Genetically modified microorganisms that increase the production of polyketide compounds or their derivatives, and methods for producing polyketide compounds or their derivatives.
Genetically modified microorganisms enhance polyketide compound production by blocking acetyl-CoA consumption and replacing carboxylase with transaminase and reductase, achieving up to 4.5 times higher yields of polyketide compounds.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- IND TECH RES INST
- Filing Date
- 2024-12-26
- Publication Date
- 2026-07-08
AI Technical Summary
Existing methods for producing polyketide compounds, such as those involving the production of carmine, fail to efficiently address the production needs of carbon emissions and the production of carbon, and existing methods for producing carmine are inefficient and lack the production of specific products, such as those involving the production of specific compounds, such as those involving the production of specific technologies, such as those involving the production of polyketide compounds.
The development of innovative methods for producing polyketide compounds or their derivatives through genetically modified microorganisms, specifically by deleting the endogenous PTA gene to block the consumption of acetyl-CoA and replacing carboxylase with transaminase and reductase to enhance the concentration of malonyl-CoA, thereby increasing the production of polyketide compounds.
The genetically modified microorganisms significantly increase the production of polyketide compounds, such as flavokermesic acid and carminic acid, by up to 4.5 times compared to wild-type strains, addressing the inefficiencies of chemical synthesis and biological production methods.
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Figure 2026114173000001_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to genetically modified microorganisms and methods for producing polyketide compounds or derivatives thereof by the same.
Background Art
[0002] Chemical synthesis usually highly relies on petrochemical raw materials and has a high carbon emission. Under the global trend of reducing carbon emissions and aiming for net-zero, biosynthesis technology has attracted attention because it conforms to the future trend of environmental protection.
[0003] Acetyl coenzyme A (acetyl-CoA) and malonyl coenzyme A (malonyl-CoA) are important initial reactants in the polyketide compound (polyketide) production pathway. In the metabolic pathway of acetyl-CoA production, phosphate acetyltransferase expressed by the pta gene consumes acetyl coenzyme to produce acetic acid, thereby reducing the concentration of acetyl-CoA in the organism. Also, in the metabolic pathway of malonyl-CoA production, carboxylase participating in the production catalytic reaction is subject to feedback inhibition by the product malonyl-CoA, and at the same time, it also needs to consume adenosine triphosphate (ATP), an energy molecule. Therefore, the concentration of malonyl-CoA in the organism cannot increase. Low concentrations of acetyl-CoA and malonyl-CoA indirectly affect the carbon flux in the polyketide synthesis metabolic pathway, limiting the production of polyketide compounds and their related derivatives.
[0004] For example, the pigment "carmine," which is used in large quantities in expensive cosmetics, is one of the derivatives of common polyketide compounds. Because it contains a complex structure in which one anthraquinone polyketide compound is linked to one glucose molecule, it is very difficult to prepare through chemical synthesis, and the production volume is not high. Furthermore, the mainstream method of producing carmine by extraction using cochineal insects is also difficult to mass-produce due to factors such as the long growth cycle of cochineal insects, geographical and climatic limitations, and the complicated extraction steps.
[0005] As mentioned above, one of the research challenges currently being addressed by the industry is to develop biosynthetic methods that can improve the production efficiency of polyketide compounds or their derivatives, thereby enhancing the market competitiveness of related products. [Overview of the project]
[0006] Some embodiments of the present disclosure provide genetically modified microorganisms that enhance the production of polyketide compounds or derivatives thereof, comprising any one or two of the following gene modifications: (a) a deleted endogenous PTA gene encoding phosphate acetyltransferase, wherein the expression level of phosphate acetyltransferase in the deleted endogenous PTA gene is lower than that of the wild type; and (b) an added first exogenous nucleotide sequence and a added second exogenous nucleotide sequence, wherein the first exogenous nucleotide sequence encodes a transaminase and the second exogenous nucleotide sequence encodes a reductase.
[0007] Some other embodiments of this disclosure provide a method for producing polyketide compounds or derivatives thereof, comprising the steps of: (a) preparing the genetically modified microorganism described above; (b) preparing a first culture medium, inoculating the genetically modified microorganism into the first culture medium, and culturing the genetically modified microorganism at a temperature of 28°C to 37°C for 12 to 24 hours; (c) inoculating the first culture medium containing the cultured genetically modified microorganism into a second culture medium, and culturing the genetically modified microorganism at a temperature of 28°C to 37°C for 24 to 80 hours to obtain a second culture medium containing polyketide compounds or derivatives thereof; and (d) separating the polyketide compounds or derivatives thereof from the second culture medium of step (c).
[0008] Furthermore, several other embodiments of this disclosure provide a novel genetically modified strain of Escherichia coli, accession number BCRC 940701. The novel genetically modified strain of Escherichia coli has a deletion of the endogenous pta gene and contains a first exogenous nucleotide sequence and a second exogenous nucleotide sequence, the endogenous pta gene encoding phosphate acetyltransferase, the first exogenous nucleotide sequence encoding transaminase, and the second exogenous nucleotide sequence encoding reductase.
