Multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol and application of multi-bacteria system

Abstract

The invention relates to a multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol. The multi-bacteria system comprises an upstream strain and a downstream strain, the upstream strain can be used for converting a substrate glucose into 2-butanone acid through pyruvic acid or converting the substrate glucose into 3-methyl-2-oxyvaleric acid through 2-butanone acid; the downstream strain can be used for converting the 2-butanone acid into the 2-methylbutyric acid / alcohol through the 3-methyl-2-oxyvaleric acid, or converting the 3-methyl-2-oxyvaleric acid into the 2-methylbutyric acid / alcohol. An upstream strain and a downstream strain in the multi-bacteria system are obtained by modifying escherichia coli, the metabolic pressure of a host is smaller, the functional specificity is higher, and more substances and energy in cells are used for synthesizing intermediate products and target products. Under the condition of the same substrate concentration, the yield of the 2-methylbutyric acid / alcohol synthesized by the multi-bacteria system is obviously higher than that of the 2-methylbutyric acid / alcohol synthesized by pure culture, which indicates that the multi-bacteria system has higher synthesis efficiency and lower cost in the 2-methylbutyric acid / alcohol.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol and application thereof. Background Art

[0002] 2-Methylbutyric acid is an important precursor for the production of various valuable products in the manufacturing, pharmaceutical and food industries, so the market demand is large. 2-Methylbutanol can be used as a synthetic raw material to introduce a rotating pentyl group, and it also has the potential to replace gasoline as the next generation of green fuel. At present, the main method for synthesizing 2-methylbutyric acid / alcohol is to oxidize it from fossil energy such as fusel oil. However, with the depletion of fossil resources, finding alternative production methods has become a new research direction. There are a variety of catalytic enzymes in microbial cell factories, which can provide suitable reaction conditions and environment for the synthesis and conversion of 2-methylbutyric acid / alcohol, and the synthesis process is more green and gentle.

[0003] Currently, the synthesis of 2-methylbutyric acid / alcohol can be completed by pure culture of microorganisms. However, in the pure culture system, the overexpression of heterologous pathways in the recombinant strain will increase the metabolic pressure of the chassis cells, leading to an imbalance in energy distribution in the cells, which in turn has an adverse effect on the normal growth of the chassis cells and the biosynthesis of the target product. Compared with the pure culture system, the multi-bacteria system is a more novel and effective biosynthetic platform. While providing a diverse and adaptive expression environment for the biosynthesis of the target product, it can also effectively distribute the metabolic burden of the chassis cells and make the global regulation of the pathway more flexible.

[0004] Therefore, there is a need to develop a multi-bacteria system with high yield, easy to transform and control, to carry out green biosynthesis of 2-methylbutyric acid / alcohol, and to promote the development of the application field of 2-methylbutyric acid / alcohol. Summary of the invention

[0005] The purpose of the present invention is to improve the yield of 2-methylbutyric acid / alcohol in biosynthesis and develop a multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol. The multi-bacteria system is easy to transform and control, and the yield of the target product 2-methylbutyric acid / alcohol is high.

[0006] The first aspect of the present invention provides a multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol, which comprises an upstream strain and a downstream strain; wherein the upstream strain is capable of converting a substrate glucose into 2-ketobutyric acid via pyruvic acid or converting a substrate glucose into 3-methyl-2-oxypentanoic acid via 2-ketobutyric acid; and the downstream strain is capable of converting 2-ketobutyric acid into 2-methylbutyric acid / alcohol via 3-methyl-2-oxypentanoic acid or converting 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol.

[0007] According to the present invention, the upstream strains include upstream strain mc2MBA-up-1 and upstream strain mc2MBA-up-2, and the downstream strains include downstream strain mc2MBA-down-1 and downstream strain mc2MBA-down-2.

[0008] In some embodiments of the present invention, the plasmid contained in the upstream strain mc2MBA-up-1 contains a gene encoding citrate synthase, a gene encoding 3-isopropylmalate dehydrogenase, and a gene encoding 3-isopropylmalate dehydratase; preferably, the upstream strain mc2MBA-up-1 can convert the substrate glucose into pyruvate, and then convert pyruvate into 2-ketobutyric acid.

[0009] In some embodiments of the present invention, the plasmid contained in the upstream strain mc2MBA-up-2 contains a gene encoding citrate synthase, a gene encoding 3-isopropylmalate dehydrogenase, a gene encoding 3-isopropylmalate dehydratase, and a gene encoding acetolactate synthase; preferably, the upstream strain mc2MBA-up-2 can convert the substrate glucose into 2-ketobutyric acid, and then convert 2-ketobutyric acid into 3-methyl-2-oxopentanoic acid.

[0010] In some embodiments of the present invention, the plasmid contained in the downstream strain mc2MBA-down-1 contains a gene encoding acetolactate synthase, a gene encoding 2-ketoacid decarboxylase, and a gene encoding aldehyde dehydrogenase; preferably, the downstream strain mc2MBA-down-1 can convert 2-ketobutyric acid into 3-methyl-2-oxopentanoic acid, and then convert 3-methyl-2-oxopentanoic acid into 2-methylbutyric acid / alcohol.

