WO2018233703A1 - 一种精细调控共聚物中4-羟基丁酸组成比例的基因盒及其应用 - Google Patents

一种精细调控共聚物中4-羟基丁酸组成比例的基因盒及其应用 Download PDF

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WO2018233703A1
WO2018233703A1 PCT/CN2018/092467 CN2018092467W WO2018233703A1 WO 2018233703 A1 WO2018233703 A1 WO 2018233703A1 CN 2018092467 W CN2018092467 W CN 2018092467W WO 2018233703 A1 WO2018233703 A1 WO 2018233703A1
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promoter
gene
seq
gene cassette
mmp1
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French (fr)
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李腾
叶健文
尹进
陈祥斌
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北京蓝晶微生物科技有限公司
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
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    • C12Y208/03—CoA-transferases (2.8.3)
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a gene cassette for finely regulating the composition ratio of 4-hydroxybutyric acid in a polyhydroxyalkanoate (PHA) copolymer, a bacterial recombinant strain containing the gene cassette, and a preparation poly A method of copolymerizing a hydroxy fatty acid ester (PHA).
  • PHA polyhydroxyalkanoate
  • Polyhydroxyalkanoate PHA is a general term for a class of bio-polyesters and is the only bio-based material synthesized entirely by microorganisms. Depending on the structure of the monomers constituting the polyester, PHA can exhibit various mechanical properties such as mechanics, stretching, and elasticity, and thus can be applied to different scenes.
  • PHA poly-3-hydroxybutyrate
  • PHBHHx poly-3-hydroxybutyrate-3-hydroxyhexanoate
  • PHBV poly-3-hydroxybutyrate-3- Hydroxyvalerate
  • P3HB4HB poly-3-hydroxybutyrate-4-hydroxybutyrate
  • PHB is the simplest PHA member, but due to its high crystallinity, low elongation at break, brittle material properties and poor toughness, these disadvantages increase the difficulty of PHB treatment and limit the application range of PHB.
  • the incorporation of other monomeric compositions in other PHA materials greatly affects the physicochemical properties of PHA.
  • the difference in the content of 4HB monomer in P3HB4HB has a great influence on its material properties, so that it can change from hard high crystal to soft elastomer, and its thermodynamic and mechanical properties will also vary with 4HB monomer content. Change and change.
  • the content of 4HB monomer increased from 64mol% to 100mol%, the tensile strength of P3HB4HB increased from 17Mpa to 104Mpa; when the content of 4HB monomer increased from 0mol% to 82mol%, the elongation at break of P3HB4HB increased from 5% to 1320. %.
  • P3HB4HB can be produced by some wild-type bacteria or genetically engineered bacteria.
  • recombinant E. coli can realize the synthesis of P3HB4HB with a composition ratio of 4HB exceeding 10 mol% (Li, ZJ, Shi, ZJ, Guo, YY, Wu, Q., Chen, GQ, 2010.
  • Production of poly(3-hydroxybutyrate-co- 4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli. Metab.Eng., 12, 4, 352-359.) but the dry weight of the cells in the fermenter reached only 23.5 g/l, making industrial production difficult.
  • Rhizoctonia solani can synthesize P3HB4HB under the conditions of adding related precursors such as ⁇ -butyrolactone and 4-hydroxybutyric acid. After 74 hours of fermentation, the dry weight of the cells reaches 51 g/L. , P3HB4HB content of 35%, 4HB ratio of 32mol% (Song, JY, Kim, BS, 2005.
  • Halomonas is a halophilic bacterium characterized by growth in a high salt concentration environment. Because other microorganisms are difficult to grow in this environment, S. monocytogenes can be used to develop a non-sterilized open continuous fermentation process.
  • Halomonas sp. TD01 has been developed as a platform strain for the production of PHB, PHBV and its PHA-related proteins PhaR and PhaP.
  • the TD01 and its derived bacteria can reach a dry weight of nearly 80 g/L in fermentation, and the PHA content is nearly 80%. It has the potential to be an industrial production strain of PHA (Tan, D., Xue, YS, Aibaidula, G., Chen, GQ, 2011. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour. Technol., 102, 17, 8130.).
  • Wild type bacterium TD01 cannot synthesize P3HB4HB with precursors such as ⁇ -butyrolactone and 4-hydroxybutyric acid because it lacks an enzyme capable of converting 4-hydroxybutyric acid to 4-hydroxybutyrate-CoA, but only Coenzyme A form can be catalyzed by PHA synthase as a polymer. Therefore, it is necessary to genetically engineer the M. sphaeroides and construct a metabolic pathway to realize the synthesis of P3HB4HB.
  • the orfZ gene from Clostridium kawachii has the function of 4-hydroxybutyrate-CoA transferase, which converts 4-hydroxybutyric acid produced by hydrolysis of ⁇ -butyrolactone into 4-hydroxybutyrate-CoA, which in turn Bacteria utilize the synthesis of P3HB4HB.
  • overexpression of the orfZ gene with a strong promoter can inhibit cell growth, so in order to achieve optimal balance between high-density cell growth, high PHA and appropriate 4HB ratio, fine control of orfZ expression in bacteria is required. .
  • the genetically modified and wild bacteria have been reported to be able to synthesize P3HB4HB, but it is difficult to satisfy both high cell dry weight and suitable 4HB ratio, which is not suitable for industrial production.
  • Halomonas TD01 has the potential to be an industrial production strain of PHA, but the wild type cannot synthesize P3HB4HB.
  • the orfZ gene to be introduced into P3HB4HB needs to be finely regulated. Otherwise, the expression level is too high to be toxic to bacterial growth, and too low will cause the ratio of 4HB to be too low.
  • An object of the present invention is to provide a gene cassette for finely regulating the composition ratio of 4-hydroxybutyric acid in a polyhydroxyalkanoate (PHA) copolymer, which comprises a promoter and a 4-hydroxybutyrate-CoA transferase gene or has Gene for the function of 4-hydroxybutyrate-CoA transferase.
  • PHA polyhydroxyalkanoate
  • Another object of the present invention is to provide a bacterial recombinant strain.
  • a gene cassette for finely regulating a composition ratio of 4-hydroxybutyric acid in a polyhydroxyalkanoate (PHA) copolymer which comprises a promoter and a 4-hydroxybutyrate-CoA transferase gene Or a gene having the function of 4-hydroxybutyrate-CoA transferase.
  • the gene having a function of 4-hydroxybutyrate-CoA transferase may be a gene which catalyzes 4-hydroxybutyric acid to 4-hydroxybutyrate-CoA, and examples thereof include from gram OrfZ gene of Clostridium kluyveri.
  • the promoter may be an inducible promoter or a constitutive promoter.
  • the gene cassette expressing orfZ from an inducible promoter can finely regulate the ratio of 4HB in P3HB4HB.
  • the gene cassette expressing orfZ from the constitutive promoter enables high-density fermentation and high-content P3HB4HB production in the fermentation process of different scales, and the ratio of 4HB is not less than 10 mol%.
  • the effect of the gene cassette expressing orfZ by the constitutive promoter is similar to that of the gene cassette of the inducible promoter expressing orfZ. More preferably, the promoter may be a tac promoter.
  • the promoter may be the DNA molecule set forth in SEQ ID NO: 6, or the DNA molecule set forth in SEQ ID NO: 14, or one or more of SEQ ID NO: 6 or SEQ ID NO: A DNA molecule derived from SEQ ID NO: 6 or SEQ ID NO: 14 with substitutions and/or deletions and/or additions of nucleotide residues and having the same function.
  • the nucleic acid sequence of the 4-hydroxybutyrate-CoA transferase may be SEQ ID NO: 7, or the SEQ ID NO: 7 may be subjected to one or several nucleotide residues.
  • a DNA molecule derived from SEQ ID NO: 7 substituted and/or deleted and/or added and having the same function.
  • the 4-hydroxybutyrate-CoA transferase may be the protein represented by SEQ ID NO: 8, or the amino acid sequence shown in SEQ ID NO: 8 may be passed through one or several A protein derived from SEQ ID NO: 8 with substitutions and/or deletions and/or additions of amino acid residues and having the same function.
  • the copolymer may include P3HB-co-4HB (3-hydroxybutyric acid-4 hydroxybutyric acid copolyester), P3HB-co-4HB and P3HB (poly-3-hydroxybutyl) a blend of acids), a blend of P3HB-co-4HB and P4HB (poly 4-hydroxybutyrate), and a blend of P3HB-co-4HB, P3HB and P4HB.
  • a bacterial recombinant strain comprising the gene cassette of the present invention.
  • the bacteria include Escherichia coli and a recombinant strain thereof.
  • the bacteria include the salt bacterium 310 and its recombinant bacteria.
  • the bacterial recombinant strain of the present invention preferably, for example, the bacterial recombinant strain further contains a lacI repressor protein, a lacO regulatory element, and the like.
  • the bacterial recombinant strain into which the gene cassette containing the inducible promoter is introduced can realize the production of P3HB-co-4HB, and the 4HB mol% is from 0.1% to 30%;
  • a bacterial recombinant strain containing a gene cassette containing a constitutive promoter, which can produce P3HB-co-4HB, has a 4HB mol% of 0.1% to 30%.
  • the above recombinant bacteria have stable performance in producing P3HB4HB in fermenters of different scales, and have potential as industrial production bacteria of P3HB4HB.
  • a process for producing a polyhydroxyalkanoate copolymer which comprises preparing a polyhydroxyalkanoate using a bacterial recombinant strain comprising the gene cassette of the present invention.