[0009] To make the features or advantages of this disclosure clearer and easier to understand, several embodiments are described below in detail, along with the accompanying drawings. [Brief explanation of the drawing]
[0010] [Figure 1] The biosynthetic pathway diagrams of carminic acid constructed by genetically modified microorganisms are shown in some examples of the present disclosure. [Figure 2] The plasmid construction maps of genetically modified microorganisms are shown in some examples of the present disclosure. [Figure 3]The charts shown here illustrate high-performance liquid chromatography (HPLC) analysis of products produced by genetically modified microorganisms via the polyketide synthesis metabolic pathway, as demonstrated in some examples of this disclosure. [Figure 4] Figures 4A and 4B show the results of tests conducted using several examples of the present disclosure to examine the effects of pta gene deletion strains on the production of acetic acid and polyketide derivatives (flavokermesic acid). The "control group" consists of strains without pta gene deletion, while the "pta gene deletion" group consists of genetically modified microbial strains with pta gene deletion. Both the "control group" and the "pta gene deletion" strains contain exogenous gene plasmids necessary for expressing the polyketide metabolic pathway. [Figure 5] Several examples of this disclosure demonstrate the effects of substituting carboxylase with transaminase and / or reductase on the production of polyketide derivatives (flavokermesic acid) by bacterial strains. The "control group" refers to strains in which the carboxylase gene has not been substituted with the transaminase and / or reductase gene; "single transaminase substitution" refers to genetically modified microbial strains in which the transaminase gene is expressed and the carboxylase gene is substituted; "single reductase substitution" refers to genetically modified microbial strains in which the reductase gene is expressed and the carboxylase gene is substituted; and "transaminase and reductase co-substitution" refers to genetically modified microbial strains in which the transaminase and reductase genes are co-expressed and the carboxylase gene is substituted. Furthermore, all of the "control group," "single transaminase substitution," "single reductase substitution," and "transaminase and reductase co-substitution" strains contain the exogenous gene plasmid necessary for expressing the polyketide metabolic pathway. [Figure 6]Several examples of this disclosure demonstrate the effects of transaminase and reductase substitution of carboxylase and pta gene deletion on the production of polyketide derivatives (flavokermesic acid) in bacterial strains. The "control group" consists of bacterial strains that do not have pta gene deletion and do not have the carboxylase gene substituted with the transaminase and / or reductase gene. The "transaminase and reductase co-substitution + pta gene deletion" consists of modified microbial strains in which the transaminase and reductase genes are co-expressed to substitute the carboxylase, and the pta gene is deleted. Both the "control group" and the "transaminase and reductase co-substitution + pta gene deletion" strains possess the exogenous gene plasmids necessary for expressing the polyketide metabolic pathway. [Modes for carrying out the invention]
[0011] The following describes in detail genetically modified microorganisms that increase the production of polyketide compounds or their derivatives according to the embodiments of this disclosure, and methods for producing polyketide compounds or their derivatives. It should be understood that the following description presents many different embodiments or examples to be used to carry out different aspects of some embodiments of this disclosure. The specific components and arrangements described below are merely for the purpose of briefly and clearly illustrating some embodiments of this disclosure. Naturally, these are for illustrative purposes only and do not limit this disclosure.
[0012] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those ordinarily understood by a person of ordinary skill in the art to which this disclosure pertains. These terms, for example, those defined in commonly used dictionaries, should be understood to have meaning consistent with the relevant technology and the context of this disclosure, and should not be interpreted in an idealized or overly strict manner. To facilitate understanding of this disclosure, definitions of technical terms and expressions are provided below.
[0013] In the biological world, acetyl-CoA and malonyl-CoA are important metabolic raw materials for the synthesis of polyketide compounds. In the genetically modified microorganisms provided in some examples of this disclosure, the endogenous pta gene is deleted, thereby blocking the synthetic metabolic pathway that consumes acetyl-CoA to produce acetic acid, reducing the conversion and use of acetyl-CoA, allowing it to enter the polyketide metabolic pathway and increasing the production of polyketide compounds or related derivatives. According to some examples of this disclosure, the provided genetically modified microorganisms replace the carboxylase with a combination of transaminase and reductase. When malonyl-CoA is produced in this manner, it is not affected by feedback inhibition of enzyme activity, thus increasing the concentration of malonyl-CoA in the microorganism and, consequently, increasing the production of polyketide compounds or related derivatives.
[0014] Examples of the present disclosure provide a genetically modified microorganism that enhances the production of polyketide compounds or derivatives thereof, comprising any one or two of the following gene modifications: (a) a deleted endogenous PTA gene, the PTA gene encoding phosphate acetyltransferase, wherein the expression level of phosphate acetyltransferase in the deleted endogenous PTA gene is lower than that of the wild type; and (b) an added first exogenous nucleotide sequence and a second exogenous nucleotide sequence, the first exogenous nucleotide sequence encoding transaminase, and the second exogenous nucleotide sequence encoding reductase. According to some examples, the genetically modified microorganism includes a gene modification that deletes the endogenous PTA gene. According to some embodiments, the genetically modified microorganisms include gene modifications that add a first exogenous nucleotide sequence and a second exogenous nucleotide sequence. According to some embodiments, the genetically modified microorganisms include gene modifications that delete the endogenous pta gene, as well as gene modifications that add a first exogenous nucleotide sequence and a second exogenous nucleotide sequence.
