CN111019874A

CN111019874A - Construction method and fermentation process of recombinant bacteria for producing artemisinin precursor

Info

Publication number: CN111019874A
Application number: CN201811176936.4A
Authority: CN
Inventors: 张聪强
Original assignee: Hangzhou Aikousi Biological Technology Co ltd
Current assignee: Hangzhou Aikousi Biological Technology Co ltd
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2020-04-17

Abstract

The invention relates to a construction method and a process of engineering bacteria for producing amorpha-4,11-diene (amorpha-4, 11-diene). The strain for efficiently producing the artemisinin precursor amorpha-4,11-diene (amorpha-4,11-diene) is characterized by comprising the following steps of: obtaining recombinant bacteria by regulating and controlling the expression and/or activity of escherichia coli and exogenous genes; the genes comprise an acetoacetyl coenzyme A thiolase atoB gene, an HMG-CoA synthase HMGs gene, an HMG-CoA reductase hmgR gene, a mevalonate kinase mevK gene, a phosphohydroxyproline kinase pmk gene, a mevalonate-5-pyrophosphate decarboxylase pmd gene, an isopentenyl pyrophosphate isomerase idi gene, a farnesyl pyrophosphate synthase ispA gene and an amorpha-4,11-diene synthase ads gene.

Description

Construction method and fermentation process of recombinant bacteria for producing artemisinin precursor

Technical Field

The invention relates to a construction method and a process of engineering bacteria for producing amorpha-4,11-diene (amorpha-4, 11-diene).

Background

Published by the World Health organization, in 2010, 2.19 million people were infected with Malaria and 66 million deaths were attributed to Malaria infection [ World Health organization. World Malaria Report 2012(WHO,2012) ]. Malaria is derived from the Plasmodium spp. Among them, Plasmodium vivax has been widely resistant to traditional antimalarial drugs such as chloroquine and sulfadoxine. Artemisinin and its derivatives are a new generation of rapid-acting and low-toxicity antimalarial drugs, and are one of the main components of the currently most effective Antimalarial Combination Therapies (ACT). Artemisinin is an antimalarial drug extracted from traditional Chinese medicine Artemisia annua in 1971 by U-yo of China pharmacy, and also has antitumor effect (hence, U-yo obtains the prize of Laske clinical medicine research and the prize of Nobel medicine). However, the content of artemisinin in natural artemisia apiacea is very low (0.1-0.5%), and the defects of large content difference, self-incompatibility and the like exist. Meanwhile, the influence of fluctuation of external natural environment (weather and the like) and market demand and the like is added, so that the supply of the sweet wormwood is unstable, the price is high and low, and the treatment of the world poor population is not facilitated. The price reaches $600/kg in the early 2012, and the price is reduced to $300/kg in the end of 2013. At low prices, farmers reduce the planting of artemisia apiacea and turn to other commercial crops, thus exacerbating market uncertainty and supply instability. In addition, the cultivation of the sweet wormwood in a large amount inevitably occupies more valuable cultivated land. In view of the goal of sustainable development, there is a need to establish a method for producing artemisinin that is sustainable, stable, less polluting, does not occupy arable land and has price advantage.

At present, artemisinin biosynthesis-related genes are introduced into microorganisms and their biosynthetic pathways are reconstituted by metabolic engineering methods. The method was first successfully constructed in yeast by Jay D.Keasling, the academy of engineering, and related technologies were assigned to Sainunflat, France, 2013, Nature 496, 528-plus 532 (2013). However, because of the problems of excessive cost and price fluctuation, the company Sonofugen stopped producing artemisinin and sold it in 2015 (synthetic biology's first large drug measures market resistance,2016, Mark Peplow, Naturenes). Therefore, there is a need to construct a more efficient and less costly strain and fermentation process. By virtue of the characteristics of fast growth and reproduction and low fermentation cost of microorganisms (such as escherichia coli) in the text, a large amount of artemisinin which is cheap and can be produced in a short time can be produced. Due to the microbial method cycle (2-3 days), production can be carried out as required without worrying about problems of over-supply, price fluctuation and the like. In recent years, the research on artemisinin metabolism engineering is rapidly advanced, and the artemisinin metabolism engineering is expected to become a main method for producing artemisinin in the future. In the artemisinin biosynthetic pathway, amorpha-4,11-diene synthase (ADS) catalyzes terpene precursors FPP (farnesyl pyrophosphate) to form artemisinin precursors amorpha-4,11-diene (AD) (fig. 1). FPP is synthesized by IPP and DMAPP, which can be synthesized by two metabolic pathways, mevalonate pathway (MVA) and non-mevalonate pathway (MEP). The MVA approach is mainly adopted to realize the high-efficiency production of amorpha-4,11-diene, and a corresponding fermentation process with low cost and easy separation is developed.