  • the copolymer may include P3HB-co-4HB (3-hydroxybutyrate-4 hydroxybutyric acid copolyester), P3HB-co-4HB and P3HB (poly-3-hydroxybutyric acid) a blend of P3HB-co-4HB and P4HB (poly 4-hydroxybutyric acid), and a blend of P3HB-co-4HB, P3HB and P4HB.
  • P3HB-co-4HB 3-hydroxybutyrate-4 hydroxybutyric acid copolyester
  • P3HB-co-4HB and P3HB poly-3-hydroxybutyric acid
  • P4HB poly 4-hydroxybutyric acid
  • the bacterium comprises Escherichia coli and a recombinant bacterium thereof.
  • the bacterium comprises Mycobacterium sphaeroides TD and recombinant bacteria thereof.
  • the gene cassette expressing orfZ constructed by the invention has a 4HB component in the PHA synthesized by the recombinant bacteria after being introduced into the Halophilus TD01 and its derivative bacteria, and the ratio of 4HB can be finely regulated; In the fermentation process, high-density growth (high cell dry weight) can be achieved, and the appropriate 4HB ratio in P3HB4HB can be ensured. Therefore, the gene cassette expressing orfZ constructed by the present invention has important application value in the industrial production of P3HB4HB.
  • Figure 1 is a schematic representation of the gene cassette of the inducible promoter expressing orfZ.
  • the invention provides a gene cassette for expressing a 4-hydroxybutyryl-CoA transferase, the gene cassette comprising a promoter, and a 4-hydroxybutyryl-coenzyme located downstream of the promoter and operably linked to the promoter A transferase gene; the promoter is a constitutive promoter or an inducible promoter; the constitutive promoter is a tac promoter, and the inducible promoter is an Mmp1 promoter.
  • the promoter is used to drive expression of a 4-hydroxybutyryl-CoA transferase gene.
  • the gene cassette comprises, in order from the 5' end to the 3' end, a Mmp1 promoter, a lacO sequence, and a 4-hydroxybutyryl-CoA transferase gene.
  • the 4-hydroxybutyryl-CoA transferase gene can be the orfZ gene, preferably the orfZ gene from Clostridium kluyveri.
  • the promoter may be the DNA molecule set forth in SEQ ID NO: 6, or the DNA molecule set forth in SEQ ID NO: 14, or a SEQ ID NO: 6 or SEQ ID NO: 14 A DNA molecule derived from SEQ ID NO: 6 or SEQ ID NO: 14 with substitutions and/or deletions and/or additions of several nucleotide residues and having the same function.
  • the nucleic acid sequence of the 4-hydroxybutyrate-CoA transferase can be SEQ ID NO: 7, or the substitution of SEQ ID NO: 7 by one or several nucleotide residues and/or A DNA molecule derived from SEQ ID NO: 7 that is deleted and/or added and has the same function.
  • the 4-hydroxybutyrate-CoA transferase may be the protein set forth in SEQ ID NO: 8, or the amino acid sequence set forth in SEQ ID NO: 8 may be subjected to one or several amino acid residues.
  • a protein derived from SEQ ID NO: 8 substituted and/or deleted and/or added and having the same function.
  • the gene cassette may further comprise additional elements for expressing the 4-hydroxybutyryl-CoA transferase gene in the gene cassette.
  • additional elements for gene expression are well known to those skilled in the art and include, but are not limited to, terminators and the like.
  • Mmp1 promoter transcription can be activated by MmP1 RNA polymerase by inducing expression of MmP1 RNA polymerase corresponding to the Mmp1 promoter in an induced manner.
  • MmP1 RNA polymerase is known in the art, for example, see Zhao, H., Zhang, HM, Chen, X., Li, T., Wu, Q., Ouyang, Q., Chen, GQ, 2016. Novel T7 MpP1 RNA polymerase as described in -like expression systems used for Halomonas.
  • the MmP1 RNA polymerase may, for example, be an RNA polymerase (Morganella phage MmP1) of Genebank Accession No.: YP_003324580.1.
  • transcription of the Mmp1 promoter can be activated by a regulatory element to express 4-hydroxybutyryl-CoA transferase.
  • the regulatory element may comprise a MmP1 RNA polymerase gene and other elements required to induce expression of MmP1 RNA polymerase.
  • expression of MmP1 RNA polymerase in an inducible manner can be used to induce expression control systems using various proteins well known in the art, including but not limited to lac lactose operon expression regulation systems that induce expression of proteins by IPTG, for example, see Zhao, H. ,Zhang,HM,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,GQ,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140. Expression regulation system used in.
  • an example of using the lac lactose operon expression regulatory system to express MmP1 RNA polymerase in an inducible manner is a regulatory element comprising a repressor protein expression cassette, an RNA polymerase expression cassette.
  • the regulatory element comprising the repressor protein expression cassette and the RNA polymerase expression cassette may include a repressor expression cassette, a nonsense random fragment, an RNA polymerase promoter, a lacO sequence, and an RNA polymerase encoding gene from the 5' end to the 3' end. Or consist of these components.
  • the repressor protein expression cassette may be composed of a repressor protein promoter and a repressor-encoding gene, and the repressor protein may preferably be lacI, and the repressor protein is expressed in the opposite direction to RNA polymerase. See, for example, Zhao, H., Zhang, HM, Chen, X., Li, T., Wu, Q., Ouyang, Q., Chen, GQ, 2016. Novel T7-like expression systems used for Halomonas. Metab. Regulatory elements containing a repressor protein expression cassette, an RNA polymerase expression cassette, used in Eng., 39, 128-140. and CN105779488A.
  • the regulatory element comprising the repressor protein expression cassette, the RNA polymerase expression cassette comprises, in order from the 5' end to the 3' end, a lacI expression cassette, a tac promoter, a lacO sequence, a MmP1 RNA polymerase encoding gene or These components are composed.
  • the repressor protein expression cassette consists of the lacI promoter and the lacI coding sequence, and the expression of lacI and MmP1 RNA polymerase is opposite.
  • the invention also provides an expression vector comprising the gene cassette of the invention.
  • the expression vector can be a plasmid.
  • the expression vector when the promoter in the gene cassette is an inducible promoter Mmp1 promoter, the expression vector may further comprise the above-described regulatory element, preferably the above-described repressor protein expression cassette, RNA polymerase expression a regulatory element of the cassette, and the gene cassette is located downstream of the regulatory element.
  • the present invention also provides a bacterial recombinant strain comprising the gene cassette of the present invention, which is a recombinant Halomonas sp.
  • Halomonas sp. also known as halophilic bacteria, is a type of Gram-negative bacteria that can grow normally at high salt concentrations.
  • the M. sphaeroides useful in the present invention include, but are not limited to, Halomonas sp. TD01 and Halomonas sp. LS21 or from the genomonas.
  • the recombinant A. faecalis of the present invention can be obtained by transferring the gene cassette of the present invention into a starting strain.
  • it can be obtained by transferring an expression vector containing the gene cassette of the present invention into a starting strain.
  • the gene cassette is integrated on the genome of the recombinant M. sphaeroides.
  • the gene cassette is located on an expression vector that is stably present in the recombinant S. genus.
  • the starting strain can be Salmonella TD01 or a salt-producing bacterium isolated from the bacterium Phytophthora TD01.
  • Phytophthora TD01 refers to a salty bacterium which is obtained by genetic modification or mutation or the like by the bacterium TD01.
  • Phytophthora TD01 refers to a salty bacterium which is obtained by genetic modification or mutation or the like by the bacterium TD01.
  • those skilled in the art will appreciate that such genetic engineering or mutations and the like should not affect the expression of the orfZ gene and the production of the polyhydroxyalkanoate copolymer.
  • the recombinant Salmonella when the promoter in the gene cassette is an inducible promoter Mmp1 promoter, can also contain the above-described regulatory elements, preferably the regulatory elements are as previously described.
  • a regulatory element comprising a repressor protein expression cassette and an RNA polymerase expression cassette.
  • the regulatory element can be integrated into a bacterial chromosome or can be located on an expression vector that is stably present in the recombinant Salmonella.
  • the starting strain can be Salmonella TD-MmP1.
  • Salmonella TD-MmP1 For example, Z., H., Zhang, HM, Chen, X., Li, T., Wu, Q., Ouyang, Q., Chen, GQ, 2016. Novel T7-like expression Systems used for Halomonas.Metab.Eng., 39, 128-140.
  • the recombinant M. aeruginosa comprises an expression vector stably present therein, the expression vector comprising the above regulatory element and a gene cassette of the present invention, the promoter contained in the gene cassette being inducible Promoter Mmp1 promoter.
  • the present invention also provides a process for producing a polyhydroxyalkanoate copolymer, which is characterized in that a polyhydroxyalkanoate copolymer is prepared by fermentation with the bacterial recombinant strain of the present invention.
  • the polyhydroxyalkanoate copolymer comprises 3-hydroxybutyrate-4 hydroxybutyric acid copolyester (P3HB-co-4HB). In some embodiments, the polyhydroxyalkanoate copolymer is a 3-hydroxybutyrate-4 hydroxybutyrate copolyester.
  • the 3-hydroxybutyric acid-4 hydroxybutyric acid copolyester may also be referred to as P3HB4HB.
  • the present invention also provides the use of the gene cassette of the present invention, the expression vector of the present invention, and the bacterial recombinant strain of the present invention for producing a polyhydroxyalkanoate copolymer.
  • the polyhydroxyalkanoate copolymer comprises 3-hydroxybutyrate-4 hydroxybutyric acid copolyester (P3HB-co-4HB). In some embodiments, the polyhydroxyalkanoate copolymer is a 3-hydroxybutyrate-4 hydroxybutyrate copolyester.
