WO2023227104A1 - 一种用于合成萜烯的共培养系统和方法 - Google Patents

一种用于合成萜烯的共培养系统和方法 Download PDF

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WO2023227104A1
WO2023227104A1 PCT/CN2023/096509 CN2023096509W WO2023227104A1 WO 2023227104 A1 WO2023227104 A1 WO 2023227104A1 CN 2023096509 W CN2023096509 W CN 2023096509W WO 2023227104 A1 WO2023227104 A1 WO 2023227104A1
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strain
culture system
production
plasmid
terpenes
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French (fr)
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潘德雷卡阿维纳什
黄侓乐
曹阳
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牛津大学(苏州)科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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 field of microorganisms, and specifically provides a co-culture system and method for synthesizing terpenes.
  • Terpenoids are widely used in different industrial applications such as flavors, fragrances, biofuels, pharmaceuticals, rubber, and pesticides, among others. These compounds are produced by plants, but often in low enough quantities to support industrial demand.
  • Terpenoids are compounds derived from mevalonate or deoxy-D-xylulose 5-phosphate, and their molecular skeleton uses isoprene units (C5 units) as the basic structural unit and their oxygen-containing derivatives. These oxygen-containing derivatives can be alcohols, aldehydes, ketones, carboxylic acids, esters, etc.
  • Terpenes are a common class of terpenoid compounds. Most of these metabolites produced during the production of terpene molecules are complex. The chemical production process has many steps and involves complex chemical reactions, resulting in low yields, incorrect stereochemistry, and high costs. In recent years, synthetic biology has become a rapidly expanding and advancing research field with a wide range of applications in the synthesis of sustainable and greener compounds.
  • a typical example of terpenoid synthesis in synthetic biology is the synthesis of artemisinin, produced from artemisinic acid, which is produced from the sesquiterpene precursor amorpha-4,11-diene artemisinin precursor. Produced by the biosynthesis of Bauhinia. It is then oxidized sequentially to alcohols and aldehydes, then acids.
  • this complex multigene system is limited by metabolic stresses and pathways, such as metabolic flux and release of intermediates into the culture medium without re-entering the cell wall.
  • both strains In one process of pinene biosynthesis, both strains must be grown in culture medium and the cells harvested and then transferred to phosphate-buffered saline to produce terpenes.
  • mevalonate-producing bacteria In a sabinene biosynthetic process, mevalonate-producing bacteria must be grown in a culture medium to produce mevalonate, and then the mevalonate-containing culture medium is combined with the growth medium of the sabinene biosynthetic strain. use.
  • terpenoid metabolic engineering Most research on terpenoid metabolic engineering has focused on engineering microbial genomes and evolving isoprenoid auxotrophic mutants to enhance terpenoid biosynthesis and metabolic intermediates. However, these intermediates are toxic and difficult to synthesize in microorganisms. exchange between.
  • the present invention developed a simple co-factor for enhancing terpene biosynthesis by partitioning the isoprenoid MVA pathway between microbial strains without changing their natural isoprene pathway. training system. Furthermore, the use of polar molecules as transfer metabolites between strains prevents partitioning into organic solvents. Toxic hydrophobic products can be extracted from microbial cultures using organic solvents. Therefore, the present invention provides a co-culture system, which includes a feeder strain that synthesizes mevalonate as an exchange metabolite and a production strain that uses mevalonate as a raw material to synthesize terpenes, providing for heterologous protein expression. An adjustable solution. The co-culture system provided by the present invention reduces the metabolic pressure of microorganisms by separating the synthesis of metabolite intermediates between microorganisms, thereby improving the conversion rate of intermediates and the production of target compounds.
  • a first aspect of the present invention provides a co-culture system for synthesizing terpenes, which includes at least one feeder strain and at least one production strain, wherein the feeder bacteria are used to provide mevalonate, and the production strain Used to synthesize terpenes using the mevalonic acid.
  • the feeder strain contains a first plasmid containing a gene for synthesizing mevalonate and not containing a gene for synthesizing isoprenyl pyrophosphate.
  • the feeding strain further contains a second plasmid, and the second plasmid is a blank plasmid that does not require any gene insertion.
  • the second plasmid is pUC19, and this pUC does not require any gene insertion.
  • the second plasmid facilitates compensation of the antibiotic with the production strain.
  • the feeder strain provides mevalonate, which is released into the culture medium and then enters the cell wall, isoprene pyrophosphate synthase and terpenes of the production strain capable of expressing the mevalonate gene.
  • the cell membrane of the synthase-producing strain isoprene pyrophosphate synthase and terpenes of the production strain capable of expressing the mevalonate gene.
  • the production strain contains a third plasmid and a fourth plasmid
  • the third plasmid contains a gene for synthesizing farnesyl pyrophosphate and does not contain a gene for synthesizing mevalonate
  • the third plasmid contains Four plasmids contain the terpene-forming enzyme gene or the fusion gene of terpene synthase and GGPP synthase.
  • the feeding strain and production strain are obtained by modifying the MVA pathway plasmid pBbA5c-MevT(CO)-T1-MBIS(CO,ispA) (pMVA) from Addgene.
  • pMVA plasmid is a medium copy number plasmid that contains two modules, one for the production of mevalonate and the other for the production of farnesyl pyrophosphate (FPP).
  • New plasmids pMevt and pMBIS were generated by cloning each module from pMVA.
  • pMevt is a medium copy number plasmid containing the mevalonate production module; pMBIS is another medium copy number plasmid containing the FPP production module.
  • pTRC-X is produced by cloning the codon-optimized terpene X synthase gene into pTRC-HisA.
  • the feeding strain is produced by transforming pMevt plasmid pUC-19 into microorganisms such as E. coli, and the production strain is produced by transforming pMBIS plasmid and pTRC-X plasmid into microorganisms. produced in organisms such as E. coli.
  • the ratio of the feeding strain and the production strain is (0.25-99.75): (99.75-0.25), such as (0.25-99.5): (99.75-0.5), preferably (1.25-75) :(98.75-25).
  • the ratio of the feeding strain and the production strain is (0.5-75): (99.5-25), such as (12.5-75): (87.5-25).
  • the range of co-culture strains is further improved by changing plasmids, inoculation ratios, heterologous enzyme expression, efficiency and genome modification, thereby further improving the terpene yield.
  • the terpenes include taxadiene, ⁇ -buniene, valenene, ⁇ -guaiene, ⁇ -patchoulene or aristolochene. one or more.
  • the feeding strain and/or the production strain are selected from one or more of bacteria, fungi or yeast. According to some specific embodiments of the invention, the feeding strain and/or the production strain is Escherichia coli.
  • the metabolic pathway of the feeder strain and/or production strain includes an endogenous isoprenoid/terpene biosynthetic pathway.
  • bacteria have an endogenous MEP pathway and yeast have an endogenous MVA pathway.
  • the metabolic pathway of the feeder strain and/or the production strain further includes a partially engineered MVA pathway to convert isoprene pyrophosphate precursors (GPP, FPP, GGPP, etc.) into terpenes.
  • this co-culture system works by dividing the many steps in the biosynthetic pathway of terpenes into two modules, where one module is carried out in one strain and the remaining steps are carried out by another strain.
  • the steps of the two strains are linked by a freely diffusing exchange metabolite (mevalonate).
  • the metabolic stress of all steps performed by a single strain is divided between the two strains, thereby reducing the metabolic stress of each strain and thereby increasing the yield of the target product.
  • the use of polar molecules (such as mevalonate) as transfer metabolites between the two strains prevents partitioning into the extraction agent (such as decane).
  • the two strains interact to balance mevalonate production and consumption to promote survival and growth of both strains.
  • a second aspect of the present invention provides a method for synthesizing terpenes using the co-culture system of the first aspect, the method comprising incubating the feeding strain and the production strain in the presence of an inducer, and for An organic phase or solid phase resin that separates terpenes from the aqueous phase.
