Co-culture system and method for synthesizing terpene

Abstract

The invention discloses a co-culture system and method for synthesizing terpenes, the system comprising at least one feed strain for providing mevalonic acid and at least one production strain for synthesizing terpenes using the mevalonic acid. The co-culture system and the co-culture method can be applied to production of any terpene, and the total yield and the valence alkene conversion rate of the terpene can be remarkably increased.

Description

A co-cultivation system and method for synthesizing terpenes Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular provides a co-cultivation system and method for synthesizing terpenes. Background Art

[0002] Terpenes are widely used in various industrial applications, such as flavors, fragrances, biofuels, pharmaceuticals, rubber, and pesticides. These compounds are produced by plants, but usually in low concentrations that do not support industrial demand.

[0003] Terpenoids are compounds derived from mevalonic acid or deoxy-D-xylulose 5-phosphate, with a molecular skeleton based on isoprene units (C5 units) and their oxygenated derivatives. These oxygenated derivatives can be alcohols, aldehydes, ketones, carboxylic acids, esters, and more. Terpenes are a common class of terpenoids. The production of these metabolites is often complex, with multi-step chemical production processes involving complex reactions, leading to low yields, incorrect stereochemistry, and high costs. In recent years, synthetic biology has become a rapidly expanding and advancing research field, with widespread applications in sustainable and more environmentally friendly compound synthesis. A typical example of terpenoid synthesis using synthetic biology is the synthesis of artemisinin, which is produced via artemisinic acid, which is produced from the biosynthesis of the sesquiterpene precursor amorpha-4,11-diene artemisinin. This acid is then oxidized sequentially to alcohols, aldehydes, and finally acids. However, this complex multigene system is subject to metabolic pressure and pathway limitations, such as metabolic flux and release of intermediates into the culture medium without re-entry through the cell wall.

[0004] In a pinene biosynthesis process, two strains must be grown in culture, the cells harvested, and then transferred to phosphate-buffered saline to produce the terpene. In a sabinene biosynthesis process, mevalonate-producing bacteria must be grown in culture to produce mevalonate, and the mevalonate-containing culture medium must then be used alongside the growth medium of the sabinene biosynthetic strain. These processes are complex and require strict process control. Furthermore, the use of isoprene units as exchange metabolites can also be stressful for the bacteria. Much of the 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 exchange between microorganisms.

[0005] However, there is still no synthesis method that can meet the requirements of industrial production with high terpene content.

[0006] Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention has developed a simple co-cultivation system for enhancing terpene biosynthesis by partitioning the isoprenoid MVA pathway between microbial strains without altering their native isoprenoid pathway. 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-cultivation system comprising a feeder strain that synthesizes mevalonic acid as an exchange metabolite and a production strain that uses mevalonic acid as a feedstock to synthesize terpenes, providing a tunable solution for heterologous protein expression. The co-cultivation system provided by the present invention reduces the metabolic stress on microorganisms by partitioning the synthesis of metabolite intermediates between microorganisms, thereby increasing the conversion rate of intermediates and the yield of target compounds.

[0008] A first aspect of the present invention provides a co-cultivation system for synthesizing terpenes, comprising at least one feeder strain and at least one production strain, wherein the feeder bacteria are used to provide mevalonic acid, and the production strain is used to synthesize terpenes using the mevalonic acid.

[0009] According to some embodiments of the present invention, the feeder strain contains a first plasmid containing a gene for synthesizing mevalonic acid and does not contain a gene for synthesizing isoprenyl pyrophosphate. According to some embodiments of the present invention, the feeder strain further contains a second plasmid, which is a blank plasmid that does not require any gene insertion. According to some specific embodiments of the present invention, the second plasmid is pUC19, which does not require any gene insertion. According to some embodiments of the present invention, the second plasmid facilitates antibiotic compensation with the production strain. In the present invention, the feeder strain provides mevalonic acid, which is released into the culture medium and then enters the cell wall of the production strain capable of expressing the mevalonic acid gene, the cell membrane of the production strain of isoprenyl pyrophosphate synthase and the terpene synthase.

[0010] According to some embodiments of the present invention, 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 mevalonic acid, and the fourth plasmid contains the gene for the terpene synthase or a fusion gene of the terpene synthase and the GGPP synthase.