[0011] In some embodiments of the present invention, the plasmid contained in the downstream strain mc2MBA-down-2 contains a gene encoding 2-ketoacid decarboxylase and a gene encoding aldehyde dehydrogenase; preferably, the downstream strain mc2MBA-down-2 can convert 3-methyl-2-oxopentanoic acid into 2-methylbutyric acid / alcohol.

[0012] Preferably, the gene encoding citrate synthase is the gene cimA encoding citrate synthase derived from Methanococcus jannaschii or the gene cimA encoding citrate synthase derived from Methanococcus jannaschii and optimized for codons.

[0013] Preferably, the gene encoding 3-isopropylmalate dehydrogenase is the gene leuB encoding 3-isopropylmalate dehydrogenase derived from Escherichia coli.

[0014] Preferably, the genes encoding 3-isopropylmalate dehydratase are the genes leuC and leuD encoding 3-isopropylmalate dehydratase derived from Escherichia coli.

[0015] Preferably, the gene encoding acetolactate synthase is the gene ilvG and ilvM encoding acetolactate synthase derived from Escherichia coli.

[0016] Preferably, the gene encoding 2-ketoacid decarboxylase is the gene kivD encoding 2-ketoacid decarboxylase derived from Lactococcus lactis or the gene kivD encoding 2-ketoacid decarboxylase derived from Lactococcus lactis and codon-optimized.

[0017] Preferably, the gene encoding aldehyde dehydrogenase is the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae or the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae and codon-optimized.

[0018] According to some embodiments of the present invention, the multi-bacteria system is a multi-bacteria system I, which comprises an upstream strain mc2MBA-upper-1 and a downstream strain mc2MBA-lower-1; wherein the upstream strain mc2MBA-upper-1 converts the substrate glucose into pyruvate, and then converts the pyruvate into 2-butanone; accordingly, the downstream strain mc2MBA-lower-1 converts the 2-butyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and then converts the 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol.

[0019] According to some other embodiments of the present invention, the multi-bacteria system is a multi-bacteria system II, which comprises an upstream strain mc2MBA-upper-2 and a downstream strain mc2MBA-lower-2; wherein the upstream strain mc2MBA-upper-2 converts the substrate glucose into 2-ketobutyric acid, and then converts 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid; accordingly, the downstream strain mc2MBA-lower-2 converts the 3-methyl-2-oxypentanoic acid synthesized by the upstream strain mc2MBA-upper-2 into 2-methylbutyric acid / alcohol.

[0020] According to some other embodiments of the present invention, the multi-bacteria system is a multi-bacteria system III, which comprises an upstream strain mc2MBA-upper-2 and a downstream strain mc2MBA-lower-1; wherein the upstream strain mc2MBA-upper-2 converts the substrate glucose into 2-ketobutyric acid, and then converts the 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid; accordingly, the downstream strain mc2MBA-lower-1 converts the excess 2-ketobutyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and then converts the 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol; preferably, in the multi-bacteria system III, the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-1 are both capable of converting 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid.

[0021] In the present invention, host bacteria used for expressing plasmids include Escherichia coli, Corynebacterium glutamicum, yeast, and modified bacteria and fungi; preferably, the host bacteria is Escherichia coli.

[0022] The second aspect of the present invention provides the use of the multi-bacteria system as described in the first aspect of the present invention in the synthesis of 2-methylbutyric acid / alcohol.

[0023] According to the present invention, the application comprises using a multi-bacteria system to carry out fermentation culture to synthesize 2-methylbutyric acid / alcohol.

[0024] Specifically, the application includes inoculating the upstream strain and the downstream strain in the multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol into the fermentation medium at an initial inoculation volume ratio of 19:1 to 1:19 for fermentation; preferably, the initial inoculation volume ratio is 9:1 to 1:9.

[0025] In some embodiments of the present invention, the fermentation culture temperature is 33-38°C.

[0026] In some embodiments of the present invention, the fermentation culture time is 60h-84h.

[0027] In the present invention, the inducers are IPTG and L-ara, and the two inducers are added simultaneously.

[0028] In some embodiments of the present invention, the IPTG induction concentration is 0.05-1 mM, preferably 0.1-1 mM.

[0029] In some embodiments of the present invention, the L-ara induction concentration is 0.05 mM-20 mM, preferably 10-20 mM.

[0030] The advantages of the present invention are:

[0031] (1) The upstream strains and downstream strains included in the multi-bacteria systems I, II, and III mentioned in the present invention are all obtained through transformation. Compared with the strains containing the complete 2-methylbutyrate / alcohol synthesis pathway, the strains containing only part of the 2-methylbutyrate / alcohol synthesis pathway have less metabolic pressure and stronger functional specificity, so there are more substances and energy in the cell for the synthesis of intermediates and target products. The synthesis of 2-methylbutyrate / alcohol can be completed by combining the above strains with partial functions of synthesizing 2-methylbutyrate / alcohol.

[0032] (2) Synthesizing 2-methylbutyric acid / alcohol in a multi-bacteria system can change the expression intensity of each module in the pathway by adjusting the initial inoculation ratio of the upstream and downstream strains. This adjustment method is simpler in operation than the transformation method of the pure culture system.