  • the gene cassette, expression vector or bacterial recombinant strain of the invention can control the proportion of 4HB in the fermentation production of polyhydroxyalkanoate copolymer, especially the ratio of 4HB in the fermentation production of P3HB4HB, and achieve high density growth in the fermentation process. (High cell dry weight) also ensures that the 4HB ratio in P3HB4HB does not affect bacterial growth.
  • the enzyme reagents were purchased from ThermoFisher and New England Biolabs (NEB).
  • the kit for extracting the plasmid was purchased from Tiangen Biotechnology (Beijing) Co., Ltd., and the kit for recovering DNA fragments was purchased from Omega, USA. In strict accordance with the product specifications, all media were prepared with deionized water unless otherwise specified.
  • LB medium 5 g/L yeast extract (purchased from OXID, UK, catalog number LP0021), 10 g/L peptone (purchased from OXID, UK, catalog number LP0042), 10 g/L NaCl, and the balance was water. Adjust the pH to 7.0-7.2 and autoclave.
  • 60 LB medium 5 g/L yeast extract (purchased from OXID, UK, catalog number LP0021), 10 g/L peptone (purchased from OXID, UK, catalog number LP0042), 60 g/L NaCl, and the balance was water. Adjust the pH to 7.0-7.2 and autoclave.
  • 60MMG medium 60g/L NaCl, 30g/L glucose, 1g/L yeast extract, 2g/L NH 4 Cl, 0.2g/L MgSO 4 , 9.65g/L Na 2 HPO 4 ⁇ 12H 2 O, 1.5g /L KH 2 PO 4 , 10 ml/L trace element solution I and 1 ml/L trace element solution II.
  • the composition of the trace element solution I was: 5 g / L ammonium ferric citrate, 2 g / L CaCl 2 , formulated with 1 M HCl.
  • the composition of trace element solution II is: 100 mg / L ZnSO 4 ⁇ 7H 2 O, 30 mg / L MnCl 2 ⁇ 4H 2 O, 300 mg / L H 3 BO 3 , 200 mg / L CoCl 2 ⁇ 6H 2 O, 10 mg / L CuSO 4 5H 2 O, 20 mg/L NiCl 2 ⁇ 6H 2 O, 30 mg/L NaMoO 4 ⁇ 2H 2 O, formulated with 1 M HCl. The final pH of the medium was adjusted to 8.5 with a 5 M NaOH solution.
  • the above reagents were purchased from Sinopharm Chemical Reagent Company.
  • a certain concentration of antibiotic may be added to the above medium to maintain the stability of the plasmid, such as 100 ⁇ g/mL ampicillin or 25 ⁇ g/mL chloramphenicol.
  • Example 1 Inducible promoter expression of the orfZ gene as a gene cassette regulates the proportion of 4HB in P3HB4HB
  • MmP1-r is p321-MmP1-gfp (Zhao, H., Zhang, HM, Chen, X., Li, T., Wu, Q., Ouyang, Q., Chen, GQ, 2016. Novel T7-like Expression systems used for Halomonas.Metab.Eng., 39, 128-140.)
  • a plasmid backbone fragment was amplified by template, and two fragments were ligated by Gibson Assembly method to obtain plasmid p321-MmP1-orfZ, and the orfZ gene was expressed by MmP1 promoter.
  • the primers used are as follows:
  • the gene cassette sequence of the obtained inducible promoter expressing orfZ is:
  • the gene cassette for expressing the orfZ of the inducible promoter consists of two parts, one of which is an inducible promoter and the other is an orfZ gene.
  • the inducible promoter sequence is SEQ ID NO: 6:
  • the orfZ gene sequence is SEQ ID NO: 7:
  • amino acid sequence of the 4-hydroxybutyrate-CoA transferase encoded by the orfZ gene is SEQ ID NO:8:
  • Halomonas TD-MmP1 is a modified strain of TD01, which integrates the RNA polymerase gene of MmP1 into the genome, expresses it with the addition of the inducer IPTG, and specifically recognizes the MmP1 promoter after expression, thereby realizing the regulation of the MmP1 promoter by an inducer. Expression of downstream genes. Therefore, the obtained recombinant strain Halomonas TD-MmP1/p321-MmP1-orfZ can regulate the expression of the orfZ gene. See Figure 1 for a schematic diagram of the relevant principle.
  • the recombinant bacteria were cultured in 60 LB medium at 200 rpm, and cultured at 37 ° C for 12 hours, and then inoculated to 50 ml of 60 MM G medium at 5%, 200 rpm, 37 ° C, and cultured for 48 hours. After 4 hours of inoculation, 5 g/L of ⁇ -butyrolactone was added; while different concentrations of IPTG were added to regulate orfZ gene expression, and the IPTG concentrations were 0 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 400 ⁇ M, 1000 ⁇ M, respectively.
  • the measuring tube was used to measure 30 ml of the bacterial liquid into a 50 ml centrifuge tube, and the cells were collected by centrifugation at 10,000 rpm for 10 minutes. The cells were resuspended in deionized water, centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. After the cells were frozen at -80 ° C for 1 h, they were vacuum freeze-dried for more than 12 h to completely remove water. The weight of the centrifuge tube before and after sampling was weighed, and the difference was the dry cell weight CDW.
  • esterification solution 485 ml of anhydrous methanol was added, 1 g/L of benzoic acid was added, and 15 ml of concentrated sulfuric acid was slowly added to prepare about 500 ml of esterified liquid.
  • Sample preparation 30 to 60 mg of freeze-dried cells were weighed, accurately weighed, placed in an esterification tube, and 2 ml of an esterified solution and 2 ml of chloroform were added. A PMA sample of about 10 mg was weighed and treated in the same manner as a standard sample. The esterification tube was sealed and sealed at 100 degrees for 4 hours. After the reaction, the esterification tube was cooled to room temperature, 1 ml of deionized water was added, and the mixture was vortexed until thoroughly mixed, and allowed to stand for stratification. After the aqueous phase and the organic phase were completely separated, the lower organic phase was taken for gas chromatography (GC) analysis.
  • GC gas chromatography
  • GC analysis of PHA composition and content using Shimadzu's GC-2014 gas chromatograph.
  • the configuration of the chromatograph is: HP-5 capillary column, hydrogen flame ionization detector FID, SPL split inlet; high purity nitrogen as carrier gas, hydrogen gas, air as combustion gas; use AOC-20S automatic Injector, acetone is the washing liquid.
  • the GC analysis program was set to an inlet temperature of 240 ° C, a detector temperature of 250 ° C, a column temperature onset temperature of 80 ° C for 1.5 minutes, and a temperature of 30 ° C / minute to 140 ° C for 0 minutes; The rate of 40 ° C / min was increased to 240 ° C and maintained for 2 minutes; the total time was 8 minutes.
  • the GC results were quantitatively calculated based on the peak area using the internal standard normalization method.
  • Example 2 Constitutive promoter expression of the orfZ gene as a gene cassette
  • the 4HB ratio in the P3HB4HB synthesized by the recombinant bacteria can be regulated by the concentration of the inducer, however, the addition of the inducer in industrial production brings about an additional cost increase.
  • the expression intensity of the promoter is comparable to the expression intensity of the MmP1 promoter at 10 ⁇ M inducing agent concentration in the above examples, and the approximate effect of orfZ expression can be achieved without the addition of an inducer.
  • the constitutive promoter used is the tac promoter derived from p321-Ptac-GFP (Zhao, H., Zhang, HM, Chen, X., Li, T., Wu, Q., Ouyang, Q., Chen, GQ, 2016. Novel T7-like expression systems used for Halomonas. Metab. Eng., 39, 128-140.).
  • Primers (Ptac-orfZ-f and Ptac-orfZ-r) were used as p68orfZ (Li, ZJ, Shi, ZJ, Guo, YY, Wu, Q., Chen, GQ, 2010.
  • the orfZ gene fragment was amplified by template, which replaced the gfp gene in plasmid p321-Ptac-GFP.
  • the plasmid p321-Ptac-orfZ was obtained, which contained a gene cassette of the constitutive tac promoter expressing the orfZ gene.
  • the primers used are as follows:
  • the gene cassette sequence of the obtained constitutive promoter expressing orfZ is:
  • the gene cassette of the constitutive promoter expressing orfZ is composed of two parts, one of which is a constitutive promoter, and the other is an orfZ gene.
  • the constitutive promoter sequence is SEQ ID NO: 14:
  • the orfZ gene sequence is the same as in Example 1.
  • the amino acid sequence of the 4-hydroxybutyrate-CoA transferase encoded by the orfZ gene is the same as in Example 1.
  • the plasmid p321-Ptac-orfZ was transferred into Escherichia coli S17-1, and then introduced into the genus Halomonas TD01 by the conjugative transformation method (preserved by the General Microbiology Center of the China Microbial Culture Collection Management Committee, the deposit number is CGMCC No. 4353).
  • the recombinant strain Halomonas TD01/p321-Ptac-orfZ was obtained.
  • the recombinant strain is introduced into the halophilic bacteria as a gene cassette in the form of a plasmid.
  • the gene cassette of Ptac-orfZ was integrated into the genome of Halomonas TD01, and the specific steps are as follows:
  • Fragment H1 and H2 were amplified by PCR using Halomonas TD01 genome as template, Ptac-orfZ fragment was amplified by PCR using p321-Ptac-orfZ as template, and overlap extension PCR (OE PCR) was performed on H1, H2 and Ptac-orfZ.
  • OE PCR overlap extension PCR
  • the H1-Ptac-orfZ-H2 fragment was obtained.
  • This fragment was ligated into plasmid pRE112-6IsceI by Gibson Assembly method (Fu, XZ, Tan, D., Aibaidula, G., Wu, Q., Chen, JC, Chen, GQ, 2014.
