WO2025044707A1 - 一种细胞形态纤长化的重组菌株及其构建方法和应用 - Google Patents

一种细胞形态纤长化的重组菌株及其构建方法和应用 Download PDF

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WO2025044707A1
WO2025044707A1 PCT/CN2024/110346 CN2024110346W WO2025044707A1 WO 2025044707 A1 WO2025044707 A1 WO 2025044707A1 CN 2024110346 W CN2024110346 W CN 2024110346W WO 2025044707 A1 WO2025044707 A1 WO 2025044707A1
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promoter
mincd
recombinant
nucleotide sequence
recombinant bacterium
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陈国强
郑爽
刘絮
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Tsinghua University
Beijing Phabuilder Biotechnology Co Ltd
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Tsinghua University
Beijing Phabuilder Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the technical field of bacterial strain transformation, and in particular to a recombinant bacterial strain with elongated cell morphology, a construction method and application thereof.
  • PHA Polyhydroxybutyrate
  • PHA exists in the form of intracellular inclusion bodies, when its content exceeds 50wt%, especially when PHA% exceeds 80wt% in industrial production, the size of the cell volume becomes an important factor affecting PHA production, and it is also the determining factor for the upper limit of PHA content.
  • One of the measures to increase PHA production and reduce production costs is to increase cell volume. The goal of increasing production and reducing costs can be achieved by modifying cell morphology. Increasing cell volume to accommodate more PHA not only helps to increase PHA content, but also helps to reduce the difficulty of cell collection after fermentation and reduce product separation costs.
  • the present invention introduces the expression model of the deformation gene minCD regulated by PHA synthesis into the chassis cells.
  • the cells are combined with ribosome binding site sequences (RBS) of different strengths to significantly increase the length of the chassis cells.
  • RBS ribosome binding site sequences
  • a recombinant bacterium comprising an expression vector, wherein the expression vector comprises a minCD gene and a promoter;
  • the promoter contains a PhaR binding site
  • the recombinant bacteria are recombinant bacteria expressing PhaR protein.
  • the nucleotide sequence of the minCD gene contains the nucleotide sequence shown in SEQ ID NO:1, or contains a nucleotide sequence with more than 80% homology to the nucleotide sequence shown in SEQ ID NO:1.
  • nucleotide sequence of the minCD gene is as shown in SEQ ID NO:1.
  • the minCD gene is expressed on an expression vector and/or on a chromosome.
  • the promoter comprises a constitutive promoter and/or an inducible promoter.
  • the promoter may be a conventional promoter in the prior art.
  • the constitutive promoter is selected from but not limited to Sp6 promoter or its variant or porin promoter or its mutant.
  • the mutant of porin promoter is selected from any one of porin-203, porin-221, porin-194, porin-278, porin-68, porin-58 or porin-42 (Ye et.al. Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas. Metabolic Engineering. 57 (2020) 85-95.).
  • the inducible promoter is selected from but not limited to luc promoter or variants thereof, lac promoter or variants thereof, trp promoter or variants thereof, tac promoter or variants thereof, phage promoter or variants thereof, araBAD promoter or variants thereof or phaP promoter or variants thereof.
  • the phage promoter includes but is not limited to a ⁇ phage promoter or a T7 phage promoter.
  • the phaP promoter includes but is not limited to phaP1 promoter, phaP2 promoter or phaP3 promoter.
  • the promoter is phaP1
  • the phaP1 promoter is located at 109 bases upstream of the open reading frame of the phaP1 gene.
  • the phaP1 promoter includes the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology thereto and having the same or similar activity.
  • the phaP1 promoter is shown as SEQ ID NO:2.
  • the recombinant bacteria can be recombinant bacteria that can express PhaR protein itself, or the recombinant bacteria can be transformed to express PhaR protein (for example, an expression vector containing a gene encoding PhaR protein is introduced into the recombinant bacteria).
  • the expression of the promoter will be inhibited.
  • PhaR will be detached from the promoter, the expression of the promoter will be restored, and the expression of the promoter will be regulated by PHA synthesis.
  • the promoter is a promoter that itself contains a PhaR binding site, or the promoter can also be modified, for example, a PhaR binding site is added to the promoter, and the promoter with the PhaR binding site still has the function of a promoter, and the expression of the promoter can be regulated by PHA synthesis.
  • the expression vector further comprises an RBS sequence.
  • RBS sequence In order to regulate and utilize the phaP1 promoter and meet the expression requirements of different strengths, the core region of the original phaP1 RBS was mutated to obtain 1024 RBS libraries with different strengths.
  • the sequence of the RBS library is as shown in SEQ ID NO: 3 (wherein N is a mutation site, which can be any one of A, T, C, G or U).
  • the nucleotide sequence of the RBS is selected from an RBS library, and the nucleotide sequence of the RBS includes SEQ ID NO:4-13 or includes a nucleotide sequence having more than 90% homology with SEQ ID NO:4-13, or a nucleotide sequence as shown in SEQ ID NO:4-13.
  • the nucleotide sequence of the RBS includes SEQ ID NO: 7-13 or a nucleotide sequence having more than 90% homology with SEQ ID NO: 7-13, or a nucleotide sequence as shown in SEQ ID NO: 7-13.
  • the promoter, minCD gene and RBS sequence contained in the expression vector are in the order of promoter, RBS sequence and minCD gene.
  • the RBS is adjacent to the start codon of the minCD gene.
