcgaacgc ctaatggggt tcggccaccg cgtgtaccgc 780

gttcgggacc cacgcgcagc ggtgctctcg acggccgccg agcgcttcta cgaggcgagt 840

tcgaatacgg agttcttcga gaccgttcaa gacctcgaag agtgcgctgt cgatatcttg 900

gcggagcaca agccagaccg gcgactggag acgaatgtcg agttttacac cgctgcgctc 960

cttcacggcc tcggtatccc gaaagacgtg ttcacggcga cgttcggcgt gtcgcgagtc 1020

ggtggctgga tggcgcactg tctcgaacaa ctggaaaata atagagtaat tcgtccgcgg 1080

gcacactacg tgggcgcgac tgggcggacg tggacaccag ttggcgaacg atag 1134

Claims

1. A method of constructing a recombinant thalassemia salt-rich bacterium comprising:

2) Introducing a crRNA expression cassette for transcriptional inhibition of citrate synthase gene expression into the cas3 knockout bacterium to obtain recombinant Mediterranean salt-rich bacteria;

The citrate synthase gene is an HFX_0432 gene and/or an HFX_6170 gene, the sequence of the HFX_0432 gene is shown as SEQ ID No.5, and the sequence of the HFX_6170 gene is shown as SEQ ID No. 6;

The expression cassette is obtained by connecting a promoter, a repeat sequence, a spacer sequence and a terminator sequence; the repeat sequence is 55-84 th bit in SEQ ID No.4 in the sequence table, and the spacer sequence targets the coding region or the promoter region of the citrate synthase gene.

2. The method according to claim 1, characterized in that: the crRNA targets the coding region or promoter region of the citrate synthase gene.

3. The method according to claim 1, characterized in that: the expression cassette is introduced into the Zhonghai salt-rich bacteria in the departure place through a recombinant vector containing the expression cassette.

4. Recombinant Mediterranean salt-rich bacteria obtained by the method of any one of claims 1-3.

5. A method of producing PHBV comprising: culturing the recombinant Mediterranean salt-rich bacterium of claim 4 in a medium containing glucose or starch to obtain PHBV.

6. Kit of parts consisting of the cas3 knockout bacterium of any one of claims 1 to 3 and the expression cassette, or of the cas3 knockout bacterium of any one of claims 1 to 3 and the recombinant vector.

7. Use of the method of any one of claims 1-3 or the recombinant mediterranean salt-rich bacterium of claim 4 or the kit of parts of claim 6 for the preparation of PHBV.