  • Recombinant plasmid pRE112-6ISceI-orfZ was transfected into E. coli S17-1, and then transferred into Halomonas TD01 by conjugative transformation method.
  • the suicide plasmid was unable to replicate in the host strain, and was resistant to 25 g/ml chloramphenicol. Positive clones were screened on 60LB plates.
  • the recombinant plasmid carrying the homologous fragment in the positive clone was integrated into the specific position where H1 and H2 on the genome were located, which was the first homologous recombinant strain.
  • Inducible plasmid pBBR1MCS1-ISceI (Fu, XZ, Tan, D., Aibaidula, G., Wu, Q., Chen, JC, Chen, GQ, 2014. Development of Halomonas TD01as a host for open production of chemicals. Metab.Eng , 23, 2, 78-91.) expressed a type of I-SceI, which is classified as endonuclease, and cleaves six I-SceI sites on the genome of the first homologous recombinant strain to produce a double-stranded DNA gap.
  • the second homologous recombination is induced to produce a mutant or wild type, and then the mutant strain is screened by a specific PCR primer.
  • the induced plasmid pBBR1MCS1-ISceI was introduced into the above-mentioned first homologous recombinant strain by a ligation transformation method, and a positive clone was obtained by screening on a 60 LB plate of 100 g/ml spectinomycin.
  • the positive clone is a genomically integrated Ptac-orfZ gene cassette capable of constitutively expressing the orfZ gene, and the resulting recombinant strain is Halomonas TD40.
  • Sites for integration of the Ptac-orfZ gene cassette in the genome in this example include, but are not limited to, the specific sites described above. The selection of other sites is determined by the specific sequence of primers H1-pre112-f/H1-pre112-r and H2-pre112-f/H2-pre112-r.
  • the recombinant strain Halomonas TD40 is expressed on the genome of the gene cassette Ptac-orfZ (constitutive promoter); the recombinant strain Halomonas TD01/p321-Ptac-orfZ is expressed on the plasmid Ptac-orfZ; the recombinant strain Halomonas TD-MmP1/p321-MmP1-orfZ is expressed on the plasmid as a gene cassette MmP1-orfZ (inducible promoter).
  • the above three recombinant bacteria and the starting bacteria Halomonas TD01 were cultured in 60 LB medium at 200 rpm, and cultured at 37 ° C for 12 hours, and then inoculated to 50 ml of 60 MM G medium at 5%, 200 rpm, 37 ° C, and cultured for 48 hours. After 4 hours of inoculation, 5 g/l of ⁇ -butyrolactone was added. Among them, 10 ⁇ M of IPTG was additionally added 4 hours after the inoculation of Halomonas TD-MmP1/p321-MmP1-orfZ.
  • the cells were collected after 48 hours, and the dry weight and PHA content of the cells were measured.
  • the method was as described in Example 1, and the results are shown in the following table:
  • the starting bacterium Halomonas TD01 can only synthesize P3HB without 4HB components, and the three recombinant strains can express P3HB4HB after introducing the gene cassette expressing orfZ.
  • the ratio of 4HB in the P3HB4HB synthesized by the gene cassette Ptac-orfZ expressing the orfZ of the constitutive promoter to the halophilic bacterium Halomonas reached 8.86 mol% or more, which was equivalent to the optimal condition for expressing the orfZ of the inducible promoter.
  • Example 3 Production of P3HB4HB by high-density fermentation of recombinant bacteria
  • the recombinant strain Halomonas TD40 was fermented in P1HB4HB in 1 liter, 7 liter and 1000 liter fermentors, respectively.
  • the specific method is as follows:
  • the volume of the liquid in the 1 liter fermenter is 600 ml; the medium is 60 MMG, wherein the yeast paste is replaced with 20 g/L corn syrup, the amount of NH 4 Cl is 3 g/L, and 2 g/L of urea is additionally added;
  • the fermentation temperature is controlled at 37 ° C, the pH is controlled at 8.5 by 5M NaOH solution; the initial stirring speed is 200 rpm, the highest is 800 rpm; the dissolved oxygen control strategy is used to keep the dissolved oxygen above 20%; the air aeration is 0.7 vvm; the feed solution It is 800 g/L of glucose, 18 g/L of NH 4 Cl and 30 g/L of ⁇ -butyrolactone, and is fed in a manner of maintaining a glucose concentration of 5-10 g/l in the fermentation broth.
  • the fermentation process was terminated at 48 hours, and the dry cell weight CDW and PHA contents and the 4HB ratio in the fermentation broth were examined, and the detection method was as described in Example 1.
  • the liquid volume in the 7 liter fermenter was 3 liters, and the remaining conditions were the same as the above 1 liter fermentor.
  • the fermentation process was terminated at 48 hours, and the dry weight of the cells in the fermentation broth was measured for CDW and PHA content and 4HB ratio.
  • the detection method was as described above.
  • the volume of the 1000 liter fermenter is 600 liters.