  • the expression vector is capable of replication, transcription and translation in the host cell, and therefore, it may also contain other conventional expression elements in the expression vector, such as terminator, restriction site and the like.
  • the expression vector comprises a promoter, a minCD gene, an RBS sequence and a terminator, and the order of the expression elements and genes in the expression vector is promoter, RBS sequence, minCD gene and terminator.
  • the expression vector may be a prokaryotic expression vector or a eukaryotic expression vector, and more preferably a prokaryotic expression vector.
  • the expression vector is a plasmid vector.
  • the plasmid vector is an Escherichia coli expression vector.
  • the expression vector integrates the minCD gene into the genome of the recombinant bacteria, or the expression vector is free in the recombinant bacteria.
  • the expression vector also integrates the promoter, RBS sequence and/or terminator into the genome of the recombinant bacteria.
  • the recombinant bacteria are recombinant bacteria that synthesize PHA or constitute monomers of PHA.
  • the recombinant bacteria may be recombinant bacteria that naturally synthesize PHA or monomers that constitute PHA, or recombinant bacteria that can synthesize PHA or monomers that constitute PHA after being modified (for example, by introducing PHA synthesis-related genes into the recombinant bacteria).
  • the expression vector may also contain genes encoding product synthesis pathways.
  • the product comprises monomers constituting PHA or PHA.
  • the monomers constituting PHA include but are not limited to one or more of 2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxyvaleric acid, 5-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid;
  • the PHA includes but is not limited to one or more of P(HB-LA), P3HP, PHB, P4HB, PHV, PHO, PHN, PHD, PHBV, P34HB, PHBHHp, PHBHHx, P3HB4HB3HV, P3HB4HB5HV or other medium- and long-chain hydroxy fatty acids.
  • the recombinant bacteria is selected from the genus Escherichia, Ralstonia, Alcaligenes, Pseudomonas, Aeromonas or Halomonas.
  • the genus Escherichia includes but is not limited to Escherichia coli or its derivatives.
  • the genus Ralstonia includes but is not limited to Ralstonia eutropha (also called Alcaligenes eutrophus, Cupriavidus necator) or its derivatives.
  • the genus Alcaligenes includes but is not limited to Alcaligenes latus or its derivatives.
  • the genus Pseudomonas includes but is not limited to Pseudomonas putida or its derivatives.
  • the genus Aeromonas includes but is not limited to Aeromonas hydrophila or its derivatives.
  • the genus Halomonas includes but is not limited to Halomonas bluephagenesis or its derivatives, Halomonas possiblynsis or its derivatives or Halomonas aydingkolgenesis or its derivatives.
  • the recombinant bacteria are of the genus Halomonas, and the recombinant bacteria include but are not limited to Halomonas bluephagenesis TD01 CGMCC.No.4353, Halomonas possiblynsis LS21 CGMCC No.6593, Halomonas aydingkolgenesis M1 CGMCC NO.19880, Halomonas bluephagenesis WZY254 or Halomonas bluephagenesis WZY278.
  • the second aspect of the present invention provides a method for preparing the recombinant bacteria described in the first aspect, characterized in that the preparation method comprises introducing the expression vector into the recombinant bacteria.
  • the expression vector integrates the minCD gene into the genome of the recombinant bacteria, or the expression vector is free in the recombinant bacteria.
  • the expression vector also integrates the promoter, RBS sequence and/or terminator into the genome of the recombinant bacteria.
  • the relevant limitations on expression vector, promoter, RBS sequence and recombinant bacteria are the same as those in the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant bacterium obtained by the preparation method described in the second aspect.
  • the fourth aspect of the present invention provides an expression vector.
  • the fifth aspect of the present invention provides a fermentation method.
  • the fermentation method comprises fermenting and culturing the recombinant bacteria described in the first aspect.
  • a method for producing PHA is provided.
  • the method comprises fermenting and culturing the recombinant bacteria described in the first aspect.
  • the seventh aspect of the present invention provides a method for promoting cell fibrillation.
  • the method comprises introducing an expression vector into a recombinant bacterium, wherein the expression vector comprises a minCD gene and a promoter; the promoter comprises a PhaR binding site; and the recombinant bacterium is a recombinant bacterium expressing a PhaR protein.
  • the nucleotide sequence of the minCD gene contains the nucleotide sequence shown in SEQ ID NO:1, or contains a nucleotide sequence with more than 80% homology to the nucleotide sequence shown in SEQ ID NO:1.
  • nucleotide sequence of the minCD gene is as shown in SEQ ID NO:1.
  • the relevant limitations on expression vectors and recombinant bacteria are the same as those in the first aspect of the present invention.
  • the present invention provides a method for isolating recombinant bacteria, the separation method comprising centrifuging the recombinant bacteria.
  • the centrifugation may be performed at any centrifugal force.
  • the centrifugal force is greater than 300g.
  • the centrifugal force of the centrifugation is any value in the range of 300-15000g, for example 300, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000 or 15000g.
  • the separation time is more than 3 minutes, for example 3, 4, 5, 6, 7, 8, 9, 10, or longer.
  • the bacteria Due to the elongation of the cells of the recombinant bacteria described in the present application, the bacteria can be collected in a short time, for example, within 10 minutes, under low centrifugal force, thereby simplifying the downstream processing flow and reducing the downstream processing cost.
  • PhaP protein is a PHA particle binding protein, and its expression level is positively correlated with the percentage of PHA.