[0033] (3) Under the condition of the same substrate concentration, the yield of 2-methylbutyric acid / alcohol synthesized by the multi-bacteria system was significantly higher than that by pure culture, and there was basically no accumulation of acetic acid in the fermentation broth without inhibiting acetic acid synthesis, indicating that the multi-bacteria system has an effective substrate utilization rate and a higher yield in the synthesis of 2-methylbutyric acid / alcohol. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the present invention easy to understand, the present invention will be described in detail below with reference to the accompanying drawings.

[0035] Figure 1 The reaction mechanisms of the synthesis of 2-methylbutyric acid / alcohol by multi-bacteria system I, multi-bacteria system II, and multi-bacteria system III are shown.

[0036] Figure 2 This is the yield diagram of 2-ketobutyric acid, 3-methyl-2-oxypentanoic acid and 2-methylbutyric acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by multi-bacteria system I (mc2MBA-upper-1-mc2MBA-lower-1).

[0037] Figure 3 This is a graph showing the production of acetic acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by the multi-bacteria system I (mc2MBA-upper-1-mc2MBA-lower-1).

[0038] Figure 4 This is the yield diagram of 2-ketobutyric acid, 3-methyl-2-oxypentanoic acid and 2-methylbutyric acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by multi-bacteria system II (mc2MBA-upper-2-mc2MBA-lower-2).

[0039] Figure 5 This is a graph showing the production of acetic acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by the multi-bacteria system II (mc2MBA-upper-2-mc2MBA-lower-2).

[0040] Figure 6 This is the yield diagram of 2-ketobutyric acid, 3-methyl-2-oxypentanoic acid and 2-methylbutyric acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by multi-bacteria system III (mc2MBA-upper-2-mc2MBA-lower-1).

[0041] Figure 7 This is a graph showing the production of acetic acid at different inoculation ratios during the fermentation production of 2-methylbutyric acid by the multi-bacteria system III (mc2MBA-upper-2-mc2MBA-lower-1). DETAILED DESCRIPTION

[0042] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and do not represent limitations.

[0043] Where a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicit exclusions in the specified range. Where the specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0044] Ⅰ. Embodiment

[0045] As mentioned above, there is currently a need to develop a multi - strain system with high productivity and easy to modify and control. For this purpose, the present inventors have studied, designed, and developed a multi - strain system for synthesizing 2 - methylbutyric acid / alcohol that can significantly increase the production of 2 - methylbutyric acid / alcohol in biosynthesis.

[0046] This multi - strain system includes an upstream strain and a downstream strain;

[0047] Among them, the upstream strain includes:

[0048] (1) The upstream strain mc2MBA - up - 1, whose plasmid contains the gene encoding citrate synthase, the gene encoding 3 - isopropylmalate dehydrogenase, and the gene encoding 3 - isopropylmalate dehydratase, which can convert the substrate glucose into pyruvate and then convert pyruvate into 2 - oxobutyric acid;

[0049] (2) The upstream strain mc2MBA - up - 2, whose plasmid contains the gene encoding citrate synthase, the gene encoding 3 - isopropylmalate dehydrogenase, the gene encoding 3 - isopropylmalate dehydratase, and the gene encoding acetolactate synthase, which can convert the substrate glucose into 2 - oxobutyric acid and then convert 2 - oxobutyric acid into 3 - methyl - 2 - oxopentanoic acid;

[0050] The downstream strain includes:

[0051] (1) The downstream strain mc2MBA - down - 1, whose plasmid contains the gene encoding acetolactate synthase, the gene encoding 2 - ketoacid decarboxylase, and the gene encoding aldehyde dehydrogenase, which can convert 2 - oxobutyric acid into 3 - methyl - 2 - oxopentanoic acid and then convert 3 - methyl - 2 - oxopentanoic acid into 2 - methylbutyric acid / alcohol;

[0052] (2) The downstream strain mc2MBA - lower - 2 contains genes encoding 2 - ketoacid decarboxylase and aldehyde dehydrogenase in its plasmid, and it can convert 3 - methyl - 2 - oxopentanoic acid into 2 - methylbutyric acid / alcohol.

[0053] Preferably, the gene encoding citrate synthase is the gene cimA encoding citrate synthase derived from Methanococcus jannaschii (the sequence is shown as SEQ ID NO.1) or the gene cimA encoding citrate synthase derived from Methanococcus jannaschii and codon - optimized (the sequence is shown as SEQ ID NO.2).

[0054] Preferably, the gene encoding 3 - isopropylmalate dehydrogenase is the gene leuB encoding 3 - isopropylmalate dehydrogenase derived from Escherichia coli, and its sequence is shown as SEQ ID NO.3.

[0055] Preferably, the genes encoding 3 - isopropylmalate dehydratase are the genes leuC (the sequence is shown as SEQ ID NO.4) and leuD (the sequence is shown as SEQ ID NO.5) encoding 3 - isopropylmalate dehydratase derived from Escherichia coli.

[0056] Preferably, the genes encoding acetolactate synthase are the genes ilvG (the sequence is shown as SEQ ID NO.6) and ilvM (the sequence is shown as SEQ ID NO.7) encoding acetolactate synthase derived from Escherichia coli.

[0057] Preferably, the gene encoding 2 - ketoacid decarboxylase is the gene kivD encoding 2 - ketoacid decarboxylase derived from Lactococcus lactis (the sequence is shown as SEQ ID NO.8) or the gene kivD encoding 2 - ketoacid decarboxylase derived from Lactococcus lactis and codon - optimized (the sequence is shown as SEQ ID NO.9).