[0011] According to some embodiments of the present invention, the MVA pathway plasmid pBbA5c-MevT(CO)-T1-MBIS(CO,ispA)(pMVA) from Addgene was modified to obtain the feeder and production strains. The pMVA plasmid is a medium-copy number plasmid containing two modules, one for producing mevalonate and the other for producing 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 another medium-copy number plasmid containing an FPP production module. pTRC-X is generated by cloning a codon-optimized terpene X synthase gene into pTRC-HisA. In the present invention, the feeder strain is generated by transforming the pMevt plasmid pUC-19 into a microorganism such as Escherichia coli, and the production strain is generated by transforming the pMBIS plasmid and the pTRC-X plasmid into a microorganism such as Escherichia coli.

[0012] According to some embodiments of the present invention, the ratio of the feeder strain to the production strain is (0.25-99.75):(99.75-0.25), for example (0.25-99.5):(99.75-0.5), preferably (1.25-75):(98.75-25). According to some preferred embodiments of the present invention, the ratio of the feeder strain to the production strain is (0.5-75):(99.5-25), for example (12.5-75):(87.5-25). In the present invention, the range of co-cultivated strains is further improved by changing the plasmid, inoculation ratio, heterologous enzyme expression, efficiency and genome modification, thereby further improving the yield of terpenes.

[0013] According to some embodiments of the invention, the terpene comprises one or more of taxadiene, α-bunielene, valencene, α-guaiene, β-patchoulene or aristolochene.

[0014] According to some embodiments of the present invention, the feeder strain and / or the production strain is selected from one or more of bacteria, fungi or yeast. According to some specific embodiments of the present invention, the feeder strain and / or the production strain is Escherichia coli.

[0015] According to some embodiments of the present invention, the metabolic pathways of the feeder strain and / or production strain include endogenous isoprenoid / terpene biosynthesis pathways. For example, bacteria have an endogenous MEP pathway, and yeast have an endogenous MVA pathway. According to some embodiments of the present invention, the metabolic pathways of the feeder strain and / or production strain further include a partially engineered MVA pathway to convert isoprenyl pyrophosphate precursors (GPP, FPP, GGPP, etc.) into terpenes.

[0016] In the present invention, the co-cultivation system works by dividing many steps in the terpene biosynthesis pathway into two modules, one of which is performed by one strain, while the remaining steps are performed by another strain. The steps of the two strains are connected by a freely diffusing exchange metabolite (mevalonate). The metabolic pressure of performing all steps by a single strain is distributed between the two strains, thereby reducing the metabolic pressure of each strain and increasing the yield of the target product. Using a polar molecule (such as mevalonate) as a transfer metabolite between the two strains prevents partitioning into the extractant (such as decane). The two strains interact to balance the production and consumption of mevalonate, promoting the survival and growth of both strains.

[0017] The second aspect of the present invention provides a method for synthesizing terpenes using the co-cultivation system described in the first aspect, the method comprising incubating the feeder strain and the production strain in the presence of an inducer, and an organic phase or solid phase resin for separating terpenes from an aqueous phase.

[0018] According to some embodiments of the present invention, an organic phase (e.g., decane) or a solid phase resin (e.g., Dionex HP20) is used to extract terpenes from the aqueous phase. In some embodiments, the organic phase is selected from decane. According to the present invention, the organic phase is not limited to decane and may be further selected from undecane and dodecane; or, alternatively, selected from isopropyl decanoate, isopropyl laurate, isopropyl n-octanoate, isopropyl myristate, and the like. In some embodiments, the organic phase comprises 5%–30% v / v of the culture, preferably 10%–20%. The solid phase resin is not limited to Dionex HP20 and may also be selected from Dowax, HP-20, XAD7HP, or HP-2MG.

[0019] According to some embodiments of the present invention, the feeder strain is present in an amount of 0.25% to 99% by volume of the total inoculum of the feeder strain and the production strain. According to a preferred embodiment of the present invention, the feeder strain is present in an amount of 0.25% to 99% by volume of the inoculum of the feeder strain and the production strain, preferably 0.5% to 75%, for example 12.5% ​​to 75%. In some embodiments, the feeder strain is present in an amount of 0.5%, 1%, 2%, 5%, 10%, 12.5%, 25%, 37.5%, 50%, 62.5%, 75% and any value therebetween by volume of the inoculum of the feeder strain and the production strain.