  • an organic phase eg, decane
  • a solid phase resin eg, Dionex HP20
  • the organic phase is selected from decane.
  • the organic phase is not limited to decane, and can be further selected from undecane and dodecane; or, selected from isopropyl decanoate, isolaurate Propyl ester, isopropyl n-octanoate, isopropyl myristate, etc.
  • the organic phase constitutes 5%-30% v/v of the culture, preferably 10%-20%.
  • the solid phase resin is not limited to Dionex HP20 and can also be selected from Dowax, HP-20, XAD7HP or HP-2MG.
  • the content of the feeding strain is 0.25% to 99% based on the total inoculum volume of the feeding strain and the production strain.
  • the content of the feeding strain is 0.25%-99%, preferably 0.5%-75%, such as 12.5%-75% .
  • the content of the feeding strain is 0.5%, 1%, 2%, 5%, 10%, 12.5%, 25%, 37.5%, 50%, 62.5%, 75% and anything in between.
  • the content of the feeding strain is 0.25%-99% based on the inoculum volume of the feeding strain and the production strain, especially 0.25% to 37.5%, such as 0.25% to 12.5% or 12.5% to 37.5%, preferably 0.25% to 12.5%, further preferably 0.25% to 5%, particularly preferably 0.5% to 5%.
  • the content of the feeding strain is 12.5%-75% based on the inoculum volume of the feeding strain and the production strain, 25% to 75% is preferred, and 37.5% to 62.5% is particularly preferred.
  • the content of the feeding strain is preferably 12.5%-75% based on the inoculum volume of the feeding strain and the production strain.
  • the separation and purification is performed by extraction with an organic solvent.
  • the organic solvent includes decane.
  • a third aspect of the present invention provides the use of the co-culture system according to the first aspect or the method according to the second aspect in synthesizing terpenes.
  • a fourth aspect of the present invention provides the use of mevalonate as an exchange metabolite in the synthesis of terpenes in microbial co-culture.
  • the co-culture system of the present invention can increase the total terpene production. Production is currently conducted in shake flasks, which can produce approximately 1g/L of terpenes. When the production system is transferred to fermenters, the output will increase significantly.
  • the co-culture system and method of the present invention can be applied to the production of any terpenes, including but not limited to (a) the production of monoterpenes for biofuels and other applications ( Linalool, geraniol and 2,6-dimethyloctane) and other sesquiterpenes (bisabolene, pentene, and isocrene); (b) terpene pathway fragmentation in coculture leads to increased expression of diterpene synthase, This results in a higher yield of diterpenes with a wider range of biological activities, including pharmaceutical effects; (c) Feeding strains can be further modified to express hydrolytic enzymes to degrade polymers such as cellulose, lignin, hemicellulose; (d) Multiple feeding strains can be added to utilize complex materials such as agricultural and food waste; (e) These multi-strain principles can be applied to any other pathway to enhance microbial community interactions, flux and product formation; (f)
  • Another key advantage of the present invention is that one strain (the feeder strain) provides mevalonate which is released into the culture medium and then enters through the cell wall of the other strain (the production strain), This strain expresses a gene that uses mevalonate, isoprene pyrophosphate synthetase (Fig. 3). By dividing the process into these steps, the metabolic stress of expressing heterologous enzymes on each organism is reduced, resulting in faster growth, increased strain viability, and higher volumetric yields. Co-culture systems reduce fermentation time and allow the ratio of feeder and production strains to be adjusted by, for example, controlling the inoculum size.
  • Figure 1 shows examples of terpenes synthesized by terpene synthases of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • Figure 2 shows some existing technologies for culture systems using more than one strain for terpene production:
  • A is a two-strain system for pinene production using phosphate buffer
  • B a two-step culture system, where An initial strain is grown to produce mevalonate and then a second strain is fed with medium containing mevalonate to produce terpenes.
  • Figure 3 shows a minimal or simple two-strain co-culture system according to the present invention.
  • Figure 4 shows the biosynthesis of taxadiene using a single strain culture system and a dual strain co-culture system in Example 1.1.
  • Figure 5 shows gas chromatography analysis of taxadiene biosynthesis in Example 1.1.
  • Figure 6 shows the biosynthesis of taxadiene using a single strain culture system and a dual strain co-culture system in Example 1.2.
  • Figure 7 shows gas chromatography analysis of taxadiene biosynthesis in Example 1.2.
  • Figure 8 shows the biosynthesis of ⁇ -buniene in a single-strain culture system and a dual-strain co-culture system in Example 2.
  • Figure 9 shows gas chromatography analysis of ⁇ -buniene synthesis in Example 2.
  • Figure 10 shows the biosynthesis of valencene in a single-strain culture system and a dual-strain co-culture system in Example 3.
  • Figure 11 shows gas chromatography analysis of valensene biosynthesis in Example 3.
  • Figure 12 shows the biosynthesis of ⁇ -patchouli in a single strain culture system and a double strain co-culture system in Example 4.
  • Figure 13 shows gas chromatography analysis of ⁇ -patchouli biosynthesis in Example 4.
  • Figure 14 shows the biosynthesis of aristolochin in a single strain culture system and a dual strain co-culture system in Example 5.
  • G3P glyceraldehyde 3-phosphate
  • ACAT acetyl-CoA C-acetyltransferase
  • HMGCS 3-hydroxy-3-methylglutaryl-CoA synthase
  • HMGCR 3-hydroxy-3 -Methylglutaryl-CoA reductase
  • MVK mevalonate-5-kinase
  • PMVK phosphomevalonate kinase
  • PMD mevalonate pyrophosphate decarboxylase
  • IPPI isopentenyl pyrophosphate Constructase
  • GPPS geranyl pyrophosphate synthase
  • FPPS farnesyl pyrophosphate synthase
  • GGPPS geranylgeranyl pyrophosphate synthase
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate Phosphate
  • GGPP geranylgeranylgeranyl
  • any lower limit can be combined with any upper limit to form an unexpressed range; and any lower limit can be combined with other lower limits to form an unexpressed range, and likewise any upper limit can be combined with any other upper limit to form an unexpressed range.
  • each individually disclosed point or single value may itself serve as a lower or upper limit in combination with any other point or single value or with other lower or upper limits to form a range not expressly recited.
  • a list of items connected by the terms "at least one of,””at least one of,” or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase “at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, then the phrase "A, "At least one of B and C” means A only; or B only; C only; A and B (excluding C); A and C (excluding B); B and C (excluding A); or A, B and All of C.
  • Item A may contain a single component or multiple components.
  • Item B may contain a single component or multiple components.
  • Item C may contain a single component or multiple components.
  • This application describes a co-culture system that includes a feeder strain that provides mevalonate as an exchange metabolite and a production strain that uses mevalonate as a feedstock.
  • the synthesis of metabolic intermediates and the exchange of such intermediates between microorganisms reduces the metabolic pressure of microorganisms, thereby increasing the conversion rate of intermediates and the production of target compounds.
  • the following numbered aspects describe various embodiments of the invention:
  • Embodiment 1 A co-culture system for synthesizing terpenes, which includes at least one feeding strain and at least one production strain, wherein the feeding bacteria are used to provide mevalonate, and the production strain is used to utilize the The mevalonic acid synthesizes terpenes.
  • Embodiment 2 The co-culture system according to Embodiment 1, characterized in that: the feeding strain contains a first plasmid, the first plasmid contains a gene for synthesizing mevalonate and does not contain isoprene synthesis. pyrophosphate gene, preferably, the feeding strain also contains a second plasmid, and the second plasmid is a blank plasmid that does not require gene insertion.
  • Embodiment 3 The co-culture system according to embodiment 1 or 2, characterized in that: the production strain contains a third plasmid and a fourth plasmid, and the third plasmid contains a gene for synthesizing isoprenyl pyrophosphate. , and does not contain the gene for synthesizing mevalonate, and the fourth plasmid contains the gene for the terpene synthase or the fusion gene of the terpene synthase and geranyl pyrophosphate (GGPP) synthase.