[0058] Preferably, the gene encoding aldehyde dehydrogenase is the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae (the sequence is shown as SEQ ID NO.10) or the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae and codon - optimized (the sequence is shown as SEQ ID NO.11).

[0059] In the present invention, the host bacteria for expressing the plasmid include Escherichia coli, Corynebacterium glutamicum, yeast, and genetically modified bacteria and fungi; preferably, the host bacterium is Escherichia coli BL21(DE3).

[0060] The study found that compared with the traditional 2-methylbutyrate / alcohol pure culture synthesis system, the above-mentioned multi-bacteria system has significantly reduced the metabolic pressure of chassis cells, can coordinate the expression intensity of each module in the pathway by adjusting the upstream and downstream inoculation ratios, and has a higher 2-methylbutyrate / alcohol production.

[0061] According to some embodiments of the present invention, the present invention provides a multi-bacteria system I, the reaction mechanism of which synthesizes 2-methylbutyric acid / alcohol is as follows: Figure 1 As shown, the multi-bacteria system I comprises:

[0062] (1) an upstream strain mc2MBA-upper-1, which converts the substrate glucose into pyruvate, and then converts the pyruvate into 2-butanone; and

[0063] (2) Downstream strain mc2MBA-lower-1; it converts 2-ketobutyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and then converts 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol.

[0064] Preferably, based on the initial inoculation ratio, the initial inoculation volume ratio of the upstream strain mc2MBA-upper-1 and the downstream strain mc2MBA-lower-1 is 19:1 to 1:19, and more preferably 9:1 to 1:9.

[0065] Preferably, the host cells of the upstream strain mc2MBA-upper-1 and the downstream strain mc2MBA-lower-1 are Escherichia coli BL21 (DE3).

[0066] According to other embodiments of the present invention, the present invention provides a multi-bacteria system II, the reaction mechanism of which for synthesizing 2-methylbutyric acid / alcohol is as follows: Figure 1 As shown, the multi-bacteria system II comprises:

[0067] (1) an upstream strain mc2MBA-upper-2, which converts the substrate glucose into 2-ketobutyrate, and further converts 2-ketobutyrate into 3-methyl-2-oxypentanoate; and

[0068] (2) The downstream strain mc2MBA-lower-2 converts the 3-methyl-2-oxypentanoic acid synthesized by the upstream strain mc2MBA-upper-2 into 2-methylbutanoic acid / alcohol.

[0069] Preferably, based on the initial inoculation ratio, the initial inoculation volume ratio of the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-2 is 19:1 to 1:19, and more preferably 9:1 to 1:9.

[0070] Preferably, the host cells of the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-2 are Escherichia coli BL21 (DE3).

[0071] According to some other embodiments of the present invention, the present invention provides a multi-bacteria system III, the reaction mechanism of which for synthesizing 2-methylbutyric acid / alcohol is as follows: Figure 1 As shown, the multi-bacteria system III comprises:

[0072] (1) an upstream strain mc2MBA-upper-2, which converts the substrate glucose into 2-ketobutyrate, and further converts 2-ketobutyrate into 3-methyl-2-oxypentanoate; and

[0073] (2) a downstream strain mc2MBA-lower-1, which converts the excess 2-ketobutyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and further converts 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol;

[0074] In particular, in the multi-bacteria system III, the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-1 both have the function of converting 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid.

[0075] Preferably, based on the initial inoculation ratio, the initial inoculation volume ratio of the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-1 is 19:1 to 1:19, and more preferably 9:1 to 1:9.

[0076] Preferably, the host cells of the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-1 are Escherichia coli BL21 (DE3).

[0077] The second aspect of the present invention provides the use of the multi-bacteria system described in the first aspect of the present invention in synthesizing 2-methylbutyric acid / alcohol, which can be understood as a method for synthesizing 2-methylbutyric acid / alcohol using the multi-bacteria system described in the first aspect of the present invention.

[0078] According to the present invention, the application / method comprises respectively connecting the above-mentioned multi-bacteria system I, multi-bacteria system II and multi-bacteria system III for synthesizing 2-methylbutyric acid / alcohol to fermentation medium, performing fermentation culture, and producing 2-methylbutyric acid / alcohol.

[0079] Preferably, in terms of initial inoculation ratio, the volume ratio of the initial inoculation of the upstream strain and the downstream strain in the multi-bacteria system is 19:1 to 1:19, more preferably 9:1 to 1:9.

[0080] In some embodiments of the present invention, the fermentation culture conditions are:

[0081] (1) The fermentation medium is M9Y medium;

[0082] (2) The fermentation culture temperature is 33-38°C, preferably 37°C;

[0083] (3) The fermentation culture time is 60h-84h, preferably 72h;

[0084] (4) The inducers are IPTG and L-ara, and the two inducers are added simultaneously when the system OD600 is 0.6; wherein the IPTG induction concentration is 0.05-1mM, preferably 0.1mM; the L-ara induction concentration is 0.05mM-20mM, preferably 10mM.