[0015] According to several examples, the source of genetically modified microorganisms includes bacteria. According to several examples, the bacteria may include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Yarrowia lipolytica, Saccharomyces cerevisiae, or Pichia pastoris. According to several examples, the Escherichia coli may include, but are not limited to, Escherichia coli BL21, K12, BW25113, DH5α, XL1-blue, W3110, or other suitable strains.
[0016] According to some embodiments, the phosphate acetyltransferase encoded by the pta gene may contain the amino acid sequence shown in SEQ ID NO:1. According to some embodiments, the pta gene may contain a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO:2, and may, but is not limited to, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO:2. According to some embodiments, the pta gene may contain a nucleotide sequence such as that shown in SEQ ID NO:2. According to some embodiments, the pta gene may be a nucleotide sequence such as that shown in SEQ ID NO:2.
[0017] Of particular note is that in genetically modified microorganisms lacking the endogenous pta gene, the expression level of phosphate acetyltransferase is lower than that of wild-type genetically modified microorganisms. As mentioned above, the pta gene encodes phosphate acetyltransferase, which consumes acetylcoenzyme A in the body to produce acetic acid, thereby lowering the concentration of acetylcoenzyme A. Therefore, by deleting the endogenous pta gene, the synthetic metabolic pathway that consumes acetylcoenzyme A to produce acetic acid in genetically modified microorganisms can be blocked, and consequently, the concentration of acetylcoenzyme A in the genetically modified microorganisms increases.
[0018] Furthermore, the first exogenous nucleotide sequence encodes a transaminase. According to some examples, the transaminase may include β-alanine-pyruvate transaminase (BauA). According to some examples, the transaminase may include an amino acid sequence such as that shown in SEQ ID NO:3. According to some examples, the first exogenous nucleotide sequence encoding the transaminase may include a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO:4, for example, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO:4, but is not limited thereto. According to some examples, the first exogenous nucleotide sequence encoding the transaminase may include a nucleotide such as that shown in SEQ ID NO:4. According to some embodiments, the first exogenous nucleotide sequence encoding the transaminase may be a nucleotide sequence such as that shown in Sequence ID No. 4.
[0019] In some embodiments, the first exogenous nucleotide sequence may be ligated to the first promoter. In some embodiments, the first promoter may include, but is not limited to, the T7 promoter, the lac promoter, the Ptac promoter, the araBAD promoter, or other suitable promoters.
[0020] Furthermore, the second exogenous nucleotide sequence encodes a reductase. According to some examples, the reductase may include methyl coenzyme M reductase operon protein C (MCR-C). According to some examples, the reductase may contain an amino acid sequence such as that shown in SEQ ID NO: 5. According to some examples, the second exogenous nucleotide sequence encoding the reductase may contain a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 6, for example, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 6, but is not limited thereto. According to some examples, the second exogenous nucleotide sequence encoding the reductase may contain a nucleotide sequence such as that shown in SEQ ID NO: 6. According to some embodiments, the second exogenous nucleotide sequence encoding the reductase may be a nucleotide sequence such as that shown in Sequence ID No. 6.
[0021] According to some embodiments, the second exogenous nucleotide sequence and the first exogenous nucleotide sequence may be linked to the same first promoter. According to some other embodiments, the second exogenous nucleotide sequence may be linked to a second promoter different from the first promoter. According to some embodiments, the second promoter may include, but is not limited to, a T7 promoter, a lac promoter, a Ptac promoter, an araBAD promoter or other suitable promoters.
[0022] In vivo, carboxylase can catalyze the chemical reaction of converting acetyl coenzyme A to malonyl coenzyme A. However, the activity of carboxylase is subject to the feedback inhibition of the product malonyl coenzyme A, so a high concentration of malonyl coenzyme A cannot be produced in vivo. It should be noted that in the genetically modified microorganism provided by the embodiments of the present disclosure, the carboxylase in vivo can be replaced by a combination of transaminase and reductase, and by converting acetyl coenzyme A in such a manner to produce malonyl coenzyme A, it may be possible to avoid the influence of feedback inhibition of enzyme activity. Therefore, the concentration of malonyl coenzyme A in the genetically modified microorganism increases.
[0023] Furthermore, according to some examples, the genetically modified microorganism may further include (c) an added third exogenous nucleotide sequence, the third exogenous nucleotide sequence encoding an enzyme involved in carmine synthesis. According to some examples, the enzyme involved in carmine synthesis may include, but is not limited to, cyclase ZhuI, aromatase ZhuJ, type II polyketide synthase complex antDEFGB, hydroxylase dnrFP217K, glucosyltransferase GtCGTV93Q / Y193F, glucosyltransferase UGT2, monooxygenase aptC, and 4'-phosphopantetheinyl transferase npgA.
[0024] According to some embodiments, the third exogenous nucleotide sequence encoding the cyclase ZhuI may include a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO:7, for example, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO:7, but is not limited thereto. According to some embodiments, the third exogenous nucleotide sequence encoding the cyclase ZhuI may include a nucleotide sequence such as that shown in SEQ ID NO:7. According to some embodiments, the third exogenous nucleotide sequence encoding the cyclase ZhuI may be a nucleotide sequence such as that shown in SEQ ID NO:7.