Disclosure of Invention

The invention aims to develop an engineering bacterium for high-yield amorpha fruticosa-4, 11-diene (artemisinin precursor) and a corresponding fermentation process with low cost and easy separation.

The vector is introduced into host cells through artificial control components, wherein one or more, one or more artificial control components exist in each host cell, the artificial control components comprise a vector, a promoter, a 5' untranslated region and an encoding module, and the encoding module is operably connected with the artificial control components. The coding module comprises a hmgS-atoB-hmgR module, a mevK-pmk-pmd-idi module, an ads-ispA module and a hmgS-atoB-hmgR-mevK-pmk-pmd-idi module. The significance of the genes in the individual modules is as follows: atoB is acetoacetyl-CoA thiolase gene, HMG S is HMG-CoA synthase gene, hmgR is HMG-CoA reductase gene, mevK is mevalonate kinase gene, pmk is phosphohydroxyproline kinase gene, pmd is mevalonate-5-pyrophosphate decarboxylase gene, idi is isopentenyl pyrophosphate isomerase gene, ispA is farnesyl pyrophosphate synthase gene, ads is amorpha-4,11-diene synthase gene. Each enzyme gene in the coding module is correspondingly provided with a Ribosome Binding Site (RBS) in a 5' untranslated region, for example, if the coding module of the artificial control component is a hmgS-atoB-hmgR module, three Ribosome Binding Sites (RBS) are respectively used for controlling the hmgS gene, the atoB gene and the hmgR gene. The optimization steps of the engineering bacteria are as follows: a. constructing a vector library, a promoter library and a 5' untranslated region library; b. global regulatory vectors and promoters and local fine-tuning of the 5' untranslated region. Combinations of three modules may be employed, the hmgrs-atoB-hmgR module, the mevK-pmk-pmd-idi and the ads-ispA module; or a combination of two modules, the hmgS-atoB-hmgR-mevK-pmk module, the pmd-idi-ads-ispA module; or a combination of two modules, the hmgS-atoB-hmgR-mevK-pmk-pmd-idi module, the ads-ispA module; or an integral module, hmgS-atoB-hmgR-mevK-pmd-pmd-idi-ads-ispA.

The module combination obtains the optimal strain through the pre-defined biological regulatory elements (vector, promoter and 5' untranslated region) and the thallus library formed by a high-dimensional method, and is assisted by a proper screening method.

Preferably, the 5' untranslated region of the present invention includes a Ribosome Binding Site (RBS). Different RBS sequences can affect the translation speed and expression amount of protein, and engineering bacteria can be optimized by selecting different Ribosome Binding Sites (RBS).

Preferably, the present invention is controlled by a vector or a promoter or a combination of both to globally regulate and search for bottlenecks in the whole biological system, and local regulation is optimized by the 5' untranslated region to locally optimize the bottleneck module.

Preferably, the host cell of the present invention is Escherichia coli.

Preferably, the vector of the invention is pBR322 or p15A or pMB1 or ColE1 or pSC101 or variants thereof.

Preferably, the promoter of the invention is T7 or TM 1-255. Wherein, TM1-255 refers to TM1 to TM255, and the details are described in the 2017110721556 patent.

Preferably, the 5 'untranslated region of the present invention includes a ribosome binding site of 5' URT-1-32. Wherein 5 ' URT-1-32 is 5 ' URT-1 to 5 ' UTR-32.