[0085] Research has found that the upstream strain and downstream strain included in the multi-bacteria system provided by the present invention are obtained by transforming Escherichia coli, and the host metabolic pressure is smaller and the functional specificity is stronger, so there are more substances and energy in the cell for the synthesis of intermediates and target products. In addition, the expression intensity of each module in the 2-methylbutyric acid / alcohol pathway can be changed by adjusting the initial inoculation ratio of the upstream and downstream strains, and this adjustment method is simpler in operation relative to the transformation method of the pure culture system. Under the condition of the same substrate concentration, the output of 2-methylbutyric acid / alcohol synthesized by the multi-bacteria system is significantly higher than the output of 2-methylbutyric acid / alcohol synthesized by pure culture, indicating that the multi-bacteria system has a higher synthesis efficiency of 2-methylbutyric acid / alcohol and a lower cost.

[0086] Example

[0087] In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are only illustrative and are not limited to the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial channels or conventional methods unless otherwise specified. The specific experimental methods not mentioned in the following examples are usually carried out according to conventional experimental methods.

[0088] Example 1: Construction of recombinant plasmids contained in upstream strains mc2MBA-upper-1, mc2MBA-upper-2 and downstream strains mc2MBA-lower-1, mc2MBA-lower-2

[0089] The primers used in this example are shown in Table 1. The plasmid contained in the upstream strain mc2MBA-up-1 contains the gene encoding citrate synthase, the gene encoding 3-isopropylmalate dehydrogenase, and the gene encoding 3-isopropylmalate dehydratase. The target genes were amplified using 1-F / 1-R and 2-F / 2-R, respectively. Then, the vector was amplified by PCR, and the amplified vector and target fragment were recovered. After that, the target gene was inserted into the Escherichia coli expression plasmid pE8a to obtain plasmid pE8a-up-1 (see Table 2).

[0090] Table 1 Related primers for constructing recombinant plasmids

[0091]

[0092] Table 2 Plasmids and strains

[0093]

[0094]

[0095] In Table 2, pE8a is a plasmid owned by the laboratory, and pETduet was obtained commercially.

[0096] The plasmid contained in the upstream strain mc2MBA-up-2 contains the gene encoding citrate synthase, the gene encoding 3-isopropylmalate dehydrogenase, the gene encoding 3-isopropylmalate dehydratase, and the gene encoding acetolactate synthase. The target gene was amplified using 3-F / 3-R. Then, the vector pE8a-up-1 was amplified by PCR, and the amplified vector and target fragment were recovered. After that, the target gene was inserted into the Escherichia coli expression plasmid pE8a to obtain plasmid pE8a-up-2 (see Table 2).

[0097] The downstream strain mc2MBA-down-1 contains the gene encoding acetolactate synthase, the gene encoding 2-keto acid decarboxylase, and the gene encoding aldehyde dehydrogenase. The target fragment was amplified using 4-F / 4-R and 5-F / 5-R. The amplified vector and target fragment were recovered, and then the target gene was inserted into the Escherichia coli expression plasmid pETduet to obtain plasmid pETduet-down-1 (see Table 2).

[0098] The downstream strain mc2MBA-down-2 contains the gene encoding 2-keto acid decarboxylase and the gene encoding aldehyde dehydrogenase. The target fragment was amplified using 5-F / 5-R. The amplified vector and target fragment were recovered, and then the target gene was inserted into the Escherichia coli expression plasmid pETduet to obtain plasmid pETduet-down-2 (see Table 2).

[0099] Example 2: Construction of upstream strains mc2MBA-upper-1, mc2MBA-upper-2 and downstream strains mc2MBA-lower-1, mc2MBA-lower-2

[0100] Prepare competent cells of Escherichia coli BL21 (DE3) and dispense 50 μL into 1.5 mL EP tubes for electroporation. Add 2-5 μL of the constructed recombinant plasmids pE8a-upper-1, pE8a-upper-2, pETduet-lower-1, and pETduet-lower-2 to 1.5 mL centrifuge tubes containing 50 μL competent cells. Then use an electroporator to electrotransform the plasmid into the competent cells. After electroporation, quickly add LB medium [yeast powder 10 g / L, peptone 20 g / L, NaCl 20 g / L, sterilized at 116°C for 25 min. Add 1.5%-2% agar to the corresponding solid medium], and transfer the mixture to a 1.5 mL centrifuge tube and resuscitate at 37°C for 20-30 min. Then apply the bacterial solution to a plate containing the corresponding antibiotics and culture at 37°C for 12 h. The upstream strains mc2MBA-upper-1 and mc2MBA-upper-2 and the downstream strains mc2MBA-lower-1 and mc2MBA-lower-2 were prepared (see Table 2).

[0101] Example 3: Preparation of a multi-bacterial system for synthesizing 2-methylbutyric acid

[0102] Single colonies were picked from the plates with upstream strains mc2MBA-upper-1 and mc2MBA-upper-2 and downstream strains mc2MBA-lower-1 and mc2MBA-lower-2, and inoculated into 5 mL of liquid LB medium with resistance, respectively, and cultured at 37°C, 200 rpm for 12 hours. Then, the upstream strain and the downstream strain were transferred to a 250 mL conical flask containing 25 mL of fermentation medium according to an inoculation amount of 1% of the total bacterial liquid.

[0103] Among them, the initial inoculation ratios of mc2MBA-upper-1:mc2MBA-lower-1 of multi-bacteria system I were 9:1, 6:1, 3:1, 1:1, 3, 1:6, and 1:9, respectively.