  • the production strain contains a third plasmid and a fourth plasmid
  • the third plasmid contains a gene for synthesizing isoprenyl pyrophosphate.
  • the fourth plasmid contains the gene for the terpene synthase or the fusion gene of the terpene synthase and geranyl pyrophosphate (GGPP) synthase.
  • Embodiment 4 The co-culture system according to any one of embodiments 1-3, characterized in that: the ratio of the feeding strain and the production strain is (0.25-99.75): (99.75-0.25), preferably ( 1.25-75): (98.75-25).
  • Embodiment 5 The co-culture system according to any one of embodiments 1-4, characterized in that: the terpenes include taxadiene, ⁇ -buniene, valenene, and ⁇ -guaiacene , one or more of ⁇ -patchoulene or aristolochene.
  • the terpenes include taxadiene, ⁇ -buniene, valenene, and ⁇ -guaiacene , one or more of ⁇ -patchoulene or aristolochene.
  • Embodiment 6 The co-culture system according to any one of embodiments 1-5, characterized in that: the feeding strain and/or the production strain are selected from one or more of bacteria, fungi or yeast. kind.
  • Embodiment 7 The co-culture system according to any one of claims 1-6, characterized in that: the metabolic pathway of the feeding strain and/or the production strain includes an endogenous isoprenoid synthesis pathway. way.
  • Embodiment 8 A method for synthesizing terpenes using the co-culture system of any one of embodiments 1-7, the method comprising incubating the feeder strain and the production strain in the presence of an inducer, and Isolation and purification of terpenes.
  • Embodiment 9 The method according to Embodiment 8, characterized in that: based on the total inoculum volume of the feeding strain and the production strain, the content of the feeding strain is 0.25% to 90%, preferably 0.5 %-75%; and/or preferably, the organic phase is selected from decane, undecane, dodecane, preferably decane; or, is selected from isopropyl caprate, isopropyl laurate, n-octanoic acid Isopropyl ester, isopropyl myristate; the solid phase resin is selected from Dionex HP20, Dowax20, XAD7HP, HP-2MG, preferably Dionex HP20; the organic phase accounts for 5%-30% v/v of the culture, preferably 10%–20%v/v.
  • Embodiment 10 Application of the co-culture system according to any one of embodiments 1-7 or the method according to embodiment 8 or 9 in the synthesis of terpenes.
  • plasmid pBbA5c-MevT(CO)-T1-MBIS(CO,ispA) was obtained from Addgene and the codon-optimized terpene synthase was cloned into the pTRC-Hisa vector (Invitrogen) middle. Both plasmids were transformed into E. coli BL21(DE3) strain. Transformed E. coli BL21(DE3) cells were grown until the OD at 600 nm reached 0.6, then induced with 0.1 mM IPTG and incubated at 16°C or 30°C.
  • the present invention has discovered through research that the division of the terpene biosynthetic pathway requires an intermediate that can be transferred between the two strains and will not accumulate in the extraction agent such as decane.
  • Isoprene units IPP and DMAPP
  • terpene hydrocarbons Zhou et al., Nature Biotechnology, 2015, 33, 377– 383; DOI: 10.1038/nbt.3095
  • Co-culture of bacteria and yeast using terpene hydrocarbons as transfer intermediates resulted in the biosynthesis of 33 mg/L oxygenated taxanes, 20 mg/L ferruginol, and 5 mg/L nootkatone (Zhou et al., Nature Biotechnology, 2015, 33, 377–383; DOI: 10.1038/nbt.3095).
  • isoprene units as transfer intermediates ( Figure 2A)
  • dual-strain co-culture produced 166 mg/L pinene (Niu et al., Frontiers in Microbiology, 2018, 9, 16232; DOI: 10.3389/fmicb.2018.01623).
  • a two-step culture system using mevalonate as an intermediate produced 150 mg/L sabinene in bacteria (Liu et al., Process Biochemistry, 2017, 62, 1–9; DOI: 10.1016/j.procbio.2017.07.021).
  • the present invention investigates the terpene biosynthetic pathway, and there is currently no prior art using mevalonate as a transfer intermediate in a co-culture system.
  • Mevalonate is a small polar molecule that diffuses easily through microbial membranes and does not partition into organic extractants such as decane.
  • the present invention provides a simple co-culture system ( Figure 3), in which one strain (the feeder strain) releases mevalonate into the growth medium and the other strain (the production strain) Biosynthesis of terpenes using mevalonate.
  • This method is easy to implement and has been demonstrated by the production of different terpenes such as taxadiene, ⁇ -buniene, valenene, ⁇ -patchoulene and aristolochene (Fig. 1).
  • Feeder and production strains contain the endogenous MEP pathway and part of the engineered heterologous MVA pathway.
  • the MEP pathway enables bacterial growth on the minimal inner side without replenishing isoprene intermediates.
  • the engineered MVA pathway increases the flux of terpene biosynthesis.
  • MVA pathway plasmid pBbA5c-MevT(CO)-T1-MBIS(CO,ispA)(pMVA) from Addgene http://www.addgene.org/35152/; US7183089 and US736882; Tsuruta et al., PLoS One, 2009, 4, e4489; DOI: 10.1371/journal.pone.0004459; Peralta-Yahya, et al., Nature Communications, 2011, 2, 483; DOI: 10.1038/ncomms1494) was modified to work in a co-culture system.
  • the pMVA plasmid is a medium copy number plasmid that contains two modules, one for the production of mevalonate and the other for the production of farnesyl pyrophosphate (FPP).
  • New plasmids pMevt and pMBIS were generated by cloning each module from pMVA.
  • pMevt is a medium copy number plasmid containing a mevalonate production module.
  • pMBIS is a medium copy number plasmid containing the FPP production module.
  • the pMevT and pMBIS plasmids in the present invention are derived from the Addgene pMVA plasmid, which is optimized for a single strain system.
  • pMevT and pMIBS plasmids can be further optimized by modifying the promoter and RBS (ribosome binding site). Feeding and production strains can be further optimized by improving heterologous enzyme expression, efficiency, and host genome modification.
  • codon-optimized genes of taxadiene synthase and geranylgeranyl pyrophosphate synthase were fused (as reported by Ajikumar, Parayil Kumaran (10.1126/science.1191652) et al.) and cloned into pTRC-HisA to generate Plasmid pTRC-TXSGPPS.
  • a single-strain system for taxadiene production was obtained by transforming E. coli BL21(DE3) strain with pTRC-TXSGPPS and pMVA plasmids. Inoculate a single colony of transformed E.coli BL21 (DE3) into 5 mL LB (Luria-Bertani) medium and culture it at 37°C for 12 hours to obtain an overnight culture. This overnight feeding culture was inoculated into 25 mL of TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD value at 600 nm reaches 0.6, induce with 0.1mM IPTG and incubate at 20°C. Add 2% w/v glucose as carbon source and 10% v/v decane as organic phase to extract terpenes.
  • the feeder strain was generated by transforming pUC-19 plasmid without any gene insertion and pTRC-Mevt plasmid into E.coli BL21(DE3), and the taxadiene-producing strain was generated by transforming pMBIS and pTRC-TXSGPPS The plasmid was transformed into E.coli BL21(DE3).
  • the colonies of the feeder strain and the production strain were independently inoculated into 5 mL LB (Luria-Bertani) medium and cultured at 37°C for 12 hours to obtain an overnight culture.
  • codon-optimized genes of taxadiene synthase and geranylgeranyl pyrophosphate synthase were fused (as reported by Ajikumar, Parayil Kumaran (10.1126/science.1191652) et al.) and cloned into pTRC-HisA to generate Plasmid pTRC-TXSGPPS.
