Genetically engineered bacterium, construction method thereof and method for preparing emodin and physcion by using genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium, a method for constructing the genetically engineered bacterium and a method for preparing emodin and physcion by using the genetically engineered bacterium. The genetically engineered bacterium carries an exogenous gene; the exogenous gene comprises a gene npgA for coding phosphopantetheinyl transferase and a gene gedC for coding polyketide synthase, further comprises at least one of a gene HyTE and a gene gedB for coding beta-lactamase thioesterase, and further comprises at least one of a decarboxylase gene TpCK and a gene AfDC. According to the invention, a complete synthetic route of the physcion is constructed from the beginning in a heterologous host microorganism for the first time; a novel microbial fermentation mode is used for replacing traditional plant extraction, and novel biological manufacturing of the emodin and the emodin monomethyl ether is developed.

Description

Technical Field

[0001] The present application relates to the fields of fermentation and genetic engineering bacteria technology, and in particular to genetic engineering bacteria and a method for preparing rhamnosin and rhamnosin methyl ether using the same. Background Art

[0002] Rhamnosin and Rhamnosin methyl ether are important biological pesticides with agricultural disease control functions, and also have extremely high medicinal value. Rhamnosin methyl ether has a good control effect on a variety of crop diseases including downy mildew, powdery mildew, gray mold, anthracnose, etc., and is safe for humans, animals and the environment. Previous work has explored the antibacterial and anti-disease effects of Rhamnosin methyl ether as a biological pesticide, and developed and optimized the extraction process from plants such as rhubarb and knotweed. Rhamnosin methyl ether has been accepted by the market as a biological pesticide with low resistance risk, high activity, broad bactericidal spectrum, low toxicity, fast degradation and low residue.

[0003] At present, the large-scale preparation and production of physaccharin methyl ether is mainly extracted from plants such as rhubarb. The disadvantages of this process include: rhubarb crops are planted in high-altitude cold areas, and the planting scale is limited; the planting cycle of rhubarb crops is as long as 2 to 5 years, and continuous cropping is prohibited; the yield of rhubarb is low, and its average yield is 300 kg / mu. The content of effective ingredients is low, and physaccharin methyl ether is about 0.01%~0.2%; finally, the plant extraction method is complicated, and it needs to go through hydrolysis, a large amount of organic solvent extraction and column chromatography and other separation steps.

[0004] With the development of genetic engineering, especially synthetic biology, it is possible to use microbial "cell factories" to construct and regulate metabolic pathways to biosynthesize natural products with diverse structures, and to produce target products more efficiently, economically and environmentally friendly. The use of microbial culture and fermentation technology to synthesize physaccharin methyl ether is also being actively explored. Although the presence of physaccharin methyl ether can be detected in some natural microorganisms, the content is extremely low. For example, the production of physaccharin methyl ether by marine fungi Microsporum sp. and Aspergillus parasiticus is 0.59 mg / L and 1.2 mg / L, respectively. By heterologously expressing the relevant genes from the ascomycete Talaromyces sp. F08Z-0631 in the fungus Aspergillus oryzae, torosachrysone was successfully synthesized. Combined with subsequent chemical transformation, the synthesis of 526 mg / L of physaccharin methyl ether was finally achieved. In 2019, a team from Guangzhou University of Chinese Medicine achieved the synthesis of 528.4 mg / L of rhamnosine (without methyl group) by synthetic biology modification of the model organism Saccharomyces cerevisiae and the combined expression of genes from different microbial sources. In 2022, Aspergillus terreus was genetically engineered to produce rhamnosine methyl ether. The overexpression of the transcriptional regulator GedR successfully activated the diazolin biosynthetic gene cluster and expressed rhamnosine-3-OH-O-methyltransferase, successfully achieving high yields of rhamnosine methyl ether. However, its fermentation cycle is long and contains a variety of byproducts with similar structures to rhamnosine.

[0005] It can be seen that microbial fermentation is expected to overcome the bottleneck of traditional plant extraction process to produce physcholin and physcholin methyl ether. Despite a series of progress, there are still the following shortcomings: because some biosynthetic pathways have not been fully elucidated, there is no recombinant strain that synthesizes physcholin methyl ether from scratch; the production of physcholin and physcholin methyl ether of most wild-type microbial strains and genetically recombinant Saccharomyces cerevisiae is far behind the industrial demand; the fungus A. terreus will form a large amount of mycelium during the fermentation process, which is difficult to control. Its fermentation cycle is as long as 14 days, resulting in a decrease in the rate of fermentation synthesis products. At the same time, the fungus A. terreus is a conditionally pathogenic fungus, which poses a potential safety risk to production and subsequent product applications. It produces a variety of secondary metabolites with similar structures, and there is a large accumulation of physcholin in the strains that synthesize physcholin methyl ether, which makes it difficult to separate and purify the subsequent products.