[0104] The initial inoculation ratios of mc2MBA-upper-2:mc2MBA-lower-2 in the multi-bacteria system II were 9:1, 6:1, 3:1, 1:1, 3, 1:6, and 1:9, respectively.

[0105] The initial inoculation ratios of mc2MBA-upper-2:mc2MBA-lower-1 of the multi-bacteria system III were 9:1, 6:1, 3:1, 1:1, 3, 1:6, and 1:9, respectively.

[0106] There is no particular limitation on the fermentation medium in the present invention, as long as it is a fermentation medium for producing 2-methylbutyric acid / alcohol. Preferably, the fermentation medium formula is: each liter of fermentation medium includes: 20g glucose, 5g yeast extract, 11.3g M9 medium, 15.7g 3-morpholinepropanesulfonic acid, 2mM MgSO4, 0.1mM CaCl2, 2.78mg FeSO4, 1mg Vitamin B1, and 1mL trace elements.

[0107] Example 4: Application of Multi-bacteria System I in the Synthesis of 2-Methylbutyric Acid

[0108] When the total OD600 in the fermentation medium containing the multibacterial system I is 0.4-0.6, L-ara is added to a final concentration of 10mM and IPTG is added to a final concentration of 0.1mM for induction. The fermentation culture conditions are 37°C, 200rpm, and the culture time is 72h. After sampling, the fermentation broth is centrifuged at 12000rpm for 1min. After aspirating the supernatant, it is filtered through a 0.22μm aqueous filter membrane. High performance liquid chromatography is then used for qualitative and quantitative detection of 2-methylbutyric acid. The 2-methylbutyric acid yield of the multibacterial system I at different initial inoculation ratios (mc2MBA-upper-1:mc2MBA-lower-1) is shown in Figure 2 Figure 2 The yield of 2-methylbutyric acid synthesized from glucose by multi-bacteria system I ranged from 0.014 g / L to 0.17 g / L.

[0109] After the same 2-methylbutyrate synthesis pathway was fully expressed in Escherichia coli BL21 (DE3), the production of 2-methylbutyrate was only 0.062 g / L (e.g. Figure 2 As shown). After the synthesis of 2-methylbutyric acid by the multi-bacteria system I, when the initial inoculation ratio of mc2MBA-upper-1:mc2MBA-lower-1 was 1:1, the yield of 2-methylbutyric acid was the highest, which was 0.17 g / L, 2.7 times the yield of pure culture. In addition, in the experimental groups with different upstream and downstream initial inoculation ratios of the multi-bacteria system I, the accumulation of the intermediate product 2-butyric acid was generally higher than that of the pure culture system, and decreased with the decrease of the inoculation amount of the upstream strain mc2MBA-upper-1. Similarly, when the initial inoculation ratio was between 9:1 and 3:1, the intermediate product 3-methyl-2-oxypentanoic acid was basically not accumulated, and when the initial inoculation ratio was between 1:1 and 1:9, the intermediate product 3-methyl-2-oxypentanoic acid increased with the increase of the downstream strain mc2MBA-lower-1. In addition, when the multi-bacteria system I was fermenting 2-methylbutyric acid, at different initial inoculation ratios, the accumulation of acetic acid in the fermentation broth was as shown Figure 3As shown. At the initial inoculation ratio with the highest 2-methylbutyric acid production (i.e., mc2MBA-upper-1: mc2MBA-lower-1 = 1:1), no acetic acid accumulated in the fermentation broth, while a large amount of acetic acid accumulated in the fermentation broth during the synthesis of 2-methylbutyric acid in pure culture. This indicates that the substrates were all used for the synthesis of 2-methylbutyric acid. In summary, the multi-bacteria system I is superior to the pure culture system in the synthesis and conversion of intermediates and the synthesis of target products.

[0110] Example 5: Application of Multi-bacteria System II in the Synthesis of 2-Methylbutyric Acid

[0111] When the total OD600 in the fermentation medium containing the multibacterial system II is 0.4-0.6, L-ara is added to a final concentration of 10mM and IPTG is added to a final concentration of 0.1mM for induction. The fermentation culture conditions are 37°C, 200rpm, and the culture time is 72h. After sampling, the fermentation broth is centrifuged at 12000rpm for 1min. After aspirating the supernatant, it is filtered through a 0.22μm aqueous filter membrane. High performance liquid chromatography is then used for qualitative and quantitative detection of 2-methylbutyric acid. The 2-methylbutyric acid yield of the multibacterial system II at different initial inoculation ratios (mc2MBA-upper-2: mc2MBA-lower-2) is shown in Figure 2. Figure 4 The yield of 2-methylbutyric acid synthesized from glucose by the multi-bacteria system ranged from 0.13 g / L to 0.35 g / L.