Drawings

FIG. 1 is a schematic diagram of a strain optimization method in example 1 of the present invention.

FIG. 2 is a library of 5 'untranslated regions (5' UTRs) of the present invention.

FIG. 3 shows the construction of different cells in example 1 of the present invention.

FIG. 4 shows the yields of different species in example 1 of the present invention.

FIG. 5 is the batch yield in shake flasks of example 2 of the invention.

FIG. 6 shows the liquid-liquid synchronous extraction of fed-batch fermentation in example 3 of the present invention, in which the organic phase is dodecane.

FIG. 7 shows the liquid-liquid synchronous extraction (different medium from example 3) of fed-batch fermentation in example 4 of the present invention, and dodecane is used as the organic phase.

FIG. 8 shows the liquid-liquid synchronous extraction of fed-batch fermentation in example 5 of the present invention, wherein the organic phase is isopropyl myristate (isopropyl myristate).

FIG. 9 shows the liquid-solid simultaneous adsorption of fed-batch fermentation in example 6 of the present invention, the solid phase being reversed phase silica gel (silicagel) C18.

Detailed Description

The present invention will be described in further detail below by way of examples with reference to the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting the present invention.

See fig. 1-7.

Example 1: strain optimization

The biosynthetic pathway of this example is shown in FIG. 1, and the construction method of engineering bacteria includes global optimization and local optimization, specifically, by using predefined regulatory elements (the regulatory elements refer to vectors, promoters and 5' untranslated regions) to finely assemble a thallus library in a high-dimensional full-combinatorial manner, and combining analytical chemistry (gas phase mass spectrometer) to screen the microorganism strains with the highest yield.

The flow chart of the engineering bacteria for producing high-value natural products is shown in figure 1:

(1) first, promoter and RBS libraries were established and characterized experimentally or mathematically. Promoter libraries (T7, TM1-255) were established experimentally, with details given in the 2017110721556 patent application. A library of 5 'untranslated regions was created and characterized by mathematical modeling (5' URT-1-32), see FIG. 2.

(2) Plasmid construction by using TM1, TM2 or TM3 to link and control the different modules (hmgS-atoB-hmgR module, mevK-pmk-pmd-idi and ads-ispA module; or a combination of two modules; hmgS-atoB-hmgR-mevK-pmk module, pmd-idi-ads-ispA module; or a combination of two modules; hmgS-atoB-hmgR-mevK-pmk-pmd-idi module, ads-ispA module; or an integral module, hmgS-atoB-hmgR-mevK-pmd-pmd-idi-ads-ispA).

(3) The 5 ' untranslated region of the mutant plasmid was transformed to be different (5 ' URT-1, 5 ' URT-2, 5 ' URT-11 or 5 ' URT-28) and transfected into E.coli cells to obtain 108 different species (FIG. 3).

(4) 108 different species were cultured in shake flasks in 2xpy medium (20g/L peptone, 10g/L yeast extract, 10g/L NaCl, 30mM HEPES, 10g/L glycerol and 0.5% (v/v) Tween 80, pH 7.0) at 28 ℃.

(5) The yield of amorpha-4,11-diene was determined by gas mass spectrometry, extracting amorpha-4,11-diene by diluting 5 μ L dodecane phase into 495 μ L ethyl acetate and analyzing on an Agilent7980A gas chromatograph equipped with an Agilent 5975C mass spectrometer (GC/MS) and scanning 189 and 204m/z ions (SIM mode) using trans-caryophyllene as internal standard.

(6) As shown in FIG. 4, the yield of strain 323-2(TM3, TM2, TM3 and 5M3-2 internal standard) in the shake flask reached 4g/L, which is 1580 times higher than that of the lowest strain (strain 123-4, 2.53 mg/L).