[0025] According to some embodiments, the third exogenous nucleotide sequence encoding aromatase ZhuJ may comprise a nucleotide sequence having at least 85% sequence similarity with SEQ ID NO:8, for example, it may comprise a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98% or 99% sequence similarity with SEQ ID NO:8, but is not limited thereto. According to some embodiments, the third exogenous nucleotide sequence encoding aromatase ZhuJ may comprise a nucleotide sequence as shown in SEQ ID NO:8. According to some embodiments, the third exogenous nucleotide sequence encoding aromatase ZhuJ may be a nucleotide sequence as shown in SEQ ID NO:8.
[0026] According to several embodiments, the third exogenous nucleotide sequence encoding the polyketide synthase antDEFGB may include, but is not limited to, a nucleotide sequence having at least 85% sequence similarity to at least one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. For example, it may include, but is not limited to, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to at least one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. According to some embodiments, the third exogenous nucleotide sequence encoding the polyketide synthase antDEFGB may include nucleotide sequences such as those shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. According to some embodiments, the third exogenous nucleotide sequence encoding the polyketide synthase antDEFGB may be a sequentially linked nucleotide sequence as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. More specifically, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 The nucleotide sequences shown in NO:11), SEQ ID NO:12, and SEQ ID NO:13 may encode the antD, antE, antF, antG, and antB units of polyketide synthase, respectively, and can become polyketide synthase after co-expression and polymerization.
[0027] According to several embodiments, the third exogenous nucleotide sequence encoding the hydroxylase dnrFP217K may include a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 14, for example, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 14, but is not limited thereto. According to several embodiments, the third exogenous nucleotide sequence encoding the hydroxylase dnrFP217K may include a nucleotide sequence such as that shown in SEQ ID NO: 14. According to several embodiments, the third exogenous nucleotide sequence encoding the hydroxylase dnrFP217K may be a nucleotide sequence such as that shown in SEQ ID NO: 14.
[0028] According to several embodiments, the third exogenous nucleotide sequence encoding the glucosyltransferase GtCGTV93Q / Y193F may include a nucleotide sequence having at least 85% sequence similarity to SEQ ID NO: 15, for example, a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 15, but is not limited thereto. According to several embodiments, the third exogenous nucleotide sequence encoding the glucosyltransferase GtCGTV93Q / Y193F may include a nucleotide sequence such as that shown in SEQ ID NO: 15. According to several embodiments, the third exogenous nucleotide sequence encoding the glucosyltransferase GtCGTV93Q / Y193F may be a nucleotide sequence such as that shown in SEQ ID NO: 15.
[0029] In some embodiments, a third exogenous nucleotide sequence may be ligated to a third promoter. In some embodiments, the third promoter may include, but is not limited to, the T7 promoter, the lac promoter, the Ptac promoter, the araBAD promoter, or other suitable promoters.
[0030] As described above, the genetically modified microorganisms provided by the embodiments of this disclosure can increase the production of polyketide compounds or derivatives thereof. According to some embodiments, the polyketide compounds may include, but are not limited to, flavokermesic acid. According to some embodiments, the production of flavokermesic acid in the genetically modified microorganisms provided by the embodiments of this disclosure may be 1.5 to 5 times, for example, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, or 4.5 times, compared to its wild type. According to some embodiments, the derivatives of the polyketide compounds may include, but are not limited to, carminic acid. According to some embodiments, the production of carminic acid in the genetically modified microorganisms provided by the embodiments of this disclosure may be 1.5 to 5 times, for example, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, or 4.5 times, compared to its wild type.
[0031] According to several examples, the genetically modified microorganism that increases the production of the aforementioned polyketide compounds or their derivatives has accession number BCRC 940701.
[0032] Furthermore, several embodiments of this disclosure provide a novel genetically modified strain of Escherichia coli, accession number BCRC 940701. The novel genetically modified strain of Escherichia coli lacks an endogenous pta gene and contains a first exogenous nucleotide sequence and a second exogenous nucleotide sequence, the endogenous pta gene encoding phosphate acetyltransferase, the first exogenous nucleotide sequence encoding transaminase, and the second exogenous nucleotide sequence encoding reductase.
