Artificial synthesis method for malonyl-coa and use of same

Abstract

Disclosed in the present invention are an artificial synthesis method for malonyl-CoA and the use of same. By means of heterologous expression of an aminotransferase and a malonyl-CoA reductase, an artificial synthesis pathway using β-alanine (β-ala) as a precursor for synthesizing malonyl-CoA is constructed, which comprises: first, under the catalysis of the aminotransferase, β-ala transferring an amino group to an α-keto acid (such as pyruvic acid, oxaloacetic acid or α-ketoglutaric acid), so as to form an intermediate product 3-oxopropanoic acid and a corresponding amino acid; and 3-oxopropanoic acid then generating malonyl-CoA under the action of the malonyl-CoA reductase. The pathway solves the defects of a natural malonyl-CoA synthesis pathway, such as low carbon utilization rates, consuming an energy substance ATP, releasing greenhouse gas CO2, and pathway enzymes pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC) therein being strictly regulated, thereby achieving high yields of products using malonyl-CoA as a precursor, including flaviolin, caprylic acid, phloroglucinol, pentadecaheptaene, natamycin, spinosad, etc.

Description

Artificial synthesis method of malonyl-CoA and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for artificially synthesizing malonyl-CoA and its application. Background Art

[0002] Malonyl-CoA is considered a central compound in biological metabolism. In primary metabolism, malonyl-CoA is an essential extension unit for the synthesis of long-chain fatty acids, precursors of cell membrane phospholipids. Furthermore, long-chain fatty acids undergo esterification with methanol / ethanol to form fatty acid methyl esters / ethyl esters, the primary components of biodiesel. Compared to traditional petrochemical diesel, biodiesel offers several significant advantages, including renewable raw materials and environmental friendliness, and is considered a new generation of bioenergy.

[0003] In secondary metabolism, malonyl-CoA is the precursor for the synthesis of nearly all polyketides and flavonoids (Figure 1A). Over 10,000 different polyketides have been discovered, and countless new products have been derived from them. According to statistics, over 20 polyketides (such as erythromycin, tetracycline, lovastatin, and avermectin) have become commercialized drugs. Erythromycin is the most commonly used macrolide antibiotic in medicine and animal husbandry, while statins, including lovastatin, account for over 80% of the total lipid-lowering drug market. Global annual sales of these 20 drugs currently exceed US$20 billion. Flavonoids are also a widespread secondary metabolite. Over 8,000 flavonoids have been discovered, most of which possess excellent antiviral, antibacterial, anti-inflammatory, anticancer, and anti-obesity activities. They hold broad application prospects in health supplements, cosmetics, and pharmaceuticals. The flavonoid market is projected to reach US$1.2 billion by 2024.

[0004] With the development of synthetic biology and metabolic engineering, the construction of efficient microbial cell factories to synthesize high-value-added malonyl-CoA derivatives (long-chain fatty acids, polyketides, flavonoids, etc.) has become a research hotspot in the field of biosynthesis (Figure 1A). However, under normal conditions, malonyl-CoA synthesis in microbial cells is relatively low and always at a low level, and malonyl-CoA cannot be added exogenously, thus becoming a key bottleneck limiting the efficient synthesis of derivatives. Based on this, enhancing the supply capacity of intracellular malonyl-CoA is crucial for achieving efficient biosynthesis of derivatives. The currently known natural synthesis pathways of malonyl-CoA mainly include the PDH-ACC pathway and the MCS pathway. The following describes these two pathways separately:

[0005] 1. PDH-ACC pathway: This is the most widespread and most widely used malonyl-CoA synthesis pathway that exists in almost all cells. In this pathway, pyruvate produced by carbon source decomposition is first converted into acetyl-CoA (acetyl-CoA), NADH and CO2 under the catalysis of pyruvate dehydrogenase complex (PDH), and then acetyl-CoA, ATP and HCO3 - Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA (Figure 1B).

[0006] Although it is the most important malonyl-CoA synthesis pathway in cells, this pathway has obvious catalytic drawbacks: First, the decarboxylation reaction catalyzed by PDH produces greenhouse gases (CO2) and also loses a molecule of carbon, reducing atom economy (Figure 1B). Second, the PDH complex is one of the most complex enzymes in nature, consisting of three different subunits: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2), and dihydrolipoyl dehydrogenase (E3). In Gram-negative bacteria, the typical composition of this complex is ([E1] 24 [E2] 24 [E3] 12 ), with a molecular weight of approximately 4600 kDa; in Gram-positive bacteria and eukaryotes, the typical composition of this complex is ([E1] 30 [E2] 60 [E3] 12 ), with a molecular weight of about 8000 to 10000 kDa. Modification and overexpression of such a complex complex will bring a huge burden to the cell. Third, PDH catalysis requires thiamine pyrophosphate (TPP), lipoic acid, FAD, NAD + , CoA, and other cofactors, which are also required for the catalysis of other intracellular enzymes. Therefore, the synthesis of malonyl-CoA is very likely to form a high degree of competition with other intracellular metabolisms. In addition, the catalysis of PDH is strictly inhibited by acetyl-CoA, NADH, etc., among which acetyl-CoA is considered to be a typical PDH inhibitor: 2mM acetyl-CoA can cause a 70% decrease in PDH activity. Finally, PDH activity is also affected by the intracellular energy status. High ATP levels severely inhibit PDH activity: when ADP / ATP is equal to 1, PDH activity is reduced by 90%.

[0007] ACC is composed of three subunits: biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier (BCCP). In addition to the above-mentioned catalytic drawbacks of PDH, ACC catalysis also has obvious drawbacks: First, ACC catalysis requires the consumption of additional ATP, wasting intracellular energy (Figure 1B). Second, the transcription of ACC encoding genes is strictly regulated by the cell. When bacterial growth enters the stable phase, the transcription of mRNA tends to stop. Third, ACC catalysis is inefficient and is susceptible to a variety of metabolites / enzymes such as long-chain fatty acyl-CoA, cyclic adenosine monophosphate (cAMP), AMP-dependent protein kinase (AMPK) and other metabolites / enzymes such as Mg. 2+ 、HCO3 - The allosteric inhibition of ACC by long-chain fatty acyl-CoA is considered a classic example of allosteric inhibition: extremely low concentrations of long-chain fatty acyl-CoA, such as 0.24 μM palmitoyl-CoA (C16-CoA), can lead to a loss of ACC activity of more than 80%.

[0008] 2. MCS pathway: This pathway is relatively narrowly distributed, primarily found in cells such as rhizobia, Arabidopsis, and Pseudomonas fluorescens, as well as in mammalian mitochondria. In this pathway, malonyl-CoA is converted from malonate under the catalysis of malonyl-CoA synthase (MCS) (Figure 1C). The main drawbacks of this pathway include: First, the MCS catalytic process consumes ATP energy. Second, the pathway requires the additional substrate malonate, increasing the cost of biosynthesis. Third, extracellular malonate requires specialized exogenous membrane transporters (such as MatC) for transport into the cell. However, exogenous membrane proteins in microbial cells often face challenges such as difficulty in soluble expression and misfolding. Finally, malonate is a structural analog of succinate and a competitive inhibitor of succinate dehydrogenase in the TCA cycle, a key central carbon metabolism activity. Adding malonate disrupts the normal TCA cycle in cells, severely impacting cell survival and growth. The existence of this series of disadvantages also makes the transformation of increasing the level of malonyl-CoA in microbial cells by introducing exogenous MCS pathways have little effect.

[0009] Summary of the Invention

[0010] In view of this, the object of the present invention is to provide a method for artificially synthesizing malonyl-CoA and its application; the method for artificial malonyl-CoA synthesis provided by the present invention constructs an artificial synthesis pathway for synthesizing malonyl-CoA using β-alanine (β-ala) as a precursor by heterologously expressing an aminotransferase and a malonyl-CoA reductase. First, under the catalysis of the aminotransferase, the amino group of β-ala is transferred to an α-keto acid (such as pyruvate, oxaloacetate or α-ketoglutarate), forming an intermediate product 3-oxopropionic acid and the corresponding α-amino acid. The 3-oxopropionic acid generates malonyl-CoA under the action of the malonyl-CoA reductase. This pathway overcomes the drawbacks of the natural malonyl-CoA biosynthesis pathway, such as low carbon utilization, consumption of the energy substance ATP, release of the greenhouse gas CO2, and strict regulation of the key enzymes pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC). This allows for the efficient synthesis of compounds using malonyl-CoA as a precursor, including flavonoids, octanoic acid, phloroglucinol, pentadecaheptaene, natamycin, and spinosad. The introduction of an artificial malonyl-CoA biosynthesis pathway has also improved cell tolerance to a range of stress conditions, including organic acids, osmotic pressure, and cytotoxic substances.

[0011] The technical solutions provided by the present invention are as follows:

[0012] <First Aspect>

[0013] The present invention provides a method for artificially synthesizing malonyl-CoA, which comprises the following steps:

[0014] S1, β-alanine and the compound represented by formula (1) form 3-oxopropionic acid and the compound represented by formula (2) under the catalysis of aminotransferase;

[0015] Wherein, R is selected from H, alkane, alkanol, and alkanoic acid.

[0016] S2, 3-oxopropionic acid, coenzyme A, and electron acceptor transfer the electrons of 3-oxopropionic acid to the electron acceptor under the catalysis of oxidoreductase; forming malonyl-CoA and 2[H].

[0017] The electron acceptor includes NAD + 、NCD + 、NTD + NGD + , FAD, CoQ(ubiquinol), PQ(plastoquinol), FMN, NADP + , NCDP +, NGDP + NTDP + , oxidized ferredoxin, oxidized cytochrome c, oxidized thioredoxins, oxidized glutaredoxins, O2, etc.

[0018] The 2[H] includes NADH, NCDH, NTDH, NGDH, FADH2, CoQH2, PQH2, FMNH2, NADPH, NCDPH, NGDPH, NTDPH, reduced ferredoxin, reduced cytochrome c, reduced thioredoxins, reduced glutaredoxins, H2O, etc.

[0019] The compound represented by formula (1) includes α-keto acids.

[0020] The α-keto acid is selected from one or more of pyruvic acid, oxaloacetic acid, and α-ketoglutaric acid.

[0021] The compound represented by formula (2) includes α-amino acids.

[0022] The α-amino acid includes α-alanine, aspartic acid or glutamic acid.

[0023] The aminotransferase includes one or more of BauA from Pseudomonas aeruginosa, AptA from Acinetobacter pittii, CCNA_03245 from Caulobacter vibrioides, PydD2 from Bacillus megaterium, YhxA from Bacillus cereus, PydD from Ureibacillus massiliensis, and Pyd4 from Saccharomyces kluyveri.

[0024] The amino acid sequence of BauA is shown in SEQ ID NO.7;

[0025] The amino acid sequence of AptA is shown in SEQ ID NO.8;

[0026] The amino acid sequence of CCNA_03245 is shown in SEQ ID NO.9;

[0027] The amino acid sequence of PydD2 is shown in SEQ ID NO.10;

[0028] The amino acid sequence of YhxA is shown in SEQ ID NO.11;

[0029] The amino acid sequence of PydD is shown in SEQ ID NO.71;

[0030] The amino acid sequence of Pyd4 is shown in SEQ ID NO.72.

[0031] The oxidoreductase includes one or more of MCR-C from Chloroflexus aurantiacu, StMCR from Sulfurisphaera tokodaii, Msed_0709 from Metallosphaera sedula, PnSCR from Pyrobaculum neutrophilum, and SucD from Clostridium kluyveri.

[0032] The amino acid sequence of MCR-C is shown in SEQ ID NO.70;

[0033] The amino acid sequence of StMCR is shown in SEQ ID NO.12;

[0034] The amino acid sequence of Msed_0709 is shown in SEQ ID NO.13;

[0035] The amino acid sequence of the PnSCR is shown in SEQ ID NO.14;

[0036] The amino acid sequence of SucD is shown in SEQ ID NO.15.

[0037] The preparation method of the MCR-C comprises the following steps:

[0038] 1) Construct the recombinant plasmid pCDF-P1-mcrC based on the amino acid sequence of MCR-C;

[0039] 2) Transform the recombinant plasmid pCDF-P1-mcrC into E. coli BL21 DE3 competent cells to obtain positive clones;

[0040] 3) Cultivate the positive monoclonal colonies under the induction of inducers;

[0041] 4) centrifuging and purifying the culture product of step 3), resuspending the bacteria in a non-denaturing lysis buffer, adding lysozyme and mixing, cooling and sonicating, and lysing the bacteria; centrifuging and collecting the supernatant to prepare a bacterial lysate;

[0042] 5) The bacterial lysate prepared in step 4) is subjected to chromatography to obtain a purified MCR-C protein sample.

[0043] As another embodiment of the present invention, the preparation method of MCR-C comprises the following steps:

[0044] 1) Transform the recombinant plasmid pCDF-P1-mcrC into E. coli BL21 (DE3) competent cells to obtain positive clones;

[0045] 2) Take the positive monoclonal colony and inoculate it into liquid LB medium containing spectinomycin and culture it until OD 550 =0.5-0.8, add inducer to induce culture to a final concentration of 0.1-0.2mM;

[0046] 3) Centrifuging the culture product of step 2), collecting the induced bacteria, purifying the protein using a protein purification kit, adding non-denaturing lysis buffer to resuspend the bacteria, adding lysozyme to mix, cooling and sonicating, and lysing the bacteria until the bacterial solution becomes translucent; centrifuging, collecting the supernatant, and preparing a bacterial lysate;

[0047] 4) The bacterial lysate prepared in step 3) is subjected to chromatography to obtain a purified MCR-C protein sample.

[0048] As another embodiment of the present invention, the preparation method of MCR-C comprises the following steps:

[0049] 1) Transform the recombinant plasmid pCDF-P1-mcrC into E. coli BL21 (DE3) competent cells to obtain positive clones;

[0050] 2) Take the positive monoclonal colony and inoculate it into liquid LB medium containing spectinomycin, and culture it with shaking for 8-12 hours until the OD 550 =2.0-2.5 as seed solution, the seed solution was diluted with an initial OD 550 = 0.1-0.2 inoculated into LB medium containing spectinomycin and cultured until OD 550 =0.5-0.8, add the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the culture to a final concentration of 0.1-0.2 mM, and the culture condition is shaking culture;

[0051] 3) Centrifuging the culture product of step 2) to collect the induced bacteria, and then purifying the protein using a His-tag protein purification kit, adding non-denaturing lysis buffer to resuspend the bacteria, adding lysozyme to mix, cooling and sonicating, and lysing the bacteria until the bacterial solution becomes translucent; centrifuging, collecting the supernatant, and preparing a bacterial lysate;

[0052] 4) Purification of proteins using nickel affinity columns: BeyoGold TM His-tag Purification Resin, centrifuge and discard the storage solution, add non-denaturing lysis buffer to the gel and mix well to balance the gel, centrifuge and discard the liquid, add the bacterial lysate prepared in step 3), mix well, load into a nickel column affinity chromatography column and elute to obtain a purified MCR-C protein sample.