  • the cell deformation-related gene minCD is placed downstream of the PhaP promoter, and the expression of minCD is regulated by the phaP promoter, so that MinCD can be produced without induction in PHA production.
  • the self-induced expression of the agent can increase the cell length to nearly ten times the original cell length during the fermentation process, while increasing the dry weight and PHA percentage.
  • the cells with increased length can be separated and collected under low centrifugal force conditions, thereby reducing the downstream processing cost of PHA and helping PHA to move towards a wider market application. It can be used in molecular biology, genetic engineering, metabolic engineering, synthetic biology, fermentation engineering and downstream separation and purification fields.
  • Figure 1 Structure diagram of the recombinant plasmid pSEVA321-P p1 -RBS-minCD.
  • the expression of the minCD gene is regulated by the phaP1 promoter.
  • oriT represents the transfer origin of the bacterial conjugative plasmid
  • Cm R represents chloramphenicol resistance
  • T represents the terminator
  • the semicircle in the figure is the ribosome binding site RBS.
  • Figure 2 Cell morphology of strains WZY278(pSEVA321-P p1 ) and WZY278(pSEVA321-P p1 -RBS3-minCD) at the end point of the shake flask fermentation experiment.
  • Figure 3 The states of the shake flask fermentation broth of strains WZY278 (pSEVA321-P p1 ) and WZY278 (pSEVA321-P p1 -RBS3-minCD) when centrifuged at 500 g for 3 min, 6 min and 10 min, respectively; wherein 1 is the control strain WZY278 (pSEVA321-P p1 ), and 2 is the deformed strain WZY278 (pSEVA321-P p1 -RBS3-minCD).
  • Escherichia coli was grown in LB medium, and the medium composition was: 10 g/L sodium chloride, 10 g/L peptone, and 5 g/L yeast extract.
  • H.bluephagenesis WZY278 and its derivative strains were cultured in LB50 or MM30 medium.
  • LB50 medium has the same ingredients as LB except that the concentration of sodium chloride is changed to 50 g/L.
  • MM30 medium has the following components: 30 g/L sodium chloride, 1 g/L yeast extract, 30 g/L glucose, 0.5 g/L urea and other trace elements.
  • the shaking growth conditions for Escherichia coli and H. bluephagenesis and their derivatives were 37°C and 200 rpm.
  • RBSs with different strengths were selected for the construction of the minCD recombinant expression plasmid, and the minCD gene was placed downstream of the phaP1 promoter, and the RBS was located upstream of minCD, close to the start codon of minCD.
  • RBS1-RBS9 were derived from the P phaP1 RBS X library constructed by the RBS library calculator of Salis Lab (https://salislab.net/software/design_rbs_library_calculator).
  • the library uses the RBS0 (i.e., P phaP1 natural RBS) homologous sequence CTCACTTAXXXXXATTATGTGTG (where X is any base of A, T, C, G or U) as a template, minCD as an expression gene, and the five X sites are randomly mutated, and each RBS corresponds to its own relative strength.
  • RBS0 i.e., P phaP1 natural RBS
  • CTCACTTAXXXXXATTATGTGTG where X is any base of A, T, C, G or U
  • the H. bluephagenesis TD01 genome was selected as a template and minCD was obtained by PCR.
  • the 10 RBS with different strengths in Table 1 were constructed to construct recombinant expression plasmids with different minCD expression intensities, namely pSEVA321-P p1 -RBS0-minCD, pSEVA321-P p1 -RBS1-minCD, pSEVA321-P p1 -RBS2-minCD, pSEVA321-P p1 -RBS3-minCD, pSEVA321-P p1 -RBS4-minCD, pSEVA321-P p1 -RBS5-minCD, pSEVA321-P p1 -RBS6-minCD, pSEVA321-P p1 p1
  • the plasmid pSEVA321-P p1 without minCD was used as a control.
  • Each plasmid was transformed into Escherichia coli S17-1 competent cells.
  • Single clones were picked and PCR identification and sequencing were performed, and the single clones with correct sequencing were selected to obtain 10 self-inducing recombinant expression plasmids with different minCD expression intensities (plasmid structure is shown in Figure 1) and a control plasmid.
  • H.bluephagenesis WZY278 was used as the host cell, and the 10 autoinducing recombinant expression plasmids and the control plasmid obtained above were respectively transferred into H.bluephagenesis WZY278 (for specific methods, see Zhao, H., et al. Novel T7-like expression systems used for Halomonas.
  • WZY278 pSEVA321-P p1 -RBS0-minCD
  • WZY278 pSEVA321-P p1 -RBS1-minCD
  • WZY278 pSEVA321-P p1 -RBS2-minCD
  • WZY278 pSEVA321-P p1 -RBS3-minCD
  • WZY278 pSEVA321-P p1 -RBS4-minCD
  • WZY278 pSEVA321-P p1 -RBS5-minCD
  • shake flask fermentation is required.
  • the 11 engineered strains obtained above were inoculated into 20 mL of LB50 medium containing 34 mg/L chloramphenicol and cultured for 12-16 hours, and then transferred to a new 20 mL of LB50 medium containing 34 mg/L chloramphenicol at a volume ratio of 1% and continued to be cultured for 8-10 hours to obtain a shake flask seed solution.
  • 2.5 mL of the seed bacterial solution was inoculated into a 500 mL conical flask containing 50 mL of MM30 medium containing 34 mg/L chloramphenicol (inoculation ratio 1:20), and fermented for 48 hours at 37 ° C and 200 rpm.