[0033] For more details, please refer to Figure 1, which shows a diagram of the biosynthetic pathway of carminic acid constructed by genetically modified microorganisms according to some examples of this disclosure. In the genetically modified microorganisms, glucose is broken down into pyruvate by glycolysis, and pyruvate can produce acetylcoenzyme A by oxidative decarboxylation. Since the endogenous pta gene is deleted in the genetically modified microorganisms, the synthetic metabolic pathway that consumes acetylcoenzyme A to produce acetic acid is blocked, thereby increasing the concentration of acetylcoenzyme A in the genetically modified microorganisms. Furthermore, pyruvate can be converted to malonylcoenzyme A by the action of transaminase and reductase. A combination of transaminase and reductase can substitute for the action of carboxylase, and when converted to malonylcoenzyme A in this manner, the carboxylase may become less susceptible to feedback inhibition of the enzymatic activity of its product, malonylcoenzyme A, thus increasing the concentration of malonylcoenzyme A in genetically modified microorganisms. The reduction in acetylcoenzyme A consumption and the increase in malonylcoenzyme A concentration can increase the carbon flow in the polyketide synthesis metabolic pathway, thereby increasing the production of polyketide compounds and their related derivatives. Specifically, acetyl coenzyme A and malonyl coenzyme A are then condensed by polyketide synthase (e.g., antDEFGB) to synthesize octaketide. Octaketide is then converted to flavokermesic acid by cyclase (e.g., ZhuI) and aromatase (e.g., ZhuJ). Next, kermesic acid is formed by the action of hydroxylase (e.g., dnrFP217K), and finally, carminic acid is formed by the action of glucosyltransferase (e.g., GtCGTV93Q / Y193F). Adding an aluminum salt or calcium salt to carminic acid yields carmine.
[0034] Furthermore, the embodiments of this disclosure also provide a method for producing polyketide compounds or derivatives thereof, comprising the steps of (a) preparing genetically modified microorganisms that increase the production amount of the aforementioned polyketide compounds or derivatives thereof, and (b) preparing a first culture medium, inoculating the genetically modified microorganisms into the first culture medium, and culturing the genetically modified microorganisms at a temperature of 28°C to 37°C for 12 to 24 hours.
[0035] According to some embodiments, the first medium may include, but is not limited to, LB medium (Lysogeny broth, LB), NB medium, M9 medium, TB medium, or a combination thereof. According to some embodiments, the first medium may contain a suitable antibiotic to allow for the selection of plasmids by the antibiotic to which the plasmids are tolerated. According to some embodiments, the pH value of the first medium may be, but is not limited to, between pH 6 and pH 8, for example, pH 6.2, pH 6.5, pH 6.8, pH 7, pH 7.2, pH 7.5, or pH 7.8. According to some embodiments, the temperature range for culturing the genetically modified microorganisms in the first medium may be, but is not limited to, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Furthermore, according to some embodiments, the time for culturing the genetically modified microorganisms in the first medium may be, but is not limited to, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.
[0036] Furthermore, a method for producing polyketide compounds or their derivatives may include step (c) inoculating a first medium containing cultured genetically modified microorganisms into a second medium, and culturing the genetically modified microorganisms at a temperature of 28°C to 37°C for 24 to 80 hours to obtain a second medium containing polyketide compounds or their derivatives.
[0037] According to several examples, the second medium may include, but is not limited to, M9 medium (M9 minimal medium), LB medium, TB medium, or a combination thereof. According to several examples, the second medium may contain a suitable antibiotic to allow for the selection of plasmids by the antibiotic to which they are resistant. According to several examples, the second medium may further contain yeast extract, monosodium glutamate, glucose, calcium chloride (CaCl2), magnesium sulfate (MgSO4), rare trace metals, or other suitable nutrients. The second medium can be used to ferment and culture genetically modified microorganisms. According to several examples, the pH value of the second medium may be between pH 6 and pH 8, for example, pH 6.2, pH 6.5, pH 6.8, pH 7, pH 7.2, pH 7.5, or pH 7.8. According to some embodiments, the temperature range for culturing the genetically modified microorganisms in the second medium may be, but is not limited to, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Furthermore, according to some embodiments, the time for culturing the genetically modified microorganisms in the second medium may be, but is not limited to, 24 hours, 28 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, or 80 hours.
[0038] Furthermore, the method for producing polyketide compounds or their derivatives may include the step of separating the polyketide compounds or their derivatives from the second culture medium of step (c).
[0039] Polyketide compounds or their derivatives can be obtained by separating them from a second culture medium by any suitable method and purifying them. According to some examples, the polyketide compounds may include, but are not limited to, flavokermesic acid. According to some examples, the derivatives of the polyketide compounds may include, but are not limited to, carminic acid.
[0040] To make the above-mentioned and other purposes, features, and benefits of this disclosure clearer and easier to understand, several examples, comparative examples, and test examples are described below in detail. However, these are not intended to limit the scope of this disclosure.
[0041] Example 1 - Construction of a strain lacking the pta gene and containing exogenous nucleotide sequences encoding transaminase and reductase.
[0042] Using a genetic modification method, plasmid pKM124 was constructed by deleting the nucleotide sequence of the pta gene (SEQ ID NO: 2) and containing a nucleotide sequence encoding β-alanine-pyruvate transaminase (BauA) (SEQ ID NO: 4) and a nucleotide sequence encoding methyl coenzyme M reductor zeoperon protein C (MCR-C) (SEQ ID NO: 6). The structure of the constructed plasmid is shown in Figure 2.