[0112] After the same 2-methylbutyrate synthesis pathway was fully expressed in Escherichia coli BL21 (DE3), the production of 2-methylbutyrate was only 0.062 g / L (e.g. Figure 4 As shown). After the synthesis of 2-methylbutyric acid with the multi-bacteria system II, when the initial inoculation ratio of mc2MBA-upper-2-mc2MBA-lower-2 was 1:1, the yield of 2-methylbutyric acid was the highest, which was 0.35 g / L, 5.6 times the yield of pure culture. In addition, in the experimental groups with different upstream and downstream initial inoculation ratios of the multi-bacteria system II, the accumulation of the intermediate product 2-butyric acid was generally higher than that of the pure culture system, and decreased with the decrease of the inoculation amount of the upstream strain mc2MBA-upper-2. Similarly, when the initial inoculation ratio was between 9:1 and 1:1, the intermediate product 3-methyl-2-oxypentanoic acid was basically not accumulated. When the initial inoculation ratio was between 1:1 and 1:9, the intermediate product 3-methyl-2-oxypentanoic acid increased with the increase of the downstream strain mc2MBA-lower-2. In addition, when the multi-bacteria system II was fermenting 2-methylbutyric acid, the accumulation of acetic acid in the fermentation broth at different initial inoculation ratios was as shown below. Figure 5As shown. At the initial inoculation ratio with the highest 2-methylbutyric acid production (i.e., mc2MBA-upper-2: mc2MBA-lower-1 = 1:1), no acetic acid accumulated in the fermentation broth, while a large amount of acetic acid accumulated in the fermentation broth during the pure culture synthesis of 2-methylbutyric acid. This indicates that at this time, all substrates are used for the synthesis of 2-methylbutyric acid. In summary, the multi-bacteria system II is superior to the pure culture system in the synthesis and conversion of intermediates and the synthesis of target products.

[0113] Example 6: Application of Multi-bacteria System III in 2-Methylbutyric Acid Synthesis

[0114] When the total OD600 in the fermentation medium containing the multibacterial system III is 0.4-0.6, L-ara is added to a final concentration of 10mM and IPTG is added to a final concentration of 0.1mM for induction. The fermentation culture conditions are 37°C, 200rpm, and the culture time is 72h. After sampling, the fermentation broth is centrifuged at 12000rpm for 1min. After aspirating the supernatant, it is filtered through a 0.22μm aqueous filter membrane. High performance liquid chromatography is then used for qualitative and quantitative detection of 2-methylbutyric acid. The 2-methylbutyric acid yield of the multibacterial system III under different initial inoculation ratios (mc2MBA-upper-2: mc2MBA-lower-1) is shown in the figure. Figure 6 The yield of 2-methylbutyric acid synthesized from glucose by the multi-bacteria system ranged from 0.063 g / L to 0.12 g / L.

[0115] After the same 2-methylbutyrate synthesis pathway was fully expressed in Escherichia coli BL21 (DE3), the production of 2-methylbutyrate was only 0.062 g / L (e.g. Figure 6 As shown). After the synthesis of 2-methylbutyric acid with the multi-bacteria system III, when the initial inoculation ratio of mc2MBA-upper-2-mc2MBA-lower-1 was 1:3, the yield of 2-methylbutyric acid was the highest, which was 0.12 g / L, 1.9 times the yield of pure culture. In addition, in the experimental groups with different upstream and downstream initial inoculation ratios of the multi-bacteria system III, the accumulation of the intermediate product 2-butyric acid was generally higher than that of the pure culture system, and decreased with the decrease in the inoculation amount of the upstream strain mc2MBA-upper-2. In addition, when the multi-bacteria system III was fermenting 2-methylbutyric acid, the accumulation of acetic acid in the fermentation broth at different initial inoculation ratios was as shown below. Figure 7 As shown in the figure, with the increase of downstream strain inoculation amount, the accumulated acetic acid in the fermentation broth increased. In the group with the highest 2-methylbutyric acid production (i.e., mc2MBA-upper-2: mc2MBA-lower-1 = 1:3), the accumulated acetic acid in the fermentation broth was slightly lower than that in the pure culture. In summary, the multi-bacteria system II is superior to the pure culture system in the synthesis and conversion of intermediates and the synthesis of target products.

[0116] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol, comprising an upstream strain and a downstream strain; wherein: The upstream strain can convert the substrate glucose into 2-ketobutyric acid via pyruvate or convert the substrate glucose into 3-methyl-2-oxopentanoic acid via 2-ketobutyric acid; the downstream strain can convert 2-ketobutyric acid into 2-methylbutyric acid / alcohol via 3-methyl-2-oxopentanoic acid, or convert 3-methyl-2-oxopentanoic acid into 2-methylbutyric acid / alcohol.

2. The multi-bacteria system according to claim 1, characterized in that: The upstream strains include upstream strain mc2MBA-up-1 and upstream strain mc2MBA-up-2, and the downstream strains include downstream strain mc2MBA-down-1 and downstream strain mc2MBA-down-2.