  • the feeder strain was generated by transforming pUC-19 plasmid without any gene insertion and pTRC-Mevt plasmid into E.coli BL21(DE3), and the taxadiene-producing strain was generated by transforming pMBIS and pTRC-TXSGPPS The plasmid was transformed into E.coli BL21(DE3).
  • the colonies of the feeder strain and the production strain were independently inoculated into 5 mL LB (Luria-Bertani) medium and cultured at 37°C for 12 hours to obtain an overnight culture.
  • the codon-optimized buninene synthase gene (NCBI-KF800046) was cloned into pTRC-HisA to generate plasmid pTRC-BS.
  • a single-strain system for ⁇ -buniene production was generated by transforming E. coli BL21(DE3) with pMVA and pTRC-BS plasmids.
  • a single colony was inoculated into 5 mL of LB (Luria-Bertani) medium and cultured at 37°C for 12 hours. The overnight culture was inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 Incubate at rpm. Once the OD value at 600 nm reaches 0.6, induce with 0.1 mM IPTG and incubate at 30 °C. Add 2% w/v glucose as carbon source and 10% v/v decane as organic phase to extract terpenes. The total terpene production reached 1345 mg/L ( Figure 8).
  • feeder strains were generated by transforming pMevt and pUC-19 plasmids into E. coli BL21(DE3).
  • the production strain for ⁇ -buniene synthesis was generated by transforming pMBIS and pTRC-BS plasmids into E. coli BL21(DE3). Inoculate colonies of the feeder strain and the production strain into separate 5 mL LB (Luria-Bertani) medium and incubate at 37°C for 12 hours to obtain an overnight culture.
  • the codon-optimized valentene synthase gene (from US9303252B2) was cloned into pTRC-HisA to generate plasmid pTRC-VS.
  • a single-strain system for valensene biosynthesis was generated by transforming E. coli BL21(DE3) with pMVA and pTRC-VS plasmids. Inoculate a single colony into 5 mL of LB (Luria-Bertani) medium and incubate at 37°C for 12 hours to obtain an overnight culture. Inoculate the overnight culture into 25mL TB (Terrific Broth) medium and incubate at 37°C and 200rpm. Once the OD value at 600 nm reaches 0.6, induce with 0.1mM IPTG and incubate at 30°C. Add 2% w/v glucose as carbon source and 10% v/v decane as organic phase to extract terpenes. The total terpene production reached approximately 336 mg/L (Figure 10).
  • feeder strains were generated by transforming pMevt and pUC-19 into E. coli BL21(DE3).
  • the production strain for valencene biosynthesis was generated by transforming plasmids pMBIS and pTRC-VS into E. coli BL21(DE3).
  • Colonies of the feeder strain and the production strain were inoculated into separate 5 mL LB (Luria-Bertani) medium and cultured at 37°C for 12 hours. Overnight cultures of feeder strains and production strains at different proportions (25% to 75%) were inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 Incubate at rpm.
  • the codon-optimized ⁇ -patchoulene synthase gene (XM_044587644) was cloned into pTRC-HisA to generate plasmid pTRC-PCL.
  • a single-strain system for ⁇ -patchoulene biosynthesis was generated by transforming E. coli BL21(DE3) with pMVA and pTRC-PCL plasmids. Inoculate a single colony into 5 mL of LB (Luria-Bertani) medium and incubate at 37°C for 12 hours to obtain an overnight culture. Inoculate the overnight culture into 25mL TB (Terrific Broth) medium and incubate at 37°C and 200rpm. Once the OD value at 600 nm reaches 0.6, induce with 0.1mM IPTG and incubate at 16°C. Add 2% w/v glucose as carbon source and 10% v/v decane as organic phase to extract terpenes. The total terpene production reached approximately 7 mg/L ( Figure 12).
  • feeder strains were generated by transforming pMevt and Puc-19 plasmids into E. coli BL21(DE3).
  • the production strain for ⁇ -patchoulene biosynthesis was generated by transforming plasmids pMBIS and pTRC-PCL into E. coli BL21(DE3). Inoculate colonies of the feeder strain and the production strain into separate 5 mL LB (Luria-Bertani) medium and incubate at 37°C for 12 hours to obtain an overnight culture.