[0006] Therefore, there is an urgent need in the art to develop novel microorganisms for fermentation production of rhamnosine and rhamnosine methyl ether. Summary of the invention

[0007] Based on this, it is necessary to provide at least a genetically engineered bacterium and a method for preparing rhamnosin and rhamnosin methyl ether using the same.

[0008] In the first aspect of the present application, a genetically engineered bacterium is provided, which carries exogenous genes; the exogenous genes include the gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase, and also include at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, and also include at least one of the decarboxylase gene TpCK and the gene AfDC.

[0009] In the second aspect of the present application, a recombinant expression vector is provided, which comprises the following genes: gene npgA encoding phosphopantetheinyl transferase and gene gedC encoding polyketide synthase, and also comprises at least one of gene HyTE and gene gedB encoding β-lactamase type thioesterase, and also comprises at least one of decarboxylase gene TpCK and gene AfDC.

[0010] In the third aspect of the present application, a recombinant expression vector combination is provided, which comprises the recombinant expression vector as described in the second aspect as a first recombinant expression vector, and a second recombinant expression vector; the second recombinant expression vector comprises gene ptaI.

[0011] In a fourth aspect of the present application, a method for preparing rhamnosine or rhamnosine methyl ether is provided, the method comprising:

[0012] Cultivating the genetically engineered bacteria as described above in a fermentation medium to obtain a culture;

[0013] Rhamnosin or Rhamnosin methyl ether is isolated from the culture.

[0014] In the fifth aspect of the present application, a method for constructing a genetically engineered bacterium for producing rhamnosin is provided, the method comprising: integrating at least one copy of the following gene into the genome of the starting strain:

[0015] The gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase also include at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, and also include at least one of the decarboxylase gene TpCK and the gene AfDC.

[0016] In the sixth aspect of the present application, a method for constructing a genetically engineered bacterium for producing physcion is provided, the method comprising: integrating at least one copy of the following gene into the genome of the starting strain:

[0017] The gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase, at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, at least one of the decarboxylase gene TpCK and the gene AfDC, the gene ptaI; and optionally the gene pEX10.

[0018] The innovation of this application is at least reflected in: for the first time, a complete synthetic pathway of physcion methyl ether was constructed from scratch in a heterologous host microorganism; a new type of microbial fermentation method was used to replace traditional plant extraction, and a new type of biomanufacturing of physcion and physcion methyl ether was developed; it is expected to eventually achieve the goal of using microbial fermentation process to achieve sustainable, renewable, low-cost and scalable production of plant natural products. It can overcome the defects of the current plant extraction process to obtain physcion and physcion methyl ether, which is conducive to reducing the use of arable land, energy conservation and emission reduction, reducing dependence on chemical pesticides, and further promoting the development of circular economy and bioeconomy.

[0019] In some embodiments, by discovering and combining genes from different fungi, the unconventional yeast Yarrowia lipolytica is used as a chassis microorganism to achieve the synthesis of rhamnosin and rhamnosin methyl ether, and to build a more efficient cell factory. The unique advantages of Yarrowia lipolytica as a starting strain are at least reflected in: it is recognized as a biosafe strain (GRAS), has strong ability to synthesize oils and organic acids, and can tolerate strong acid and alkaline environments. Yarrowia lipolytica can naturally accumulate microbial oils, and by redirecting the carbon metabolic flow of the original synthetic oils, the target product can be efficiently synthesized in Yarrowia lipolytica, which is a first in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0021] Figure 1 This is a synthetic route for rhamnosin and rhamnosin methyl ether in one embodiment of the present application.

[0022] Figure 2 Schematic diagram of plasmid pEM15 in one embodiment of the present application.

[0023] Figure 3Schematic diagram of the plasmid pPEX10-ptaI in one embodiment of the present application.

[0024] Figure 4 The present invention is a construction process of a genetically recombinant Yarrowia lipolytica for synthesizing rhamnocin and rhamnocin methyl ether in one embodiment of the present application.

[0025] Figure 5 In one embodiment of the present application, the genetically recombinant strain synthesizes rhamnosin.

[0026] Figure 6 In one embodiment of the present application, the genetically recombinant strain GEN3 synthesizes physcion methyl ether.