  • the bacterial liquid of WZY278(pSEVA321-P p1 ) and WZY278(pSEVA321-P p1 -RBS7-minCD) were taken for microscopic cell morphology observation and bacterial liquid centrifugation analysis.
  • the cell morphology of the two strains is shown in Figure 2.
  • the cell length of WZY278(pSEVA321-P p1 -RBS7-minCD) increased significantly, reaching 10 ⁇ m, which is about 10 times the length of the control strain cell.

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Abstract

涉及菌种改造技术领域，具体涉及一种细胞形态纤长化的重组菌株及其构建方法和应用。对底盘细胞进行细胞形变基因rmnCD自诱导表达构建重组菌株，使得重组菌株的细胞长度显著增长，同时可以提高细胞干重及PHA产量，所述的重组菌株在较低离心力时即可实现菌体收集，大大简化下游处理流程，降低下游处理成本。

Description

一种细胞形态纤长化的重组菌株及其构建方法和应用 技术领域
本发明涉及菌种改造技术领域，具体涉及一种细胞形态纤长化的重组菌株及其构建方法和应用。
背景技术
聚羟基脂肪酸酯PHA是一种可由多种细菌天然产生的胞内聚酯，是细菌在营养或代谢不平衡时用于储存碳和能量的一种自然方式，在菌体中以不溶于水的包涵体形式存在(Shang,L.,et al.Poly(3-Hydroxybutyrate)Synthesis in Fed-Batch Culture of Ralstoniaeutropha with Phosphate Limitation under Different Glucose Concentrations.Biotechnol.Lett.2003,25(17),1415-1419.)。PHA具有与传统石油基塑料相当的材料性能，其已被用于包装、食品、医疗、化妆品、农业、生物能源等领域。PHA的市场需求存在巨大潜力，而PHA产量和生产成本是制约PHA走向市场应用的最大障碍。
由于PHA以胞内包涵体形式存在，当其含量超过50wt％时，尤其是工业化生产中PHA％超过80wt％时，细胞体积的大小成为PHA产量的重要影响因素，其也是PHA含量上限的决定性因素。提高PHA产量和降低生产成本的措施之一为增大细胞体积。通过改造细胞形态可以实现增加产量和降低成本的目标，增大细胞体积以容纳更多的PHA，既有助于PHA含量的提高，又助于减轻发酵结束后的细胞收集难度，降低产物分离成本。
虽然在现有技术(Tan D,Wu Q,Chen J C,et al.Engineering Halomonas TD01for the low-cost production of polyhydroxyalkanoates[J].Metab Eng,2014,26:34-47.)中公开过表达minCD使细胞纤长化，在降低了下游分离成本的同时增高了PHA产量，但是此过程需要添加诱导剂IPTG，IPTG的使用使得生产成本提高。PHA的市场需求存在巨大潜力，而PHA产量和生产成本是制约PHA走向市场应用的最大障碍。现有技术(CN113999869A)中公开了使用PhaP系列启动子过表达MinCD可以自诱导细胞纤长化，但原本的表达强度过高，对细胞的生长不利。
发明内容
本发明通过在底盘细胞中引入受PHA合成调控的形变基因minCD表达模 块，搭配不同强度的核糖体接合位点序列(Ribosome Binding Site,RBS)，使得底盘细胞的细胞长度显著增长，长度增长的细胞可以在低离心力的条件下分离收集，并提高PHA的产量。具体为：
本发明的第一方面，提供了一种重组菌，所述的重组菌中包含表达载体，所述的表达载体中包含minCD基因和启动子；
所述的启动子中包含PhaR的结合位点；
所述的重组菌为表达PhaR蛋白的重组菌。
优选的，所述的minCD基因的核苷酸序列包含SEQ ID NO:1所示的核苷酸序列，或包含与SEQ ID NO:1所示的核苷酸序列具有80％以上同源性。
优选的，所述的minCD基因的核苷酸序列如SEQ ID NO:1所示。
优选的，所述的minCD基因在表达载体上表达和/或在染色体上表达。
优选的，所述的启动子包括组成型启动子和/或诱导型启动子。
优选的，所述的minCD基因由所述的启动子调控表达。
优选的，所述的启动子可以是现有技术中常规的启动子。
优选的，所述的组成型启动子选自但不限于Sp6启动子或其变体或porin启动子或其突变体。
其中，porin启动子的突变体选自porin-203、porin-221、porin-194、porin-278、porin-68、porin-58或porin-42中的任一种(Ye et.al.Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas.Metabolic Engineering.57(2020)85-95.)。
优选的，所述的诱导型启动子选自但不限于luc启动子或其变体、lac启动子或其变体、trp启动子或其变体、tac启动子或其变体、噬菌体启动子或其变体、araBAD启动子或其变体或phaP启动子或其变体。