[0053] The construction method of the recombinant plasmid pCDF-P1-mcrC comprises the following steps:

[0054] 1) Using the pCDF-duet plasmid containing a 6×His tag as a template, primers P1-R / P1-F were used to amplify a pCDF vector backbone fragment containing a T7 promoter, an Ori sequence, and a spectinomycin resistance gene Smr, referred to as fragment I; wherein the sequence of P1-R is shown in SEQ ID NO.1, the sequence of P1-F is shown in SEQ ID NO.2, and the sequence of fragment I is shown in SEQ ID NO.3;

[0055] 2) Using the pMCR-C plasmid as a template and primers MCR-C-pCDF-P1-up / MCR-C-pCDF-P1-down, the MCR-C coding gene was amplified, referred to as fragment II; wherein the sequence of MCR-C-pCDF-P1-up is shown in SEQ ID NO.4, the sequence of MCR-C-pCDF-P1-down is shown in SEQ ID NO.5, and the sequence of fragment II is shown in SEQ ID NO.6;

[0056] 3) Remove the template from fragments I and II using the endonuclease DpnI, respectively. After electrophoresis, use a PCR purification kit to clean the fragments I and II after template removal, and add 2X Seamless Cloning Mix to react for 20-30 minutes.

[0057] 4) Take the reaction product of step 3) and transform it into Trans-T1 competent cells. Select positive single colonies and perform colony PCR verification with primers P1-YZ-up (SEQ ID NO.54) / MCR-C-YZ-down (SEQ ID NO.55); obtain the correct plasmid pCDF-P1-mcrC; wherein the sequence of P1-YZ-up is shown in SEQ ID NO.54; the sequence of MCR-C-YZ-down is shown in SEQ ID NO.55.

[0058] The preparation method of BauA comprises the following steps:

[0059] Step 1, constructing a recombinant plasmid pCDF-P1-bauA according to the amino acid sequence of bauA;

[0060] Step 2: Transform the recombinant plasmid pCDF-P1-bauA into E. coli BL21 DE3 competent cells to obtain positive clones;

[0061] Step 3: Cultivate the positive monoclonal colonies obtained in step 3 under the induction of an inducer;

[0062] Step 4: centrifuge and purify the culture product of step 3, add non-denaturing lysis buffer to resuspend the bacteria, add lysozyme to mix, cool and sonicate, and lyse the bacteria; centrifuge and collect the supernatant to prepare a bacterial lysate;

[0063] Step 5: The bacterial lysate prepared in step 4 is subjected to chromatography to obtain a purified BauA protein sample.

[0064] <Second Aspect>

[0065] The present invention provides a recombinant plasmid comprising BauA and MCR-C, wherein the amino acid sequence of BauA is shown as SEQ ID NO.7; and the amino acid sequence of MCR-C is shown as SEQ ID NO.70.

[0066] <Third Aspect>

[0067] A method for synthesizing fatty acids using the recombinant plasmid described above, wherein the recombinant plasmid uses pCDF as a vector and is named pCDF-bauA-mcrC; the method comprises the following steps:

[0068] The recombinant plasmid pCDF-bauA-mcrC and the thioesterase expression plasmid were transformed into Escherichia coli competent cells to obtain a positive clone strain, and the fatty acid was obtained after fermentation and culture.

[0069] The fatty acid includes caprylic acid, capric acid, lauric acid, myristic acid or hexadecanoic acid.

[0070] The method for preparing octanoic acid comprises the following steps: transforming recombinant plasmids pCDF-bauA-mcrC and pTrc-TE10 into competent Escherichia coli cells to obtain positive clone strains, and obtaining the octanoic acid after fermentation and culture.

[0071] The construction method of the recombinant plasmid pCDF-bauA-mcrC comprises the following steps:

[0072] Step 1. Using the pCDF-P1-bauA plasmid as a template and primers P2-MCRC-R / P2-MCRC-F, amplify the pCDF-P1-bauA vector backbone fragment, which contains the T7 promoter, the Ori sequence, the bauA coding sequence, and the spectinomycin resistance gene Smr, referred to as fragment III; wherein the sequence of P2-MCRC-R is shown in SEQ ID NO. 25, the sequence of P2-MCRC-F is shown in SEQ ID NO. 26, and the sequence of fragment III is shown in SEQ ID NO. 27;

[0073] Step 2: Using the pCDF-P1-McrC plasmid as a template, primers MCR-C-pCDF-P2-up / MCR-C-pCDF-P2-down were used to amplify the McrC coding gene, referred to as fragment IV; wherein the sequence of fragment IV is shown in SEQ ID NO. 28; the sequence of MCR-C-pCDF-P2-up is shown in SEQ ID NO. 29; and the sequence of MCR-C-pCDF-P2-down is shown in SEQ ID NO. 30;

[0074] Step 3: Remove the template from fragment III and fragment IV using the endonuclease DpnI; after electrophoresis, use a PCR purification kit to clean the fragments; take the template-removed fragments III and IV and add them to the 2X Seamless Cloning Mix for reaction;

[0075] Step 4: Take the reaction product of step 3 and transform it into Trans-T1 competent cells. Select positive single colonies and perform colony PCR verification with primers P2-YZ-up / MCR-C-YZ-down; the verified correct plasmid pCDF-bauA-mcrC is obtained; the sequence of MCR-C-YZ-down is shown in SEQ ID NO. 55; the sequence of P2-YZ-up is shown in SEQ ID NO. 56.

[0076] The recombinant plasmid pCDF-P1-mcrC was constructed based on the amino acid sequence of mcrC, and the amino acid sequence of mcrC is shown in SEQ ID NO.70; the recombinant plasmid pCDF-P1-bauA was constructed based on the amino acid sequence of bauA, and the amino acid sequence of bauA is shown in SEQ ID NO.7.

[0077] <Fourth Aspect>

[0078] A method for synthesizing fatty acids using the recombinant plasmid as described above, wherein the recombinant plasmid uses pCDF as a vector and is named pCDF-bauA-mcrC; the method comprises the following steps: transferring the recombinant plasmid pCDF-bauA-mcrC and a polyketide synthase encoding gene into competent Escherichia coli cells to obtain a positive clone strain, and obtaining the polyketide compound after fermentation and culture.

[0079] The polyketide compounds include phloroglucinol, phloroglucinol, and pentadecene.

[0080] The synthesis method of phloroglucinol comprises the following steps: transforming plasmids pCDF-bauA-mcrC and pCum-phlD into competent Escherichia coli cells to obtain positive clone strains, and obtaining the phloroglucinol after fermentation and culture.

[0081] The synthesis method of flavonoids comprises the following steps: transforming plasmids pCDF-bauA-mcrC and pCum-rppA into competent Escherichia coli cells to obtain positive clone strains, and obtaining the flavonoids after fermentation and culture.

[0082] The synthesis method of pentadecene comprises the following steps: transforming plasmids pCDF-bauA-mcrC and pQL1 into competent Escherichia coli cells to obtain positive clone strains, and obtaining the pentadecene after fermentation and culture.

[0083] The construction method of pCDF-bauA-mcrC is the same as that of the above <Part 3>

[0084] <Fifth Aspect>

[0085] A method for synthesizing natamycin or spinosad using the recombinant plasmid, wherein the recombinant plasmid uses SET152 as a vector and uses strong promoters KasOP and SPL42 to express BauA and MCR-C, respectively, and is named SET152-KasOP-bauA-SPL42-mcrC;

[0086] The synthesis method of natamycin comprises the following steps:

[0087] The recombinant plasmid SET152-KasOP-bauA-SPL42-mcrC was transformed into Streptomyces chrysoporus to obtain a positive clone strain, and the natamycin was obtained after fermentation and culture;

[0088] The synthesis method of the spinosad comprises the following steps:

[0089] The recombinant plasmid SET152-KasOP-bauA-SPL42-mcrC was transformed into Saccharopolyspora spinosa to obtain a positive clone strain, and the spinosad was obtained after fermentation and culture.

[0090] The construction method of plasmid pSET152-KasOP-bauA-SPL42-mcrC is as follows:

[0091] 1) Plasmid pSET152 was digested with XbaI and BamHI, and a 5709 bp pSET152 backbone fragment was recovered after agarose gel electrophoresis, designated as fragment F (SEQ ID NO. 41);

[0092] 2) Using the pLH10 plasmid as a template, primers KasOP-F / R were used to amplify the KasOP promoter fragment, designated Fragment G (SEQ ID NO. 42); using the pLH2 plasmid as a template, primers SPL42-F / R were used to amplify the SPL42 promoter fragment, designated Fragment H (SEQ ID NO. 43); the sequence of KasOP-F is shown in SEQ ID NO. 46; the sequence of KasOP-R is shown in SEQ ID NO. 47; the sequence of SPL42-F is shown in SEQ ID NO. 48; and the sequence of SPL42-R is shown in SEQ ID NO. 49;

[0093] 3) Using the pCDF-bauA-mcrC plasmid as a template, primers BauA-F / R were used to amplify the BauA gene fragment, designated Fragment J (SEQ ID NO. 44), and primers McrC-F / R were used to amplify the McrC gene fragment, designated Fragment K (SEQ ID NO. 45). The sequence of BauA-F is shown in SEQ ID NO. 50; the sequence of BauA-R is shown in SEQ ID NO. 51; the sequence of McrC-F is shown in SEQ ID NO. 52; and the sequence of McrC-R is shown in SEQ ID NO. 53.

[0094] 4) Using KasOP-F / BauA-R as primers and fragments H and M as templates, fragments H and M were connected by OE-PCR, and the result was called fragment P. Using SPL42-F / McrC-R as primers and fragments K and N as templates, fragments K and N were connected by OE-PCR, and the result was called fragment Q.

[0095] 5) Take appropriate amounts of fragment G, fragment P, and fragment Q and add 2x Seamless Cloning Mix for reaction.

[0096] 6) Take the reaction product of step 5) and add it to Trans-T1 competent cells to obtain a positive single colony, and perform colony PCR verification with primers BauA-F / R and McrC-F / R; obtain the verified correct plasmid pSET152-KasOP-bauA-SPL42-mcrC.

[0097] Compared with the prior art, the present invention has the following beneficial effects:

[0098] The present invention constructs an artificial synthesis pathway for synthesizing malonyl-CoA using β-alanine (β-ala) as a precursor by heterologously expressing an aminotransferase and a malonyl-CoA reductase. First, under the catalysis of the aminotransferase, the amino group of β-ala is transferred to an α-keto acid (such as pyruvate, oxaloacetate, or α-ketoglutarate), forming the intermediate product 3-oxopropionic acid and the corresponding amino acid. The 3-oxopropionic acid is then converted to malonyl-CoA under the action of the malonyl-CoA reductase. This pathway overcomes the drawbacks of the natural malonyl-CoA synthesis pathway, such as low carbon utilization, consumption of the energy substance ATP, release of the greenhouse gas CO2, and strict regulation of the key enzymes pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC). This achieves the purpose of increasing the production of flavolin, octanoic acid, phloroglucinol, pentadecaheptaene, natamycin, and spinosad, which use malonyl-CoA as a precursor. The introduction of an artificial malonyl-CoA synthesis pathway improved the cells' tolerance to organic acids, osmotic pressure, and cytotoxic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0100] Figure 1 is a diagram showing the current status of research on malonyl-CoA synthesis pathways and their derivatives in the background art, wherein A represents a compound with malonyl-CoA as a precursor; B represents the PDH-ACC pathway for synthesizing malonyl-CoA; and C represents the MCS pathway for synthesizing malonyl-CoA.

[0101] FIG2 is a schematic diagram of the NCM pathway of the present invention;

[0102] Figure 3 Transaminases and reductases heterologously expressed and purified in Escherichia coli; M: standard protein reference; 1: AptA; 2: BauA; 3: CCNA_03245; 4: PydD2; 5: YhxA; 6: PydD; 7: Pyd4; 8: StMCR; 9: Msed_0709; 10: PnSCR; 11: SucD; 12: MCR-C;

[0103] Figure 4 shows the in vitro activity test of transaminase and reductase, with the left figure showing transaminase and the right figure showing reductase;

[0104] Figure 5 is a schematic diagram of 2[H](NADPH) synthesis when transaminase uses α-ketoglutarate (A) and oxaloacetate (B) as substrates;

[0105] Figure 6 shows the effects of inhibitors of the natural synthesis pathway on the artificial NCM synthesis pathway; A. Effects of adding different concentrations of ATP on the NCM pathway (lower ADP / ATP ratios indicate higher ATP content); B. Effects of adding different concentrations of acetyl-CoA on the NCM pathway; C. Effects of adding different concentrations of long-chain fatty acyl-CoA (palmitoyl-CoA) on the NCM pathway;

[0106] Figure 7 shows that the NCM pathway replaces the PDH-ACC pathway in synthesizing malonyl-CoA; A: Schematic diagram of CRISPRi technology; B: Growth curve after the accBC gene is inhibited by CRISPRi technology;

[0107] Figure 8 shows that the NCM pathway improves the strain's ability to synthesize fatty acids (octanoic acid, C8). Schematic diagram of the octanoic acid synthesis pathway (left), and histogram of octanoic acid production (right);

[0108] Figure 9: The NCM pathway improves the strain's ability to synthesize phloroglucinol. Schematic diagram of the phloroglucinol synthesis pathway (left) and histogram of phloroglucinol production (right).

[0109] Figure 10 shows that the NCM pathway improves the strain's ability to synthesize flavonoids. Schematic diagram of the flavonoids synthesis pathway (left), and a histogram of flavonoids production (right).

[0110] Figure 11. The NCM pathway improves the strain's ability to synthesize pentadecaheptaene. Schematic diagram of the pentadecaheptaene synthesis pathway (left), and histogram of pentadecaheptaene production (right).

[0111] Figure 12: The NCM pathway improves the strain's ability to synthesize natamycin. Schematic diagram of the natamycin synthesis pathway (left), and histogram of natamycin production (right).

[0112] Figure 13. The NCM pathway enhances the ability of the strain to synthesize spinosyns. Schematic diagram of the spinosyn synthesis pathway (left), and histogram of spinosyn production (right).

[0113] Figure 14 The NCM pathway improves the synthesis of NADPH in the strain.