[0043] The plasmid pKM124 was transformed into the E. coli BL-21 strain, which has a polyketide synthesis metabolic pathway, by electroporation. This strain has three plasmids: pKM124, pFA, and pCA. The pKM124 plasmid has a nucleotide sequence encoding BauA (SEQ ID NO: 4) and a nucleotide sequence encoding MCR-C (SEQ ID NO: 6). The pFA plasmid has nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), a nucleotide sequence encoding zhuI (SEQ ID NO: 7), and a nucleotide sequence encoding zhuJ (SEQ ID NO: 8). The pCA plasmid has a nucleotide sequence encoding dnrFP217K (SEQ ID NO: 14) and a nucleotide sequence encoding GtCGTV93Q / Y193F (SEQ ID NO: 15). Single colonies of the transformed strains, grown on plates, were inoculated into 2 mL of LB (Lysogeny broth) medium supplemented with a suitable antibiotic. The cultures were incubated at 28°C to 37°C and a rotation speed of 150 rpm to 250 rpm for 12 to 24 hours to obtain a starter culture. A 0.1% to 5% starter culture was inoculated into a 250 mL shaking Erlenmeyer flask containing a fermentation culture medium (M9 medium, 2-20 g / L yeast extract, 0-10 g / L monosodium glutamate, 5-20 g / L glucose, 100 μM CaCl2, 1 mM MgSO4, rare trace metals, and a suitable antibiotic). Fermentation production was carried out in an incubator at 28°C to 37°C and a rotation speed of 150 rpm to 250 rpm. Once the strain concentration grew to the point where the absorbance (OD600) exceeded 0.3, 0.1 mM to 1 mM IPTG was added to the culture medium to induce protein production. The culture was then fermented and cultured for 72 hours, after which samples were taken and analyzed.
[0044] First, the solid matter was removed from the extracted sample by centrifugation, and then the supernatant was filtered through a 0.22 μm filtration membrane. The product was then analyzed using a high-performance liquid chromatography (HPLC) system (SHIMADZU, LC-20A series). The HPLC analysis conditions were set as follows. Mobile phase: Ratio of acetonitrile and 1-5% acetic acid = 40 / 60 Flow rate: 0.3~1mL / min Temperature: 35~45℃ Detector: UV light Wavelength: 460nm
[0045] The HPLC analysis chart of the products produced after fermentation of the aforementioned strain is shown in Figure 3. As shown in Figure 3, the products produced by the polyketide synthesis metabolic pathway of the aforementioned strain contained polyketide compounds, such as flavokermesic acid.
[0046] Example 2 - Test on the effect of pta gene deletion strains on polyketide compound production.
[0047] A strain was constructed in which the nucleotide sequence of the pta gene (SEQ ID NO: 2) was deleted in isolation, similar to the method described in Example 1. Plasmids of the relevant carmine metabolic pathway were then transformed into the pta gene-deleting E. coli BL-21 strain. This pta gene-deleting strain had three plasmids: pACC, pFA, and pCA. The pACC plasmid had nucleotide sequences encoding the carboxylase accBCD (SEQ ID NO: 16 and SEQ ID NO: 17; the nucleotide sequences shown in SEQ ID NO: 16 and SEQ ID NO: 17 encode the accBC unit and the accD unit of the carboxylase, respectively). The pFA plasmid had nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), zhuI (SEQ ID NO: 7), and zhuJ (SEQ ID NO: 8). The pCA plasmid had nucleotide sequences encoding dnrFP217K (SEQ ID NO: 14) and GtCGTV93Q / Y193F (SEQ ID NO: 15). Next, the bacterial strains were grown on plates, and single colonies were selected for fermentation culture. The production amounts of acetic acid and flavokermesic acid were then analyzed by HPLC. The results are shown in Figures 4A and 4B.
[0048] Figure 4A shows the effect of pta gene deletion on acetic acid production, and Figure 4B shows the effect of pta gene deletion on polyketide derivative (flavokermesic acid) production. In the figures, the "control group" is a strain without pta gene deletion, and the "pta gene deletion" group is a strain in which the pta gene has been deleted.
[0049] As shown in Figure 4A, in the group of strains lacking the pta gene, acetic acid production decreased by approximately 68% compared to the wild type. As shown in Figure 4B, in the group of strains lacking the pta gene, flavokermous acid production increased by approximately 61% compared to the wild type. This indicates that deletion of the pta gene reduces the synthetic metabolic pathway that consumes acetylcoenzyme A to produce acetic acid, thereby increasing the concentration of acetylcoenzyme A in the microbial cells and consequently increasing the production of flavokermous acid.
[0050] Example 3 - Test on the effect of carboxylase substitution with transaminase and reductase on polyketide compound production in bacterial strains.