其中，所述的噬菌体启动子包括但不限于λ噬菌体启动子或T7噬菌体启动子。
优选的，所述的phaP启动子包括但不限于phaP1启动子、phaP2启动子或phaP3启动子。
在本发明的一个具体实施方式中，所述的启动子为phaP1，phaP1启动子在phaP1基因开放阅读框上游109位碱基处。
优选的，phaP1启动子包括SEQ ID NO：2所示核苷酸序列或与其具有80％以上同源性且具有相同或相似的活性的核苷酸序列。
在本发明的一个具体实施方式中，所述的phaP1启动子如SEQ ID NO：2所示。
优选的，所述的重组菌可以为本身可以表达PhaR蛋白的重组菌，或者，所述的重组菌可以经过改造后表达PhaR蛋白(例如向重组菌中导入包含PhaR蛋白的编码基因的表达载体)。
优选的，当重组菌表达的PhaR蛋白与启动子上的PhaR的结合位点结合时，会抑制启动子的表达，而当体系中合成的PHA与PhaR结合，使PhaR从启动子上脱离，启动子的表达恢复，启动子的表达受PHA合成的调控。
优选的，所述的启动子为本身包含PhaR的结合位点的启动子，或者，所述的启动子还可以经过改造，例如在启动子上加入PhaR的结合位点，加上PhaR的结合位点后的启动子仍具有启动子的功能，并使启动子的表达可以受PHA合成的调控。
优选的，所述的表达载体中还包含RBS序列。针对phaP1启动子的调控利用，为了适应不同强度的表达需求，针对原本的phaP1的RBS的核心区域进行了突变，获得了1024个具有不同强度的RBS库。
优选的，该RBS库的序列如SEQ ID NO：3所示(其中，N为突变位点，可以为A、T、C、G或U中任一种)。
优选的，所述的RBS的核苷酸序列为RBS库中选取获得，所述的RBS的核苷酸序列包括SEQ ID NO:4-13或包括与SEQ ID NO:4-13具有90％以上同源性的核苷酸序列，或如SEQ ID NO:4-13所示核苷酸序列。
在本发明的一个具体实施方式中，所述的RBS的核苷酸序列包括SEQ ID NO:7-13或与SEQ ID NO:7-13具有90％以上同源性的核苷酸序列，或如SEQ ID NO:7-13所示核苷酸序列。
在本发明的一个具体实施方式中，所述的表达载体中包含的启动子、minCD基因和RBS序列在表达载体中的顺序为启动子、RBS序列、minCD基因。
优选的，在所述的表达载体中，所述的RBS紧邻minCD基因的起始密码子。
优选的，所述的表达载体能够在宿主细胞中复制、转录和翻译，因此，其还可以包含表达载体中常规的其他表达原件，例如终止子、酶切位点等等。
在本发明的一个具体实施方式中，所述的表达载体中包含启动子、minCD基因、RBS序列和终止子，各表达元件和基因在表达载体中的顺序为启动子、RBS序列、minCD基因、终止子。
优选的，所述的表达载体可以为原核表达载体或真核表达载体。进一步优选为原核表达载体。
在本发明的一个具体实施方式中，所述的表达载体为质粒载体。
在本发明的一个具体实施方式中，所述的质粒载体为大肠杆菌表达载体。
优选的，所述的表达载体将minCD基因整合至重组菌的基因组中，或者，所述表达载体游离在重组菌中。
优选的，所述的表达载体还将启动子、RBS序列和/或终止子也整合至重组菌的基因组中。
优选的，所述的重组菌为合成PHA或组成PHA的单体的重组菌。
优选的，所述的重组菌可以为天然合成PHA或组成PHA的单体的重组菌，或经过改造(例如向重组菌中导入PHA合成相关基因)后可以合成PHA或组成PHA的单体的重组菌。
优选的，所述的表达载体中还可以包含编码产物合成途径相关的基因。
优选的，所述的产物包含组成PHA的单体或PHA。
优选的，所述的组成PHA的单体包含但不限于2-羟基丙酸、3-羟基丙酸、3-羟基丁酸、4-羟基丁酸、3-羟基戊酸、5-羟基戊酸、3-羟基己酸、3-羟基庚酸、3-羟基辛酸、3-羟基壬酸、3-羟基癸酸、3-羟基十二酸中的一种或两种以上；
优选的，所述PHA包含但不限于P(HB-LA)、P3HP、PHB、P4HB、PHV、PHO、PHN、PHD、PHBV、P34HB、PHBHHp、PHBHHx、P3HB4HB3HV、P3HB4HB5HV或其他中长链羟基脂肪酸中的一种或两种以上。
优选的，所述的重组菌选自埃希氏菌属(Escherichia)、罗氏菌属(Ralstonia)、产碱杆菌属(Alcaligenes)、假单胞菌属(Pseudomonas)、气生单胞菌属(Aeromonas)或盐单胞菌属(Halomonas)。
优选的，所述的埃希氏菌属(Escherichia)包括但不限于大肠杆菌(Escherichia coli)或其衍生菌。
优选的，所述的罗氏菌属(Ralstonia)包括但不限于罗氏真养杆菌(Ralstonia eutropha，也叫Alcaligenes eutrophus，Cupriavidus necator)或其衍生菌。
优选的，所述的产碱杆菌属(Alcaligenes)包括但不限于巨大产碱杆菌(Alcaligenes latus)或其衍生菌。
优选的，所述的假单胞菌属(Pseudomonas)包括但不限于恶臭假单胞菌(Pseudomonas putida)或其衍生菌。
优选的，所述的气生单胞菌属(Aeromonas)包括但不限于嗜水气单胞菌(Aeromonas hydrophila)或其衍生菌。
优选的，所述的盐单胞菌属(Halomonas.)包括但不限于Halomonas bluephagenesis或其衍生菌、Halomonas campaniensis或其衍生菌或Halomonas aydingkolgenesis或其衍生菌。