[0114] Figure 15 The NCM pathway improves cell tolerance to various growth inhibitory and adverse growth environments. DETAILED DESCRIPTION

[0115] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0116] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0117] In the following embodiments:

[0118] His-tag protein purification kit: Beyuntian (His-tag Protein Purification Kit), commonly known as nickel column kit.

[0119] Table 1. Primers used in the present invention

[0120] Table 3. Plasmids used in the present invention

[0121] Table 3. Strains used in the present invention

[0122] Example 1 Design of a new synthetic pathway for Malonyl-CoA

[0123] Since pyruvate is the most common product of carbon source catabolism, we attempted to introduce a new connecting molecule between pyruvate (C3) and malonyl-CoA (C3). Unlike the C3 (pyruvate)-C2 (acetyl-CoA)-C3 (malonyl-CoA) pattern of the natural pathway, the present invention adopted a new pattern of "C3-C3-C3" (Figure 2), thereby avoiding the ineffective reaction of "decarboxylation and carboxylation" (Figure 1A). Among the numerous C3 connecting molecules, we selected 3-oxopropanoate. Specifically, pyruvate and β-alanine first form 3-oxopropanoate and α-alanine under the catalysis of transaminases or aminotransferases, and 3-oxopropanoate subsequently forms malonyl-CoA (Figure 2) under the catalysis of oxidoreductases. In this design, since the artificial pathway does not release CO2, does not lose carbon sources, and does not consume ATP, it avoids the problems of carbon source and energy waste inherent in the natural pathway (Figure 1A). In addition, unlike the central metabolite acetyl-CoA, 3-oxopropionic acid is not a central metabolite in the vast majority of living cells, so the artificial pathway does not have the problem of high competition with the cellular background metabolic network. We call this design the NCM (non-carboxylative malonyl-CoA) pathway. The advantages of artificial synthesis compared to the natural synthesis pathway are detailed in Table 1.

[0124] Table 4. Comparison of artificial and natural pathways Note: a. Pyruvate dehydrogenase contains three subunits (E1, E2 and E3), acetyl-CoA carboxylase contains four subunits (AccA, AccB, AccC and AccD), while the NCM pathway enzymes BauA and MCR-C are both single subunits.

[0125] b. The molecular weight of pyruvate dehydrogenase is approximately 4600 kDa in Gram-negative bacteria, while it is approximately 8000-10000 kDa in Gram-positive bacteria and fungi. The molecular weight of BauA in the NCM pathway is approximately 200 kDa, and the molecular weight of MCR-C is approximately 150 kDa.

[0126] c. The natural malonyl-CoA synthesis pathway requires seven cofactors, namely thiamine pyrophosphate, FAD, coenzyme A, and NAD + , lipoic acid, biotin and ATP. The NCM pathway only requires three cofactors, namely pyridoxal phosphate, NADP + and coenzyme A.

[0127] d.+ represents production, - represents consumption.

[0128] e. Carbon utilization efficiency is the ratio of the number of carbon atoms in the acyl backbone of the product malonyl-CoA derived from the substrate pyruvate to the number of carbon atoms in the acyl backbone of the malonyl-CoA.

[0129] f. Standard Δ r G'° is calculated from the difference in Gibbs free energy between the product and substrate of a biochemical reaction.

[0130] g. Natural pathways compete with various metabolic pathways, such as the TCA cycle and amino acid and sterol synthesis pathways. In contrast, artificial pathways do not compete with important cellular metabolic pathways. Competition intensity is categorized as high (+++), medium (++), and low (+).

[0131] h. The natural pathway is tightly regulated by multiple factors, such as allosteric inhibition of key enzymes, product feedback inhibition, and inhibition by the energy substance ATP. These inhibitors have no significant inhibitory effect on the artificial pathway. The degree of tight regulation is categorized as high (+++), medium (++), and low (+).

[0132] i. The feasibility of implementation is estimated based on factors such as enzyme availability, the amount of enzyme required, the ability to express the enzyme, and the complexity of the enzyme. The feasibility is divided into three levels: difficult (+++), moderate (++), and easy (+).

[0133] Example 2 Mining and testing of candidate enzymes for the new Malonyl-CoA synthesis pathway

[0134] Since the NCM pathway is composed of transaminases and oxidoreductases, we attempted to screen candidate enzymes from different sources and test them separately or in combination. Specifically:

[0135] In the mining of transaminases, we selected BauA (from Pseudomonas aeruginosa), AptA (from Acinetobacter pittii), CCNA_03245 (from Caulobacter vibrioides), PydD2 (from Bacillus megaterium), YhxA (from Bacillus cereus), PydD (from Ureibacillus massiliensis), and Pyd4 (from Saccharomyces kluyveri);

[0136] In the mining of oxidoreductases, we selected MCR-C (from Chloroflexus aurantiacu), StMCR (from Sulfurisphaera tokodaii), Msed_0709 (from Metallosphaera sedula), PnSCR (from Pyrobaculum neutrophilum), and SucD (from Clostridium kluyveri);

[0137] The following is the operational process for the construction of candidate enzyme expression plasmids, purification of candidate enzymes, and in vitro reactions:

[0138] 1. Construction of candidate enzyme expression plasmid

[0139] 1.1 Construction of plasmid pCDF-P1-mcrC

[0140] The plasmid was constructed according to the amino acid sequence of MCR-C, wherein the amino acid sequence of MCR-C is shown in SEQ ID NO.70.

[0141] 1) Using the pCDF-duet plasmid containing a 6×His tag as a template, primers P1-R / P1-F were used to amplify a pCDF vector backbone fragment of 3781 bp, which contained the T7 promoter, the Ori sequence, and the spectinomycin resistance gene Smr (accession number UKC63634.1), referred to as fragment I; wherein the sequence of P1-R is shown in SEQ ID NO. 1, the sequence of P1-F is shown in SEQ ID NO. 2, and the sequence of fragment I is shown in SEQ ID NO. 3;

[0142] The amplification system was as follows: Phanta 2× buffer 25 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phanta Max Super-Fidelity DNA polymerase 1 μl, distilled water 18 μl, and the total volume was 50 μl.

[0143] Amplification conditions were as follows: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 120 s (30 cycles); and extension at 72°C for 5 min (1 cycle).

[0144] 2) Using the pMCR-C plasmid as a template and primers MCR-C-pCDF-P1-up / MCR-C-pCDF-P1-down, the MCR-C coding gene was amplified. The gene fragment was 2048 bp in size and was referred to as fragment II. The sequence of MCR-C-pCDF-P1-up is shown in SEQ ID NO. 4, the sequence of MCR-C-pCDF-P1-down is shown in SEQ ID NO. 5, and the sequence of fragment II is shown in SEQ ID NO. 6.

[0145] The amplification system was as follows: Phanta 2× buffer 25 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phanta Max Super-Fidelity DNA polymerase 1 μl, distilled water 18 μl, and the total volume was 50 μl.

[0146] Amplification conditions were as follows: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 60 s (30 cycles); and extension at 72°C for 5 min (1 cycle).

[0147] 3) The fragment I obtained in the first step and the fragment II obtained in the second step were respectively digested with endonuclease DpnI (NEB) to eliminate the template at 37°C for 30 min; after agarose electrophoresis, they were cleaned using a PCR purification kit (FastPure Gel DNA Extraction Mini Kit, purchased from Novozyme Biotechnology Co., Ltd.); appropriate amounts of fragment I and fragment II (V I +V II =5μl, n I / n II =1 / 3), 5 μl 2X Seamless Cloning Mix (Biyuntian Company) was added, and the reaction was carried out at 50°C for 30 min.

[0148] 4) Take 2 μl of the reaction product from step 3) and add it to 50 μl of Trans-T1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds and immediately place on ice for 2 minutes. Add 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 220 rpm at 37°C for 1 hour. Centrifuge and remove the supernatant, leaving approximately 50 μl. Mix and spread the entire amount on an LB plate containing spectinomycin (final concentration of 50 μg / ml). After incubation for 12 hours, select 5 positive single colonies and perform colony PCR verification using primers P1-YZ-up (SEQ ID NO.54) / MCR-C-YZ-down (SEQ ID NO.55). The clone with a 488 bp PCR fragment was identified as a positive clone. Two positive clones were selected to extract plasmids for sequencing analysis. The sequencing results were compared to obtain the correct plasmid pCDF-P1-mcrC.

[0149] 1.2. The construction of plasmids pCDF-P1-bauA, pCDF-P1-aptA, pCDF-P1-CCNA_03245, pCDF-P1-pydD2, pCDF-P1-yhxA, pCDF-P1-StMcr, pCDF-P1-Msed_0709, pCDF-P1-PnScr, and pCDF-P1-sucD was completed by GENEWIZ and constructed according to the corresponding amino acids (the construction method is the same as that of plasmid pCDF-P1-mcrC). The amino acid sequences involved are as follows:

[0150] 2. Purification of candidate enzymes

[0151] 2.1 Purification of MCR-C

[0152] In the first step, 50 ng of the plasmid pCDF-P1-mcrC was added to 50 μl of E. coli BL21(DE3) competent cells and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. Then, 500 μl of SOC medium was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and plated onto an LB plate containing spectinomycin (final concentration 50 μg / ml). After overnight incubation, positive clones were isolated.

[0153] In the second step, five positive monoclonal colonies were taken and inoculated into 5 mL of liquid LB seed medium containing spectinomycin (final concentration of 50 μg / ml), and cultured at 220 rpm and 37°C for 8 hours until the OD 550 =2.0. The seed solution was heated to an initial OD 550= 0.1 was inoculated into 50 mL of liquid LB medium (containing spectinomycin at a final concentration of 50 μg / ml), and cultured at 220 rpm and 37°C until OD 550 =0.5-0.8, add inducer isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and continue culturing at 220 rpm and 37°C for 4-5 h.

[0154] In the third step, centrifuge at 6000g for 10 minutes to collect the induced bacteria. Next, use a His-tag protein purification kit (purchased from Biyuntian Biotechnology Co., Ltd.) to purify the protein, add lysis buffer at a ratio of 4ml of non-denaturing lysis buffer per gram of bacterial pellet wet weight, and fully resuspend the bacteria. Add lysozyme to a final concentration of 1mg / ml and mix well, and place on ice for 30 minutes. Ultrasonicate the bacteria under cooling with an ice-water mixture: ultrasonic power 150-250W, ultrasonic treatment for 2s each time, interval 2s, a total of ultrasonic treatment for 5-10 minutes (until the bacterial solution becomes translucent); centrifuge at 4℃10000g for 20-30min, collect the supernatant, prepare the bacterial lysate, and place on ice.

[0155] The fourth step is to purify the protein using nickel affinity column. Specifically, take 1 ml of 50% BeyoGold TM His-tag Purification Resin, centrifuge at 4℃ (1000g×10s) and discard the storage solution. Add 0.5ml of non-denaturing lysis buffer to the gel and mix well to balance the gel. Centrifuge at 4℃ (1000g×10s) and discard the liquid. Repeat the balance 1-2 times and discard the liquid. Add about 4ml of the bacterial lysate prepared in the third step and shake slowly at 4℃ for 60min. Then, mix the lysate with BeyoGold TM Load the His-tag Purification Resin mixture onto a nickel affinity chromatography column. Open the bottom cap of the purification column and allow the liquid to drain by gravity. Wash the column five times, adding 0.5-1 ml of native wash buffer each time. Elute the target protein 6-10 times, using 0.5 ml of native wash buffer each time. Collect each eluate into separate centrifuge tubes. The collected eluate is the purified His-tagged protein sample.

[0156] The fifth step is to add the above eluate to a 2 mL ultrafiltration tube (Sartorius), place the ultrafiltration tube in a centrifuge, and centrifuge at 4°C, 13000r until all the eluate is concentrated to 50 μl, then add 500 μl TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), 4°C, 13000r centrifugation until the solution is concentrated to 50 μl, then add 500 μl TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), 4°C, 13000r centrifugation until about 50-100 μL protein solution remains in the ultrafiltration tube, pipette and mix well, and then transfer to a pre-cooled EP tube.

[0157] Step 6: Determine the concentration of the purified MCR-C protein using a BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd.). Based on the sample quantity, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. Add 20 μL of a 25 mg / mL protein standard to 980 μL of diluent to prepare a 0.5 mg / mL protein standard. Dilute the 0.5 mg / mL protein standard to 0.05 (10→90), 0.1 (20→80), 0.2 (40→60), 0.3 (60→40), and 0.4 (80→20) mg / mL, respectively. Add 20 μL of each sample to a 96-well plate (triplicate). After initial analysis on the Nano-300, dilute the sample to the desired concentration, and add 20 μL to each well of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Create a standard curve based on the absorbance at 562 nm and the concentration of the standard protein solution. Calculate the concentration of the purified protein based on the standard curve.

[0158] The purification methods of other reductases StMCR, Msed_0709, PnSCR and SucD were the same as those of MCRC.

[0159] 2.2 Purification of BauA

[0160] The method for preparing the BauA protein sample comprises the following steps:

[0161] In the first step, 50 ng of the plasmid pCDF-P1-bauA was added to 50 μl of E. coli BL21(DE3) competent cells and placed on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. Then, 500 μl of SOC medium was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and plated onto an LB plate containing spectinomycin (final concentration 50 μg / ml). After overnight incubation, positive clones were isolated.

[0162] In the second step, five positive monoclonal colonies were taken and inoculated into 5 mL of liquid LB seed medium containing spectinomycin (final concentration of 50 μg / ml), and cultured at 220 rpm and 37°C for 8 hours until the OD 550 =2.0. The seed solution was heated to an initial OD 550 = 0.1 was inoculated into 50 mL of liquid LB medium (containing spectinomycin at a final concentration of 50 μg / ml), and cultured at 220 rpm and 37°C until OD 550 =0.5-0.8, add inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, and continue culturing at 220 rpm and 37°C for 4-5 h.

[0163] In the third step, centrifuge at 6000g for 10 minutes to collect the induced bacteria. Next, use a His-tag protein purification kit (purchased from Biyuntian Biotechnology Co., Ltd.) to purify the protein, add lysis buffer at a ratio of 4ml of non-denaturing lysis buffer per gram of bacterial pellet wet weight, and fully resuspend the bacteria. Add lysozyme to a final concentration of 1mg / ml and mix well, and place on ice for 30 minutes. Ultrasonicate the bacteria under cooling with an ice-water mixture: ultrasonic power 150-250W, ultrasonic treatment for 2s each time, interval 2s, a total of ultrasonic treatment for 5-10 minutes (until the bacterial solution becomes translucent); centrifuge at 4℃10000g for 20-30min, collect the supernatant, prepare the bacterial lysate, and place on ice.