Example 2: basal media optimization

Step 1) preparation of 5L of chemically defined Medium comprising 10g/L glucose, 2g/L (NH)₄)₂SO₄，4.2g/LKH₂PO₄And 11.24g/LK₂HPO₄1.7g/L citric acid, 0.5g/L MgSO₄And 10mL/L of the trace element solution. Wherein the trace element solution contains 0.25g/L CoCl₂·6H₂O，1.5g/L MnSO₄·4H₂O，0.15g/L CuSO₄·2H₂O，0.3g/LH₃BO₃，0.25g/L Na₂MoO₄·2H₂O，0.8g/L Zn(CH₃COO)₂5g/L of iron (III) citrate and 0.84g/L of EDTA, the pH of the microelement solution was 8.0.

Step 2) sterilizing the culture medium for 15-20 minutes at 121 ℃. (Note that the glucose solution is sterilized separately and then mixed with the other ingredients).

Step 3) Next, the strain 323-2 was pre-cultured in the chemically defined medium of step 2, and cultured at 37 ℃ for about 14 hours to prepare a bacterial solution.

Step 4) 1% of strain 323-2 was inoculated into 25mL of the medium of step 2 and the effect of different concentrations of glucose on the product was compared. After 6 hours at 37 ℃ for fermentation, when OD reached 0.8-1.5, cells were induced with 0.1mmol/LIPTG and the temperature was reduced to 28-30 ℃ for further 72 hours. After induction, the product was extracted by adding 5ml of dodecane.

And 5) centrifugally separating the dodecane from the fermentation liquor after the fermentation is finished, taking the dodecane, diluting the dodecane by 100 times in hexane, and injecting the dodecane into a gas phase mass spectrometer for analyzing products.

As shown in FIG. 5, the yield of amorpha-4,11-diene decreased with increasing glucose concentration despite the increased cell mass, with the optimum glucose concentration being 10 g/L. Meanwhile, the yield of amorpha-4,11-diene is gradually increased and reaches the maximum within 72 hours. At 72 hours, 10g/L glucose produced 1.8g/L amorpha-4,11-diene in chemically defined medium with a carbon yield of 18% (slightly higher than Gejie. Baselin optimized strain 16.98%, Proc. Natl. Acad. Sci. U.S. A.109, E111-E118 (2012)).

Example 3: feed batch fermentation sample injection medium optimization

Step 1) preparation of 5L of chemically defined Medium comprising 10g/L glucose, 2g/L (NH)₄)₂SO₄，4.2g/LKH₂PO₄And 11.24g/L K₂HPO₄1.7g/L citric acid, 0.5g/L MgSO₄And 10mL/L of the trace element solution. Wherein the trace element solution contains 0.25g/L CoCl₂·6H₂O，1.5g/L MnSO₄·4H₂O，0.15g/L CuSO₄·2H₂O，0.3g/LH₃BO₃，0.25g/L Na₂MoO₄·2H₂O，0.8g/L Zn(CH₃COO)₂5g/L of iron (III) citrate and 0.84g/L of EDTA, the pH of the microelement solution was 8.0.

Step 4) 2% of the strain 323-2 was inoculated into a fermenter, the pH was controlled at 7.1 using 15% ammonia solution and the starting temperature was controlled at 37 ℃. The initial glucose concentration of the culture medium is 10-15 g/L.

Step 5) Once OD reaches about 6-10, feeding is started, either continuously or at intervals (e.g., every 6 hours), typically with glucose concentration controlled between 2-10 g/L. Whether to continue feeding is determined by directly measuring the glucose concentration or indirectly analyzing the dissolved oxygen value. The feed medium contained 500g/L glucose and 5g/L MgSO₄。

And 6) the induction time is that OD reaches 30-60 (about 15-18 hours after inoculation), the temperature is controlled at 30 ℃ after induction, air is used as an oxygen source, the ventilation amount is controlled at about 1vvm, the oxygen concentration is ensured to be more than 1mg/L as far as possible, and the inducer is 0.1mmol/L IPTG.

And 7) after induction, adding dodecane serving as an extracting agent, wherein the volume ratio of the extracting agent to fermentation broth of the fermentation tank is 15 to 100. The whole fermentation process is about 72 hours.

And 8) centrifugally separating dodecane from the fermentation liquid after the fermentation is finished, taking an organic phase (dodecane), diluting the organic phase in hexane by 1000 times, and then injecting the organic phase into a gas phase mass spectrometer for analyzing products.