在本发明的一个具体实施方式中，所述的重组菌为盐单胞菌属，所述的重组菌包括但不限于Halomonas bluephagenesis TD01 CGMCC.No.4353，Halomonas campaniensis LS21 CGMCC No.6593、Halomonas aydingkolgenesis M1 CGMCC NO.19880、Halomonas bluephagenesis WZY254或Halomonas bluephagenesis WZY278。
本发明的第二方面，提供了一种上述第一方面所述的重组菌的制备方法，其特征在于，所述的制备方法包括将表达载体导入重组菌中。
优选的，所述的表达载体将minCD基因整合至重组菌的基因组中，或者，所述表达载体游离在重组菌中。
优选的，所述的表达载体还将启动子、RBS序列和/或终止子也整合至重组菌的基因组中。
优选的，对表达载体、启动子、RBS序列、重组菌的相关限定，同本发明的第一方面。
本发明的第三方面，提供了一种上述第二方面所述制备方式获得的重组菌。
本发明的第四方面，提供了一种表达载体。
对表达载体的相关限定，同本发明的第一方面。
本发明的第五方面，提供了一种发酵方法。
所述的发酵方法包括发酵培养上述第一方面所述的重组菌。
本发明的第六方面，提供了一种生产PHA的方法。
所述的方法包括发酵培养上述第一方面所述的重组菌。
本发明的第七方面，提供了一种促进细胞纤长化的方法。
所述的方法包括将表达载体向导入重组菌中，所述的表达载体中包含minCD基因和启动子；所述的启动子中包含PhaR的结合位点；所述的重组菌为表达PhaR蛋白的重组菌。
优选的，所述的minCD基因的核苷酸序列包含SEQ ID NO:1所示的核苷酸序列，或包含与SEQ ID NO:1所示的核苷酸序列具有80％以上同源性。
优选的，所述的minCD基因的核苷酸序列如SEQ ID NO:1所示。
优选的，对表达载体、重组菌的相关限定，同本发明的第一方面。
本发明的第八方面，提供了一种重组菌的分离方法，所述的分离方法包括离心分离上述的重组菌。
优选的，所述的离心可以为在任意离心力下离心。
优选的，所述的离心力大于300g。
优选的，所述的离心的离心力为300-15000g中任一数值，例如300、500、800、1000、2000、3000、4000、5000、6000、7000、8000、9000、10000、11000、12000、13000、14000或15000g。
优选的，所述分离的时间为3分钟以上，例如3、4、5、6、7、8、9、10，或更长时间。
由于本申请所述的重组菌的细胞纤长化，在低离心力下就可以在短时间内，例如在10分钟内完成菌体收集，简化下游处理流程，降低下游处理成本。
有益效果：
在PHA的生产菌株中，PhaP蛋白作为PHA颗粒结合蛋白，其表达量与PHA的百分含量具有正相关关系。将细胞形变相关基因minCD置于PhaP启动子下游，由phaP启动子调控表达minCD，实现MinCD在PHA生产中实现不需诱导 剂的自诱导表达，在发酵过程中，实现细胞长度增加至原本细胞长度的近十倍，同时增加干重和PHA百分含量的效果，且长度增长的细胞可以在低离心力的条件下分离收集，从而降低PHA的下游处理成本，有助于PHA走向更广泛的市场应用，可用于分子生物学、基因工程、代谢工程、合成生物学、发酵工程领域及下游分离纯化领域。
附图说明
图1：重组质粒pSEVA321-Pp1-RBS-minCD的结构图，minCD基因受phaP1启动子调控表达，其中，oriT表示细菌接合质粒的转移原点，CmR表示氯霉素抗性，T表示终止子，图中半圆为核糖体结合位点RBS。
图2：菌株WZY278(pSEVA321-Pp1)和WZY278(pSEVA321-Pp1-RBS3-minCD)摇瓶发酵实验终点时的细胞形态。
图3：菌株WZY278(pSEVA321-Pp1)和WZY278(pSEVA321-Pp1-RBS3-minCD)的摇瓶发酵菌液在500g离心力条件下分别离心3min、6min、10min时的状态；其中，①为对照菌株WZY278(pSEVA321-Pp1)，②为形变菌株WZY278(pSEVA321-Pp1-RBS3-minCD)。
具体实施方式
下面结合实施例、序列表和说明书附图对本发明作进一步说明，但并不仅限于此。实施例中所使用的实验方法未作具体说明的，均为常规方法、常规操作或说明书条件进行；未作具体说明的试剂或药品，均为普通市售产品，均可从商业途径获得。
大肠杆菌生长在LB培养基上，培养基成分组成：10g/L的氯化钠，10g/L的蛋白胨，5g/L的酵母提取物。
盐单胞菌H.bluephagenesis WZY278及其衍生菌株在LB50或MM30的培养基上培养。LB50培养基，其成分除氯化钠的浓度变成50g/L外，其它成分同LB一样。MM30培养基的组分如下：30g/L的氯化钠，1g/L的酵母提取物，30g/L浓度葡萄糖，0.5g/L的尿素及其它微量元素组成。
大肠杆菌和盐单胞菌H.bluephagenesis及其衍生菌株的摇床生长条件均为37℃，200rpm。
实施例：细胞形态纤长化的重组菌株的构建
首先，针对phaP1启动子的调控利用，为了适应不同强度的表达需求，针对原本的phaP1的RBS的核心区域进行了饱和突变，获得了1024个具有不同强度的RBS库。该RBS库的序列如SEQ ID NO：3所示(其中，N为饱和突变位点)。