3. The multi-bacterial system according to claim 2, wherein the plasmid contained in the upstream strain mc2MBA-up-1 contains the gene encoding citrate synthase, the gene encoding 3-isopropylmalate dehydrogenase, and the gene encoding 3-isopropylmalate dehydratase; preferably, the upstream strain mc2MBA-up-1 can convert the substrate glucose into pyruvate and then convert pyruvate into 2-ketobutyric acid; and / or, the plasmid contained in the upstream strain mc2MBA-up-2 contains the gene encoding citrate synthase, the gene encoding 3-isopropylmalate dehydrogenase, the gene encoding 3-isopropylmalate dehydratase, and the gene encoding acetolactate synthase; preferably, the upstream strain mc2MBA-up-2 can convert the substrate glucose into 2-ketobutyric acid and then convert 2-ketobutyric acid into 3-methyl-2-oxopentanoic acid; and / or, the plasmid contained in the downstream strain mc2MBA-down-1 contains the gene encoding acetolactate synthase, the gene encoding 2-ketoacid decarboxylase, and the gene encoding aldehyde dehydrogenase; preferably, the downstream strain mc2MBA-down-1 can convert 2-ketobutyric acid into 3-methyl-2-oxopentanoic acid and then convert 3-methyl-2-oxopentanoic acid into 2-methylbutyric acid / alcohol; and / or, the plasmid contained in the downstream strain mc2MBA-down-2 contains the gene encoding 2-ketoacid decarboxylase and the gene encoding aldehyde dehydrogenase; preferably, the downstream strain mc2MBA-down-2 can convert 3-methyl-2-oxopentanoic acid into 2-methylbutyric acid / alcohol.

4. The multi-bacterial system according to claim 3, wherein the gene encoding citrate synthase is the gene cimA encoding citrate synthase derived from Methanococcus jannaschii or the gene cimA encoding citrate synthase derived from Methanococcus jannaschii and codon-optimized; and / or, the gene encoding 3-isopropylmalate dehydrogenase is the gene leuB encoding 3-isopropylmalate dehydrogenase derived from Escherichia coli; and / or, the gene encoding 3-isopropylmalate dehydratase is the genes leuC and leuD encoding 3-isopropylmalate dehydratase derived from Escherichia coli; and / or, the gene encoding acetolactate synthase is the genes ilvG and ilvM encoding acetolactate synthase derived from Escherichia coli; and / or, the gene encoding 2-ketoacid decarboxylase is the gene kivD encoding 2-ketoacid decarboxylase derived from Lactococcus lactis or the gene kivD encoding 2-ketoacid decarboxylase derived from Lactococcus lactis and codon-optimized; And / or, the gene encoding aldehyde dehydrogenase is the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae or the gene ALD2 encoding aldehyde dehydrogenase derived from Saccharomyces cerevisiae and subjected to codon optimization.

5. The multi-bacteria system according to any one of claims 2 to 4, characterized in that: The multi-bacteria system is a multi-bacteria system I, which comprises an upstream strain mc2MBA-upper-1 and a downstream strain mc2MBA-lower-1; wherein the upstream strain mc2MBA-upper-1 converts the substrate glucose into pyruvate, and then converts the pyruvate into 2-butanone; correspondingly, the downstream strain mc2MBA-lower-1 converts the 2-butyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and then converts the 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol.

6. The multi-bacteria system according to any one of claims 2 to 4, characterized in that: The multi-bacteria system is a multi-bacteria system II, which comprises an upstream strain mc2MBA-upper-2 and a downstream strain mc2MBA-lower-2; wherein the upstream strain mc2MBA-upper-2 converts the substrate glucose into 2-ketobutyric acid, and then converts 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid; correspondingly, the downstream strain mc2MBA-lower-2 converts the 3-methyl-2-oxypentanoic acid synthesized by the upstream strain mc2MBA-upper-2 into 2-methylbutyric acid / alcohol.

7. The multi-bacteria system according to any one of claims 2 to 4, characterized in that: The multi-bacteria system is a multi-bacteria system III, which comprises an upstream strain mc2MBA-upper-2 and a downstream strain mc2MBA-lower-1; wherein the upstream strain mc2MBA-upper-2 converts the substrate glucose into 2-ketobutyric acid, and then converts the 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid; accordingly, the downstream strain mc2MBA-lower-1 converts the excess 2-ketobutyric acid synthesized by the upstream strain mc2MBA-upper-1 into 3-methyl-2-oxypentanoic acid, and then converts the 3-methyl-2-oxypentanoic acid into 2-methylbutyric acid / alcohol; preferably, in the multi-bacteria system III, the upstream strain mc2MBA-upper-2 and the downstream strain mc2MBA-lower-1 are both capable of converting 2-ketobutyric acid into 3-methyl-2-oxypentanoic acid.

8. The multi-bacteria system according to any one of claims 1 to 7, characterized in that: Host bacteria used for expressing plasmids include Escherichia coli, Corynebacterium glutamicum, yeast, and modified bacteria and fungi; preferably, the host bacteria is Escherichia coli.

9. Use of the multi-bacteria system according to any one of claims 1 to 8 in synthesizing 2-methylbutyric acid / alcohol; preferably, the use comprises using the multi-bacteria system for fermentation culture to synthesize 2-methylbutyric acid / alcohol.

10. The use according to claim 9, characterized in that: The application comprises inoculating the upstream strain and the downstream strain in the multi-bacteria system for synthesizing 2-methylbutyric acid / alcohol into the fermentation medium at an initial inoculation volume ratio of 19:1 to 1:19 for fermentation; preferably, the initial inoculation volume ratio is 9:1 to 1:

9.

11. The use according to claim 10, characterized in that: The fermentation culture temperature is 33-38°C; And / or, the fermentation culture time is 60h-84h; And / or, the inducers are IPTG and L-ara, and the two inducers are added simultaneously; preferably, the IPTG induction concentration is 0.05-1mM, preferably 0.1-1mM; the L-ara induction concentration is 0.05mM-20mM, preferably 10-20mM.

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