[0020] According to some embodiments of the present invention, when the terpene is taxadiene, the content of the feeding strain is 0.25%-99%, especially 0.25% to 37.5%, for example 0.25%-12.5% ​​or 12.5% ​​to 37.5%, preferably 0.25% to 12.5%, further preferably 0.25% to 5%, and particularly preferably 0.5% to 5%, based on the inoculum volume of the feeding strain and the production strain.

[0021] According to some embodiments of the present invention, when the terpene is β-patchouli, the content of the feeder strain is 12.5%-75%, preferably 25%-75%, and particularly preferably 37.5%-62.5%, based on the inoculum volume of the feeder strain and the production strain.

[0022] According to some embodiments of the present invention, when the terpene is aristolochicine, the content of the feeder strain is preferably 12.5%-75% based on the inoculum volume of the feeder strain and the production strain.

[0023] According to some embodiments of the present invention, the separation and purification is performed by extraction with an organic solvent, and preferably the organic solvent comprises decane.

[0024] The third aspect of the present invention provides use of the co-cultivation system according to the first aspect or the method according to the second aspect in synthesizing terpenes.

[0025] A fourth aspect of the present invention provides the use of mevalonic acid as an exchange metabolite in the synthesis of terpenes by co-cultivation of microorganisms.

[0026] Beneficial effects of the present invention:

[0027] 1. The co-cultivation system of the present invention can increase the total terpene yield. Currently, production is carried out in shake flasks, which can produce about 1g / L of terpene. When the production system is transferred to fermenters, the yield will be significantly increased.

[0028] 2. By expressing the corresponding terpene synthases, the co-cultivation system and method of the present invention can be applied to the production of any terpene, including but not limited to (a) the production of monoterpenes (linalool, geraniol and 2,6-dimethyloctane) and other sesquiterpenes (bisabolene, amylene and isocornein) for biofuels and other applications; (b) the splitting of the terpene pathway in the co-cultivation leads to increased expression of diterpene synthases, resulting in higher production of diterpenes with a wider range of biological activities, including pharmaceutical effects; (c) the feeder strains can be further modified to express hydrolases to degrade polymers such as cellulose, lignin, hemicellulose, etc.; (d) multiple feeder 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 the interactions, flux and product formation of microbial communities; (f) different microorganisms such as yeast, fungi and bacteria can be used to create multi-strain co-cultures or consortia.

[0029] 3. 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 another strain (the production strain), which expresses a gene that uses mevalonate, namely isoprene pyrophosphate synthase (Figure 3). By separating the process into these steps, the metabolic pressure of expressing heterologous enzymes in each organism is reduced, thereby achieving faster growth, increased strain viability, and higher volumetric yield. The co-culture system reduces fermentation time and allows the ratio of feeder and production strains to be adjusted, for example, by controlling the inoculum size. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 shows examples of terpenes synthesized by terpene synthases that synthesize farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).

[0031] Figure 2 shows some prior art culture systems using more than one strain for terpene production: (A) 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 grown on a medium containing mevalonate to produce terpenes.

[0032] FIG3 shows a minimal or simple two-strain co-cultivation system according to the present invention.

[0033] FIG4 shows the biosynthesis of taxadiene using the single-strain culture system and the two-strain co-culture system in Example 1.1.

[0034] FIG5 shows the gas chromatography analysis of taxadiene biosynthesis in Example 1.1.

[0035] FIG6 shows the biosynthesis of taxadiene using the single-strain culture system and the dual-strain co-culture system in Example 1.2.

[0036] FIG7 shows the gas chromatography analysis of taxadiene biosynthesis in Example 1.2.

[0037] FIG8 shows the biosynthesis of α-buniene in the single-strain culture system and the dual-strain co-culture system in Example 2.

[0038] FIG. 9 shows the gas chromatography analysis of the α-bunylene synthesis in Example 2.

[0039] FIG10 shows the biosynthesis of valencene in the single-strain culture system and the dual-strain co-culture system in Example 3.

[0040] FIG11 shows gas chromatography analysis of valencene biosynthesis in Example 3.

[0041] FIG. 12 shows the biosynthesis of β-patchouli in the single-strain culture system and the dual-strain co-culture system in Example 4.

[0042] FIG. 13 shows the gas chromatography analysis of β-patchouli biosynthesis in Example 4.

[0043] FIG14 shows the biosynthesis of aristolochicine in the single-strain culture system and the dual-strain co-culture system in Example 5.