[0164] The fourth step is to purify the protein using nickel affinity column. Specifically, take 1 ml of 50% BeyoGold TM His-tag Purification Resin, centrifuge at 4℃ (1000g×10s) and discard the storage solution. Add 0.5ml of non-denaturing lysis buffer to the gel and mix well to balance the gel. Centrifuge at 4℃ (1000g×10s) and discard the liquid. Repeat the balance 1-2 times and discard the liquid. Add about 4ml of bacterial lysate supernatant to it and shake slowly at 4℃ for 60min. Then, mix the lysate and BeyoGold TMLoad the His-tag Purification Resin mixture onto a nickel affinity chromatography column. Open the bottom cap of the purification column and allow the liquid to drain by gravity. Wash the column five times, adding 0.5-1 ml of native wash buffer each time. Elute the target protein 6-10 times, using 0.5 ml of native wash buffer each time. Collect each eluate into separate centrifuge tubes. The collected eluate is the purified His-tagged protein sample.

[0165] The fifth step is to add the above eluate to a 2 mL ultrafiltration tube (Sartorius), place the ultrafiltration tube in a centrifuge, and centrifuge at 4°C, 13000r until all the eluate is concentrated to 50 μl, then add 500 μl TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), 4°C, 13000r centrifugation until the solution is concentrated to 50 μl, then add 500 μl TEG buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, pH 7.9), 4°C, 13000r centrifugation until about 50-100 μL protein solution remains in the ultrafiltration tube, pipette and mix well, and then transfer to a pre-cooled EP tube.

[0166] Step 6: Determine the concentration of the purified MCR-C protein using a BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd.). Based on the sample quantity, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. Add 20 μL of a 25 mg / mL protein standard to 980 μL of diluent to prepare a 0.5 mg / mL protein standard. Dilute the 0.5 mg / mL protein standard to 0.05 (10→90), 0.1 (20→80), 0.2 (40→60), 0.3 (60→40), and 0.4 (80→20) mg / mL, respectively. Add 20 μL of each sample to a 96-well plate (triplicate). After initial analysis on the Nano-300, dilute the sample to the desired concentration, and add 20 μL to each well of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Create a standard curve based on the absorbance at 562 nm and the concentration of the standard protein solution. Calculate the concentration of the purified protein based on the standard curve.

[0167] The purification methods of other transaminases, AptA, CCNA_03245, PydD2, YhxA, PydD, and Pyd4, were the same as those for BauA.

[0168] The results of SDS-PAGE electrophoresis of transaminase and reductase are shown in Figure 3

[0169] Example 3 In vitro reaction (artificial synthesis of malonyl-CoA)

[0170] Transaminase activity assay using pyruvate as substrate: In vitro reactions were performed in a 200 μL total reaction volume containing 100 mM Tris buffer (pH 7.8), 2 mM MgCl2, 3 mM DTT, 5 mM pyruvate, 5 mM β-alanine, 2.5 mM NADP + , 2.5 mM Coenzyme A, 1 μM purified transaminase, and 10 μM MCR-C enzyme. The reaction mixture was incubated at 37°C and the increase in NADPH generated by the reaction was monitored at 340 nm (the absorption peak at 340 nm). The transaminase activities were as follows: BauA (1.60 μmol / min / mg), PydD2 (0.96 μmol / min / mg), AptA (0.90 μmol / min / mg), YhxA (0.15 μmol / min / mg), and CCNA_03245 (0.13 μmol / min / mg). BauA showed the best effect (Figure 4).

[0171] Transaminase activity assay using α-ketoglutarate as substrate: In vitro reactions were performed in a 200 μL total reaction volume containing 100 mM Tris buffer (pH 7.8), 2 mM MgCl2, 3 mM DTT, 5 mM α-ketoglutarate, 5 mM β-alanine, 2.5 mM NADP + , 2.5 mM Coenzyme A, 10 μM purified transaminase and 10 μM MCR-C enzyme. The reaction mixture was incubated at 37°C, and the increase at 340 nm (NADPH generated in the reaction has an absorption peak at 340 nm) was monitored; the transaminase activities were pydD (0.825 μmol / min / mg) and Pyd4 (1.15 μmol / min / mg), respectively ( Figure 5A ).

[0172] Transaminase activity assay using oxaloacetate as substrate: In vitro reactions were performed in a 200 μL total reaction volume containing 100 mM Tris buffer (pH 7.8), 2 mM MgCl2, 3 mM DTT, 5 mM oxaloacetate, 5 mM β-alanine, 2.5 mM NADP +, 2.5 mM Coenzyme A, 10 μM purified transaminase and 10 μM MCR-C enzyme. The reaction mixture was incubated at 37°C and the increase at 340 nm (NADPH generated in the reaction has an absorption peak at 340 nm) was monitored; the transaminase activities were BauA (0.46 μmol / min / mg), ApaT (0.42 μmol / min / mg), and PydD2 (0.49 μmol / min / mg), respectively ( Figure 5B ).

[0173] For the oxidoreductase activity test, the in vitro reaction was performed in a total reaction volume of 200 μL, which contained 100 mM Tris buffer (pH 7.8), 2 mM MgCl2, 3 mM DTT, 5 mM pyruvate, 5 mM β-alanine, 2.5 mM NADP + , 2.5 mM Coenzyme A, 10 μM BauA, and 1 μM purified oxidoreductase. The reaction mixture was incubated at 37°C and the increase at 340 nm was monitored. In vitro reactions revealed that both MCR-C and Msed_0709 exhibited the desired enzyme activity, with MCR-C exhibiting the highest activity (2.0 μmol / min / mg) (Figure 4).

[0174] Example 4: A new Malonyl-CoA synthesis pathway relieves multiple feedback inhibitions

[0175] The natural PDH-ACC pathway is tightly regulated by numerous factors. For example, high intracellular ATP levels severely reduce PDH activity. When ADP / ATP = 1, PDH activity is only 10% of that at ADP / ATP = 20 (Hansford RG et al., 1976). Acetyl-CoA is also a PDH inhibitor; at 2 mM, PDH activity decreases by 70%. Long-chain acyl-CoAs, such as palmitoyl-CoA, are inhibitors of ACC; 0.24 μM palmitoyl-CoA can reduce ACC activity by 80%. In the above-mentioned NCM in vitro reaction system, ATP and ADP (ATP+ADP=3.75mM, ADP / ATP were +∞, 10, 1 and 0.1, respectively) (Figure 6A), acetyl-CoA (0, 0.5, 1, 2mM) (Figure 6B) and palmitoyl-CoA (0, 0.24, 0.48, 0.96, 2.4 and 24μM) (Figure 6C) were added respectively. The in vitro experimental results showed that these typical inhibitors of natural synthesis pathways had no inhibitory effect on the artificial NCM pathway.

[0176] Example 5: New synthetic pathway can replace the natural pathway for intracellular malonyl-CoA synthesis

[0177] (I) CRISPR interference (CRISPRi) technology was used to inhibit the expression of the key accBC gene in the natural malonyl-CoA biosynthesis pathway in Escherichia coli MG1655(DE3) ( Figure 7A ).

[0178] 1. Construction of plasmids pACYC-dCas9 and Sg-accBC

[0179] 1.1. Construction of plasmid pACYC-dCas9

[0180] Step 1: Using the pCF-tesA plasmid as a template and primers pCF-R / pCF-F, the pACYC-dCas9 backbone fragment was amplified. The fragment size was 8184 bp and contained the Ori sequence, the chloramphenicol resistance gene (SEQ ID NO. 16), and the dCas9 protein coding sequence (SEQ ID NO. 17), referred to as fragment A (SEQ ID NO. 18). The pCF-R sequence is shown in SEQ ID NO. 19; the pCF-F sequence is shown in SEQ ID NO. 20.

[0181] Step 2: Remove the template from Fragment A using the endonuclease DpnI; perform electrophoresis and clean using a PCR purification kit; take an appropriate amount of Fragment A and add 2X Seamless Cloning Mix to react for 30-60 minutes;

[0182] Step 3: Take the reaction product of the second step, add it to Trans-T1 competent cells, ice bath, heat shock, and place on ice for 2-5 minutes; add SOC medium and incubate; centrifuge to remove the supernatant, mix well and spread on LB plate containing chloramphenicol, culture for 12-20 hours, select single colonies, extract plasmid to obtain pACYC-dCas9.

[0183] 1.2 Construction of plasmid Sg-accBC

[0184] Step 1: Using the Sg-O-RNA plasmid as a template, primers pecg-accB-F / pecg-accB-R were used to amplify the Sg-accBC backbone fragment. The fragment size was 2239 bp and contained the Ori sequence, sgRNA-scaffold, and ampicillin resistance gene coding sequence (SEQ ID NO. 21), referred to as fragment B (SEQ ID NO. 22). The pecg-accB-F sequence is shown in SEQ ID NO. 23, and the pecg-accB-R sequence is shown in SEQ ID NO. 24.

[0185] Step 2: Remove the template from fragment B using the endonuclease DpnI; perform electrophoresis and clean using a PCR purification kit; take an appropriate amount of fragment I and add 2X Seamless Cloning Mix to react for 30-60 minutes;

[0186] Step 3: Take the reaction product of step 2, add it to Trans-T1 competent cells, ice bath, heat shock, and place on ice for 2-5 minutes; add SOC medium and incubate; centrifuge to remove the supernatant, mix well, and spread on LB plates containing ampicillin. After culturing for 12-20 hours, select single colonies and extract the plasmid to obtain Sg-accBC.

[0187] 2. Construction of strains containing pACYC-dCas9 and Sg-accBC expression vectors

[0188] 1 μL of pACYC-dCas9 and Sg-accBC were added to 50 μL of MG1655 (DE3) competent cells and placed on ice for 30 minutes. Heat shock was performed at 42°C for 30 seconds, and the cells were immediately placed on ice for 2 minutes. 500 μL of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 37°C for 1 hour at 220 rpm. 20 μL of the bacterial solution was spread on an LB plate containing chloramphenicol and ampicillin (final concentration of 50 μg / ml). After overnight incubation, 5 positive single colonies were selected and verified by colony PCR to obtain MG1655 (DE3), pACYC-dCas9, and Sg-accB-positive bacteria (strain A).

[0189] 3. CRISPRi Experiment

[0190] Seed culture: Pick 3-5 single clones of strain A and inoculate them into a 15 mL test tube filled with 5 mL MOPS medium (containing 20 g / L glucose) containing chloramphenicol and ampicillin (final concentration of 50 μg / mL). Cultivate at 37°C for 220 rpm until the OD550 reaches about 1.5-1.8, which is used as the seed solution.

[0191] The seed solution was inoculated into MOPS medium (containing 20 g / L glucose, 0.1 mM IPTG, and a final concentration of 50 μg / mL chloramphenicol and ampicillin) at an initial OD550 of 0.1. After inoculation, the mixture was shaken and 200 μL was pipetted into a 96-well plate. A continuous kinetic assay was performed using a microplate reader (Biotek), with OD550 values ​​measured every 10 minutes to determine the strain growth curve.

[0192] The results showed that when the interference vectors pACYC-dCas9 and Sg-accBC were introduced into MG1655(DE3), the strain could not grow in MOPS + 2% (w / v) glucose medium due to malonyl-CoA nutritional deficiency ( Figure 7B ).

[0193] The preparation method of MOPS medium is as follows:

[0194] MOPS medium: 25 mL of 40×M stock solution, 1 mL of 0.528 M MgCl2, 1 mL of 0.276 M K2SO4, 1 mL of 1 M K2HPO4, 100 mL of 200 g / L glucose, distilled water to 1 L, filter with a 0.22 μM sterile filter membrane and set aside.

[0195] To prepare the 40×M stock solution, weigh 334.8g MOPS powder, 28.2g Tricine, 116.8g NaCl, 20.4g NH₄Cl, and 32g KOH, add distilled water to 900mL, and stir thoroughly. Adjust the pH to 7.3-7.4 with 10M HCl or 10M KOH. While stirring, add 2ml of the "trace metal stock solution," 0.2ml of ZnCl₂ solution (34g / L), 0.2ml of Na₂SeO₃ solution (43g / L), and 0.2ml of Na₂MoO₄ solution (60.5g / L), and bring the volume to 1L with distilled water. Dispense the prepared solution into 20 50mL centrifuge tubes.

[0196] The preparation method of "trace metal mother liquor" is as follows: weigh 5g Fecl2·4H2O, 184mg CaCl2·2H2O, 62mg H3BO3, 40mg Mncl2·4H2O, 18mg Cocl2·6H2O, and 4mg Cucl2·2H2O, add 70mL distilled water, add 8ml concentrated hydrochloric acid (37% HCl), stir evenly, transfer to a 100ml graduated cylinder, and dilute to 100ml with ddH2O.

[0197] (II) Construction and introduction of NCM pathway plasmid (pCDF-bauA-mcrC)

[0198] 1. Construction of pCDF-bauA-mcrC plasmid

[0199] Step 1. Using the pCDF-P1-bauA plasmid as a template and primers P2-MCRC-R / P2-MCRC-F, a pCDF-P1-bauA vector backbone fragment was amplified. The fragment was 5129 bp in size and contained the T7 promoter, Ori sequence, bauA coding sequence, and spectinomycin resistance gene Smr, referred to as fragment III. The sequence of P2-MCRC-R is shown in SEQ ID NO. 25, the sequence of P2-MCRC-F is shown in SEQ ID NO. 26, and the sequence of fragment III is shown in SEQ ID NO. 27.

[0200] The amplification system was as follows: Phanta 2X buffer 25 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phanta Max Super-Fidelity DNA polymerase 1 μl, distilled water 18 μl, and the total volume was 50 μl.

[0201] The amplification conditions were as follows: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 150 s (30 cycles); and extension at 72°C for 5 min (1 cycle).

[0202] Step 2. Using the pCDF-P1-McrC plasmid as a template, primers MCR-C-pCDF-P2-up / MCR-C-pCDF-P2-down were used to amplify the McrC encoding gene. The gene fragment was 2051 bp in size and was referred to as fragment IV. The sequence of fragment IV is shown in SEQ ID NO. 28; the sequence of MCR-C-pCDF-P2-up is shown in SEQ ID NO. 29; and the sequence of MCR-C-pCDF-P2-down is shown in SEQ ID NO. 30.

[0203] The amplification system was as follows: Phanta 2X buffer 25 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phanta Max Super-Fidelity DNA polymerase 1 μl, distilled water 18 μl, and the total volume was 50 μl.

[0204] Amplification conditions were as follows: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 60 s (30 cycles); and extension at 72°C for 5 min (1 cycle).