[0027] Figure 7 In one embodiment of the present application, high performance liquid chromatography (HPLC) was used to detect the synthesis of emodin and physcion products by the recombinant bacteria GEN2 and GEN3, wherein the wild bacteria Po1f was used as a control; wherein "Emodin" represents emodin, "Physcion" represents physcion, "Intensity" represents intensity, and "Time" represents time.

[0028] Figure 8 This is a plasmid map of pLTinX in one embodiment of the present application.

[0029] Fig. 9 This is the plasmid map of pUTinX in one embodiment of the present application. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0032] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0033] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0034] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0035] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0036] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0037] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0038] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0039] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0040] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0041] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0042] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0043] This application successfully constructed the biosynthetic pathway of physaccharin and physaccharin methyl ether through fermentation and expression optimization of heterologous genes, and the genetically recombinant strain can efficiently synthesize the target product. By comparing the effects of the expression of candidate genes from different sources on the accumulation of the target product, the functional gene was determined. Metabolic engineering strategies such as increasing the supply of physaccharin methyl ether synthesis precursors ("push") and optimizing the synthesis pathway of the target product ("pull") were used to improve the accumulation of the target product.

[0044] One aspect of the present application provides a genetically engineered bacterium carrying exogenous genes; the exogenous genes include the gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase, and also include at least one of the gene HyTE and the gene gedB encoding β-lactamase-type thioesterase, and also include at least one of the decarboxylase gene TpCK and the gene AfDC.

[0045] In some embodiments, the genetically engineered bacteria carry four of the above exogenous genes. Exemplarily, the genetically engineered bacteria contain genes npgA, genes gedC, genes HyTE and genes AfDC; or, the genetically engineered bacteria contain genes npgA, genes gedC, genes HyTE and decarboxylase gene TpCK; or, the genetically engineered bacteria contain genes npgA, genes gedC, genes gedB and genes AfDC; or, the genetically engineered bacteria contain genes npgA, genes gedC, genes gedB and decarboxylase gene TpCK.

[0046] The gene gedC in the present application may be derived from Aspergillus terreus. It should be understood that the gene gedC sequence in different strains may be different. In some embodiments, the gene gedC nucleic acid sequence comprises the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the gene gedC nucleic acid sequence is shown in SEQ ID NO: 1.

[0047] The gene gedB in the present application may be derived from Aspergillus terreus. In some embodiments, the nucleic acid sequence of the gene gedB comprises the nucleic acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleic acid sequence of the gene gedB is shown in SEQ ID NO: 2.

[0048] The gene HyTE in the present application may be derived from Hypoxylon sp. EC3. In some embodiments, the nucleic acid sequence of the gene HyTE comprises the nucleic acid sequence shown in SEQ ID NO: 3. In some embodiments, the nucleic acid sequence of the gene HyTE is shown in SEQ ID NO: 3.

[0049] The decarboxylase gene TpCK in the present application may be derived from Aspergillus fumigatus. In some embodiments, the nucleic acid sequence of the decarboxylase gene TpCK comprises the nucleic acid sequence shown in SEQ ID NO: 4. Exemplarily, the nucleic acid sequence of the decarboxylase gene TpCK is shown in SEQ ID NO: 4.

[0050] The gene AfDC in the present application may be derived from Aspergillus fischeri NRRL 181. In some embodiments, the nucleic acid sequence of the gene AfDC comprises the nucleic acid sequence shown in SEQ ID NO: 5. Exemplarily, the nucleic acid sequence of the gene AfDC is shown in SEQ ID NO: 5.

[0051] The gene npgA in the present application may be derived from Aspergillus nidulans. In some embodiments, the nucleic acid sequence of the gene npgA comprises the nucleic acid sequence shown in SEQ ID NO: 6. Exemplarily, the nucleic acid sequence of the gene npgA is shown in SEQ ID NO: 6.

[0052] The existence form of the foreign gene described in the present application in the genetically engineered bacteria can be a conventional existence form. Exemplarily, the foreign gene can be integrated into the chromosome or genome of the starting strain by genetic engineering technology, and replicated along with cell division. For example, the foreign gene can be integrated into the chromosome or genome of the starting strain by plasmid transformation.

[0053] Alternatively, the exogenous gene is contained in a recombinant expression vector and exists outside the chromosome in the form of a plasmid or the like, and replicates independently.

[0054] The plasmid used in the present application is, for example, a linear plasmid or a circular plasmid.

[0055] The exogenous genes in the present application may be contained in the same expression cassette of the plasmid described above; or, each of the exogenous genes may be independently contained in different expression cassettes of the plasmid described above.