[0044] In Figure 2-3, 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 isomerase; GPPS: geranyl pyrophosphate synthase; FPPS: farnesyl pyrophosphate synthase; GGPPS: geranylgeranyl pyrophosphate synthase; GPP: geranyl pyrophosphate; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; TS: terpene synthase. DETAILED DESCRIPTION

[0045] The present application will be further described below in conjunction with specific embodiments. It should be understood that these specific embodiments are only used to illustrate the present application and are not used to limit the scope of the present application.

[0046] For the sake of clarity, only some numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0047] Unless otherwise specified, the terms used in this application have the commonly understood meanings commonly understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the examples of this application).

[0048] 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, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and 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.

[0049] This application describes a co-cultivation system comprising 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 these intermediates between microorganisms reduce metabolic stress on the microorganisms, thereby increasing intermediate conversion and target compound yield. The following numbered aspects describe various embodiments of the invention:

[0050] Embodiment 1. A co-cultivation system for synthesizing terpenes, comprising at least one feeder strain and at least one production strain, wherein the feeder bacteria are used to provide mevalonic acid, and the production strain is used to synthesize terpenes using the mevalonic acid.

[0051] Embodiment 2. The co-cultivation system according to embodiment 1 is characterized in that: the feeder strain contains a first plasmid, the first plasmid contains a gene for synthesizing mevalonic acid and does not contain a gene for synthesizing isoprenyl pyrophosphate. Preferably, the feeder strain also contains a second plasmid, which is a blank plasmid that does not require gene insertion.

[0052] Embodiment 3. The co-cultivation system according to embodiment 1 or 2 is characterized in that: the production strain contains a third plasmid and a fourth plasmid, the third plasmid contains a gene for synthesizing isoprenyl pyrophosphate and does not contain a gene for synthesizing mevalonic acid, and the fourth plasmid contains the gene for the terpene synthase or a fusion gene of the terpene synthase and geranyl pyrophosphate (GGPP) synthase.

[0053] Embodiment 4. The co-cultivation system according to any one of embodiments 1-3, characterized in that the ratio of the feeder strain to the production strain is (0.25-99.75): (99.75-0.25), preferably (1.25-75): (98.75-25).

[0054] Embodiment 5. The co-cultivation system according to any one of embodiments 1 to 4, characterized in that the terpene comprises one or more of taxadiene, α-bunielene, valencene, α-guaiacene, β-patchoulene, or aristolochene.

[0055] Embodiment 6. The co-cultivation system according to any one of embodiments 1 to 5, characterized in that the feeder strain and / or the production strain is selected from one or more of bacteria, fungi or yeast.

[0056] Embodiment 7. The co-cultivation system according to any one of claims 1-6, wherein the metabolic pathway of the feeder strain and / or the production strain comprises an endogenous isoprenoid synthesis pathway.

[0057] Embodiment 8. A method for synthesizing terpenes using the co-cultivation 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 isolating and purifying the terpenes.

[0058] Embodiment 9. The method according to embodiment 8 is characterized in that: the content of the feeder strain is 0.25% to 90%, preferably 0.5%-75%, based on the total inoculum volume of the feeder strain and the production strain; and / or preferably, the organic phase is selected from decane, undecane, dodecane, preferably decane; or, selected from isopropyl decanoate, isopropyl laurate, isopropyl n-octanoate, isopropyl myristate; the solid phase resin is selected from Dion HP20, Dowax20, XAD7HP, HP-2MG, preferably Dion HP20; the organic phase accounts for 5%-30% v / v of the culture, preferably 10%-20% v / v.

[0059] Embodiment 10. Use of the co-cultivation system according to any one of embodiments 1 to 7 or the method according to embodiment 8 or 9 in synthesizing terpenes.

[0060] Previous reports have demonstrated the production of terpenes in microorganisms. The plasmid pBbA5c-MevT(CO)-T1-MBIS(CO, ispA) was obtained from Addgene, and a codon-optimized terpene synthase was cloned into the pTRC-Hisa vector (Invitrogen). Both plasmids were transformed into Escherichia coli BL21(DE3). Transformed E. coli BL21(DE3) cells were grown until an OD at 600 nm reached 0.6, then induced with 0.1 mM IPTG and incubated at 16°C or 30°C. 2% w / v glucose was added as the carbon source, and 10% v / v decane was added as the organic phase for terpene extraction. This system produced reasonable levels of terpenes for soluble enzymes and lower levels for insoluble enzymes. This lower terpene production may be due to the stress associated with heterologous enzyme expression. This obstacle can be overcome by dividing or sharing metabolic stress between different strains. Co-culture or dual-strain metabolic engineering is becoming an important research area to reduce the number of heterologously expressed genes in a single strain. With this approach in mind, the present invention investigates methods for partitioning the combined terpene biosynthetic pathway among microorganisms and provides examples of two bacterial strain culture systems.