[0051] Using a method similar to that described in Example 1, strains possessing a nucleotide sequence encoding β-alanine-pyruvate transaminase (BauA) (SEQ ID NO: 4) alone, a strain possessing a nucleotide sequence encoding methyl coenzyme M reductor zeoperon protein C (MCR-C) (SEQ ID NO: 6) alone, and a strain possessing the nucleotide sequences of both BauA and MCR-C were constructed, and the corresponding carmine metabolic pathway plasmids were used to transform Escherichia coli BL-21 strains. A strain possessing a single nucleotide sequence encoding BauA has three plasmids: pKM129, pFA, and pCA. The pKM129 plasmid contains the nucleotide sequence encoding BauA (SEQ ID NO: 4), the pFA plasmid contains the nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), the nucleotide sequence encoding zhuI (SEQ ID NO: 7), and the nucleotide sequence encoding zhuJ (SEQ ID NO: 8), and the pCA plasmid contains the nucleotide sequence encoding dnrFP217K (SEQ ID NO: 14) and the nucleotide sequence encoding GtCGTV93Q / Y193F (SEQ ID NO: 15). A strain possessing a single nucleotide sequence encoding MCR-C has three plasmids: pKM130, pFA, and pCA. The pKM130 plasmid contains the nucleotide sequence encoding MCR-C (SEQ ID NO: 6), the pFA plasmid contains the nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), zhuI (SEQ ID NO: 7), and zhuJ (SEQ ID NO: 8), and the pCA plasmid contains the nucleotide sequence encoding dnrFP217K (SEQ ID NO: 14) and GtCGTV93Q / Y193F (SEQ ID NO: 15).Strains possessing both BauA and MCR-C nucleotide sequences have three plasmids: pKM124, pFA, and pCA. The pKM124 plasmid contains nucleotide sequences encoding BauA (SEQ ID NO: 4) and MCR-C (SEQ ID NO: 6). The pFA plasmid contains nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), zhuI (SEQ ID NO: 7), and zhuJ (SEQ ID NO: 8). The pCA plasmid contains nucleotide sequences encoding dnrFP217K (SEQ ID NO: 14) and GtCGTV93Q / Y193F (SEQ ID NO: 15). The strains were then grown on plates, single colonies were selected and fermented, and the amount of flavokermesic acid produced was analyzed by HPLC. The results are shown in Figure 5.
[0052] Figure 5 shows the results of a study on the effect of carboxylase substitution with transaminase and / or reductase on the flavokermesic acid production of bacterial strains. In the figure, the "control group" refers to strains in which the carboxylase gene was not substituted with the transaminase and / or reductase gene; "single transaminase substitution" refers to strains in which the transaminase gene was expressed and the carboxylase was substituted; "single reductase substitution" refers to strains in which the reductase gene was expressed and the carboxylase was substituted; and "transaminase and reductase co-substitution" refers to strains in which the transaminase and reductase genes were co-expressed and the carboxylase was substituted.
[0053] As shown in Figure 5, the effect of expressing transaminase or reductase alone to substitute for carboxylase was not significant in terms of the amount of flavokermic acid produced by the strain. It should be noted that compared to the wild type, the amount of flavokermic acid produced by strains co-expressing transaminase and reductase to substitute for carboxylase increased by approximately 43.9%. This indicates that carboxylase can be substituted using a combination of transaminase and reductase. When acetylcoenzyme A is converted to malonylcoenzyme A in this manner, the carboxylase is no longer susceptible to feedback inhibition of its enzymatic activity by its product, malonylcoenzyme A. Therefore, the concentration of malonylcoenzyme A in the genetically modified microorganism can be increased, and consequently, the amount of flavokermic acid produced can be increased.
[0054] Example 4 - Test on the effect of carboxylase substitution with transaminase and reductase, and deletion of the pta gene, on the production of polyketide compounds in bacterial strains.
[0055] Using a method similar to that described in Example 1, a plasmid was constructed in which the nucleotide sequence of the pta gene (SEQ ID NO: 2) was deleted, and which contained a nucleotide sequence encoding β-alanine-pyruvate transaminase (BauA) (SEQ ID NO: 4) and a nucleotide sequence encoding methyl coenzyme M reductor zeoperon protein C (MCR-C) (SEQ ID NO: 6). This plasmid was then used to transform E. coli BL-21 strain lacking the pta gene. The strain lacking this pta gene possessed three plasmids: pKM124, pFA, and pCA. The pKM124 plasmid contained nucleotide sequences encoding BauA (SEQ ID NO: 4) and MCR-C (SEQ ID NO: 6). The pFA plasmid contained nucleotide sequences encoding antDEFGB (SEQ ID NOs: 9 to 13), zhuI (SEQ ID NO: 7), and zhuJ (SEQ ID NO: 8). The pCA plasmid contained nucleotide sequences encoding dnrFP217K (SEQ ID NO: 14) and GtCGTV93Q / Y193F (SEQ ID NO: 15). The strains were then grown on plates, single colonies were selected and fermented, and the amount of flavokermesic acid produced was analyzed by HPLC. The results are shown in Figure 6.
[0056] Figure 6 shows the results of tests investigating the effects of transaminase and reductase substitution of carboxylase, as well as pta gene deletion, on the flavokermesic acid production of bacterial strains. In the figure, the "control group" consists of strains that do not have pta gene deletion and do not have the carboxylase gene substituted with the transaminase and / or reductase gene, while the "transaminase and reductase substitution + pta gene deletion" group consists of strains that co-express transaminase and reductase genes to substitute for carboxylase, and also have the pta gene deleted.
[0057] As shown in Figure 6, compared to the wild type, the production of flavokermous acid in strains co-expressing transaminase and reductase to substitute for carboxylase and deleting the pta gene was approximately 2.78 times higher. This indicates that by substituting carboxylase with a combination of transaminase and reductase and deleting the pta gene, the concentrations of malonylcoenzyme A and acetylcoenzyme A in the genetically modified microorganism can be increased, and consequently, the production of flavokermous acid can be increased.