筛选其中10个不同强度RBS(见表1，其中RBS0为天然RBS，RBS1-RBS9为改造后的RBS，RBS0的表达强度过高，对细胞的生长不利)用于构建minCD重组表达质粒，将minCD基因置于phaP1启动子的下游，RBS位于minCD上游，紧邻minCD的起始密码子。其中，RBS1-RBS9来源于由Salis Lab的RBS library calculator构建的PphaP1 RBSX库(https://salislab.net/software/design_rbs_library_calculator)。该库以RBS0(即PphaP1天然RBS)同源序列CTCACTTAXXXXXATTATGTG(其中X为A、T、C、G或U中任一碱基)为模板，minCD为表达基因，将五个X位点随机突变而得，每个RBS对应其各自的相对强度。然后，以菌株H.bluephagenesis WZY278(H.bluephagenesis WZY278菌株：将4HB-CoA转移酶编码基因整合进重组H.bluephagenesis TD1.0基因组，ΔphaP1，ΔlpxL，ΔlpxM，将表达phaCAB的PMmp1启动子替换为Pporin启动子，再过表达与必需基因ompW相关联的额外的phaCAB，具体详见文章Ji,M.,et al.PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.Metabolic Engineering,2023,77,12-20.)为宿主，将重组表达质粒结合到宿主中，得到相应工程菌株。将各菌株进行摇瓶发酵实验，分析细胞形态、干重和PHB％的变化，发现各菌株细胞长度显著增长，并且其中一株的PHB产量和CDW同时提升。
表1：RBS0-RBS9的核酸序列及相对强度

1.自诱导minCD重组表达质粒的构建
首先，选取H.bluephagenesis TD01基因组为模板，PCR得到minCD。然后，以pSEVA321质粒为载体，以phaP1启动子(Pp1)为基因表达的启动子，将表1中的10个不同强度的RBS，构建不同minCD表达强度的重组表达质粒，分别为pSEVA321-Pp1-RBS0-minCD、pSEVA321-Pp1-RBS1-minCD、pSEVA321-Pp1-RBS2-minCD、pSEVA321-Pp1-RBS3-minCD、pSEVA321-Pp1-RBS4-minCD、pSEVA321-Pp1-RBS5-minCD、pSEVA321-Pp1-RBS6-minCD、pSEVA321-Pp1-RBS7-minCD、pSEVA321-Pp1-RBS8-minCD、pSEVA321-Pp1-RBS9-minCD。其中，以不含minCD的质粒pSEVA321-Pp1为对照。将各质粒转化至大肠杆菌S17-1感受态细胞。挑取单克隆并进行PCR鉴定和测序，选择测序正确的单克隆，即得到10个不同minCD表达强度的自诱导重组表达质粒(质粒结构如图1所示)及对照质粒。
2.细胞形态纤长化菌株的构建
以H.bluephagenesis WZY278为宿主细胞，将其分别与上述得到的10个自诱导重组表达质粒和对照质粒转至H.bluephagenesis WZY278中(具体方法见Zhao,H.,et al.Novel T7-like expression systems used for Halomonas.Metabolic Engineering,2017,39,128-140.)，即得WZY278(pSEVA321-Pp1-RBS0-minCD)、WZY278(pSEVA321-Pp1-RBS1-minCD)、WZY278(pSEVA321-Pp1-RBS2-minCD)、WZY278(pSEVA321-Pp1-RBS3-minCD)、WZY278(pSEVA321-Pp1-RBS4-minCD)、WZY278(pSEVA321-Pp1-RBS5-minCD)、WZY278(pSEVA321-Pp1-RBS6-minCD)、WZY278(pSEVA321-Pp1-RBS7-minCD)、WZY278(pSEVA321-Pp1-RBS8-minCD)、WZY278(pSEVA321-Pp1-RBS9-minCD)以及对照菌株WZY278(pSEVA321-Pp1)。
3.菌株CDW和PHB％的测定
首先要进行摇瓶发酵。将上述得到的11株工程菌株分别接种到20mL含34mg/L氯霉素的LB50培养基中培养12-16h，然后按1％的体积比转接到新的20mL含34mg/L氯霉素的LB50培养基中继续培养8-10h，得摇瓶种子液。将2.5mL的种子菌液接种到含有50mL含34mg/L氯霉素的MM30培养基的500mL锥形瓶中(接种比例1：20)，于37℃、200rpm的条件下，发酵培养48h。发酵结束后，收集样品，对样品进行CDW和PHB％分析(具体的实验方法见Ye,J.,et al.Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas.Metabolic Engineering,2020,57,85-95)，结果如表2。由表2可以看出，与对照相比，含有自诱导minCD重组表达质粒的部分菌株的干重和PHB％均有所提高，且因其RBS强度不同而不同，其中，RBS7的效果最好，WZY278(pSEVA321-Pp1-RBS7-minCD)的干重提高约15.5％，同时PHB％提高约6.7％。
表2：phaP1启动子驱动PHB合成基因提高菌株PHB产量

4.细菌形态及菌液离心情况分析
取上述摇瓶发酵实验结束时WZY278(pSEVA321-Pp1)、WZY278(pSEVA321-Pp1-RBS7-minCD)的菌液，分别进行显微镜细胞形态观察和菌液离心情况分析。两种菌株的细胞形态如图2所示。由图2可以看出，WZY278(pSEVA321-Pp1-RBS7-minCD)的细胞长度显著增长，长度达10μm，约为对照菌株细胞长度的10倍。