  • the codon-optimized aristolochene synthase gene (PDB-3M01_A) was cloned into pTRC-HisA to generate plasmid pTRC-TEAS.
  • a single-strain system for aristolochene biosynthesis was generated by transforming E. coli BL21(DE3) with pMVA and pTRC-TEAS plasmids. Inoculate a single colony into 5mL LB (Luria-Bertani) medium and incubate at 37°C Incubate for 12 hours to obtain an overnight culture. Inoculate the overnight culture into 25 mL of TB medium and incubate at 37 °C and 200 rpm. Once the OD value at 600 nm reaches 0.6, induce with 0.1 mM IPTG and incubate at 30 °C. Add 2% w/v glucose as carbon source and 10% v/v decane as organic phase to extract terpenes. The total terpene production reached approximately 3 mg/L ( Figure 14).
  • feeder strains were generated by transforming pMevt and Puc-19 plasmids into E. coli BL21(DE3).
  • the production strain for ⁇ -ristolochiin biosynthesis was generated by transforming plasmids pMBIS and pTRC-TEAS into E. coli BL21(DE3).
  • Colonies of the feeder strain and the production strain were inoculated into separate 5 mL LB (Luria-Bertani) medium and cultured at 37°C for 12 hours.

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Abstract

一种合成萜烯的共培养系统和方法，该系统包括至少一种饲养菌株和至少一种生产菌株，其中，所述饲养细菌用于提供甲羟戊酸，所述生产菌株用于利用所述甲羟戊酸合成萜类化合物。该共培养系统和方法可应用于任何萜烯的生产，能够提高萜烯的总产量和萜烯转化率。

Description

一种用于合成萜烯的共培养系统和方法 技术领域
本发明涉及微生物领域，具体提供一种用于合成萜烯的共培养系统和方法。
背景技术
萜类化合物广泛用于不同的工业用途，例如香精、香料、生物燃料、药物、橡胶和杀虫剂等。这些化合物由植物生产，但通常含量低，不足以支持工业需求。
萜类化合物是由甲羟戊酸或脱氧-D-木酮糖5-磷酸衍生而来，分子骨架以异戊二烯单元(C5单元)为基本结构单元的化合物及其含氧衍生物。这些含氧衍生物可以是醇类、醛类、酮类、羧酸类、酯类等。萜烯是一类常见的萜类化合物。萜类分子生产过程中产生的这些代谢物大多是复杂的，化学生产过程步骤多，涉及复杂的化学反应，导致收率低、立体化学不正确、成本高的问题。近年来，合成生物学已成为一个快速扩展和推进的研究领域，在可持续和更环保的化合物合成中有着广泛的应用。典型的合成生物学合成萜类化合物的例子是青蒿素的合成，通过青蒿酸产生青蒿素，青蒿酸是由倍半萜前体amorpha-4,11-二烯青蒿素前体紫荆的生物合成产生的。然后依次氧化为醇和醛，然后是酸。然而，这种复杂的多基因系统受到代谢压力和途径的限制，例如代谢通量和中间体释放到培养基中而不通过细胞壁重新进入细胞壁。
在一种蒎烯生物合成的过程中，必须在培养基中培养两种菌株并收获细胞，然后将其转移到磷酸盐缓冲盐水中以产生萜烯。在一个桧烯生物合成过程中，产生甲羟戊酸的细菌必须在培养基中生长以产生甲羟戊酸，然后将含有甲羟戊酸的培养基与桧烯生物合成菌株的生长培养基一起使用。这些过程很复杂，需要严格的过程控制，同时，使用异戊二烯单元作为交换代谢物也可能对细菌造成压力。萜类代谢工程的大部分研究都集中在改造微生物基因组，进化出类异戊二烯营养缺陷型突变体以增强萜类化合物的生物合成和代谢中间体，然而，这些中间体有毒且难以在微生物之间交换。
然而，目前仍没有一种能够满足萜烯含量大且足以支持工业需求的合成方法。
发明内容
为了克服现有技术存在的不足，本发明通过在微生物菌株之间划分类异戊二烯MVA途径而不改变它们的天然异戊二烯途径，开发了一种用于增强萜生物合成的简单共培养系统。此外，使用极性分子作为菌株之间的转移代谢物可防止分配到有机溶剂中。可以用有机溶剂从微生物培养物中提取有毒的疏水产物。因此，本发明提供一种共培养系统，其包括一种合成甲羟戊酸作为交换代谢物的饲养菌株和一种使用甲羟戊酸作为原料合成萜烯的生产菌株，为异源蛋白表达提供了一种可调节的解决方案。本发明提供的共培养系统通过在微生物之间分离代谢物中间体的合成来减少微生物的代谢压力，从而提高中间体的转化率和目标化合物的产量。
本发明的第一方面提供了一种合成萜烯的共培养系统，其包括至少一种饲养菌株和至少一种生产菌株，其中，所述饲养细菌用于提供甲羟戊酸，所述生产菌株用于利用所述甲羟戊酸合成萜烯。
根据本发明的一些实施方式，所述饲养菌株含有第一质粒，所述第一质粒含有合成甲羟戊酸的基因，且不含合成异戊二烯基焦磷酸的基因。根据本发明的一些实施方式，所述饲养菌株还含有第二质粒，所述第二质粒为无需任何基因插入的空白质粒。根据本发明的一些具体实施方式，所述第二质粒为pUC19，该pUC无需任何基因插入。根据本发明的一些实施方式，所述第二质粒有助于用生产菌株补偿抗生素。本发明中，饲养菌株提供甲羟戊酸，该甲羟戊酸被释放到培养基中，然后进入能够表达甲羟戊酸基因的生产菌株的细胞壁、异戊二烯焦磷酸合酶和萜烯合酶的生产菌株的细胞膜。
根据本发明的一些实施方式，所述生产菌株含有第三质粒和第四质粒，所述第三质粒含有合成法呢基焦磷酸的基因，且不含合成甲羟戊酸的基因，所述第四质粒含有所述萜烯成酶的基因或萜烯合成酶与GGPP合成酶的融合基因。
根据本发明的一些具体实施方式，对来自Addgene的MVA途径质粒pBbA5c-MevT(CO)-T1-MBIS(CO,ispA)(pMVA)进行修饰改造获得所述饲养菌株和生产菌株。其中，pMVA质粒是一种中等拷贝数的质粒，包含两个模块，一个用于生产甲羟戊酸，另一个用于生产法呢基焦磷酸(FPP)。通过从pMVA克隆每个模块产生新的质粒pMevt和pMBIS，pMevt是一种中等拷贝数的质粒，含有甲羟戊酸生产模块；pMBIS是另一种中等拷贝数的质粒，含有FPP生产模块。pTRC-X是通过密码子优化的萜烯X的合成酶基因克隆到pTRC-HisA中产生。本发明中，饲养菌株通过将pMevt质粒pUC-19转换到微生物如大肠杆菌种产生，生产菌株通过将pMBIS质粒和pTRC-X质粒转化到微生 物如大肠杆菌中产生。
根据本发明的一些实施方式，所述饲养菌株和所述生产菌株比例为(0.25-99.75)：(99.75-0.25)，例如(0.25-99.5)：(99.75-0.5)，优选(1.25-75)：(98.75-25)。根据本发明的一些优选实施方式，所述饲养菌株和所述生产菌株比例为(0.5-75)：(99.5-25)，例如(12.5-75)：(87.5-25)。本发明中，通过改变质粒、接种比例、异源酶表达、效率和基因组修饰进一步改进共培养菌株的范围，从而进一步提高萜烯的产率。
根据本发明的一些实施方式，所述萜烯包括紫杉二烯、α-布尼烯、瓦伦烯、α-愈创木烯(guaiene)，β-广藿烯或马兜铃烯中的一种或多种。
根据本发明的一些实施方式，所述饲养菌株和/或所述生产菌株选自细菌、真菌或酵母菌中的一种或多种。根据本发明的一些具体实施方式，所述饲养菌株和/或所述生产菌株为大肠杆菌。
根据本发明的一些实施方式，所述饲养菌株和/或生产菌株的代谢途径包括内源性类异戊二烯/萜生物合成通路。例如，细菌具有内源性MEP途径，酵母具有内源性MVA途径。根据本发明的一些实施方式，饲养菌株和/或生产菌株的代谢途径进一步包括部分工程化MVA途径，以使得异戊二烯焦磷酸前体(GPP、FPP、GGPP等)转化为萜烯。
在本发明中，该共培养系统通过将萜烯的生物合成途径中的许多步骤分为两个模块来工作，其中一个模块在一种菌株中进行，而其余步骤由另一种菌株进行。这两种菌株的步骤通过自由扩散的交换代谢物(甲羟戊酸)连接起来。由单菌株执行所有步骤的代谢压力在两种菌株之间分配，从而减少每种菌株的代谢压力，进而提高目标产物的产量。使用极性分子(如甲羟戊酸)作为两种菌株之间的转移代谢物可防止分配到萃取剂(如癸烷)中。这两种菌株相互作用以平衡甲羟戊酸的生产和消耗，以促进两种菌株的生存和生长。