[0058] In summary, the genetically modified microorganisms provided by the embodiments of this disclosure can increase the concentration of acetylcoenzyme A within the microorganism by blocking the synthetic metabolic pathway that consumes acetylcoenzyme A to produce acetic acid, and can also produce malonylcoenzyme A through a synthetic metabolic pathway that is not subject to feedback inhibition of enzyme activity, thereby increasing the concentration of malonylcoenzyme A within the microorganism, and consequently increasing the production of polyketide compounds (e.g., flavokermesic acid) or related derivatives (e.g., carminic acid).
[0059] While embodiments and their advantages have been disclosed above, it should be understood that those with ordinary skill in the art may modify, substitute, or alter them without departing from the spirit and scope of this disclosure. Each claim constitutes an individual embodiment, and the scope of protection of this disclosure also includes any combination of claims and embodiments. The scope of protection of this disclosure is defined in the appended claims. Deposit of biological materials
[0060] 1. Strain of Escherichia coli BL-21 ITRI-FA that increases the production of polyketide compounds or their derivatives. Center for Biological Resource Conservation and Research, Institute for Food Industry Development, Republic of China October 24, 2024 BCRC 940701
Claims
1. A genetically modified microorganism that increases the production of polyketide compounds or their derivatives, wherein the microorganism has any one or two of the following gene modifications: (a) A deleted endogenous PTA gene which encodes phosphate acetyltransferase, wherein the expression level of phosphate acetyltransferase in the deleted endogenous PTA gene is lower than that of the wild type, (b) an added first exogenous nucleotide sequence and a second exogenous nucleotide sequence, wherein the first exogenous nucleotide sequence encodes a transaminase and the second exogenous nucleotide sequence encodes a reductase, Genetically modified microorganisms that include [specific organisms / conditions].
2. The genetically modified microorganism according to claim 1, wherein the transaminase includes β-alanine-pyruvate transaminase (BauA).
3. The genetically modified microorganism according to claim 1, wherein the reductase contains methyl coenzyme M reductase operon protein C (MCR-C).
4. The genetically modified microorganism according to claim 1, wherein the source of the genetically modified microorganism includes bacteria.
5. The genetically modified microorganism according to claim 4, wherein the bacteria include Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Yarrowia lipolytica, Saccharomyces cerevisiae, or Pichia pastoris.
6. The following gene modifications: (c) The genetically modified microorganism according to claim 1, further comprising a third exogenous nucleotide sequence added, wherein the third exogenous nucleotide sequence encodes an enzyme related to carmine synthesis.
7. The genetically modified microorganism according to claim 6, wherein the enzymes related to carmine synthesis include at least one of the following: cyclase ZhuI, aromatase ZhuJ, type II polyketide synthase complex antDEFGB, hydroxylase dnrFP217K, glucosyltransferase GtCGTV93Q / Y193F, glucosyltransferase UGT2, monooxygenase aptC, and 4'-phosphopantetheinyl transferase npgA.
8. The genetically modified microorganism according to claim 1, wherein the polyketide compound includes flavokermesic acid.
9. The genetically modified microorganism according to claim 8, wherein the amount of flavokermesic acid produced by the genetically modified microorganism is 1.5 to 5 times higher than that of the wild type.
10. The genetically modified microorganism according to claim 1, wherein the derivative of the polyketide compound includes carminic acid.
11. A genetically modified microorganism according to claim 1, wherein the accession number is BCRC 940701.
12. A method for producing polyketide compounds or their derivatives, comprising the following steps: (a) the step of preparing a genetically modified microorganism according to any one of claims 1 to 11, (b) A step of preparing a first culture medium, inoculating the genetically modified microorganism into the first culture medium, and culturing the genetically modified microorganism at a temperature of 28°C to 37°C for 12 to 24 hours, (c) The first medium containing the cultured genetically modified microorganism is inoculated into a second medium, and the genetically modified microorganism is cultured at a temperature of 28°C to 37°C for 24 to 80 hours to obtain the second medium containing a polyketide compound or a derivative thereof. (d) A step of separating the polyketide compound or its derivative from the second culture medium of step (c), A method for producing polyketide compounds or derivatives thereof containing [the specified substance].
13. A method for producing a polyketide compound or a derivative thereof according to claim 12, wherein the pH value of the first culture medium is between pH 6 and pH 8, and the pH value of the second culture medium is between pH 6 and pH 8.
14. A method for producing a polyketide compound or a derivative thereof according to claim 12, wherein the first culture medium includes LB medium, NB medium, M9 medium, TB medium, or a combination thereof.
15. A method for producing a polyketide compound or a derivative thereof according to claim 12, wherein the second culture medium includes M9 medium, LB medium, TB medium, or a combination thereof.
16. A novel genetically modified strain of Escherichia coli, having accession number BCRC 940701, wherein the novel genetically modified strain of Escherichia coli has a deletion of the endogenous pta gene and contains a first exogenous nucleotide sequence and a second exogenous nucleotide sequence, wherein the endogenous pta gene encodes phosphate acetyltransferase, the first exogenous nucleotide sequence encodes transaminase, and the second exogenous nucleotide sequence encodes reductase.