[0205] Step 3: Fragment III and Fragment IV were digested with endonuclease DpnI (NEB) at 37°C for 30 min; after agarose electrophoresis, they were cleaned using a PCR purification kit (FastPure Gel DNA Extraction Mini Kit, purchased from Novozyme Biotechnology Co., Ltd.); appropriate amounts of Fragment III and Fragment IV (V I +V II =5μl, n I / n II =1 / 3), 5 μl 2X Seamless Cloning Mix (Biyuntian Company) was added, and the reaction was carried out at 50°C for 30 min.

[0206] In step 4, take 2 μl of the product from step 3 and add it to 50 μl of Trans-T1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds and immediately place on ice for 2 minutes. Add 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 220 rpm at 37°C for 1 hour. Centrifuge and remove the supernatant, leaving approximately 50 μl. Mix and spread the entire amount on an LB plate containing spectinomycin (final concentration of 50 μg / ml). After overnight incubation, select five positive single colonies and perform colony PCR verification using primers P2-YZ-up / MCR-C-YZ-down. The clone with a 662 bp PCR fragment was identified as a positive clone. Two positive clones were selected to extract plasmids for sequencing analysis. The sequencing results were compared to obtain the correct plasmid, pCDF-bauA-mcrC. The sequence of MCR-C-YZ-down is shown in SEQ ID NO.55; the sequence of P2-YZ-up is shown in SEQ ID NO.56.

[0207] 2. Introduction of malonyl-CoA-deficient strains into NCM pathway

[0208] Take 1 μL of pCDF-bauA-mcrC plasmid and add 50 μL of MG1655 (DE3) competent cells containing the interference vectors pACYC-dCas9 and Sg-accBC. Incubate on ice for 30 minutes. Heat shock at 42°C for 30 seconds and immediately place on ice for 2 minutes. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 37°C for 1 hour at 220 rpm. Take 20 μL of the bacterial solution and spread it on an LB plate containing chloramphenicol, ampicillin, and spectinomycin (final concentration of 50 μg / ml). After overnight culture, the target strain (strain B) containing MG1655 (DE3), pACYC-dCas9, Sg-accB, and pCDF-bauA-mcrC was obtained.

[0209] Then the growth experiment was carried out:

[0210] Seed culture: Pick 3-5 single colonies (of strain B) and inoculate them into a 15 mL test tube containing 5 mL of MOPS medium (containing 20 g / L glucose) containing chloramphenicol, ampicillin, and spectinomycin (final concentration of 50 μg / mL). Cultivate at 37°C for 220 rpm until the OD550 reaches approximately 1.5-1.8. This serves as the seed solution.

[0211] The seed solution was inoculated into MOPS medium (containing 20 g / L glucose, 0.1 mM IPTG, and a final concentration of 50 μg / mL of chloramphenicol, ampicillin, and spectinomycin) at an initial OD550 of 0.1. After inoculation, the mixture was shaken and 200 μL was transferred to a 96-well plate. Continuous kinetic analysis was performed using a microplate reader (Biotek), with OD550 values ​​measured every 10 minutes to determine the strain growth curve.

[0212] The results showed that after the introduction of the NCM pathway (pCDF-bauA-mcrC), the growth impaired by accBC interference was greatly restored (Figure 7B), indicating that the artificial pathway can replace the natural pathway in the cell for malonyl-CoA synthesis.

[0213] Example 6 Application of the new Malonyl-CoA pathway in fatty acid synthesis

[0214] In the biosynthesis of fatty acids, malonyl-CoA serves as an essential elongation unit. In this example, octanoic acid was selected as a representative fatty acid to explore the application of a novel malonyl-CoA synthesis pathway in fatty acid synthesis. The plasmid used for octanoic acid synthesis was pTrc-TE10, which contains the thioesterase TE10 from Anaerococcus tetradius, which specifically hydrolyzes octanoyl-ACP to form octanoic acid.

[0215] The fermentation of caprylic acid products is divided into three steps:

[0216] In the first step, 50 ng each of the plasmid pCDF-bauA-mcrC (constructed in Example 4) and the thioesterase expression plasmid (pTrc-TE10) were added to 50 μl of MG1655(DE3) competent cells (purchased from Pure Biosciences) and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The supernatant was mixed and plated onto an LB plate containing spectinomycin (final concentration 50 μg / ml) and ampicillin (final concentration 50 μg / ml). After overnight incubation, positive clones, MG1655(DE3), pCDF-bauA-McrC, and pTrc-TE10 (strain C), were obtained.

[0217] 50 ng each of the plasmids pCDF-duet and pTrc-TE10 were added to 50 μl of MG1655(DE3) competent cells (purchased from Yu Pure Biosciences) and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 37°C for 1 hour at 220 rpm. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and plated onto LB plates containing spectinomycin (final concentration 50 μg / ml) and ampicillin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for MG1655(DE3), pCDF-duet, and pTrc-TE10 (serving as control strain C).

[0218] Step 2: Seed culture: strain C and control strain C were selected and inoculated into 50 mL shake tubes containing 10 mL LB medium containing spectinomycin (final concentration of 50 μg / ml) and ampicillin (final concentration of 50 μg / ml), respectively, and cultured at 37°C overnight at 220 rpm.

[0219] Step 3: Fermentation culture:

[0220] Fermentation: Prepare 50 mL of M9 fermentation medium containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the seed solution at an initial OD550 of 0.1 and incubate at 30°C for 220 days to an OD550 of approximately 0.6. Add IPTG to a final concentration of 0.25 mM. Continue fermentation at 30°C for 220 days for up to 72 hours. Measure OD550 and sample the culture every 24 hours.

[0221] The fermentation medium used was M9 medium, which has the following formulation: 20 g / L glucose, 12.8 g / L Na2HPO4·7H2O, 0.5 g / L NaCl, 1 g / L NH4Cl, 3 g / L KH2PO4, 2 mM MgSO4, and 0.1 mM CaCl2. The medium was prepared by taking 10 mL of 200 g / L glucose, 20 mL of 5× M9, 200 μL of 1 M MgSO4, and 10 μL of 1 M CaCl2, and then adding distilled water to 100 mL.

[0222] Among them, 5×M9: 64g / L Na2HPO4·7H2O, 15g / L KH2PO4, 2.5g / L NaCl and 5g / L NH4Cl.

[0223] Fatty acid analysis: After fermentation, 1 ml of fermentation broth was mixed with 125 μl of 10% NaCl (wt / v) and 125 μl of acetic acid. Then, 10 μl of 5 mg / L internal standard (C7:0 / C15:0, final concentration 50 mg / L) was added. After brief mixing, 500 μl of ethyl acetate was added. The sample was vortexed for 15 seconds and centrifuged at 16,000 × g for 10 minutes. 250 μl of the upper organic phase was transferred to a new glass tube, and 2.25 mL of ethanol:hydrogen chloride (30:1 v / v) was added. The mixture was incubated at 55°C for 1 hour, cooled to room temperature, and 1.25 mL of ddH2O and 1.25 mL of n-hexane were added. The mixture was vortexed and centrifuged at 2,000 × g for 2 minutes. The upper n-hexane layer was used for GC-MS analysis, and malonyl-CoA was quantified using a gas chromatography-flame ionization detector / mass spectrometer (GC-FID / MS) equipped with an Agilent 5975 mass spectrometer and an Agilent 7890 gas chromatograph. The analysis procedure was as follows: initial temperature of 50°C for 2 minutes, ramping to 200°C at 25°C / min for 1 minute, then ramping to 315°C at 25°C / min for 2 minutes. Helium was used as the carrier gas at a flow rate of 1 ml / min, and a DB-5MS column (30 m, 0.25 mm ID, 0.25 μm, Agilent) was used for separation. GC-MS detection was performed.

[0224] Results: Compared with the control strain C (99±3 mg / L octanoic acid), the engineered strain containing the new malonyl-CoA synthesis pathway (strain C) had an octanoic acid production of 157±9 mg / L, which was 58% higher (Figure 8).

[0225] Example 6 Application of the new Malonyl-CoA pathway in the synthesis of polyketide compound - phloroglucinol

[0226] Phloroglucinol is a phenolic compound used as an analgesic. It can also serve as a precursor for the synthesis of bioactive ingredients such as hyperforin, hyperforin, and rhotomensone, and is widely used in the pharmaceutical, leather, and dye industries (Singh et al. 2009). Type III PKS (Phloroglucinol synthase, PhlD) catalyzes the biosynthesis of phloroglucinol. The synthesis of one molecule of phloroglucinol requires three molecules of malonyl-CoA (Figure 9). A schematic diagram of the phloroglucinol biosynthesis pathway (left) and a bar chart of phloroglucinol production (right) are shown.

[0227] This experiment requires the use of pCDF-bauA-mcrC and pCum-phlD plasmids

[0228] The construction of plasmid pCDF-bauA-mcrC is the same as in Example 4

[0229] The construction of plasmid pCum-phlD is divided into four steps:

[0230] In the first step, using the pCum plasmid as a template and primers pCum-R / pCum-F, we amplified a 4461-bp pCum backbone fragment containing the Ori sequence and the kanamycin resistance gene coding sequence (SEQ ID NO. 31), designated Fragment C (SEQ ID NO. 32). The sequence of pCum-R is shown in SEQ ID NO. 33, and the sequence of pCum-F is shown in SEQ ID NO. 34.

[0231] In the second step, the pCDF-phlD plasmid (Genwizhi) was used as a template and primers phlD-up / phlD-down were used to amplify the PhlD coding gene. The gene fragment was 1092 bp in size and contained the PhlD coding sequence, referred to as fragment D (SEQ ID NO. 35). The sequence of phlD-up is shown in SEQ ID NO. 36, and the sequence of phlD-down is shown in SEQ ID NO. 37.

[0232] In the third step, fragments C and D were treated with the endonuclease DpnI to remove the template, followed by electrophoresis and cleanup using a PCR purification kit. Fragments C and D were then added to 2X Seamless Cloning Mix and reacted for 30-60 minutes.

[0233] Step 4: The reaction product from step 3 was added to Trans-T1 competent cells, ice-bathed, heat-shocked, and placed on ice for 2-5 minutes. SOC medium was added and incubated. The supernatant was removed by centrifugation, mixed, and then spread on an LB plate containing spectinomycin. After incubation for 12-20 hours, positive single colonies were selected and verified by colony PCR using primers Cum-YZ-up / Cum-YZ-down. The verified plasmid pCum-phlD was obtained. The sequence of Cum-YZ-up is shown in SEQ ID NO. 57; the sequence of Cum-YZ-down is shown in SEQ ID NO. 58.

[0234] The fermentation of phloroglucinol products is divided into three steps:

[0235] In the first step, 50 ng each of the plasmids pCDF-bauA-mcrC and pCum-phlD were added to 50 μl of MG1655(DE3) competent cells and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and spread onto an LB plate containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for MG1655(DE3), pCDF-bauA-mcrC, and pCum-phlD (strain D).

[0236] 50 ng each of the plasmids pCDF-duet and pCum-phlD were added to 50 μl of MG1655(DE3) competent cells and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and spread onto an LB plate containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for MG1655(DE3), pCDF-duet, and pCum-phlD (serving as control strain D).

[0237] Step 2: Seed culture: strain D and control strain D were selected and inoculated into 50 mL shake tubes containing 10 mL LB medium containing spectinomycin (final concentration of 50 μg / ml) and kanamycin (final concentration of 50 μg / ml), and cultured overnight at 37°C at 220 rpm to prepare seed solution.

[0238] Step 3, Fermentation: Prepare 50 mL of M9 fermentation medium containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the culture medium with the seed liquid of strain D and control strain D at an initial OD550 of 0.1. Cultivate at 37°C for 220 days to an OD550 of approximately 0.6. Add IPTG to a final concentration of 0.1 mM and cumate to 0.25 mM. Continue fermentation at 37°C for 220 days for 24 hours.

[0239] The preparation of M9 culture medium is the same as that in Example 6.

[0240] Analytical Methods: After fermentation, take a 2 mL sample and centrifuge at 12,000 × g for 2 minutes. The supernatant is then analyzed by HPLC using a C18 column, a flow rate of 0.8 mL / min, a column temperature of 30°C, a mobile phase of 40% acetonitrile, and a UV detector at 254 nm.

[0241] Results: Compared with the phloroglucinol production of the control strain D (187±33 mg / L), the engineered bacteria (strain D) containing the new malonyl-CoA synthesis pathway produced phloroglucinol of 363±21 mg / L, which was 94% higher than that of the control strain D (Figure 9).

[0242] Example 7 Application of the new Malonyl-CoA pathway in the synthesis of polyketide compound - flavonoids

[0243] Flaviolin is an important quinone compound and an essential precursor for the synthesis of a class of polyketide-isoprenoid hybrid compounds (PIHCs), which are used as antioxidants, nonsteroidal estrogen receptor antagonists, and antitumor and anticancer drugs. Flaviolin biosynthesis is catalyzed by a type III PKS (RppA), with the production of one molecule of flavolin consuming five molecules of malonyl-CoA (Figure 10, including a schematic diagram of the flavolin biosynthesis pathway (left) and a bar chart of flavolin production (right)).

[0244] This experiment requires the use of pCDF-bauA-mcrC and pCum-rppA plasmids

[0245] The construction of plasmid pCDF-bauA-mcrC is shown in Example 4

[0246] The construction of plasmid pCum-rppA is divided into four steps:

[0247] In the first step, using the pCum plasmid as a template and primers pCum-R / pCum-F, a pCum backbone fragment was amplified. The fragment was 4461 bp in size and contained the Ori sequence and the kanamycin resistance gene coding sequence (SEQ ID NO. 31), referred to as fragment C (SEQ ID NO. 32). The sequence of pCum-R is shown in SEQ ID NO. 33, and the sequence of pCum-F is shown in SEQ ID NO. 34.

[0248] In the second step, the pCDF-rppA plasmid (Jin Weizhi) was used as a template and the primers rppA-Cum-up / rppA-Cum-down were used to amplify the RppA coding gene. The gene fragment was 1169 bp in size and contained the RppA coding sequence, called fragment E (SEQ ID NO.38); the sequence of rppA-Cum-up is shown in SEQ ID NO.39; the sequence of rppA-Cum-down is shown in SEQ ID NO.40.

[0249] Step 3: Remove the template from fragments C and E using the endonuclease DpnI. After electrophoresis, clean the fragments using a PCR purification kit. Take fragments C and E and add 2X Seamless Cloning Mix to react for 30-60 minutes.