  • the first-stage seed liquid is filled with 120 ml of medium in a 250 ml shake flask, and cultured at 37 ° C for 200 h at 12 °C; into a 5 liter shake flask containing 1.2 liters of medium, and cultured at 37 ° C and 200 rpm for 12 hours to obtain a second-stage seed liquid; It is connected to a 100 liter fermenter containing 60 liters of medium, which is a grade 3 seed solution.
  • the fermentation temperature is controlled at 37 ° C, the pH is controlled at 8.5 by 5M NaOH solution, the initial stirring speed is 200 rpm, and the highest is 800 rpm.
  • Control strategy to keep dissolved oxygen above 20% and air ventilation at 0.7vvm The medium of the grade 3 seed solution was inoculated into a 1000 liter fermentor, and the culture conditions were the same as in the above 1 liter and 7 liter fermentors.
  • the fermentation process was terminated at 48 hours, and the dry weight of the cells in the fermentation broth was measured for CDW and PHA content and 4HB ratio.
  • the detection method was as described above.
  • the recombinant strain Halomonas TD40 can achieve high-density fermentation, high content of P3HB4HB production in different scale fermentation processes, and the ratio of 4HB is not less than 10mol%, which has the potential as an industrial production strain of P3HB4HB.

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Abstract

提供一种精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，含有该基因盒的细菌重组菌，以及制备聚羟基脂肪酸酯(PHA)共聚物的方法。所述基因盒包括启动子和4-羟基丁酸辅酶A转移酶基因或具有4-羟基丁酸辅酶A转移酶的功能的基因。

Description

一种精细调控共聚物中4-羟基丁酸组成比例的基因盒及其应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，含有该基因盒的细菌重组菌，以及制备聚羟基脂肪酸酯(PHA)共聚物的方法。
背景技术
全球的塑料产量一直以来都在增加，近年来产量达到了每年3亿吨。如此多的塑料累积在环境中带来了白色污染问题，不仅是影响了陆地环境，更是严重影响了海洋环境。为了解决白色污染问题，生物可降解塑料替代石油基不可降解塑料被认为是减轻塑料对环境负担的出路之一。
聚羟基脂肪酸酯PHA是一类生物聚酯的统称，是唯一完全由微生物合成的生物基材料。根据组成聚酯的单体的结构不同，PHA可以表现出多种力学、拉伸、弹性等材料性能，因此可应用于不同的场景。
在众多种类的PHA之中，目前仅有聚3-羟基丁酸酯(PHB)、聚-3-羟基丁酸-3-羟基己酸酯(PHBHHx)、聚-3-羟基丁酸-3-羟基戊酸酯(PHBV)和聚-3-羟基丁酸-4-羟基丁酸酯(P3HB4HB)实现了商业化。PHB是结构最简单的PHA成员，但是因其结晶度高、断裂延伸率低，材料性能脆、韧性差，这些缺点加大了PHB处理的难度，限制了PHB的应用范围。相比而言，其他PHA材料中由于有其他单体组成的掺入，大大影响了PHA的理化性质。例如P3HB4HB中4HB单体含量的不同对其材料学性能有很大的影响，使之可以从硬的高度结晶体到软的弹性体之间变化，其热力学和机械性能也会随4HB单体含量的改变而改变。当4HB单体含量从64mol％上升到100mol％时，P3HB4HB拉伸强度由17Mpa上升至104Mpa；当4HB单体含量从0mol％上升到82mol％时，P3HB4HB的断裂伸长率从5％上升至1320％。这些优良性能使P3HB4HB被认为是最有应用前景的PHA材料之一。
P3HB4HB可通过一些野生型细菌或者经遗传改造的细菌来生产。例如重组大肠杆菌可实现4HB组成比例超过10mol％的P3HB4HB的合成(Li,Z.J.,Shi,Z.J.,Guo,Y.Y.,Wu,Q.,Chen,G.Q.,2010.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)from unrelated carbon sources by metabolically engineered Escherichia coli.Metab.Eng.,12,4,352-359.)，但是发酵罐中细胞干重只达到23.5克/升，难以实现工业化生产。相比而言，一些野生型细菌在发酵过程中容易达到高密度的细胞生长和高含量的PHA，更适合大规模生产PHA。例如在PHA生产领域研究最广泛的罗氏真养菌，其在添加γ-丁内酯、4-羟基丁酸等相关前体的条件下能够合成P3HB4HB，发酵74h后细胞干重达到51克/升，P3HB4HB含量35％，4HB的比例为32mol％(Song,J.Y.,Kim,B.S.,2005.Characteristics of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)Production by Ralstonia eutropha NCIMB 11599 and ATCC 17699.Biotechnology and Bioprocess Engineering,10,6,603-606.)。由于相关前体会抑制细菌生长，虽然4HB的比例很高，但是细胞干重和P3HB4HB含量偏低，生产强度只有0.24克/升/小时。目前仍鲜有报道能够同时实现高密度发酵和P3HB4HB中合适的4HB比例(>10mol％)。
盐单胞菌Halomonas是一种嗜盐菌，其特点是生长在高盐浓度环境，由于这种环境下其他微生物难以生长，因此盐单胞菌可用于开发无灭菌开放式连续发酵工艺。盐单胞菌Halomonas sp.TD01已被开发作为平台菌株生产PHB、PHBV及其PHA相关蛋白PhaR和PhaP等。盐单胞菌TD01及其衍生菌可在发酵中达到近80克/升的细胞干重，PHA含量近80％，有潜力作为PHA的工业生产菌(Tan,D., Xue,Y.S.,Aibaidula,G.,Chen,G.Q.,2011.Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01.Bioresour.Technol.,102,17,8130.)。
盐单胞菌中有相应的遗传操作工具和手段可实现外源质粒转化、基因敲除、基因组上整合外源基因等基因改造。另外，类T7系统的开发实现了基因的可控表达(Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.)。
野生型盐单胞菌TD01无法以γ-丁内酯、4-羟基丁酸等前体合成P3HB4HB，因为其缺乏能够将4-羟基丁酸转化为4-羟基丁酸辅酶A的酶，而只有辅酶A形态才能被PHA合酶催化为聚合物。因此需要对盐单胞菌进行基因改造，构建代谢通路才能实现P3HB4HB的合成。来自克氏梭状芽胞杆菌的orfZ基因具有4-羟基丁酸辅酶A转移酶的功能，能够将γ-丁内酯水解产生的4-羟基丁酸转化为4-羟基丁酸辅酶A，进而被细菌利用于P3HB4HB的合成。但是用强启动子过表达orfZ基因会对细胞生长产生抑制作用，因此为了在高密度细胞生长、高含量PHA和合适的4HB比例之间取得最佳平衡，需要精细控制orfZ在细菌中的表达量。
现有技术的缺点在于：
1.已有报道的基因改造菌和野生菌能够合成P3HB4HB，但是难以同时满足高细胞干重和合适的4HB比例，不适合工业生产。
2.盐单胞菌Halomonas TD01有潜力作为PHA的工业生产菌，但是野生型无法合成P3HB4HB。
3.合成P3HB4HB所需要导入的orfZ基因，其表达量需要精细调控，否则表达量过高会对细菌生长产生毒性，过低会导致4HB的比例过低。
发明内容
针对现有技术的不足，本发明人进行广泛深入的研究，并最终完成本发明。
本发明的一个目的是提供一种精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其包括启动子和4-羟基丁酸辅酶A转移酶基因或具有4-羟基丁酸辅酶A转移酶的功能的基因。
本发明的另一个目的是提供一种细菌重组菌。
本发明的再一个目的是提供一种聚羟基脂肪酸酯共聚物的制备方法。
根据本发明的一个方面，提供了一种精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其包括启动子和4-羟基丁酸辅酶A转移酶基因或具有4-羟基丁酸辅酶A转移酶的功能的基因。
本发明的基因盒中，所述具有4-羟基丁酸辅酶A转移酶的功能的基因可为将4-羟基丁酸催化为4-羟基丁酸辅酶A的酶的基因，其实例包括来自克氏梭状芽胞杆菌(Clostridium kluyveri)的orfZ基因。
本发明的基因盒中，优选地，所述启动子可为诱导型启动子或组成型启动子。由诱导型启动子表达orfZ的基因盒能够精细调控P3HB4HB中4HB的比例。由组成型启动子表达orfZ的基因盒能够使重组菌在不同尺度发酵过程中均能实现高密度发酵、高含量P3HB4HB生产，且4HB比例不低于10mol％。由组成型启动子表达orfZ的基因盒的效果与诱导型启动子表达orfZ的基因盒的最佳条件效果接近。更优选地，所述启动子可为tac启动子。最优选地，所述启动子可为SEQ ID NO:6所示的 DNA分子，或SEQ ID NO:14所示的DNA分子，或将SEQ ID NO:6或SEQ ID NO:14经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:6或SEQ ID NO:14衍生的DNA分子。
本发明的基因盒中，优选地，所述4-羟基丁酸辅酶A转移酶的核酸序列可为SEQ ID NO:7，或将SEQ ID NO:7经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:7衍生的DNA分子。
本发明的基因盒中，优选地，所述4-羟基丁酸辅酶A转移酶可为SEQ ID NO:8所示的蛋白质，或将SEQ ID NO:8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:8衍生的蛋白质。
本发明的基因盒中，优选地，所述共聚物可以包括P3HB-co-4HB(3-羟基丁酸-4羟基丁酸共聚酯)，P3HB-co-4HB和P3HB(聚3-羟基丁酸)的掺和物，P3HB-co-4HB和P4HB(聚4-羟基丁酸)的掺和物，以及P3HB-co-4HB、P3HB和P4HB的掺和物。