[0061] The present invention discovered that the division of the terpene biosynthesis pathway requires an intermediate that can be transferred between the two strains and does not accumulate in the extractant, such as decane. Isoprene units (IPP and DMAPP) (Niu et al., Frontiers in Microbiology, 2018, 9, 1623; DOI: 10.3389 / fmicb.2018.01623) and terpene hydrocarbons (Zhou et al., Nature Biotechnology, 2015, 33, 377–383; DOI: 10.1038 / nbt.3095) have been used as transfer intermediates between strains, but production efficiency is low. Co-cultivation of bacteria and yeast using terpene hydrocarbons as transfer intermediates resulted in the biosynthesis of 33 mg / L of oxygenated taxanes, 20 mg / L of ferruginol, and 5 mg / L of nootkatone (Zhou et al., Nature Biotechnology, 2015, 33, 377–383; DOI: 10.1038 / nbt.3095). Co-cultivation of two strains using isoprene units as transfer intermediates (Figure 2A) produced 166 mg / L of pinene (Niu et al., Frontiers in Microbiology, 2018, 9, 16232; DOI: 10.3389 / fmicb.2018.01623). A two-step culture system using mevalonic acid as an intermediate (Figure 2B) produced 150 mg / L sabinene in bacteria (Liu et al., Process Biochemistry, 2017, 62, 1–9; DOI: 10.1016 / j.procbio.2017.07.021). This study investigated the terpene biosynthesis pathway, and no prior art utilizes mevalonic acid as a transfer intermediate in a co-culture system. Mevalonic acid is a small, polar molecule that readily diffuses through microbial membranes and does not partition into organic extractants such as decane.

[0062] During the biosynthesis of pinene, two strains must be cultured in a culture medium and the cells harvested and then transferred to a phosphate buffer to produce terpenes (Niu et al., Frontiers in Microbiology, 2018, 9, 16232; DOI: 10.3389 / fmicb.2018.01623) (Figure 2A). Isoprene units are used as exchange metabolites, which may cause stress to the bacteria. During the biosynthesis of sabinene, bacteria that produce mevalonate must be grown in a culture medium; the resulting mevalonate-containing culture medium is used together with the growth medium of the sabinene biosynthesis strain. To solve this problem, the present invention provides a simple co-cultivation system (Figure 3), in which one strain (feeder strain) releases mevalonate into the growth medium and the other strain (production strain) uses mevalonate to biosynthesize terpenes. The method is easy to implement and has been demonstrated with the production of different terpenes, such as taxadiene, α-bunielene, valencene, β-patchoulene, and aristolochene ( Figure 1 ).

[0063] Feeder and production strains contain the endogenous MEP pathway and a partially engineered heterologous MVA pathway. The MEP pathway enables bacterial growth in minimal media without supplementation of isoprene intermediates. In addition, the engineered MVA pathway increases the flux of terpene biosynthesis.

[0064] The technical solution of the present application is described below by way of examples.

[0065] The 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 containing two modules, one for mevalonate production and the other for farnesyl pyrophosphate (FPP) production. The 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, and pMBIS is a medium-copy number plasmid containing an FPP production module.

[0066] The pMevT and pMBIS plasmids in this invention are derived from the Addgene pMVA plasmid, which is optimized for single-strain systems. pMevT and pMIBS plasmids can be further optimized by modifying the promoter and RBS (ribosome binding site). By improving heterologous enzyme expression, efficiency, and host genome modification, the breeding and production strains can be further optimized.

[0067] [Example 1.1] Taxadiene biosynthesis

[0068] Codon-optimized genes for taxadiene synthase and geranylgeranyl pyrophosphate synthase were fused (as reported by Ajikumar, Parayil Kumaran et al. (10.1126 / science.1191652)) and cloned into pTRC-HisA to generate plasmid pTRC-TXSGPPS.