[0250] Step 4: Take the reaction product from step 3 and add it to Trans-T1 competent cells, ice-bath, heat shock, and place on ice for 2-5 minutes; add SOC medium and incubate; centrifuge to remove the supernatant, mix well, and spread on LB plates containing kanamycin. After incubation for 12-20 hours, select positive single colonies and perform colony PCR verification using primers Cum-YZ-up / Cum-YZ-down to obtain the verified correct plasmid pCum-rppA;

[0251] Flaviolin product fermentation is divided into three steps:

[0252] In the first step, 50 ng each of the plasmids pCDF-bauA-mcrC and pCum-rppA were added to 50 μl of MG1655(DE3) competent cells and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and spread onto an LB plate containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for MG1655(DE3), pCDF-bauA-mcrC, and pCum-rppA (strain E).

[0253] 50 ng each of the plasmids pCDF-duet and pCum-rppA were added to 50 μl of MG1655(DE3) competent cells and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) was added and incubated at 37°C for 1 hour at 220 rpm. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and spread onto an LB plate containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for MG1655(DE3), pCDF-duet, and pCum-rppA (serving as control strain E).

[0254] Step 2: Seed culture: strain E and control strain E were selected and inoculated into 50 mL shake tubes containing 10 mL LB medium containing spectinomycin (final concentration of 50 μg / ml) and kanamycin (final concentration of 50 μg / ml), respectively, and cultured at 37°C overnight at 220 r.

[0255] Step 3, Fermentation: Prepare 50 mL of M9 fermentation medium containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the culture medium with a seed of strain E and a control strain E at an initial OD550 of 0.1. Cultivate at 37°C for 220 days to an OD550 of approximately 0.6. Add IPTG to a final concentration of 0.1 mM and cumate to 0.25 mM. Continue fermentation at 37°C for 220 days for 24 hours.

[0256] The preparation of M9 culture medium is the same as that in Example 6.

[0257] Analytical method: After the fermentation is completed, take 2 mL of sample, centrifuge at 12,000 × g for 2 min, take the supernatant, and measure the absorbance value (A520) of the supernatant at 520 nm using a spectrophotometer.

[0258] Results: Compared with the control strain E (A520 = 1.81 ± 0.04), the engineered bacteria (strain E) containing a new malonyl-CoA synthesis pathway increased its flavonoid production (A520 = 2.57 ± 0.09) by 43% ( FIG. 10 ).

[0259] Example 8. Application of the new Malonyl-CoA pathway in the synthesis of polyketide-pentadecenes

[0260] Pentadecaheptaene, a class of olefinic compounds, is not only a key intermediate in the biosynthesis of alkyne-dienes (well-known anticancer antibiotics), but also a promising biofuel with high energy density. Pentadecaheptaene is synthesized by the type I PKS SgcE and its cognate thioesterase SgcE10. Each pentadecaheptaene molecule requires one acetyl-CoA molecule and seven malonyl-CoA molecules as precursors (Figure 11, schematic diagram of the pentadecaheptaene biosynthesis pathway (left), bar graph of pentadecaheptaene yield (right)). pQL1 is a previously constructed plasmid containing the pentadecaheptaene biosynthesis pathway (Q Liu et al., 2015).

[0261] Pentadecaheptaene production fermentation is divided into three steps:

[0262] In the first step, 50 ng each of the plasmids pCDF-bauA-mcrC and pQL1 were added to 50 μl of BL21(DE3) competent cells (purchased from BioPure) and placed on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and plated onto LB plates containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for BL21(DE3), pCDF-bauA-mcrC, and pQL1 (strain F).

[0263] 50 ng each of the plasmids pCDF-duet and pQL1 were added to 50 μl of BL21(DE3) competent cells (purchased from BioPure) and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. 500 μl of SOC medium was added and incubated at 220 rpm at 37°C for 1 hour. The supernatant was removed by centrifugation, leaving approximately 50 μl. The remaining 50 μl was mixed and plated onto LB plates containing spectinomycin (final concentration 50 μg / ml) and kanamycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained for BL21(DE3), pCDF-bauA-mcrC, and pQL1 (control strain F).

[0264] Step 2: Seed culture: Single clones of strain F and control strain F were selected and inoculated into 50 mL shake tubes containing 10 mL LB medium containing spectinomycin (final concentration of 50 μg / ml) and kanamycin (final concentration of 50 μg / ml), and cultured at 37°C overnight at 220 rpm.

[0265] Step 3: Fermentation: Prepare 50 mL of LB fermentation medium containing spectinomycin (final concentration 50 μg / mL) and kanamycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the culture medium with a seed of strain F and a control strain F at an initial OD550 of 0.1. Cultivate the culture at 37°C for 220 days to an OD550 of approximately 0.6. Add IPTG to a final concentration of 0.1 mM. Add the inducer, set the timer at zero, and continue fermentation at 18°C ​​for 24 hours.

[0266] Analytical method: Take 5 mL of sample, extract with ethyl acetate, and measure the absorbance of the supernatant at 395 nm (A395) using a spectrophotometer.

[0267] Results: Compared with the control strain F (A395 = 1.21 ± 0.05), the engineered bacteria (strain F) containing a new malonyl-CoA synthesis pathway increased pentadecaheptaene production (A395 = 1.92 ± 0.02) by 59% ( FIG. 11 ).

[0268] Case Study 9. Application of the New Malonyl-CoA Pathway in the Synthesis of Polyketide-Natamycin

[0269] Natamycin is a polyketide compound and a representative aminoglycoside antibiotic. It is the only antifungal drug approved by the U.S. Food and Drug Administration (FDA). Streptomyces gilvosporeus is the primary natamycin producer. In this strain, natamycin biosynthesis is initiated by a type I PKS, requiring one methylmalonyl-CoA molecule and 12 malonyl-CoA molecules as precursors for each natamycin molecule (Figure 12, schematic diagram of the natamycin biosynthesis pathway (left), bar graph of natamycin production (right)).

[0270] 1. Construction of plasmid pSET152-KasOP-BauA-SPL42-McrC:

[0271] 1.1. Plasmid pSET152 was digested with XbaI and BamHI at 37°C for 2 h. The pSET152 backbone fragment of 5709 bp was recovered by agarose gel electrophoresis and designated as fragment F (SEQ ID NO. 41).

[0272] 1.2. Using the pLH10 plasmid as a template, primers KasOP-F / R were used to amplify the KasOP promoter fragment, designated Fragment G (SEQ ID NO. 42). Using the pLH2 plasmid as a template, primers SPL42-F / R were used to amplify the SPL42 promoter fragment, designated Fragment H (SEQ ID NO. 43). Using the pCDF-bauA-McrC plasmid as a template, primers BauA-F / R were used to amplify the BauA gene fragment, designated Fragment J (SEQ ID NO. 44), and primers McrC-F / R were used to amplify the McrC gene fragment, designated Fragment K (SEQ ID NO. 45). The sequence of KasOP-F is shown in SEQ ID NO.46; the sequence of KasOP-R is shown in SEQ ID NO.47; the sequence of SPL42-F is shown in SEQ ID NO.48; the sequence of SPL42-R is shown in SEQ ID NO.49; the sequence of BauA-F is shown in SEQ ID NO.50; the sequence of BauA-R is shown in SEQ ID NO.51; the sequence of McrC-F is shown in SEQ ID NO.52; and the sequence of McrC-R is shown in SEQ ID NO.53.

[0273] The amplification system was as follows: 25 μL of Phanta 2x buffer, 1 μL of dNTP (10 mM each), 20 ng of DNA template, 2 μL of each primer (10 μM), 1 μL of Phanta Max Super-Fidelity DNA polymerase, and 18 μL of distilled water, with a total volume of 50 μL.

[0274] The amplification conditions were as follows: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 60 s (30 cycles); and extension at 72°C for 5 min (1 cycle).

[0275] 1.3. Using KasOP-F / BauA-R as primers and fragments H and M as templates, fragments H and M were connected by OE-PCR, and the result was named fragment P. Using SPL42-F / McrC-R as primers and fragments K and N as templates, fragments K and N were connected by OE-PCR, and the result was named fragment Q.

[0276] 1.4. Take appropriate amounts of Fragment G, Fragment P, and Fragment Q, add 5 μL of 2x Seamless Cloning Mix (Biyuntian Company), and react at 50°C for 30 min.

[0277] 1.5. Take 2 μL of the product from step 4 and add it to 50 μL of Trans-T1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and incubate on ice for 30 minutes. Heat shock the mixture at 42°C for 30 seconds and immediately place on ice for 2 minutes. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 37°C for 1 hour at 220 rpm. Centrifuge and remove the supernatant, reserving approximately 50 μL. Mix and spread the entire amount onto an LB plate containing apramycin (final concentration of 50 μg / mL). After overnight incubation, select five positive colonies and perform colony PCR verification using primers BauA-F / R and McrC-F / R. Clones with PCR fragments of 1368 bp and 2057 bp were identified as positive clones. Plasmids from two positive clones were extracted and sequenced. Comparison of the sequencing results revealed the correct plasmid, pSET152-KasOP-BauA-SPL42-McrC.

[0278] Construction of strains overexpressing the NCM pathway:

[0279] 2. Treatment of E. coli:

[0280] 2.1. Add 50 ng of plasmid pSET152-KasOP-BauA-SPL42-McrC to competent E. coli ETU12567 / pUZ8002 and incubate on ice for 30 minutes. Heat shock the culture at 42°C for 30 seconds, then immediately place on ice for 2 minutes. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 37°C for 1 hour at 220 rpm. Centrifuge and remove the supernatant, reserving approximately 50 μL. Mix thoroughly and spread the entire volume onto an LB plate containing apramycin (final concentration of 50 μg / mL), kanamycin (final concentration of 50 μg / mL), and chloramphenicol (final concentration of 34 μg / mL). Culture overnight to obtain strain H.

[0281] 2.2. Select 3-5 single colonies of strain H and culture them in 5 mL of LB medium containing apramycin (final concentration of 50 μg / mL), kanamycin (final concentration of 50 μg / mL), and chloramphenicol (final concentration of 34 μg / mL) at 220 rpm and 37°C overnight.

[0282] 2.3. Take the above bacterial suspension of strain H and inoculate it into 5 mL LB medium containing apramycin (final concentration of 50 μg / mL), kanamycin (final concentration of 50 μg / mL) and chloramphenicol (final concentration of 34 μg / mL) at a 10% inoculum volume. Cultivate for about 2.5 h and shake to OD = 0.6-0.8.

[0283] 2.4. Centrifuge at 3500 rpm for 5 min to collect the cells, add 5 mL of LB medium to wash 2-3 times, remove the supernatant, and add 1-2 mL of LB to resuspend the cells for later use. This is the standby bacterial solution of strain H.

[0284] 3. Treatment of Streptomyces chrysoporus J1-002 strain:

[0285] 3.1. Spread 100 μL of Streptomyces chrysoporus J1-002 bacterial suspension on a SFM plate and culture in a 30°C incubator for about 7 days.

[0286] 3.2. Scrape all spores with a cotton swab into a sterile 50 mL centrifuge tube, add 6 mL of TES to resuspend, and heat shock at 50°C for 10 min.

[0287] 3.3. Add 6 mL of 2x spore pre-germination solution and a final concentration of 10 mM CaCl2. Pre-germinate at 220 rpm and 37°C for 2-3 h. Aliquot 2 mL / tube into EP tubes, centrifuge, remove the supernatant, and set aside.

[0288] 3.4. Mix the prepared bacterial suspension of strain H and spore suspension of Streptomyces chrysoporus J1-002 in different proportions and apply to a solution containing 10 mM Mg 2+ The SFM plates were placed in a 30°C incubator for 14-16 hours.

[0289] 3.5. For a 25 mL solid plate, add trimethoprim to a final concentration of 50 μg / mL and Apr to a final concentration of 50 μg / mL in 2 mL of sterile water. Mix thoroughly and cover the plate. Incubate the plate in a 30°C incubator for 7-9 days to allow zygotes to grow.

[0290] SFM medium: 20 g / L soybean meal, 20 g / L mannitol, and 16 g / L Qingdao agar, pH 7.2, sterilized at 115°C for 30 min. 2x spore pre-germination solution: 0.1 g / L Difco Yeast Extract, 0.1 g / L Difco Casmino acid, sterilized at 115°C for 30 min.

[0291] Screening of engineered strains:

[0292] In the first step, the conjugates from step 3.5 were picked and transferred to SFM plates containing a final concentration of 50 μg / mL trimethoprim and 50 μg / mL apramycin for expansion culture and cultured in a 30°C incubator for 5-7 days.

[0293] The second step was to scrape off some spores to extract the genome for PCR verification. Genome extraction: Scrape off some spores and add them to 500 μL SET Buffer, followed by the addition of 20 μL 100 mg / mL lysozyme and lysis in a 37°C waterbath for 60 minutes. Add 100 μL 10% SDS solution and mix by inverting. Add 600 μL DNA extraction phenol reagent, mix by inverting, and centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 600 μL DNA extraction phenol reagent, mix by inverting, and centrifuge at 12,000 rpm for 5 minutes. Add 50 μL 3 M NaAc, mix by inverting, and then add 500 μL pre-cooled isopropanol, mix thoroughly, and centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and wash twice with 75% ethanol. After the ethanol evaporates, add an appropriate amount of distilled water to dissolve the genome. Apr-F / R primers were used, and the genome was used as the template. When a 411 bp band was amplified, it indicated the correct engineered strain, J1-002, pSET152-KasOP-BauA-SPL42-McrC (strain G). J1-002 served as the control strain G. The sequence of Apr-F is shown in SEQ ID NO. 59; the sequence of Apr-R is shown in SEQ ID NO. 60.

[0294] SET Buffer formula: 75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0.

[0295] Natamycin fermentation is divided into three steps:

[0296] Step 1: Plate culture: 100 μL of strain G and control strain G bacterial solution were spread on SFM plates containing apramycin (final concentration of 50 μg / mL) and cultured in a 30°C incubator for about 10 days until the spores turned grayish white.

[0297] Step 2: Seed culture: Take out about 1cm from the plate 2 The large and small agar blocks were inoculated into 100 mL (500 mL shake flask) of seed culture medium and cultured at 200 rpm and 28°C for 24 h.

[0298] The third step is shake flask fermentation: the seed liquid is inoculated into a fermentation shake flask at a 5% inoculum size, the volume of the fermentation shake flask is 80 mL (500 mL shake flask), and the culture is carried out at 200 rpm and 28° C. for 5 days.

[0299] Seed culture medium: yeast powder 12 g / L, NaCl 10 g / L, glucose 15 g / L, defoamer 0.2 g / L, pH 6.8-7.0, sterilization at 115°C for 30 min; fermentation medium: yeast powder 8 g / L, soy protein isolate 30 g / L, glucose 40 g / L, biotin 0.001 g / L, pH 7.1, sterilization at 115°C for 30 min.