本发明的第二方面提供了一种采用第一方面所述的共培养系统合成萜烯的方法，所述方法包括在诱导物的存在下孵育所述饲养菌株和所述生产菌株，以及用于从水相中分离萜烯的有机相或固相树脂。
根据本发明的一些实施方式，使用有机相(例如癸烷)或固相树脂(例如戴安HP20)用于从水相中萃取萜烯。在一些实施方式中，所述有机相选自癸烷。根据本发明，所述有机相不限于癸烷，还可进一步选自十一烷和十二烷；或者，选自,癸酸异丙酯、月桂酸异 丙酯、正辛酸异丙酯、肉豆蔻酸异丙酯等。在一些实施方式中，所述有机相占培养物的5％–30％v/v，优选10％–20％。所述固相树脂不限于戴安HP20且还可选自Dowax,HP-20,XAD7HP或HP-2MG。
根据本发明的一些实施方式，以所述饲养菌株和所述生产菌株的总接种量体积计，所述饲养菌株的含量为0.25％至99％。根据本发明的优选实施方式，以所述饲养菌株和所述生产菌株的接种量体积计，所述饲养菌株的含量为0.25％-99％，优选0.5％-75％，例如12.5％-75％。在一些实施方式中，以所述饲养菌株和所述生产菌株的接种量体积计，所述饲养菌株的含量为0.5％、1％、2％、5％、10％、12.5％、25％、37.5％、50％、62.5％、75％以及它们之间的任意值。
根据本发明的一些实施方式，当所述萜烯为紫衫二烯时，以所述饲养菌株和所述生产菌株的接种量体积计，所述饲养菌株的含量为0.25％-99％，尤其0.25％至37.5％，例如0.25％-12.5％或12.5％至37.5％，优选0.25％至12.5％，进一步优选0.25％至5％，特别优选0.5％至5％。
根据本发明的一些实施方式，当所述萜烯为β-广藿香时，以所述饲养菌株和所述生产菌株的接种量体积计，所述饲养菌株的含量为12.5％-75％，优选25％-75％，特别优选37.5％-62.5％。
根据本发明的一些实施方式，当所述萜烯为马兜铃素时，以所述饲养菌株和所述生产菌株的接种量体积计，所述饲养菌株的含量优选为12.5％-75％。
根据本发明的一些实施方式，所述分离纯化采用有机溶剂萃取进行，优选所述有机溶剂包括癸烷。
本发明的第三方面提供了根据第一方面所述的共培养系统或第二方面所述的方法在合成萜烯中的应用。
本发明的第四方面提供了甲羟戊酸在作为交换代谢物在微生物共培养合成萜烯中的应用。
本发明的有益效果：
1.本发明的共培养系统能够提高萜烯总产量。目前生产均在摇瓶中进行，可产生约1g/L的萜烯，当生产系统转移到发酵罐时，产量将显著增加。
2.通过表达相应的萜烯合酶，本发明的共培养系统和方法可应用于任何萜烯的生产，这些萜烯包括但不限于(a)生产用于生物燃料和其他应用的单萜(芳樟醇、香叶醇 和2,6-二甲基辛烷)和其他倍半萜(红没药烯、戊烯和异柯烯)；(b)共培养中的萜烯途径分裂导致二萜合酶的表达增加，从而导致二萜产量更高，具有更广泛的生物活性，包括药物作用；(c)可以进一步修饰饲养菌株以表达水解酶，以降解纤维素、木质素、半纤维素等聚合物；(d)可以添加多种饲喂菌株以利用农业和食物垃圾等复杂材料；(e)这些多菌株原则可应用于任何其他途径，以增强微生物群落的相互作用、通量和产物形成；(f)酵母、真菌和细菌等不同微生物可用于创建多菌株共培养或联合体(consortia)。
3.本发明的另一个关键优势是，一种菌株(饲养菌株)提供甲羟戊酸，该甲羟戊酸被释放到培养基中，然后通过另一种菌株(生产菌株)的细胞壁进入，该菌株表达使用甲羟戊酸的基因，即异戊二烯焦磷酸合成酶(图3)。通过将过程分成这些步骤，降低了在每个生物体上表达异源酶的代谢压力，从而实现更快的生长、增加的菌株活力和更高的体积产量。共培养系统减少了发酵时间，并可以通过例如控制接种量来调整饲养菌株和生产菌株的比例。
附图说明
图1示出了由法呢基焦磷酸(FPP)和香叶基香叶基焦磷酸(GGPP)的萜合酶合成的萜烯示例。
图2显示了用于萜烯生产的采用一种以上菌株的培养系统的一些现有技术：(A)是使用磷酸盐缓冲液生产蒎烯的双菌株系统，(B)两步培养系统，其中初始菌株生长以产生甲羟戊酸，然后用含有甲羟戊酸的培养基饲养第二菌株以产生萜烯。
图3示出了根据本发明的最小的或简单的双菌株共培养系统。
图4示出了实施例1.1中采用单菌株培养系统和双菌株共培养系统的紫杉二烯生物合成。
图5示出了实施例1.1中紫杉二烯生物合成的气相色谱分析。
图6示出了实施例1.2中采用单菌株培养系统和双菌株共培养系统的紫杉二烯生物合成。
图7示出了实施例1.2中紫杉二烯生物合成的气相色谱分析。
图8示出了实施例2中采用单菌株培养系统和双菌株共培养系统中的α-布尼烯生物合成。
图9示出了实施例2中α-布尼烯合成的气相色谱分析。
图10示出了实施例3中采用单菌株培养系统和双菌株共培养系统中的瓦伦烯生物合成。
图11示出了实施例3中瓦伦烯生物合成的气相色谱分析。
图12示出了实施例4中采用单菌株培养系统和双菌株共培养系统中的β-广藿香生物合成。
图13示出了实施例4中β-广藿香生物合成的气相色谱分析。
图14示出了实施例5中采用单菌株培养系统和双菌株共培养系统中的马兜铃素生物合成。
图2-3中，G3P：3-磷酸甘油醛；ACAT：乙酰辅酶A C-乙酰转移酶；HMGCS：3-羟基-3-甲基戊二酰-CoA合酶；HMGCR：3-羟基-3-甲基戊二酰-CoA还原酶；MVK：甲羟戊酸-5-激酶；PMVK：磷酸甲羟戊酸激酶；PMD：甲羟戊酸焦磷酸脱羧酶；IPPI：异戊烯焦磷酸异构酶；GPPS：香叶基焦磷酸合酶；FPPS：法呢基焦磷酸合酶；GGPPS：香叶基香叶基焦磷酸合酶；GPP：香叶基焦磷酸；FPP：法呢基焦磷酸；GGPP：香叶基香叶基焦磷酸；TS：萜烯合酶。
具体实施方式
下面结合具体实施方式，进一步阐述本申请。应理解，这些具体实施方式仅用于说明本申请而不用于限制本申请的范围。
为了简明，本文仅具体地公开了一些数值范围。然而，任意下限可以与任何上限组合形成未明确记载的范围；以及任意下限可以与其它下限组合形成未明确记载的范围，同样任意上限可以与任意其它上限组合形成未明确记载的范围。此外，每个单独公开的点或单个数值自身可以作为下限或上限与任意其它点或单个数值组合或与其它下限或上限组合形成未明确记载的范围。
除非另有说明，本申请中使用的术语具有本领域技术人员通常所理解的公知含义。除非另有说明，本申请中提到的各参数的数值可以用本领域常用的各种测量方法进行测量(例如，可以按照在本申请的实施例中给出的方法进行测试)。
术语“中的至少一个”、“中的至少一种”或其他相似术语所连接的项目的列表可意味着所列项目的任何组合。例如，如果列出项目A及B，那么短语“A及B中的至少一者”意味着仅A；仅B；或A及B。在另一实例中，如果列出项目A、B及C，那么短语“A、 B及C中的至少一者”意味着仅A；或仅B；仅C；A及B(排除C)；A及C(排除B)；B及C(排除A)；或A、B及C的全部。项目A可包含单个组分或多个组分。项目B可包含单个组分或多个组分。项目C可包含单个组分或多个组分。
本申请描述了一种共培养系统，其包括一种提供甲羟戊酸作为交换代谢物的饲养菌株和一种使用甲羟戊酸作为原料的生产菌株。代谢中间体的合成以及微生物之间的这种中间体的交换降低了微生物的代谢压力，从而提高中间体的转化率和目标化合物的产量。以下编号的方面描述了本发明的各种实施方式：
实施方式1.一种合成萜烯的共培养系统，其包括至少一种饲养菌株和至少一种生产菌株，其中，所述饲养细菌用于提供甲羟戊酸，所述生产菌株用于利用所述甲羟戊酸合成萜烯。
实施方式2.根据实施方式1所述的共培养系统，其特征在于：所述饲养菌株含有第一质粒，所述第一质粒含有合成甲羟戊酸的基因，且不含合成异戊二烯基焦磷酸的基因，优选地，所述饲养菌株还含有第二质粒，所述第二质粒为无需基因插入的空白质粒。
实施方式3.根据实施方式1或2所述的共培养系统，其特征在于：所述生产菌株含有第三质粒和第四质粒，所述第三质粒含有合成异戊二烯基焦磷酸的基因，且不含合成甲羟戊酸的基因，所述第四质粒含有所述萜烯合成酶的基因或萜烯合成酶与香叶基焦磷酸(GGPP)合成酶的融合基因。
实施方式4.根据实施方式1-3中任一项所述的共培养系统，其特征在于：所述饲养菌株和所述生产菌株比例为(0.25-99.75)：(99.75-0.25)，优选(1.25-75)：(98.75-25)。
实施方式5.根据实施方式1-4中任一项所述的共培养系统，其特征在于：所述萜烯包括紫杉二烯、α-布尼烯、瓦伦烯、α-愈创烯、β-广藿烯或马兜铃烯中的一种或多种。
实施方式6.根据实施方式1-5中任一项所述的共培养系统，其特征在于：所述饲养菌株和/或所述生产菌株选自细菌、真菌或酵母菌中的一种或多种。
实施方式7.根据权利要求1-6中任一项所述的共培养系统，其特征在于：所述饲养菌株和/或所述生产菌株的代谢途径包含内源性类异戊二烯合成通路途径。
实施方式8.一种采用实施方式1-7中任一项所述的共培养系统合成萜烯的方法，所述方法包括在诱导物的存在下孵育所述饲养菌株和所述生产菌株，以及分离和纯化萜烯。
实施方式9.根据实施方式8所述的方法，其特征在于：以所述饲养菌株和所述生产菌株的总接种量体积计，所述饲养菌株的含量为0.25％至90％，优选为0.5％-75％；和/或优选地，所述有机相选自癸烷、十一烷、十二烷，优选癸烷；或者，选自癸酸异丙酯、月桂酸异丙酯、正辛酸异丙酯、肉豆蔻酸异丙酯；固相树脂选自戴安HP20、Dowax20、XAD7HP,HP-2MG，优选为戴安HP20；有机相占培养物的5％–30％v/v，优选10％–20％v/v。
实施方式10.根据实施方式1-7中任一项所述的共培养系统或根据实施方式8或9所述的方法在合成萜烯中的应用。