根据本发明的另一个方面，提供了一种细菌重组菌，其含有本发明的基因盒。
本发明的细菌重组菌中，优选地，所述细菌包括大肠杆菌及其重组菌。
本发明的细菌重组菌中，优选地，所述细菌包括盐单胞菌TD及其重组菌。
本发明的细菌重组菌中，优选地，例如，所述细菌重组菌进一步含有lacI阻遏蛋白、lacO调控元件等。
从实施例结果可以看出，本发明的细菌重组菌中，导入了含有诱导型启动子的基因盒的细菌重组菌能实现P3HB-co-4HB的生产，4HB mol％从0.1％-30％；导入了含有组成型启动子的基因盒的细菌重组菌，其能实现P3HB-co-4HB的生产，4HB mol％为0.1％-30％。上述重组菌在不同尺度发酵罐中生产P3HB4HB表现稳定，具有作为P3HB4HB工业生产菌的潜力。
根据本发明的再一个方面，提供了一种聚羟基脂肪酸酯共聚物的制备方法，其特征在于，利用含有本发明的基因盒的细菌重组菌制备聚羟基脂肪酸酯。
本发明的方法中，优选地，所述共聚物可以包括P3HB-co-4HB(3-羟基丁酸-4羟基丁酸共聚酯)，P3HB-co-4HB和P3HB(聚3-羟基丁酸)的掺和物，P3HB-co-4HB和P4HB(聚4-羟基丁酸)的掺和物，以及P3HB-co-4HB、P3HB和P4HB的掺和物。
本发明的方法中，优选地，所述细菌包括大肠杆菌及其重组菌。
本发明的方法中，优选地，所述细菌包括盐单胞菌TD及其重组菌。
本发明构建的表达orfZ的基因盒，在导入到嗜盐菌Halomonas TD01及其衍生菌后使得重组菌合成的PHA中含有4HB的组分，且4HB的比例可以被精细调控；同时不影响细菌生长，在发酵过程中既能实现高密度生长(细胞干重高)，也能保证P3HB4HB中合适的4HB比例。因此本发明所构建的表达orfZ的基因盒在P3HB4HB的工业生产上具有重要的应用价值。
附图说明
图1为诱导型启动子表达orfZ的基因盒示意图。
具体实施方式
本发明提供用于表达4-羟基丁酰辅酶A转移酶的基因盒，所述基因盒包括启动子，和位于所述启动子下游并与所述启动子可操作相连的4-羟基丁酰辅酶A转移酶基因；所述启动子是组成型启动子或诱导型启动子；所述组成型启动子是tac启动子，所述诱导型启动子是Mmp1启动子。
本发明中，所述启动子用于驱动4-羟基丁酰辅酶A转移酶基因的表达。
在一些实施方案中，所述基因盒由5'端至3'端依次包括Mmp1启动子、lacO序列和4-羟基丁酰辅酶A转移酶基因。
在一些实施方案中，所述4-羟基丁酰辅酶A转移酶基因可以是orfZ基因，优选是来自克氏梭状芽胞杆菌(Clostridium kluyveri)的orfZ基因。
在一些实施方案中，所述启动子可为SEQ ID NO:6所示的DNA分子，或SEQ ID NO:14所示的DNA分子，或将SEQ ID NO:6或SEQ ID NO:14经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:6或SEQ ID NO:14衍生的DNA分子。
在一些实施方案中，所述4-羟基丁酸辅酶A转移酶的核酸序列可为SEQ ID NO:7，或将SEQ ID NO:7经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:7衍生的DNA分子。
在一些实施方案中，所述4-羟基丁酸辅酶A转移酶可为SEQ ID NO:8所示的蛋白质，或将SEQ ID NO:8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:8衍生的蛋白质。
在一些实施方案中，所述基因盒还可以包含用于表达该基因盒中的4-羟基丁酰辅酶A转移酶基因的其它元件。用于基因表达的其它元件是本领域技术人员公知的，包括但不限于终止子等。
本发明中，当所述基因盒中的启动子为诱导型启动子Mmp1启动子时，可以通过以诱导方式诱导表达与Mmp1启动子相对应的MmP1RNA聚合酶，由MmP1RNA聚合酶激活Mmp1启动子转录，从而驱动4-羟基丁酰辅酶A转移酶基因。MmP1RNA聚合酶是本领域已知的，例如可以参见Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.中所述的MmP1RNA聚合酶。MmP1RNA聚合酶例如可以是Genebank登录号：YP_003324580.1的RNA聚合酶(Morganella phage MmP1)。
因此，本发明中，可以通过调控元件激活Mmp1启动子起始转录，从而表达4-羟基丁酰辅酶A转移酶。所述调控元件可以包含MmP1RNA聚合酶基因和诱导表达MmP1RNA聚合酶所需的其它元件。
本发明中，以诱导方式表达MmP1RNA聚合酶可以使用本领域熟知的各种蛋白诱导表达调控系统，包括但不限于通过IPTG诱导表达蛋白的lac乳糖操纵子表达调控系统，例如可以参见Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.中使用的表达调控系统。例如，利用lac乳糖操纵子表达调控系统以诱导方式表达MmP1RNA聚合酶的一种实例是含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件。含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件可以是从5'端至3'端依次包括阻遏蛋白表达盒、无义随机片段、RNA聚合酶启动子、lacO序列和RNA聚合酶编码基因或由这些元件组成。阻遏蛋白表达盒可以由阻遏蛋白启动子和阻遏蛋白编码基因组成，阻遏蛋白优选可以是lacI，阻遏蛋白与RNA聚合酶表达方向相反。例如可以参见Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.和CN105779488A中使用的含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件。
在一个特定的实施方案中，含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件从5'端至3'端依次包括lacI表达盒、tac启动子、lacO序列、MmP1RNA聚合酶编码基因或由这些元件组成。阻遏蛋白表达盒由lacI启动子和lacI编码序列组成，lacI与MmP1RNA聚合酶的表达方向相反。
本发明还提供了表达载体，其含有本发明的基因盒。
在一些实施方案中，所述表达载体可以是质粒。
在一些实施方案中，当所述基因盒中的启动子为诱导型启动子Mmp1启动子时，所述表达载体中还可以含有上述的调控元件，优选上述含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件，并且所述基因盒位于所述调控元件下游。
本发明还提供了细菌重组菌，其含有本发明的基因盒，所述细菌重组菌是重组盐单胞菌(Halomonas sp.)。
本发明中，盐单胞菌(Halomonas sp.)也被称为嗜盐菌，是一类革兰氏阴性菌，可以在高盐浓度下正常生长。可用于本发明的盐单胞菌包括但不限于Halomonas sp.TD01和Halomonas sp.LS21或衍生自它们的盐单胞菌。
本发明的重组盐单胞菌可以通过将本发明的基因盒转入起始菌株获得。例如可以通过将含有本发明的基因盒的表达载体转入起始菌株获得。
在一些实施方案中，所述基因盒被整合在所述重组盐单胞菌的基因组上。
在一些实施方案中，所述基因盒位于表达载体上，所述表达载体稳定存在于所述重组盐单胞菌内。
在一些实施方案中，所述起始菌株可以是盐单胞菌TD01或衍生自盐单胞菌TD01的盐单胞菌。
本发明中，“衍生自盐单胞菌TD01的盐单胞菌”是指由盐单胞菌TD01经过基因改造或突变等获得的盐单胞菌。为本发明的目的，本领域技术人员应当知道，所述基因改造或突变等应当不影响orfZ基因的表达和聚羟基脂肪酸酯共聚物的生产。
在一些实施方案中，当所述基因盒中的启动子为诱导型启动子Mmp1启动子时，所述重组盐单胞菌中还可以含有上述的调控元件，优选所述调控元件是如前所述的含有阻遏蛋白表达盒、RNA聚合酶表达盒的调控元件。
在一些实施方案中，所述调控元件可以被整合到细菌染色体上，也可以位于表达载体上，所述表达载体稳定存在于所述重组盐单胞菌内。
在一些实施方案中，起始菌株可以是盐单胞菌TD-MmP1。盐单胞菌TD-MmP1例如可以参见Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140。
在一些实施方案中，所述重组盐单胞菌包含稳定存在于其中的表达载体，所述表达载体上含有上述调控元件和本发明的基因盒，所述基因盒中包含的启动子是诱导型启动子Mmp1启动子。
本发明还提供了聚羟基脂肪酸酯共聚物的制备方法，其特征在于，用本发明的细菌重组菌进行发酵，制备聚羟基脂肪酸酯共聚物。
在一些实施方案中，所述聚羟基脂肪酸酯共聚物包括3-羟基丁酸-4羟基丁酸共聚酯(P3HB-co-4HB)。在一些实施方案中，所述聚羟基脂肪酸酯共聚物是3-羟基丁酸-4羟基丁酸共聚酯。
本申请中，3-羟基丁酸-4羟基丁酸共聚酯也可被称为P3HB4HB。
本发明还提供了本发明的基因盒、本发明的表达载体、本发明的细菌重组菌在生产聚羟基脂肪酸酯共聚物中的应用。
在一些实施方案中，所述聚羟基脂肪酸酯共聚物包括3-羟基丁酸-4羟基丁酸共聚酯(P3HB-co-4HB)。在一些实施方案中，所述聚羟基脂肪酸酯共聚物是3-羟基丁酸-4羟基丁酸共聚酯。
本发明的基因盒、表达载体或细菌重组菌能够控制聚羟基脂肪酸酯共聚物发酵生产中4HB的比例，尤其是控制P3HB4HB的发酵生产中4HB的比例，在发酵过程中既能实现高密度生长(细胞干重高)，也能保证P3HB4HB中4HB比例不会影响细菌生长。
在下文中，将通过以下实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。
下述实施例所使用的实验方法如无特殊说明，均为常规方法。
下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
所用酶试剂采购自ThermoFisher公司和New England Biolabs(NEB)公司，提取质粒所用的试剂盒购自天根生化科技(北京)有限公司，回收DNA片段的试剂盒购自美国omega公司，相应的操作步骤严格按照产品说明书进行，所有培养基如无特殊说明均用去离子水配制。
培养基配方：
1)大肠杆菌培养基
LB培养基：5g/L酵母提取物(购自英国OXID公司，产品目录号LP0021)，10g/L蛋白胨(购自英国OXID公司，产品目录号LP0042),10g/L NaCl，其余为水。调pH值至7.0-7.2，高压蒸汽灭菌。
2)嗜盐菌培养基
60LB培养基：5g/L酵母提取物(购自英国OXID公司，产品目录号LP0021)，10g/L蛋白胨(购自英国OXID公司，产品目录号LP0042),60g/L NaCl，其余为水。调pH值至7.0-7.2，高压蒸汽灭菌。
60MMG培养基：60g/L NaCl，30g/L葡萄糖，1g/L酵母提取物，2g/L NH 4Cl，0.2g/L MgSO 4，9.65g/L Na 2HPO 4·12H 2O，1.5g/L KH 2PO 4，10ml/L微量元素溶液I和1ml/L微量元素溶液II。其中，微量元素溶液I的组成为：5g/L柠檬酸铁铵，2g/L CaCl 2，用1M HCl配制。微量元素溶液II的组成为：100mg/L ZnSO 4·7H 2O，30mg/L MnCl 2·4H 2O，300mg/L H 3BO 3，200mg/L CoCl 2·6H 2O，10mg/L CuSO 4·5H 2O，20mg/L NiCl 2·6H 2O，30mg/L NaMoO 4·2H 2O，用1M HCl配制。培养基的最终pH用5M NaOH溶液调至8.5。上述试剂购自国药集团化学试剂公司。