[0069] Single strain system:

[0070] A single-strain system for taxadiene production was generated by transforming Escherichia coli BL21(DE3) with the pTRC-TXSGPPS and pMVA plasmids. A single colony of the transformed E. coli BL21(DE3) was inoculated into 5 mL of Luria-Bertani (LB) medium and cultured at 37°C for 12 hours to generate an overnight culture. This overnight fed culture was inoculated into 25 mL of Terrific Broth (TB) medium and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, the culture was induced with 0.1 mM IPTG and incubated at 20°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as the organic phase to extract terpenes.

[0071] Dual strain co-culture system:

[0072] For the dual-strain co-culture system, the feeder strain was generated by transforming E. coli BL21(DE3) with the pUC-19 plasmid and the pTRC-Mevt plasmid without any gene insertions. The taxadiene-producing strain was generated by transforming E. coli BL21(DE3) with the pMBIS and pTRC-TXSGPPS plasmids. Colonies of the feeder and producer strains were independently inoculated into 5 mL of Luria-Bertani (LB) medium and incubated at 37°C for 12 hours to generate overnight cultures. Overnight cultures of the feeder and producer strains were inoculated into 25 mL of Terrific Broth (TB) medium at varying ratios (50% to 12.5%) and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, the cultures were induced with 0.1 mM IPTG and incubated at 20°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as an organic phase to extract terpenes.

[0073] The results are shown in Figures 4 and 5. They demonstrate that taxadiene production reached nearly 6 mg / L in the single-strain system. In the dual-strain co-culture system, taxadiene production increased as the feeder strain ratio decreased. In the dual-strain co-culture system with 12.5% ​​of the feeder strain in the initial inoculum, a maximum production level of ~16 mg / L was observed, representing a nearly three-fold increase in taxadiene production.

[0074] [Example 1.2] Taxadiene biosynthesis

[0075] Codon-optimized genes for taxadiene synthase and geranylgeranyl pyrophosphate synthase were fused (as reported by Ajikumar, Parayil Kumaran et al. (10.1126 / science.1191652)) and cloned into pTRC-HisA to generate plasmid pTRC-TXSGPPS.

[0076] Single strain system:

[0077] A single-strain system for taxadiene production was generated by transforming Escherichia coli BL21(DE3) with the pTRC-TXSGPPS and pMVA plasmids. A single colony of the transformed E. coli BL21(DE3) was inoculated into 5 mL of Luria-Bertani (LB) medium and cultured at 37°C for 12 hours to generate an overnight culture. This overnight fed culture was inoculated into 25 mL of Terrific Broth (TB) medium and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, the culture was induced with 0.1 mM IPTG and incubated at 20°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as the organic phase to extract terpenes. The culture was induced twice with 0.1 mM IPTG at 24-hour intervals and supplemented with 2% glucose and 2% tryptone. After 72 hours, the decane phase was collected by centrifugation and analyzed by GC.

[0078] Dual strain co-culture system:

[0079] For the dual-strain co-culture system, the feeder strain was generated by transforming E. coli BL21(DE3) with the pUC-19 plasmid and the pTRC-Mevt plasmid without any gene insertions. The taxadiene-producing strain was generated by transforming E. coli BL21(DE3) with the pMBIS and pTRC-TXSGPPS plasmids. Colonies of the feeder and producer strains were independently inoculated into 5 mL of Luria-Bertani (LB) medium and incubated at 37°C for 12 hours to generate overnight cultures. Overnight cultures of the feeder and producer strains were inoculated into 25 mL of Terrific Broth (TB) medium at varying ratios (0.125 to 12.5%) and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, the cultures were induced with 0.1 mM IPTG and incubated at 20°C. Terpene extraction was performed by adding 2% w / v glucose as a carbon source and 10% v / v decane as the organic phase. The culture medium was induced twice with 0.1 mM IPTG at 24-hour intervals and supplemented with 2% glucose and 2% tryptone. After 72 hours, the decane phase was collected by centrifugation and analyzed by GC.

[0080] The results are shown in Figures 6 and 7. They show that taxadiene production reached nearly 13 mg / L in the single-strain system. In the dual-strain co-culture system, taxadiene production increased as the feeder strain ratio decreased. In the dual-strain co-culture system with 1.25% feeder strain in the initial inoculum, a maximum production level of ~340 mg / L was observed, representing a nearly 25-fold increase in taxadiene production.