[0300] Analytical Method: Add 9 mL of methanol to 1 mL of fermentation broth. After thorough shaking, sonicate for 20 minutes, and then centrifuge at 8000 rpm for 15 minutes. Dilute the supernatant with methanol to an appropriate ratio and filter through a 0.22 μm microporous filter. HPLC Detection: Mobile phase: methanol:water = 6:4, flow rate 0.7 mL / min, detection wavelength: 303 nm, column temperature: 30°C.

[0301] Results: Compared with the control strain G (168±25 mg / L), the engineered bacteria (strain G) containing the new malonyl-CoA synthesis pathway increased the natamycin production (282±42 mg / L) by 67% ( FIG12 ).

[0302] Example 10 Application of the new Malonyl-CoA pathway in the synthesis of polyketide compounds - spinosad

[0303] Spinosad is a highly effective, safe, and broad-spectrum biopesticide. Saccharopolyspora spinosa is the primary strain producing spinosad. In this strain, spinosad biosynthesis is initiated by a type I PKS, with the synthesis of each spinosad molecule requiring one propionyl-CoA molecule, one methylmalonyl-CoA molecule, nine malonyl-CoA molecules, and one propionyl-CoA molecule as precursors (Figure 13, Schematic diagram of the spinosad biosynthesis pathway (left), and histogram of spinosad production (right).

[0304] Construction of Saccharopolyspora spinosa overexpressing the NCM pathway:

[0305] 1. Treatment of E. coli:

[0306] 1.1. Take 50 ng of plasmid pSET152-KasOP-BauA-SPL42-McrC (construction method, see Example 9) and add it to competent E. coli DH10B cells. Incubate on ice for 30 min. Heat shock at 42°C for 30 s and immediately place on ice for 2 min. Add 500 μL of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 37°C for 1 h at 220 rpm. Centrifuge and remove the supernatant, leaving approximately 50 μL. Mix and spread the entire amount onto an LB plate containing apramycin (final concentration of 50 μg / mL) to obtain strain J. Streak E. coli ET pUB307 onto an LB plate containing kanamycin (final concentration of 50 μg / mL) and chloramphenicol (final concentration of 34 μg / mL) and culture overnight.

[0307] 1.2. Select 3-5 single colonies of strain J and incubate in 5 mL of LB medium containing apramycin (final concentration of 50 μg / mL). Select 3-5 single colonies of ET pUB307 and incubate in 5 mL of LB medium containing kanamycin (final concentration of 50 μg / mL) and chloramphenicol (final concentration of 34 μg / mL). Incubate at 220 rpm and 37°C overnight.

[0308] 1.3. Take the above bacterial suspension and inoculate it into 50 mL LB medium containing apramycin (final concentration of 50 μg / mL) and 50 mL LB medium containing kanamycin (final concentration of 50 μg / mL) and chloramphenicol (final concentration of 34 μg / mL) at a 10% inoculum volume, respectively. Cultivate for about 2.5 h and shake to OD = 0.6-0.8.

[0309] 1.4. Centrifuge at 3500 rpm for 5 min to collect the cells, wash 2-3 times with 20 mL of LB medium, remove the supernatant, and resuspend in 3-4 mL of LB for later use.

[0310] 2. Treatment with Saccharopolyspora spinosa WHU1107:

[0311] 2.1. Spread 100 μL of the bacterial solution of Saccharopolyspora spinosa WHU1107 prepared in step 1.4 onto an ABB13 plate and incubate in a 28°C incubator for 7 days.

[0312] 2.2. Use the gun tip to pick up 1cm 2 The bacterial blocks were cultured in TSB-M medium for primary seed culture at 220 rpm and 28 °C for 72 h;

[0313] 2.3. Transfer 2% of the primary seed solution to the secondary seed solution and incubate at 220 rpm and 28°C for 48 h.

[0314] 2.4. Pour 50 mL of the secondary seed solution into a 50 mL centrifuge tube and centrifuge at 4500 rpm for 10 minutes to harvest the bacteria. Wash the cells 2-3 times with 20 mL of LB medium and resuspend them in approximately 5-8 mL of LB medium for later use.

[0315] 2.5. Mix the bacterial suspension of Saccharopolyspora spinosa WHU1107 with strain H and ET pUB307 in different proportions and evenly spread them on a plate containing 10 mM Mg 2+ Place the plate in an ABB13 plate in a 28°C incubator for 22 hours.

[0316] 2.6. For a 25 mL solid plate volume, add trimethoprim to a final concentration of 50 μg / mL and Apr to a final concentration of 15 μg / mL in 2 mL of sterile water. Mix thoroughly and cover the plate. Incubate the plate in a 28°C incubator for 10 days to allow zygotes to grow.

[0317] ABB13 medium: 5 g / L soluble starch, 5 g / L soy peptone, 2.1 g / L 3-CN-morpholinepropanesulfonic acid, 3 g / L calcium carbonate, 0.01 g / L thiamine hydrochloride, 0.046 g / L ferrous sulfate heptahydrate, 20 g / L agar, pH 7.0, sterilized at 115°C for 30 min. TSB-M medium: 30 g / L Tryptone Soy Broth, 50 g / L mannitol; aliquot 50 mL of medium into 250 mL shake flasks and sterilize at 115°C for 30 min.

[0318] 3. Screening of engineered strains:

[0319] In the first step, the conjugates from step 2.6 were picked and transferred to ABB13 plates containing a final concentration of 50 μg / mL trimethoprim and 15 μg / mL apramycin for expansion culture and cultured in a 28°C incubator for 5-7 days.

[0320] The second step was to scrape a portion of the spores to extract the genome for PCR verification. To extract the genome, scrape a portion of the spores and add them to 500 μL of SET Buffer. Then, add 20 μL of 100 mg / mL lysozyme and lyse the cells in a 37°C waterbath for 60 minutes. Add 100 μL of 10% SDS solution and mix by inverting. Add 600 μL of DNA extraction phenol reagent, mix by inverting, and centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 600 μL of DNA extraction phenol reagent, mix by inverting, and centrifuge at 12,000 rpm for 5 minutes. Add 50 μL of 3 M NaAc, mix by inverting, and then add 500 μL of pre-cooled isopropanol. Mix thoroughly and centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and rinse twice with 75% ethanol. After the ethanol evaporates, add an appropriate amount of distilled water to dissolve the genome. Apr-F / R is the primer and the genome is the template. When the amplified band is 411bp, it indicates the correct engineered strain WHU1107, pSET152-KasOP-BauA-SPL42-McrC (strain K). (WHU1107 is the control strain K)

[0321] SET Buffer formula: 75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0.

[0322] Spinosad fermentation is divided into four steps:

[0323] Step 1: Plate culture: 100 μL of strain K and control strain K bacterial solution were spread on fermentation plates containing apramycin resistance (final concentration of 15 μg / mL) and cultured in a 28°C incubator for 7 days;

[0324] Step 2: Primary seed culture: Cut out 1cm 2 The bacterial blocks of about 100 μg / cm were cultured in seed culture medium at 250 rpm, 28°C, and 60% humidity for 96 h.

[0325] Step 3: Secondary seed culture: transfer 1% of the transfer volume to the secondary seed and culture at 250 rpm, 28°C, and 60% humidity for 60 h;

[0326] Step 4: Fermentation: 5% of the transferred volume was transferred to the fermentation medium and cultured at 250 rpm, 28° C., and 60% humidity for 12 days.

[0327] Fermentation plate: 5 g / L glucose, 3 g / L yeast extract, 10 g / L enzymatic casein (NZ amine type A), 20 g / L agar, pH 7.0, sterilized at 115°C for 30 min; seed medium: 10 g / L glucose, 10 g / L yeast extract, 2 g / L enzymatic casein (NZ amine type A), 25 g / L cottonseed meal, 20 g / L corn starch, 2 g / L magnesium sulfate heptahydrate, 1 g / L ammonium sulfate, pH 7.0, 25 mL of medium was dispensed into 250 mL shake flasks, sterilized at 121°C for 30 min; fermentation medium: 80 g / L glucose, 20 g / L cottonseed meal, 10 g / L Dolphin brand protein powder, 5 g / L yeast powder, 4 g / L trisodium citrate, 2 g / L dipotassium hydrogen phosphate, 3 g / L calcium carbonate, 2 g / L ammonium sulfate, 50 g / L rapeseed oil, pH 7.0, 25 mL of culture medium was dispensed into 250 mL shake flasks and sterilized at 121°C for 30 min.

[0328] Detection method: Take 1 mL of fermentation broth, add 4 mL of ethanol, shake thoroughly, and then centrifuge at 8000 rpm for 15 minutes. Dilute the supernatant with ethanol to an appropriate ratio and filter through a 0.22 μm microporous filter. HPLC detection method: Mobile phase: methanol: acetonitrile: water (containing 5% ammonium acetate) = 45:45:10, flow rate: 1 mL / min, detection wavelength: 250 nm, column temperature: 30°C.

[0329] Results: Introducing the novel NCM pathway into Saccharopolyspora spinosa WHU1107 (CCTCC NO: M 2021307) increased the yield by 52% and altered the product composition of spinosyn A and spinosyn D from the original spinosyn A:spinosyn D ratio of 97:3 to 65:35 ( Figure 13 ).

[0330] Example 11 New Pathway Improves Production of NADPH Cofactor

[0331] 1. Strain Preparation: 50 ng each of the plasmids pCDF-bauA-mcrC and pCDF-duet were added to 50 μl of MG1655(DE3) competent cells (purchased from Yu Pure Biosciences) and incubated on ice for 30 minutes. Heat shock was performed at 42°C for 60 seconds, followed by immediate placement on ice for 2 minutes. Add 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 220 rpm at 37°C for 1 hour. Centrifuge and discard the supernatant, reserving approximately 50 μl. Mix and spread the remaining 50 μl onto LB plates containing spectinomycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained, yielding the engineered strain MG1655(DE3) with pCDF-bauA-mcrC (strain L) and the control strain MG1655(DE3) with pCDF-duet (strain M).

[0332] 2. Preparation of bacterial solution

[0333] Seed Culture: After sterilizing each fermentation medium component separately, prepare the seed culture medium in a clean hood. Single colonies of strain M and strain L were selected and inoculated into 50 mL shake tubes containing 5 mL of seed culture medium containing spectinomycin (final concentration 50 μg / mL). Culture overnight at 37°C at 220 rpm.

[0334] Fermentation: Prepare 50 mL of fermentation medium containing spectinomycin (final concentration 50 μg / mL) in a 250 mL Erlenmeyer flask. Inoculate the seed culture at an initial OD550 of 0.1, add IPTG to a final concentration of 0.1 mM, and incubate in a constant temperature shaker at 37°C for 220 days until the OD550 reaches approximately 1. Pipette 1 mL of each culture medium from strains M and L.

[0335] 3. Determination of NADPH

[0336] Using NADPH / NADP + The NADPH / NADP of the bacterial solution was determined by using a WST-8 assay kit (purchased from Biyuntian Biotechnology Co., Ltd.) + The results showed that compared with the control strain M (0.247±0.022), the engineered strain L containing a new malonyl-CoA synthesis pathway had a higher intracellular NADPH / NADP + (0.393±0.005) increased by 59% (Figure 14).

[0337] Case Study 12: No cytotoxicity with the new approach

[0338] Numerous studies have shown that overexpression of ACC in the natural biosynthesis pathway often leads to cytotoxicity, thereby inhibiting cell growth. Next, we examined the effects of ACC and NCM on the growth of a representative Escherichia coli MG1655(DE3).

[0339] The culture medium used for the tolerance test was MOPS medium (containing 20 g / L glucose) with IPTG (final concentration of 0.1 mM) and spectinomycin (final concentration of 50 μg / ml) added.

[0340] This experiment requires the use of pCDF-ACC plasmid

[0341] The construction of plasmid pCDF-ACC is divided into four steps:

[0342] In the first step, using the pCDF-duet plasmid as a template and primers P1-R / P1-F, a pCDF vector backbone fragment was amplified. The fragment was 3781 bp in size and contained the T7 promoter, Ori sequence, and spectinomycin resistance gene Smr, referred to as fragment I. The sequence of P1-R is shown in SEQ ID NO. 1, the sequence of P1-F is shown in SEQ ID NO. 2, and the sequence of fragment I is shown in SEQ ID NO. 3.

[0343] In the second step, the accBC coding gene was amplified using the genome of Corynebacterium glutamicum as a template using primers accBC-up / accBC-down. The gene fragment was 1811 bp in size and contained the accBC coding sequence, referred to as fragment R (SEQ ID NO.61); the sequence of accBC-up is shown in SEQ ID NO.62; the sequence of accBC-down is shown in SEQ ID NO.63.

[0344] Step 3: Remove the template from fragments I and R using the endonuclease DpnI. After electrophoresis, clean the fragments using a PCR purification kit. Add fragments I and R to 2X Seamless Cloning Mix and react for 30-60 minutes.

[0345] Step 4: Take the reaction product from step 3 and add it to Trans-T1 competent cells, ice-bath, heat shock, and place on ice for 2-5 minutes. Add SOC medium and incubate. Centrifuge to remove the supernatant, mix well, and spread on LB plates containing spectinomycin. After incubation for 12-20 hours, select positive single colonies, extract plasmids, and send for sequencing to obtain the correct pCDF-accBC plasmid.

[0346] Step 5: Using the pCDF-accBC plasmid as a template and primers P2-R / P2-F, a vector backbone fragment was amplified. The fragment was 5557 bp in size and contained the T7 promoter, accBC gene, Ori sequence, and spectinomycin resistance gene Smr, referred to as fragment S. The sequence of P2-R is shown in SEQ ID NO. 64, the sequence of P2-F is shown in SEQ ID NO. 65, and the sequence of fragment S is shown in SEQ ID NO. 66.

[0347] In the sixth step, the accD coding gene was amplified using the genome of Corynebacterium glutamicum as a template using primers accD-up / accD-down. The gene fragment was 1683 bp in size and contained the accD coding sequence, referred to as fragment T (SEQ ID NO. 67). The sequence of accD-up is shown in SEQ ID NO. 68; the sequence of accD-down is shown in SEQ ID NO. 69.

[0348] Step 7: Remove the template from fragments S and T using the endonuclease DpnI. After electrophoresis, clean the fragments using a PCR purification kit. Take fragments S and T and add 2X Seamless Cloning Mix to react for 30-60 minutes.