在先报告表明，在微生物中生产萜烯，从Addgene获得质粒pBbA5c-MevT(CO)-T1-MBIS(CO,ispA)，并将密码子优化的萜合酶克隆到pTRC-Hisa载体(Invitrogen)中。两种质粒都被转化到大肠杆菌BL21(DE3)菌株中。使转化的大肠杆菌BL21(DE3)细胞生长直到600nm的OD达到0.6，然后用0.1mM IPTG诱导并在16℃或30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以提取萜烯。该系统对可溶性酶产生合理水平的萜烯，对不溶性酶产生较低水平的萜烯。这种较低的萜烯产生的原因可能是与异源酶表达有关的压力。这个障碍可以通过在不同菌株之间划分或共享代谢压力来消除。共培养或双菌株代谢工程正在成为减少单菌株中异源表达基因数量的重要研究领域。考虑到此方法，本发明研究了在微生物之间划分联合萜生物合成途径的方法，并提供了两种细菌菌株培养系统的实例。
本发明经研究发现，萜烯生物合成途径的划分需要一种中间体，该中间体可以在两种菌株之间转移，并且不会积累到萃取剂如癸烷中。异戊二烯单元(IPP和DMAPP)(Niu et al.,Frontiers in Microbiology,2018,9,1623；DOI:10.3389/fmicb.2018.01623)和萜烯烃(Zhou et al.,Nature Biotechnology,2015,33,377–383；DOI:10.1038/nbt.3095)已被用作菌株之间的转移中间体，但生产效率较低。以萜烯烃作为转移中间体的细菌和酵母共培养导致生物合成33mg/L含氧紫杉烷、20mg/L铁黄醇(ferruginol)和5mg/L诺卡酮(nootkatone)(Zhou et al.,Nature Biotechnology,2015,33,377–383；DOI:10.1038/nbt.3095)。用异戊二烯单元作为转移中间体(图2A)双菌株共培养产生166mg/L的蒎烯(Niu et al.,Frontiers in Microbiology,2018,9,16232；DOI:10.3389/fmicb.2018.01623)。以甲羟戊酸为中间体的两步培养系统(图2B)在细菌中产生150mg/L桧木烯(Liu et al.,Process Biochemistry,2017,62,1–9；DOI: 10.1016/j.procbio.2017.07.021)。本发明研究了萜烯生物合成途径，并且目前并没有现有技术采用甲羟戊酸没有用作共培养系统中的转移中间体。甲羟戊酸是一种极性小分子，很容易通过微生物膜扩散，并且不会分配到有机萃取剂(如癸烷)中。
在蒎烯生物合成过程中，必须在培养基中培养两种菌株并收获细胞，然后转移到磷酸盐缓冲液中以生产萜烯(Niu et al.,Frontiers in Microbiology,2018,9,16232；DOI:10.3389/fmicb.2018.01623)(图2A)。异戊二烯单元用作交换代谢物，可能对细菌造成压力。在桧烯(sabinene)生物合成过程中，产生甲羟戊酸的细菌必须在培养基中生长；所得的含甲羟戊酸的培养基与桧烯生物合成菌株的生长培养基一起使用。为了解决这一问题，本发明提供了一种简单的共培养系统(图3)，其中一种菌株(饲养菌株)将甲羟戊酸释放到生长培养基中，另一种菌株(生产菌株)使用甲羟戊酸来生物合成萜烯。该方法易于实施，并已通过不同的萜烯的生产得到证明，例如紫杉二烯、α-布尼烯、瓦伦烯、β-广藿香烯和马兜铃烯(图1)。
饲养菌株和生产菌株包含内源性MEP途径和部分工程异源MVA途径。MEP途径能够使细菌在最小内侧生长，而不补充异戊二烯中间体。此外，工程化的MVA途径增加萜烯生物合成的通量。
以下通过实施例对本申请的技术方案做示例性描述。
来自Addgene的MVA途径质粒pBbA5c-MevT(CO)-T1-MBIS(CO,ispA)(pMVA)(http://www.addgene.org/35152/；US7183089和US736882；Tsuruta et al.,PLoS One,2009,4,e4489；DOI:10.1371/journal.pone.0004459；Peralta-Yahya,et al.,Nature Communications,2011,2,483；DOI:10.1038/ncomms1494)被修改为在共培养系统中工作。pMVA质粒是一种中等拷贝数的质粒，包含两个模块，一个用于生产甲羟戊酸，另一个用于生产法呢基焦磷酸(FPP)。新的质粒pMevt和pMBIS是通过从pMVA克隆每个模块生成的。pMevt是一种中等拷贝数的质粒，含有甲羟戊酸生产模块。pMBIS是含有FPP生产模块的中等拷贝数的质粒。
本发明中的pMevT和pMBIS质粒来源于Addgene pMVA质粒，该质粒针对单菌株系统进行了优化。pMevT和pMIBS质粒可以通过修饰启动子和RBS(核糖体结合位点)进一步优化。通过提高异源酶表达、效率和宿主基因组修饰，可以进一步优化饲养和生产菌株。
【实施例1.1】紫杉二烯生物合成
紫杉二烯合酶和香叶基香叶基焦磷酸合酶的密码子优化基因被融合(如Ajikumar、Parayil Kumaran(10.1126/science.1191652)等人报道)，并克隆到pTRC-HisA中以生成质粒pTRC-TXSGPPS。
单菌株系统：
通过用pTRC-TXSGPPS和pMVA质粒转化大肠杆菌BL21(DE3)菌株得到用于紫杉二烯生产的单菌株系统。将转化的E.coli BL21(DE3)单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将该过夜饲喂培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在20℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将无任何基因插入的pUC-19质粒和pTRC-Mevt质粒转化到E.coli BL21(DE3)中生成，紫杉二烯生产菌株通过将pMBIS和pTRC-TXSGPPS质粒转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落分别独立接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将不同比例(50％到12.5％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在20℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。
结果见图4和图5。结果表明，紫杉二烯产量在单菌株系统中达到近6mg/L。对于双菌株共培养系统，随着饲养菌株比例的降低，紫杉二烯的产量增加。在初始接种物中12.5％的饲养菌株的双菌株共培养系统中，观察到最大产量水平为～16mg/L，代表紫杉二烯产量增加近3倍。
【实施例1.2】紫杉二烯生物合成
紫杉二烯合酶和香叶基香叶基焦磷酸合酶的密码子优化基因被融合(如Ajikumar、Parayil Kumaran(10.1126/science.1191652)等人报道)，并克隆到pTRC-HisA中以生成质粒pTRC-TXSGPPS。
单菌株系统：
通过用pTRC-TXSGPPS和pMVA质粒转化大肠杆菌BL21(DE3)菌株得到用于紫 杉二烯生产的单菌株系统。将转化的E.coli BL21(DE3)单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将该过夜饲喂培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在20℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。其中，培养基在24小时的间隔中以0.1mM IPTG诱导两次并补充2％的葡萄糖和2％的胰蛋白胨。在72小时候后通过离心收集癸烷相并通过GC分析。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将无任何基因插入的pUC-19质粒和pTRC-Mevt质粒转化到E.coli BL21(DE3)中生成，紫杉二烯生产菌株通过将pMBIS和pTRC-TXSGPPS质粒转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落分别独立接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将不同比例(0.125到12.5％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在20℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。其中，培养基在24小时的间隔中以0.1mM IPTG诱导两次并补充2％的葡萄糖和2％的胰蛋白胨。在72小时候后通过离心收集癸烷相并通过GC分析。
结果见图6和图7。结果表明，紫杉二烯产量在单菌株系统中达到近13mg/L。对于双菌株共培养系统，随着饲养菌株比例的降低，紫杉二烯的产量增加。在初始接种物中1.25％的饲养菌株的双菌株共培养系统中，观察到最大产量水平为～340mg/L，代表紫杉二烯产量增加近25倍。