在实际培养过程中，可向上述培养基中加入一定浓度的抗生素以维持质粒的稳定性，如100μg/mL氨苄青霉素或25μg/mL的氯霉素。
实施例1：诱导型启动子表达orfZ基因作为基因盒调控P3HB4HB中4HB的比例
用引物(MmP1-orfZ-f和MmP1-orfZ-r)以p68orfZ(Li,Z.J.,Shi,Z.J.,Guo,Y.Y.,Wu,Q.,Chen,G.Q.,2010.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)from unrelated carbon sources by metabolically engineered Escherichia coli.Metab.Eng.,12,4,352-359.)为模板扩增得到orfZ基因片段，用另一对引物(p321-MmP1-f和p321-MmP1-r)以p321-MmP1-gfp(Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.)为模板扩增得到质粒骨架片段，通过Gibson Assembly方法连接两个片段，即得到质粒p321-MmP1-orfZ，用MmP1启动子表达orfZ基因。使用的引物如下表：
Figure PCTCN2018092467-appb-000001
所得到的诱导型启动子表达orfZ的基因盒序列为：
Figure PCTCN2018092467-appb-000002
所述诱导型启动子表达orfZ的基因盒由两部分组成，其一是诱导型启动子，其二是orfZ基因。
所述诱导型启动子序列为SEQ ID NO:6：
Figure PCTCN2018092467-appb-000003
所述orfZ基因序列为SEQ ID NO:7：
Figure PCTCN2018092467-appb-000004
所述orfZ基因所编码的4-羟基丁酸辅酶A转移酶的氨基酸序列为SEQ ID NO:8：
MEWEEIYKEKLVTAEKAVSKIENHSRVVFAHAVGEPVDLVNALVKNKDNYIGLEIVHMVAMGKGEYTKEGMQRHFRHNALFVGGCTRDAVNSGRADYTPCFFYEVPSLFKEKRLPVDVALIQVSEPDKYGYCSFGVSNDYTKPAAESAKLVIAEVNKNMPRTLGDSFIHVSDIDYIVEASHPLLELQPPKLGDVEKAIGENCASLIEDGATLQLGIGAIPDAVLLFLKNKKNLGIHSEMISDGVMELVKAGVINNKKKTLHPGKIVVTFLMGTKKLYDFVNNNPMVETYSVDYVNNPLVIMKNDNMVSINSCVQVDLMGQVCSESIGLKQISGVGGQVDFIRGANLSKGGKAIIAIPSTAGKGKVSRITPLLDTGAAVTTSRNEVDYVVTEYGVAHLKGKTLRNRARALINIAHPKFRESLMNEFKKRF*(*代表蛋白质的终止密码子)
将质粒p321-MmP1-orfZ转入大肠杆菌S17-1(ATCC编号:47055，可购自美国菌种保藏中心American Type Culture Collection)，再通过接合转化方法(Fu XZ,Tan D,Aibaidula G,Wu Q,Chen JC,Chen GQ(2014)Development of Halomonas TD01as a host for open production of chemicals.Metab Eng23:78–91)将质粒最终转入嗜盐菌Halomonas TD-MmP1(Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.)。Halomonas TD-MmP1为TD01的改造菌，其基因组上整合了MmP1的RNA聚合酶基因，在添加诱导剂IPTG的情况下表达，表达后特异性识别MmP1启动子，从而实现通过诱导剂调控MmP1启动子下游的基因的表达。因此所得到的重组菌Halomonas TD-MmP1/p321-MmP1-orfZ可调控orfZ基因的表达。相关原理示意图见附图1。
重组菌在60LB培养基中200rpm，37℃培养12小时后按5％接种至50ml 60MMG培养基，200rmp，37℃，培养48小时。接种4小时后添加5克/升的γ-丁内酯；同时添加不同浓度的IPTG调控orfZ基因表达，IPTG浓度分别为0μM，1μM，5μM，10μM，50μM，100μM，200μM，400μM，1000μM。
48小时后收集菌体，检测细胞干重和PHA含量，方法如下：
用量筒量取30ml菌液放入50ml离心管，10000rpm离心10min收集菌体。用去离子水重悬细胞洗涤一次，10000rpm离心10min，弃掉上清。将菌体在-80℃冷冻1h后，进行真空冷冻干燥12h以上至完全去除水分。称量取样前后离心管重量，其差值为细胞干重CDW。
酯化液的配制：取485ml无水甲醇，加入1g/L苯甲酸，缓慢加入15ml浓硫酸，即制成约500ml的酯化液。
样品制备：称取30至60mg冷冻干燥后的菌体，精确称重后置于酯化管中，加入2ml酯化液和2ml氯仿。称取10mg左右的PHA样品，同样方式处理作为标准样品。酯化管加盖密封后100度反应4小时。反应结束后待酯化管冷却至室温，加入1ml去离子水，漩涡震荡至充分混合，静置分层。水相和有机相完全分离后，取下层有机相用于气相色谱法(GC)分析。
GC分析PHA组成及含量：使用岛津公司的GC-2014型气相色谱仪。色谱仪的配置为：HP-5型毛细管色谱柱，氢火焰离子化检测器FID，SPL分流进样口；高纯氮气作为载气，氢气为燃气，空气为助燃气；使用AOC-20S型自动进样器，丙酮为洗涤液。GC分析程序的设置为：进样口温度240℃，检测器温度250℃，柱温起始温度为80℃，维持1.5分钟；以30℃/分钟的速率升至140℃并维持0分钟；以40℃/分钟的速率升至240℃并维持2分钟；总计时间为8分钟。GC结果采用内标归一法根据峰面积进行定量计算PHA组成及含量。
重组菌Halomonas TD-MmP1/p321-MmP1-orfZ在不同浓度IPTG诱导下合成P3HB4HB的结果如下表所示：
Figure PCTCN2018092467-appb-000005
IPTG浓度从0增加到10μM时，重组菌Halomonas TD-MmP1/p321-MmP1-orfZ所合成的P3HB4HB中4HB的比例逐渐增加；当IPTG浓度大于10M时，4HB比例呈下降趋势，且细胞干重CDW也有下降趋势，说明orfZ基因表达量过高影响细胞生长和P3HB4HB中4HB成分的合成。通过诱导剂精细调控orfZ的表达，可控制P3HB4HB中4HB的比例从2.2mol％到11.20mol％的变化范围。
实施例2：组成型启动子表达orfZ基因作为基因盒
上述实施例中可通过诱导剂的浓度来调控重组菌合成的P3HB4HB中的4HB比例，然而在工业生产中诱导剂的添加会带来额外的成本增加。选用合适的组成型启动子，该启动子的表达强度与上述实施例中MmP1启动子在10μM诱导剂浓度下的表达强度相当，则能够在无需添加诱导剂的条件下达到orfZ表达的近似效果。
所用的组成型启动子为tac启动子，来源于p321-Ptac-GFP(Zhao,H.,Zhang,H.M.,Chen,X.,Li,T.,Wu,Q.,Ouyang,Q.,Chen,G.Q.,2016.Novel T7-like expression systems used for Halomonas.Metab.Eng.,39,128-140.)。用引物(Ptac-orfZ-f和Ptac-orfZ-r)以p68orfZ(Li,Z.J.,Shi,Z.J.,Guo,Y.Y.,Wu,Q.,Chen,G.Q.,2010.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)from unrelated carbon sources by metabolically engineered Escherichia coli.Metab.Eng.,12,4,352-359.)为模板扩增得到orfZ基因片段，该片段替换质粒p321-Ptac-GFP中的gfp基因，得到质粒p321-Ptac-orfZ，该质粒含有组成型的tac启动子表达orfZ基因的基因盒。使用的引物如下表：
Figure PCTCN2018092467-appb-000006
所得到的组成型启动子表达orfZ的基因盒序列为：
Figure PCTCN2018092467-appb-000007
Figure PCTCN2018092467-appb-000008
所述组成型启动子表达orfZ的基因盒由两部分组成，其一是组成型启动子，其二是orfZ基因。
所述组成型启动子序列为SEQ ID NO:14：
Figure PCTCN2018092467-appb-000009
所述orfZ基因序列同实施例1。
所述orfZ基因所编码的4-羟基丁酸辅酶A转移酶的氨基酸序列同实施例1。
将质粒p321-Ptac-orfZ转入大肠杆菌S17-1，再通过接合转化方法导入盐单胞菌Halomonas TD01(保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.4353)，得到重组菌Halomonas TD01/p321-Ptac-orfZ。该重组菌为基因盒以质粒形式导入到嗜盐菌中。
质粒载体表达基因的时候需要向培养基中添加抗生素来维持质粒稳定性，这在工业生产中也会带来额外成本。将基因整合到细菌的基因组上则无需添加抗生素也能使基因稳定存在，因此更适合工业生产的需求。本实施例中将Ptac-orfZ的基因盒整合到了Halomonas TD01的基因组上，具体步骤如下：
以Halomonas TD01基因组为模板进行PCR扩增得到片段H1和H2，以p321-Ptac-orfZ为模板PCR扩增得到Ptac-orfZ片段，将H1、H2和Ptac-orfZ进行overlap extension PCR(OE PCR)，得到H1-Ptac-orfZ-H2片段。将该片段通过Gibson Assembly方法连入质粒pRE112-6IsceI(Fu,X.Z.,Tan,D.,Aibaidula,G.,Wu,Q.,Chen,J.C.,Chen,G.Q.,2014.Development of Halomonas TD01 as a host for open production of chemicals.Metab.Eng.,23,2,78-91.)的XbaI和SphI位点之间，得到重组质粒pRE112-6ISceI-orfZ。使用的引物如下表：
Figure PCTCN2018092467-appb-000010
将重组质粒pRE112-6ISceI-orfZ转入大肠杆菌S17-1中，再通过接合转化方法转入Halomonas TD01中，利用自杀质粒无法在宿主菌内复制的特性，用25g/ml氯霉素抗性的60LB平板筛选出阳性克隆。该阳性克隆中带有同源片段的重组质粒整合到基因组上的H1和H2所在的特定位置，为第一次同源重组菌。