As a result, as shown in FIG. 6, the yield of amorpha-4,11-diene reached 13.2g/L after 72 hours (carbon yield was around 7%). .

Example 4: feed batch fermentation sample injection medium optimization

Step 4) 2% of the strain 323-2 E.coli strain E was inoculated into a fermenter, the pH was controlled at 7.1 using 15% ammonia solution and the starting temperature was controlled at 37 ℃. The initial glucose concentration of the culture medium is 10-15 g/L.

Step 5) Once OD reaches about 6-10, feeding is started, either continuously or at intervals (e.g., every 6 hours), typically with glucose concentration controlled between 2-10 g/L. Whether to continue feeding is determined by directly measuring the glucose concentration or indirectly analyzing the dissolved oxygen value. The feed medium contained 500g/L glucose, 5g/L MgSO₄And 25g/L peptone.

As a result, as shown in FIG. 7, the yield of amorpha-4,11-diene reached about 30g/L after 70 hours (carbon yield was about 15%).

Example 5: selection of extractant for fed-batch fermentation

And 6) the induction time is that OD reaches 30-60 (about 15-18 hours after inoculation), the temperature is controlled at 30 ℃ after induction, air is used as an oxygen source, the air flow is controlled at about 1vvm, the oxygen concentration is ensured to be more than 1mg/L as far as possible, and the inducer is 0.1 mmol/LIPTG.

And 7) after induction, adding isopropyl myristate as an extractant, wherein the volume ratio of the extractant to fermentation broth of the fermentation tank is 15 to 100. The whole fermentation process is about 72 hours.

And 8) centrifugally separating isopropyl myristate from fermentation liquor after fermentation is finished, taking an organic phase (isopropyl myristate), diluting the organic phase in hexane by 1000 times, and then feeding the organic phase into a gas phase mass spectrometer for analyzing products.

As a result, as shown in FIG. 8, the yield of amorpha-4,11-diene reached about 25g/L after 70 hours (carbon yield was about 13%).

Example 6: use of solid sorbents for fed-batch fermentation

Step 1) preparation of 5L of chemically defined Medium comprising 10g/L glucose, 2g/L (NH)₄)₂SO₄，4.2g/LKH₂PO₄And 11.24g/LK₂HPO₄1.7g/L citric acid, 0.5g/L MgSO₄And 10mL/L of the trace element solution. Wherein the trace element solution contains 0.25g/L CoCl₂·6H₂O，1.5g/L MnSO₄·4H₂O，0.15g/LCuSO₄·2H₂O，0.3g/LH₃BO₃，0.25g/L Na₂MoO₄·2H₂O，0.8g/L Zn(CH₃COO)₂5g/L of iron (III) citrate and 0.84g/L of EDTA, the pH of the microelement solution was 8.0.

And 7) after induction, adding reversed phase silica gel C18 (solid particles) as a solid sorbent, wherein the addition amount of the sorbent is 0.5-1 g/L. The whole fermentation process was 72 hours.

And 8) filtering or settling to separate solid phase reverse phase silica gel C18 and fermentation liquor after fermentation is finished, washing the reverse phase silica gel C18 with water, and extracting amorpha fruticosa-4, 11-diene adsorbed on the reverse phase silica gel with hexane. The dried reverse phase silica gel was reused.

The gas phase mass spectrometer analyzed the product concentration in hexane.

As a result, as shown in FIG. 8, the yield of amorpha-4,11-diene reached about 13.2g/L after 70 hours.

The fermentation liquid obtained by synchronous extraction and fermentation of the organic phase is centrifuged to separate the organic phase and the water phase (containing bacteria), wherein the product is in the organic phase, so that the product can be separated by distillation, extraction and the like and used as a medicinal product for synthesizing artemisinin or other medicines. The organic phase can be recovered by evaporation or rectification.

The fermentation liquid after solid phase simultaneous adsorption fermentation is centrifuged and membrane shared to separate water phase, solid phase and thallus, wherein the product is in solid phase and is further extracted with solvent to obtain product for use in medicine product to synthesize artemisinin or other medicines. The solid sorbent is directly recycled after being washed and dried. The sorbent can also be fixed inside by devices such as filler, so that the separation and the reutilization are simpler.