【实施例2】α-布尼烯生物合成
将密码子优化的布尼烯合酶基因(NCBI-KF800046)克隆到pTRC-HisA中以生成质粒pTRC-BS。
单菌株系统：
通过用pMVA和pTRC-BS质粒转化大肠杆菌BL21(DE3)生成了用于生产α-布尼烯的单菌株系统。将单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时。将过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200 rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。萜烯总产量达到1345mg/L(图8)。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将pMevt和pUC-19质粒转化E.coli BL21(DE3)中生成。用于α-布尼烯合成的生产菌株通过将pMBIS和pTRC-BS质粒转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落接种到单独的5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将不同比例(25％至75％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦600nm处的OD达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。含25％饲养菌株的双菌株共培养系统产生1500mg/L的总萜烯，比单菌株系统高10％(图8)。
【实施例3】瓦伦烯生物合成
将密码子优化的瓦伦烯合酶基因(来自US9303252B2)克隆到pTRC-HisA中以生成质粒pTRC-VS。
单菌株系统：
通过用pMVA和pTRC-VS质粒转化E.coli BL21(DE3)生成了用于瓦伦烯生物合成的单菌株系统。将单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。萜烯总产量达到约336mg/L(图10)。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将pMevt和pUC-19转化到E.coli BL21(DE3)中生成。用于瓦伦烯生物合成的生产菌株通过将质粒pMBIS和pTRC-VS转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落接种到单独的5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时。将不同比例(25％至75％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200 rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v的癸烷以提取萜烯。含25％饲养菌株的双菌株共培养系统产生540mg/L的总萜烯，比单菌株系统高35％(图10)。
【实施例4】β-广藿香生物合成
将密码子优化的β-广藿香烯合酶基因(XM_044587644)克隆到pTRC-HisA中以生成质粒pTRC-PCL。
单菌株系统：
通过用pMVA和pTRC-PCL质粒转化E.coli BL21(DE3)生成了用于β-广藿香烯生物合成的单菌株系统。将单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在16℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。萜烯总产量达到约7mg/L(图12)。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将pMevt和Puc-19质粒转化到E.coli BL21(DE3)中生成。用于β-广藿香烯生物合成的生产菌株通过将质粒pMBIS和pTRC-PCL转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落接种到单独的5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时，得到过夜培养物。将不同比例(37.5％到62.5％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦600nm处的OD达到0.6，用0.1mM IPTG诱导并在16℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v的癸烷以提取萜烯。含37.5％饲养菌株的双菌株共培养系统产生25mg/L的总萜烯，是单菌株系统的三倍(图12)。
【实施例5】马兜铃素生物合成
将密码子优化的马兜铃烯合酶基因(PDB-3M01_A)克隆到pTRC-HisA中以生成质粒pTRC-TEAS。
单菌株系统：
通过用pMVA和pTRC-TEAS质粒转化E.coli BL21(DE3)生成了用于马兜铃烯生物合成的单菌株系统。将单菌落接种到5mL LB(Luria-Bertani)培养基中，并在37℃下 培养12小时，得到过夜培养物。将过夜培养物接种到25mL TB培养基中，并在37℃和200rpm下孵育。一旦在600nm处的OD值达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v癸烷作为有机相以萃取萜烯。萜烯总产量达到约3mg/L(图14)。
双菌株共培养系统：
对于双菌株共培养系统，饲养菌株通过将pMevt和Puc-19质粒转化到E.coli BL21(DE3)中生成。用于β-马兜铃素生物合成的生产菌株通过将质粒pMBIS和pTRC-TEAS转化到E.coli BL21(DE3)中生成。将饲养菌株和生产菌株的菌落接种到单独的5mL LB(Luria-Bertani)培养基中，并在37℃下培养12小时。将不同比例(25％到62.5％)的饲养菌株和生产菌株的过夜培养物接种到25mL TB(Terrific Broth)培养基中，并在37℃和200rpm下孵育。一旦600nm处的OD达到0.6，用0.1mM IPTG诱导并在30℃下孵育。添加2％w/v的葡萄糖作为碳源，添加10％v/v的癸烷以提取萜烯。含25％饲养菌株的双菌株共培养系统产生15mg/L的总萜烯，是单菌株系统的5倍(图14)。
虽然已经说明和描述了本申请的一些示例性实施方式，然而本申请不限于所公开的实施方式。相反，本领域普通技术人员将认识到，在不脱离如所附权利要求中描述的本申请的精神和范围的情况下，可对所描述的实施方式进行一些修饰和改变。

Claims (10)

  1. 一种用于合成萜烯的共培养系统，其包括至少一种饲养菌株和至少一种生产菌株，其中，所述饲养细菌用于提供甲羟戊酸，所述生产菌株用于利用所述甲羟戊酸合成萜烯。
  2. 根据权利要求1所述的共培养系统，其特征在于：所述饲养菌株含有第一质粒，所述第一质粒含有合成甲羟戊酸的基因且不含合成异戊二烯基焦磷酸的基因，优选地，所述饲养菌株还含有第二质粒，所述第二质粒为无需基因插入的空白质粒。
  3. 根据权利要求1或2所述的共培养系统，其特征在于：所述生产菌株含有第三质粒和第四质粒，所述第三质粒含有合成法呢基焦磷酸的基因且不含合成甲羟戊酸的基因，所述第四质粒含有所述萜烯合成酶的基因或萜烯合成酶与GGPP合成酶的融合基因。
  4. 根据权利要求1-3中任一项所述的共培养系统，其特征在于：所述饲养菌株和所述生产菌株比例为(0.25-99.75)：(99.75-0.25)，优选(1.25-75)：(98.75-25)。
  5. 根据权利要求1-4中任一项所述的共培养系统，其特征在于：所述萜烯包括紫杉二烯、α-布尼烯、瓦伦烯、α-愈创烯、β-广藿烯或马兜铃烯中的一种或多种。
  6. 根据权利要求1-5中任一项所述的共培养系统，其特征在于：所述饲养菌株和/或所述生产菌株选自细菌、真菌或酵母菌中的一种或多种。
  7. 根据权利要求1-6中任一项所述的共培养系统，其特征在于：所述饲养菌株和/或所述生产菌株的代谢途径包含内源性类异戊二烯合成通路途径。
  8. 一种采用权利要求1-7中任一项所述的共培养系统用于合成萜烯的方法，所述方法包括在诱导物的存在下孵育所述饲养菌株和所述生产菌株，以及用于从水相中分离萜烯的有机相或固相树脂。
  9. 根据权利要求8所述的方法，其特征在于：以所述饲养菌株和所述生产菌株的总接种量体积计，所述饲养菌株的含量为0.25％至90％，优选为0.5％-75％；
    和/或所述有机相选自癸烷、十一烷、十二烷、癸酸异丙酯、月桂酸异丙酯、正辛酸异丙酯、肉豆蔻酸异丙酯，优选癸烷；所述固相树脂选自戴安HP20，Dowax 20，XAD7HP,HP-2MG，优选为戴安HP20；其中，所述有机相占培养物的5％–30％v/v，优选10％–20％v/v。
  10. 根据权利要求1-7中任一项所述的共培养系统或根据权利要求8-9中任一项所述的方法在合成萜烯中的应用。
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