[0081] [Example 2] α-Buniene Biosynthesis

[0082] The codon-optimized bunene synthase gene (NCBI-KF800046) was cloned into pTRC-HisA to generate plasmid pTRC-BS.

[0083] Single strain system:

[0084] A single-strain system for α-buniene production was generated by transforming Escherichia coli BL21(DE3) with the 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 of TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD value at 600 nm reached 0.6, the culture was induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as the organic phase to extract the terpenes. Total terpene yield reached 1345 mg / L (Figure 8).

[0085] Dual strain co-culture system:

[0086] For the dual-strain co-cultivation system, the feeder strain was generated by transforming E. coli BL21(DE3) with the pMevt and pUC-19 plasmids. The production strain for α-buniene synthesis was generated by transforming E. coli BL21(DE3) with the pMBIS and pTRC-BS plasmids. Colonies of the feeder and production strains were inoculated into separate 5 mL LB (Luria-Bertani) medium and incubated at 37°C for 12 hours to generate overnight cultures. Overnight cultures of the feeder and production strains at varying ratios (25% to 75%) were inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, the cultures were induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as the organic phase to extract terpenes. The dual-strain co-culture system containing 25% of the feeder strain produced 1500 mg / L of total terpenes, which was 10% higher than that of the single-strain system ( FIG8 ).

[0087] [Example 3] Valencene Biosynthesis

[0088] The codon-optimized valencene synthase gene (from US9303252B2) was cloned into pTRC-HisA to generate plasmid pTRC-VS.

[0089] Single strain system:

[0090] A single-strain system for valencene biosynthesis was generated by transforming E. coli BL21 (DE3) with the pMVA and pTRC-VS plasmids. A single colony was inoculated into 5 mL of LB (Luria-Bertani) medium and cultured at 37°C for 12 hours to obtain an overnight culture. The overnight 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 reached 0.6, it was induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added as the organic phase to extract the terpenes. The total terpene yield reached approximately 336 mg / L (Figure 10).

[0091] Dual strain co-culture system:

[0092] For the dual-strain co-cultivation system, the feeder strain was generated by transforming pMevt and pUC-19 into E. coli BL21 (DE3). The production strain for valencene biosynthesis was generated by transforming the 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 the feeder strain and the production strain at different ratios (25% to 75%) were inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD value at 600 nm reached 0.6, it was induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source, and 10% v / v decane was added to extract terpenes. The two-strain co-culture system containing 25% of the feeder strain produced 540 mg / L of total terpenes, which was 35% higher than that of the single-strain system ( FIG10 ).

[0093] [Example 4] β-patchouli biosynthesis

[0094] The codon-optimized β-patchoulene synthase gene (XM_044587644) was cloned into pTRC-HisA to generate plasmid pTRC-PCL.

[0095] Single strain system:

[0096] A single-strain system for β-patchoulene biosynthesis was generated by transforming E. coli BL21 (DE3) with pMVA and pTRC-PCL plasmids. A single colony was inoculated into 5 mL of LB (Luria-Bertani) medium and cultured at 37°C for 12 hours to obtain an overnight culture. The overnight 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 reached 0.6, it was induced with 0.1 mM IPTG and incubated at 16°C. 2% w / v glucose was added as a carbon source and 10% v / v decane was added as an organic phase to extract terpenes. The total terpene yield reached approximately 7 mg / L (Figure 12).

[0097] Dual strain co-culture system:

[0098] For the dual-strain co-cultivation system, the feeder strain was generated by transforming the pMevt and Puc-19 plasmids into E. coli BL21 (DE3). The production strain for β-patchoulene biosynthesis was generated by transforming the plasmids pMBIS and pTRC-PCL 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 to obtain overnight cultures. Overnight cultures of the feeder strain and the production strain at different ratios (37.5% to 62.5%) were inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, it was induced with 0.1 mM IPTG and incubated at 16°C. 2% w / v glucose was added as a carbon source and 10% v / v decane was added to extract terpenes. The dual-strain co-culture system containing 37.5% of the feeder strain produced 25 mg / L of total terpenes, which was three times that of the single-strain system ( FIG12 ).

[0099] [Example 5] Aristolochicine Biosynthesis

[0100] The codon-optimized aristolochene synthase gene (PDB-3M01_A) was cloned into pTRC-HisA to generate plasmid pTRC-TEAS.