The above description of the present invention is intended to be illustrative. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.

Claims

1. A strain for efficiently producing an artemisinin (artemisinine) precursor amorpha-4,11-diene (amorpha-4,11-diene) is characterized in that: obtaining recombinant bacteria by regulating and controlling the expression and/or activity of escherichia coli and exogenous genes; the genes comprise an acetoacetyl coenzyme A thiolase atoB gene, an HMG-CoA synthase HMGs gene, an HMG-CoA reductase hmgR gene, a mevalonate kinase mevK gene, a phosphohydroxyproline kinase pmk gene, a mevalonate-5-pyrophosphate decarboxylase pmd gene, an isopentenyl pyrophosphate isomerase idi gene, a farnesyl pyrophosphate synthase ispA gene and an amorpha-4,11-diene synthase ads gene.

2. The method for constructing engineering bacteria according to claim 1, wherein:

1) global regulation of all enzymes throughout the biosynthetic pathway is modularized in groups to reduce the number of combinations, each controlled by a quantitative vector or promoter or a combination of both. The optimal species was obtained by screening combinations of different vectors, promoters and grouping modules.

2) The grouping of modules may result in a single or several modules becoming a bottleneck for the overall biological system. On the basis of global regulation, the internal optimization of the bottleneck module is realized by adopting 5' untranslated regions with different strengths, so that the efficiency of the whole biological system is further improved.

3. The engineered bacteria of claims 1 and 2, characterized in that: the gene coding composition module is connected with the regulation and control assembly, and the coding module comprises:

a combination of three modules, the hmgrs-atoB-hmgR module, the mevK-pmk-pmd-idi and the ads-ispA module;

or a combination of two modules, the hmgS-atoB-hmgR-mevK-pmk module and the pmd-idi-ads-ispA module;

or a combination of two modules, the hmgS-atoB-hmgR-mevK-pmk-pmd-idi module and the ads-ispA module;

or an integral module, hmgS-atoB-hmgR-mevK-pmd-pmd-idi-ads-ispA.

4. The regulatory method and regulatory components of claims 1 and 2 comprising different vectors, promoters, 5' untranslated regions comprising Ribosome Binding Sites (RBSs), or combinations thereof.

5. The method for constructing engineering bacteria according to claims 1 and 2, wherein: the vector is pBR322 or p15A or pMB1 or ColE1 or pSC101 or their variants.

6. The method for constructing engineering bacteria according to claims 1 and 2, wherein: the promoter is T7, TM1-TM 255; the 5 'untranslated region includes a ribosome binding site of 5' URT-1-32.

7. The yield of the precursor amorpha fruticosa-4, 11-diene is improved by adopting a two-phase synchronous extraction fermentation or synchronous adsorption fermentation process so as to reduce the subsequent separation cost.

8. The two phases are characterized in that:

1) the combination of water phase and organic phase, the water phase is used for thallus growth and product synthesis, the organic phase is used for extracting lipophilic product amorpha fruticosa-4, 11-diene;

2) or the combination of water phase and solid phase, wherein the water phase is used for thallus growth and product synthesis, and the solid phase is used for extracting hydrophobic product amorpha-4, 11-diene.

9. The process of claim 8, wherein: the organic phase is one of or a combination of alkanes such as decane, undecane or dodecane, which have no obvious inhibition on the growth of the thallus, alcohols such as decanol or lauryl alcohol and other alcohols which have no obvious inhibition on the growth of the thallus, esters such as isopropyl myristate, methyl myristate and methyl laurate, which have no obvious inhibition on the growth of the thallus, and lipids such as edible oils such as peanut oil, sunflower seed oil, soybean oil, butter or lard. The addition of the organic phase can improve the yield and yield of the product.

10. The process of claim 8, wherein: the solid phase can selectively adsorb amorpha-4,11-diene such as reversed phase silica gel. The reversed phase silica gel can not only adsorb the product, but also simplify downstream separation, thereby effectively reducing the production cost.