关于菌液离心情况的分析，首先各取35ml菌液于50ml EP管中，500g离心力下分别离心3min、6min和10min，每次离心结束后观察两管菌液的不透明程度，结果如图3所示，其中①为对照菌株WZY278(pSEVA321-Pp1)，②为形变菌株WZY 278(pSEVA321-Pp1-RBS7-minCD)。由图3可见，在500g的离心力下，对照菌株WZY278(pSEVA321-Pp1)分离10min后，上清液中仍然比较浑浊，而形变菌株WZY278(pSEVA321-Pp1-RBS7-minCD)分离10min后，上清液明显较对照菌株组清澈，即形变菌株可以实现更快速地离心分离，有助于提高下游分离处理的效率。

Claims (19)

  1. 一种重组菌，其特征在于，所述的重组菌中包含表达载体，所述的表达载体中包含minCD基因和启动子；
    所述的启动子中包含PhaR的结合位点；
    所述的重组菌为表达PhaR蛋白的重组菌。
  2. 根据权利要求1所述的重组菌，其特征在于，所述的minCD基因的核苷酸序列包含SEQ ID NO:1所示的核苷酸序列，或包含与SEQ ID NO:1所示的核苷酸序列具有80％以上同源性。
  3. 根据权利要求1所述的重组菌，其特征在于，所述的minCD基因在表达载体上表达和/或在染色体上表达。
  4. 根据权利要求1-3任一所述的重组菌，其特征在于，所述的启动子包括组成型启动子和/或诱导型启动子，
    优选的，所述的组成型启动子选自Sp6启动子或其变体或porin启动子或其变体；
    优选的，所述的诱导型启动子选自luc启动子或其变体、lac启动子或其变体、trp启动子或其变体、tac启动子或其变体、噬菌体启动子或其变体、araBAD启动子或其变体或phaP启动子或其变体。
  5. 根据权利要求1-4任一所述的重组菌，其特征在于，所述的phaP启动子包括phaP1启动子、phaP2启动子或phaP3启动子。
  6. 根据权利要求5所述的重组菌，其特征在于，所述的phaP1启动子包括SEQ ID NO：2或包括与SEQ ID NO：2所示核苷酸序列具有80％以上同源性的核苷酸序列。
  7. 根据权利要求1-6任一所述的重组菌，其特征在于，所述的表达载体中还包含RBS序列；
    优选的，所述的表达载体中包含的启动子、minCD基因和RBS序列在表达载体中的顺序为启动子、RBS序列、minCD基因。
  8. 根据权利要求7所述的重组菌，其特征在于，在所述的表达载体中，所述的RBS紧邻minCD基因的起始密码子。
  9. 根据权利要求7或8所述的重组菌，其特征在于，所述的RBS序列如
    GCGTGTCGAATCGCTAGCTCACTTANNNNNATTATGTG(SEQ ID NO:3)所示，
    其中，N选自A、T、C、G或U中任一种。
  10. 根据权利要求7-9任一所述的重组菌，其特征在于，所述的RBS序列包括SEQ ID NO：4-13或包括与SEQ ID NO:4-13具有90％以上同源性的核苷酸序列。
  11. 根据权利要求10所述的重组菌，其特征在于，所述的RBS序列包括SEQ ID NO：7-13或包括与SEQ ID NO:7-13具有90％以上同源性的核苷酸序列。
  12. 根据权利要求1-11任一所述的重组菌，其特征在于，所述的重组菌为合成PHA或组成PHA的单体的重组菌。
  13. 根据权利要求1-12任一所述的重组菌，其特征在于，所述的重组菌选自埃希氏菌属(Escherichia)或其衍生菌、罗氏菌属(Ralstonia)或其衍生菌、产碱杆菌属(Alcaligenes)或其衍生菌、假单胞菌属(Pseudomonas)或其衍生菌、气生单胞菌属(Aeromonas)或其衍生菌或盐单胞菌属(Halomonas)或其衍生菌；
    所述的盐单胞菌属(Halomonas)优选包括Halomonas bluephagenesis或其衍生菌，Halomonas campaniensis或其衍生菌或Halomonas aydingkolgenesis或其衍生菌。
  14. 一种权利要求1-13任一所述的重组菌的制备方法，其特征在于，所述的制备方法包括将表达载体导入重组菌中。
  15. 一种促进细胞纤长化的方法，其特征在于，所述的方法包括将表达载体导入重组菌中；所述的表达载体中包含minCD基因和启动子；
    所述的启动子中包含PhaR的结合位点；
    所述的重组菌为表达PhaR蛋白的重组菌。
  16. 根据权利要求15所述的方法，其特征在于，所述的minCD基因的核苷酸序列包含SEQ ID NO:1所示的核苷酸序列，或包含与SEQ ID NO:1所示的核苷酸序列具有80％以上同源性。
  17. 一种重组菌的分离方法，其特征在于，所述的分离方法包括离心分离权利要求1-13任一所述的重组菌；
    优选的，所述离心的离心力大于300g。
  18. 一种发酵方法，其特征在于，所述的发酵方法包括发酵培养权利要求1-13任一所述的重组菌。
  19. 一种生产PHA的方法，其特征在于，所述的方法包括发酵培养权利要求1-13任一所述的重组菌。
PCT/CN2024/110346 2023-08-30 2024-08-07 一种细胞形态纤长化的重组菌株及其构建方法和应用 Pending WO2025044707A1 (zh)

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