[0349] Step 8: Take the reaction product from step 3 and add it to Trans-T1 competent cells. Place them on ice for 2-5 minutes after heat shock. Add SOC medium and incubate. Centrifuge to remove the supernatant, mix well, and spread on LB plates containing spectinomycin. After incubation for 12-20 hours, select positive single colonies, extract plasmids, and send for sequencing to obtain the correct pCDF-ACC plasmid.

[0350] To obtain the strain: 50 ng of the plasmid pCDF-ACC was added to 50 μl of MG1655(DE3) competent cells (purchased from Yu Pure Biosciences) and placed on ice for 30 minutes. Heat shock the cells at 42°C for 60 seconds and immediately place them on ice for 2 minutes. Add 500 μl of SOC medium (purchased from Shanghai Sangon Biotechnology Co., Ltd.) and incubate at 37°C for 1 hour at 220 rpm. Centrifuge and remove the supernatant, reserving approximately 50 μl. Mix and spread the remaining 50 μl onto LB plates containing spectinomycin (final concentration 50 μg / ml). After overnight incubation, positive clones were obtained, thus obtaining the engineered strain MG1655(DE3) and pCDF-ACC (strain N).

[0351] Single clones of MG1655(DE3), pCDF-bauA-mcrC (strain L), MG1655(DE3), pCDF-duet (strain M), and MG1655(DE3), pCDF-ACC (strain N) were picked and inoculated into a 15 mL test tube containing 5 mL of MOPS medium containing spectinomycin (final concentration of 50 μg / ml) and cultured at 37°C for 220 rpm until OD550 was about 1.5-1.8 as seed solution.

[0352] The seed solution was inoculated into MOPS medium (containing 20 g / L glucose, 0.1 mM IPTG, and a final concentration of 50 μg / ml chloramphenicol and ampicillin) at an initial OD550 of 0.1. After inoculation, the mixture was shaken and 200 μL was transferred to a 96-well plate. A continuous kinetic assay was performed using a microplate reader (Biotek), and the OD550 was measured every 10 minutes to determine the growth curve of the strain. The OD550 of the exponential growth phase was taken. T , culture time T and initial OD550 O The specific growth rate μ can be calculated as ln(OD550 T -OD550 O ) / T.

[0353] Results: Compared with the control strain M, the specific growth rate (μ, h -1 ) significantly reduced: from 0.50h -1 Down to 0.25h -1 , the decrease was 50% (Figure 15). Compared with ACC, the specific growth rate of E. coli strain L overexpressed by NCM strain decreased slightly (μ = 0.41h -1 ), indicating that overexpression of the NCM pathway did not lead to severe cytotoxicity ( FIG. 15 ).

[0354] Example 13 New approach improves strain tolerance

[0355] 1. Tolerance test method

[0356] Seed culture: Single clones of MG1655(DE3), pCDF-bauA-mcrC (strain L), MG1655(DE3), pCDF-duet (strain M), and MG1655(DE3), pCDF-ACC (strain N) were picked and inoculated into 15 mL test tubes containing 5 mL of MOPS medium containing spectinomycin (final concentration of 50 μg / ml). The culture was incubated at 37°C for 220 rpm until the OD550 reached about 1.5-1.8, which served as the seed solution.

[0357] Culture medium used for tolerance test: IPTG (final concentration of 0.1 mM) and spectinomycin (final concentration of 50 μg / ml) were added to MOPS medium (containing 20 g / L glucose), and then inhibitors were added. The selected inhibitors and their final concentrations were formic acid (100 mM), acetic acid (50 mM), hexanoic acid (10 mM), lactic acid (500 mM), succinic acid (200 mM), glucose (100 g / L), sucrose (480 mM), octanoic acid (10 mM), citric acid (200 mM), vanillic acid (3 g / L) and triacetic acid lactone (TAL, 1.25 g / L).

[0358] 2. Tolerance test method

[0359] The seed solution was inoculated into the medium used for tolerance testing at an initial OD550 of 0.1. After inoculation, the mixture was shaken and 200 μL was transferred to a 96-well plate. A microplate reader (Biotek) was used to perform a continuous kinetic determination, and the OD550 was measured every 10 minutes to determine the growth curve of the strain. The OD550 value during the exponential growth phase was taken. T , culture time T and initial OD550 O The specific growth rate μ can be calculated as ln(OD550 T -OD550O ) / T.

[0360] Results: Overexpression of the NCM pathway increased E. coli's tolerance to a variety of toxic chemicals, such as organic acids. Among them, E. coli overexpressing the NCM pathway showed significantly improved tolerance to succinate (200 mM succinate [23.6 g / L]), with its biomass (OD550 = 0.60) increasing by 100% compared to the control strain (OD550 = 0.30). In contrast, the growth ability of E. coli overexpressing the ACC pathway at this concentration was significantly reduced or even completely inhibited (OD550 = 0.20) (Figure 15). Similar phenomena were observed when the strain was challenged with other organic acids, including lactic acid, acetic acid, hexanoic acid, and citric acid (Figure 15).

[0361] In addition to organic acids, overexpression of the NCM pathway improved E. coli's tolerance to polyketides (Figure 15): Taking triacetic acid lactone, the simplest polyketide compound, as an example (10 mM, [1.26 g / L]), the biomass of the NCM E. coli strain (OD550 = 0.81) increased by 30% compared with the control E. coli strain (OD550 = 0.62) and increased by 180% compared with the ACC-overexpressing E. coli strain (OD550 = 0.29).

[0362] In addition, the overexpression of the NCM pathway also improves the tolerance of E. coli to adverse environmental stress (Figure 15). Under higher osmotic pressure (300mM NaCl [~17.5g / L]), the biomass of the overexpression E. coli strain of the NCM pathway (OD550=0.76) increased by 29% and 214% respectively compared with the control (OD550=0.59) and the ACC overexpression strain (OD550=0.24). In the presence of high concentrations of sugar (480mM sucrose [~164g / L]), the biomass of the overexpression E. coli strain L of the NCM pathway (OD550=0.96) increased by 26% and 320% respectively compared with the control strain M (OD550=0.77) and the ACC strain N (OD550=0.23).

[0363] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for artificially synthesizing malonyl-CoA, characterized in that: The steps include: S1, β-alanine and the compound represented by formula (1) form 3-oxopropionic acid and the compound represented by formula (2) under the catalysis of aminotransferase; wherein R is selected from H, alkanes, alkanols, and alkanoic acids; S2, 3-oxopropionic acid, Coenzyme A, electron acceptor: Under the catalysis of oxidoreductase, the electrons of 3-oxopropionic acid are transferred to the electron acceptor to form malonyl-CoA and 2[H].

2. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: The compound represented by formula (1) includes α-keto acids.

3. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: The α-keto acid is selected from one or more of pyruvic acid, oxaloacetic acid, and α-ketoglutaric acid.

4. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: The compound represented by formula (2) includes α-amino acids.

5. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: The α-amino acid includes α-alanine, aspartic acid or glutamic acid.

6. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: In step S1, the aminotransferase includes one or more of BauA from Pseudomonas aeruginosa, AptA from Acinetobacter pittii, CCNA_03245 from Caulobacter vibrioides, PydD2 from Bacillus megaterium, YhxA from Bacillus cereus, PydD from Ureibacillus massiliensis and Pyd4 from Saccharomyces kluyveri.

7. The method for artificially synthesizing malonyl-CoA according to claim 2, characterized in that: The amino acid sequence of BauA is shown in SEQ ID NO.7; The amino acid sequence of AptA is shown in SEQ ID NO.8; The amino acid sequence of CCNA_03245 is shown in SEQ ID NO.9; The amino acid sequence of PydD2 is shown in SEQ ID NO.10; The amino acid sequence of YhxA is shown in SEQ ID NO.11; The amino acid sequence of PydD is shown in SEQ ID NO.71; The amino acid sequence of Pyd4 is shown in SEQ ID NO.

72.

8. The method for artificially synthesizing malonyl-CoA according to claim 1, characterized in that: The oxidoreductase comprises one or more of MCR-C from Chloroflexus aurantiacu, StMCR from Sulfurisphaera tokodaii, Msed_0709 from Metallosphaera sedula, PnSCR from Pyrobaculum neutrophilum and SucD from Clostridium kluyveri.

9. The method for artificially synthesizing malonyl-CoA according to claim 8, characterized in that: The amino acid sequence of MCR-C is shown in SEQ ID NO.70; The amino acid sequence of StMCR is shown in SEQ ID NO.12; The amino acid sequence of Msed_0709 is shown in SEQ ID NO.13; The amino acid sequence of the PnSCR is shown in SEQ ID NO.14; The amino acid sequence of SucD is shown in SEQ ID NO.

15.

10. The method for artificially synthesizing malonyl-CoA according to claim 9, characterized in that: The preparation method of the MCR-C comprises the following steps: 1) Construct the recombinant plasmid pCDF-P1-mcrC according to the amino acid sequence of MCR-C; 2) Transform the recombinant plasmid pCDF-P1-mcrC into E.coli BL21 DE3 competent cells to obtain positive clones; 3) Cultivating the positive monoclonal colonies under the induction of an inducer; 4) centrifuging and purifying the culture product of step 3), adding a non-denaturing lysis solution to resuspend the bacteria, adding lysozyme to mix, cooling and ultrasonicating, and lysing the bacteria; centrifuging, collecting the supernatant, and preparing a bacterial lysate; 5) The bacterial lysate prepared in step 4) is subjected to chromatography to obtain a purified MCR-C protein sample.

11. The method for artificially synthesizing malonyl-CoA according to claim 10, characterized in that: The construction method of the recombinant plasmid pCDF-P1-mcrC comprises the following steps: 1) Using the pCDF-duet plasmid containing 6×His tag as a template and primers P1-R / P1-F, a pCDF vector backbone fragment was amplified, which contained T7 promoter, Ori sequence and spectinomycin resistance gene Smr, referred to as fragment I; wherein the sequence of P1-R is shown in SEQ ID NO.1, the sequence of P1-F is shown in SEQ ID NO.2, and the sequence of fragment I is shown in SEQ ID NO.3; 2) Using pMCR-C plasmid as template and primers MCR-C-pCDF-P1-up / MCR-C-pCDF-P1-down, The MCR-C coding gene was amplified and named as fragment II; wherein the sequence of MCR-C-pCDF-P1-up is shown in SEQ ID NO.4, the sequence of MCR-C-pCDF-P1-down is shown in SEQ ID NO.5, and the sequence of fragment II is shown in SEQ ID NO.6; 3) Remove the template from fragment I and fragment II using endonuclease DpnI, respectively, and after electrophoresis, use a PCR purification kit to clean the fragments. Remove the template-eliminated fragments I and II, and add 2X Seamless Cloning Mix to react; 4) Take the reaction product of step 3) and transfer it into Trans-T1 competent cells, select positive single colonies, and perform colony PCR verification with primers P1-YZ-up / MCR-C-YZ-down (SEQ ID NO.55); obtain the correct plasmid pCDF-P1-mcrC; wherein the sequence of P1-YZ-up is shown in SEQ ID NO.54; the sequence of MCR-C-YZ-down is shown in SEQ ID NO.

55.

12. The method for artificially synthesizing malonyl-CoA according to claim 9, characterized in that: The preparation method of BauA comprises the following steps: Step 1, constructing a recombinant plasmid pCDF-P1-bauA according to the amino acid sequence of bauA; Step 2, the recombinant plasmid pCDF-P1-bauA was transformed into E.coli BL21 DE3 competent cells to obtain positive clone colonies; Step 3, culturing the positive monoclonal colonies in step 3 under the induction of an inducer; Step 4, centrifuging and purifying the culture product of step 3, adding non-denaturing lysis buffer to resuspend the bacteria, adding lysozyme to mix, cooling and ultrasonicating, and lysing the bacteria; centrifuging, collecting the supernatant, and preparing a bacterial lysate; Step 5: The bacterial lysate prepared in step 4 is subjected to chromatography to obtain a purified BauA protein sample.

13. A recombinant plasmid comprising BauA and MCR-C, characterized in that: The amino acid sequence of BauA is shown in SEQ ID NO.7; the amino acid sequence of MCR-C is shown in SEQ ID NO.

70.

14. A method for synthesizing fatty acids using the recombinant plasmid according to claim 13, characterized in that: The recombinant plasmid uses pCDF as a vector and is named pCDF-bauA-mcrC; the method comprises the following steps: The recombinant plasmid pCDF-bauA-mcrC and the thioesterase expression plasmid were transferred into competent Escherichia coli cells to obtain positive clone strains, and the fatty acid was obtained after fermentation culture.

15. The method according to claim 14, characterized in that The fatty acid includes caprylic acid, capric acid, lauric acid, myristic acid or hexadecanoic acid.

16. A method for synthesizing a polyketide compound using the recombinant plasmid according to claim 13, characterized in that: The recombinant plasmid uses pCDF as a vector and is named pCDF-bauA-mcrC; the method comprises the following steps: transferring the recombinant plasmid pCDF-bauA-mcrC and a polyketide synthase encoding gene into competent Escherichia coli cells to obtain a positive clone strain, and obtaining the polyketide compound after fermentation and culture.

17. The method according to claim 16, characterized in that The polyketide compounds include phloroglucinol, flavonoids, and pentadecene.

18. The method according to claim 17, characterized in that The synthesis method of phloroglucinol comprises the following steps: transferring plasmids pCDF-bauA-mcrC and pCum-phlD into competent Escherichia coli cells to obtain positive clone strains, and obtaining the phloroglucinol after fermentation and culture.

19. The method according to claim 17, characterized in that The synthesis method of flavonoids comprises the following steps: transferring plasmids pCDF-bauA-mcrC and pCum-rppA into competent Escherichia coli cells to obtain positive clone strains, and obtaining flavonoids after fermentation and culture.

20. The method according to claim 17, characterized in that The synthesis method of the pentadecene comprises the following steps: transferring plasmids pCDF-bauA-mcrC and pQL1 into competent cells of Escherichia coli to obtain positive clone strains, and obtaining the pentadecene after fermentation and culture.

21. A method for synthesizing natamycin or spinosad using the recombinant plasmid according to claim 13, characterized in that: The recombinant plasmid uses SET152 as a vector and uses strong promoters KasOP and SPL42 to express BauA and MCR-C, respectively, and is named SET152-KasOP-bauA-SPL42-mcrC; The synthesis method of natamycin comprises the following steps: The recombinant plasmid SET152-KasOP-bauA-SPL42-mcrC was transferred into Streptomyces chrysoporus to obtain a positive clone strain, and the natamycin was obtained after fermentation and culture; The synthesis method of spinosad comprises the following steps: The recombinant plasmid SET152-KasOP-bauA-SPL42-mcrC was transferred into Saccharopolyspora spinosa to obtain a positive clone strain, and the spinosad was obtained after fermentation and culture.

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