[0101] Single strain system:

[0102] A single strain system for aristolochene biosynthesis was generated by transforming E. coli BL21 (DE3) with pMVA and pTRC-TEAS plasmids. A single colony was inoculated into 5 mL LB (Luria-Bertani) medium and cultured at 37°C for 12 hours to obtain an overnight culture. The overnight culture was inoculated into 25 mL TB medium and incubated at 37°C and 200 rpm. Once the OD value at 600 nm reached 0.6, it was induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source and 10% v / v decane was added as an organic phase to extract terpenes. The total terpene yield reached approximately 3 mg / L (Figure 14).

[0103] Dual strain co-culture system:

[0104] For the dual-strain co-cultivation system, the feeder strain was generated by transforming the pMevt and Puc-19 plasmids into E.coli BL21 (DE3). The production strain for β-aristolochicine biosynthesis was generated by transforming the 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. Overnight cultures of the feeder strain and the production strain at different ratios (25% to 62.5%) were inoculated into 25 mL TB (Terrific Broth) medium and incubated at 37°C and 200 rpm. Once the OD at 600 nm reached 0.6, it was induced with 0.1 mM IPTG and incubated at 30°C. 2% w / v glucose was added as a carbon source and 10% v / v decane was added to extract terpenes. The dual-strain co-culture system containing 25% feeder strain produced 15 mg / L of total terpenes, which was five times that of the single-strain system ( FIG14 ).

[0105] Although some exemplary embodiments of the present application have been illustrated and described, the present application is not limited to the disclosed embodiments. On the contrary, those skilled in the art will recognize that some modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present application as described in the appended claims.

Claims

1. A co-cultivation system for synthesizing terpenes, comprising at least one feeder strain and at least one production strain, wherein: The feeder bacteria are used to provide mevalonic acid, and the production strain is used to synthesize terpenes using the mevalonic acid.

2. The co-cultivation system according to claim 1, characterized in that: The feeder strain contains a first plasmid, which contains a gene for synthesizing mevalonic acid and does not contain a gene for synthesizing isoprenyl pyrophosphate. Preferably, the feeder strain further contains a second plasmid, which is a blank plasmid that does not require gene insertion.

3. The co-cultivation system according to claim 1 or 2, characterized in that: The production strain contains a third plasmid and a fourth plasmid, wherein the third plasmid contains a gene for synthesizing farnesyl pyrophosphate and does not contain a gene for synthesizing mevalonic acid, and the fourth plasmid contains a gene for the terpene synthase or a fusion gene of the terpene synthase and the GGPP synthase.

4. The co-cultivation system according to any one of claims 1 to 3, characterized in that: The ratio of the feeder strain to the production strain is (0.25-99.75): (99.75-0.25), preferably (1.25-75): (98.75-25).

5. The co-cultivation system according to any one of claims 1 to 4, characterized in that: The terpene comprises one or more of taxadiene, α-bunielene, valencene, α-guaiacene, β-patchoulene or aristolochene.

6. The co-cultivation system according to any one of claims 1 to 5, characterized in that: The feeder strain and / or the production strain are selected from one or more of bacteria, fungi or yeast.

7. The co-cultivation system according to any one of claims 1 to 6, characterized in that: The metabolic pathway of the feeder strain and / or the production strain comprises an endogenous isoprenoid synthesis pathway.

8. A method for synthesizing terpenes using the co-cultivation system according to any one of claims 1 to 7, the method comprising incubating the feeder strain and the production strain in the presence of an inducer, and an organic phase or solid phase resin for separating terpenes from an aqueous phase.

9. The method according to claim 8, characterized in that: The content of the feeder strain is 0.25% to 90%, preferably 0.5% to 75%, based on the total inoculum volume of the feeder strain and the production strain; And / or the organic phase is selected from decane, undecane, dodecane, isopropyl decanoate, isopropyl laurate, isopropyl octanoate, isopropyl myristate, preferably decane; the solid phase resin is selected from Dionex HP20, Dowax 20, XAD7HP, HP-2MG, preferably Dionex HP20; wherein the organic phase accounts for 5%-30% v / v of the culture, preferably 10%-20% v / v.

10. Use of the co-cultivation system according to any one of claims 1 to 7 or the method according to any one of claims 8 to 9 in synthesizing terpenes.

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