诱导质粒pBBR1MCS1-ISceI(Fu,X.Z.,Tan,D.,Aibaidula,G.,Wu,Q.,Chen,J.C.,Chen,G.Q.,2014.Development of Halomonas TD01as a host for open production of chemicals.Metab.Eng.,23,2,78-91.)表达一种归为内切酶I-SceI，将第一次同源重组菌的基因组上的6个I-SceI位点进行切割，产生双链DNA缺口，从而诱发第二次同源重组的发生，产生突变型或者野生型，然后通过特异的PCR引物将突变株筛选出来。将诱导质粒pBBR1MCS1-ISceI通过接合转化方法导入上述第一次同源重组菌，在100g/ml壮观霉素的60LB平板上筛选得到阳性克隆。该阳性克隆为基因组上整合了Ptac-orfZ基因盒，能够组成型表达orfZ基因，得到的重组菌为Halomonas TD40。
本实施例中Ptac-orfZ基因盒在基因组上整合的位点包括但不限于上文所述的特定位点。其他位点的选择由引物H1-pre112-f/H1-pre112-r和H2-pre112-f/H2-pre112-r的具体序列所决定。
综上所述，重组菌Halomonas TD40为基因盒Ptac-orfZ(组成型启动子)在基因组上表达；重组菌Halomonas TD01/p321-Ptac-orfZ为基因盒Ptac-orfZ在质粒上表达；重组菌Halomonas TD-MmP1/p321-MmP1-orfZ为基因盒MmP1-orfZ(诱导型启动子)在质粒上表达。上述3个重组菌与出发菌Halomonas TD01均在60LB培养基中200rpm，37℃培养12小时后按5％接种至50ml 60MMG培养基，200rmp，37℃，培养48小时。接种4小时后添加5克/升的γ-丁内酯。其中Halomonas TD-MmP1/p321-MmP1-orfZ接种4小时后额外添加10μM的IPTG。
48小时后收集菌体，检测细胞干重和PHA含量，方法同实施例1所述，结果如下表所示：
Figure PCTCN2018092467-appb-000011
出发菌Halomonas TD01只能合成P3HB而不含4HB组分，3个重组菌导入了表达orfZ的基因盒后均能合成P3HB4HB。组成型启动子表达orfZ的基因盒Ptac-orfZ导入到嗜盐菌Halomonas后合成的P3HB4HB中4HB比例达到8.86mol％以上，与诱导型启动子表达orfZ的最优条件相当。
实施例3：重组菌通过高密度发酵生产P3HB4HB
重组菌Halomonas TD40分别在1升、7升、1000升发酵罐中进行P3HB4HB的发酵生产，具体方法如下：
1升发酵罐中装液量为600毫升；培养基为60MMG，其中酵母膏替换为20克/升的玉米浆，NH 4Cl的用量为3克/升，另外添加2克/升的尿素；发酵温度控制在37℃，pH通过5M NaOH溶液控制在8.5；初始搅拌速度为200rpm，最高为800rpm；采用溶氧控制策略，保持溶氧在20％以上；空气通气量为0.7vvm；补料溶液为800克/升葡萄糖、18克/升NH 4Cl和30克/升γ-丁内酯，采用流加方式并保持发酵液中葡萄糖浓度在5-10克/升。48小时结束发酵过程，检测发酵液中的细胞干重CDW和PHA含量及4HB比例，检测方法同实施例1中所述。
7升发酵罐中装液量为3升，其余条件同上述1升发酵罐。48小时结束发酵过程，检测发酵液中的细胞干重CDW和PHA含量及4HB比例，检测方法同上所述。
1000升发酵罐中装液量为600升。1级种子液为250毫升摇瓶中装有120毫升培养基，37℃200rpm培养12h；接入装有1.2升培养基的5升摇瓶中，37℃200rpm培养12h，为2级种子液；接入装有60升培养基的100升发酵罐中，为3级种子液，发酵温度控制在37℃，pH通过5M NaOH溶液控制在8.5，初始搅拌速度为200rpm，最高为800rpm，采用溶氧控制策略，保持溶氧在20％以上，空气通气量为0.7vvm。3级种子液的培养基全部接种至1000升发酵罐中，培养条件同上1升和7升发酵罐。48小时结束发酵过程，检测发酵液中的细胞干重CDW和PHA含量及4HB比例，检测方法同上所述。
1升、7升、1000升发酵罐中重组菌Halomonas TD40生产P3HB4HB的相关检测结果如下表所示：
Figure PCTCN2018092467-appb-000012
重组菌Halomonas TD40在不同尺度发酵过程中均能实现高密度发酵、高含量P3HB4HB生产，且4HB比例不低于10mol％，具有作为P3HB4HB工业生产菌的潜力。

Claims (29)

  1. 一种精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其包括启动子和4-羟基丁酸辅酶A转移酶基因(优选来自克氏梭状芽胞杆菌的orfZ基因)或具有4-羟基丁酸辅酶A转移酶的功能的基因。
  2. 根据权利要求1所述的精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其中，所述启动子为诱导型启动子或组成型启动子，优选地，所述启动子为tac启动子，更优选地，所述启动子为SEQ ID NO:6所示的DNA分子，或SEQ ID NO:14所示的DNA分子，或将SEQ ID NO:6或SEQ ID NO:14经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:6或SEQ ID NO:14衍生的DNA分子。
  3. 根据权利要求1所述的精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其中，所述4-羟基丁酸辅酶A转移酶的核酸序列为SEQ ID NO:7，或将SEQ ID NO:7经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:7衍生的DNA分子。
  4. 根据权利要求1所述的精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其中，所述4-羟基丁酸辅酶A转移酶为SEQ ID NO:8所示的蛋白质，或将SEQ ID NO:8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:8衍生的蛋白质。
  5. 根据权利要求1所述的精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其中，所述基因盒的序列如SEQ ID NO:5或SEQ ID NO:13所示。
  6. 根据权利要求1所述的精细调控聚羟基脂肪酸酯(PHA)共聚物中4-羟基丁酸组成比例的基因盒，其中，所述共聚物可以包括P3HB-co-4HB(3-羟基丁酸-4羟基丁酸共聚酯)，P3HB-co-4HB和P3HB(聚3-羟基丁酸)的掺和物，P3HB-co-4HB和P4HB(聚4-羟基丁酸)的掺和物，以及P3HB-co-4HB、P3HB和P4HB的掺和物。
  7. 一种细菌重组菌，其含有权利要求1所述的基因盒。
  8. 根据权利要求7所述的细菌重组菌，其中，所述细菌包括大肠杆菌及其重组菌，或者所述细菌包括盐单胞菌TD及其重组菌。
  9. 一种聚羟基脂肪酸酯共聚物的制备方法，其特征在于，利用含有权利要求7或8所述的细菌重组菌制备聚羟基脂肪酸酯。
  10. 根据权利要求8所述的聚羟基脂肪酸酯共聚物的制备方法，其中，所述共聚物包括P3HB-co-4HB(3-羟基丁酸-4羟基丁酸共聚酯)，P3HB-co-4HB和P3HB(聚3-羟基丁酸)的掺和物，P3HB-co-4HB和P4HB(聚4-羟基丁酸)的掺和物，以及P3HB-co-4HB、P3HB和P4HB的掺和物。
  11. 一种用于表达4-羟基丁酰辅酶A转移酶的基因盒，所述基因盒包括启动子，和位于所述启动子下游并与所述启动子可操作相连的4-羟基丁酰辅酶A转移酶基因；所述启动子是组成型启动子或诱导型启动子；所述组成型启动子是tac启动子，所述诱导型启动子是Mmp1启动子。
  12. 根据权利要求11所述的基因盒，其中所述基因盒由5'端至3'端依次包括Mmp1启动子、lacO序列和4-羟基丁酰辅酶A转移酶基因。
  13. 根据权利要求11或12所述的基因盒，其中所述4-羟基丁酰辅酶A转移酶基因是orfZ基因。
  14. 根据权利要求11-13任一项所述的基因盒，其中所述4-羟基丁酸辅酶A转移酶为SEQ ID NO:8所示的蛋白质，或将SEQ ID NO:8所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:8衍生的蛋白质。
  15. 根据权利要求11-14任一项所述的基因盒，其中所述启动子为SEQ ID NO:6所示的DNA分子，或SEQ ID NO:14所示的DNA分子，或将SEQ ID NO:6或SEQ ID NO:14经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:6或SEQ ID NO:14衍生的DNA分子。
  16. 根据权利要求11-15任一项所述的基因盒，其中所述4-羟基丁酸辅酶A转移酶的核酸序列为SEQ ID NO:7，或将SEQ ID NO:7经过一个或几个核苷酸残基的取代和/或缺失和/或添加且具有相同功能的由SEQ ID NO:7衍生的DNA分子。
  17. 一种表达载体，其含有权利要求11-16任一项所述的基因盒。
  18. 根据权利要求17的表达载体，其中所述基因盒中的启动子为Mmp1启动子，所述表达载体中还含有调控元件，所述调控元件包含MmP1 RNA聚合酶基因和诱导表达MmP1 RNA聚合酶所需的其它元件，且所述基因盒位于所述调控元件下游。
  19. 根据权利要求18的表达载体，其中所述调控元件从5'端至3'端依次包括阻遏蛋白表达盒、无义随机片段、RNA聚合酶启动子、lacO序列和Mmp1 RNA聚合酶编码基因；所述阻遏蛋白表达盒由阻遏蛋白启动子和阻遏蛋白编码基因组成，所述阻遏蛋白与所述Mmp1 RNA聚合酶表达方向相反。
  20. 一种细菌重组菌，其含有权利要求11-16任一项所述的基因盒，所述细菌重组菌是重组盐单胞菌(Halomonas sp.)。
  21. 根据权利要求20所述的细菌重组菌，其中所述基因盒被整合在所述重组盐单胞菌的基因组上，或所述基因盒位于表达载体上，所述表达载体稳定存在于所述重组盐单胞菌内。
  22. 根据权利要求20或21所述的细菌重组菌，其中所述基因盒中的启动子为Mmp1启动子，所述重组盐单胞菌中还含有调控元件，所述调控元件包含MmP1 RNA聚合酶基因和诱导表达MmP1 RNA聚合酶所需的其它元件。
  23. 根据权利要求22所述的细菌重组菌，其中所述调控元件从5'端至3'端依次包括阻遏蛋白表达盒、无义随机片段、RNA聚合酶启动子、lacO序列和Mmp1 RNA聚合酶编码基因；所述阻遏蛋白表达盒由阻遏蛋白启动子和阻遏蛋白编码基因组成，所述阻遏蛋白与所述Mmp1 RNA聚合酶表达方向相反。
  24. 根据权利要求22或23所述的细菌重组菌，其中所述调控元件被整合到细菌染色体上，或者所述调控元件位于表达载体上，所述表达载体稳定存在于所述重组盐单胞菌内。
  25. 根据权利要求22或23所述的细菌重组菌，其中所述重组盐单胞菌包含稳定存在于其中的表达载体，所述表达载体上含有所述调控元件和所述基因盒。
  26. 聚羟基脂肪酸酯共聚物的制备方法，其特征在于，用权利要求20-25任一项所述的细菌重组菌进行发酵，制备聚羟基脂肪酸酯共聚物。
  27. 根据权利要求26的制备方法，其中所述聚羟基脂肪酸酯共聚物是P3HB4HB。
  28. 权利要求1-6、11-16任一项所述的基因盒、权利要求17-19任一项所述的表达载体或权利要求7-8、20-25任一项所述的细菌重组菌在发酵制备聚羟基脂肪酸酯共聚物中的应用。
  29. 根据权利要求28的应用，其中所述聚羟基脂肪酸酯共聚物是P3HB4HB。
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