[0056] The expression cassette in the present application comprises a promoter and a terminator. TEF1N , P GMP , P TEF and P FBA1N The terminator is exemplarily selected from T XPR2 , T OCT1 and T LIP1 The group formed.

[0057] Exemplarily, the genetically engineered bacteria also contain an expression cassette, P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 , P TEF1N- HyTE-T xpr2 and P TEF1N- AfDC-T xpr2 .

[0058] Exemplarily, the genetically engineered bacteria also contain an expression cassette P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 , P TEF1N- HyTE-T xpr2 and P TEF1N- TpCK-T xpr2 .

[0059] Exemplarily, the genetically engineered bacteria also contain an expression cassette P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 , P TEF1N- gedB-T xpr2 and P TEF1N- AfDC-T xpr22 .

[0060] Exemplarily, the genetically engineered bacteria also contain an expression cassette P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 , P TEF1N- gedB-T xpr2 and P TEF1N- TpCK-T xpr2 .

[0061] In the present application, the backbone of the plasmid is, for example, selected from pUTinX (see Fig. 9 ) and pLTinX (see Figure 8 ).

[0062] It should be understood that the genetically engineered bacteria described in the present application may contain multiple copies of exogenous genes, for example, 2 copies or more.

[0063] In some embodiments, the genetically engineered bacteria further include one or more of the gene leu2, the gene ura3, the gene ptaI encoding a methyltransferase, and the gene pEX10.

[0064] For example, the genetically engineered bacteria further comprises one or more of the gene leu2 and the gene ura3.

[0065] The gene leu2 and / or gene ura3 may be contained in another plasmid different from the plasmid containing the above exogenous gene, and the relevant information about the other plasmid can be found in the above content. Alternatively, the gene leu2 and / or gene ura3 are both contained in the same plasmid as the exogenous gene as described above as screening genes.

[0066] It should be understood that the introduction of gene leu2 and / or gene ura3 can compensate for some genetic defects of the starting strain, such as leu2 deficiency or ura3 deficiency.

[0067] In some embodiments, the genetically engineered bacteria further comprises one or more of the gene ptaI and the gene pEX10. Exemplarily, the nucleic acid sequence of the gene ptaI comprises the nucleic acid sequence shown in SEQ ID NO: 7, or as shown in SEQ ID NO: 7.

[0068] The gene ptal and the gene pEX10 may be contained in another plasmid different from the plasmid containing the above exogenous genes and another plasmid containing the gene leu2 and / or the gene ura3. For relevant information about the other plasmid, please refer to the above content.

[0069] Similar to the above exogenous genes, the gene pEX10 and the gene ptaI can be integrated into the chromosome or genome of the genetically engineered bacteria, or can be contained in a recombinant expression vector and exist outside the chromosome in the form of a plasmid or the like, and replicate independently.

[0070] In some embodiments, the gene pEX10 and the gene ptal are each independently contained in different expression cassettes of the same plasmid. For relevant information about the expression cassette and plasmid, please refer to the above content.

[0071] In some embodiments, the genetically engineered bacteria further comprises the following expression cassette: TEF1N- ptaI-T xpr2 and P TEF1N- pEX10-T xpr2 .

[0072] The starting strain of the genetically engineered bacteria described in the present application may be Yarrowia lipolytica.

[0073] In some embodiments, the Yarrowia lipolytica is the Yarrowia lipolytica with deposit number ATCC MYA-2613.

[0074] The present application also provides a recombinant expression vector, which comprises the following genes: gene npgA encoding phosphopantetheinyl transferase and gene gedC encoding polyketide synthase, and also comprises at least one of gene HyTE and gene gedB encoding β-lactamase type thioesterase, and also comprises at least one of decarboxylase gene TpCK and gene AfDC.

[0075] In some embodiments, the recombinant expression vector comprises gene npgA, gene gedC, gene HyTE and gene AfDC.

[0076] In some embodiments, the recombinant expression vector comprises gene npgA, gene gedC, gene HyTE and decarboxylase gene TpCK.

[0077] In some embodiments, the recombinant expression vector comprises gene npgA, gene gedC, gene gedB and gene AfDC.

[0078] In some embodiments, the recombinant expression vector comprises gene npgA, gene gedC, gene gedB and decarboxylase gene TpCK.

[0079] In some embodiments, the genes in the recombinant expression vector are each independently contained in a different expression cassette.

[0080] See above for information on the expression cassette.

[0081] The recombinant expression vector may be a conventional recombinant expression vector in the art, for example, a linear plasmid.

[0082] In some embodiments, the recombinant expression vector further comprises one or more of gene leu2 and gene ura3.

[0083] The present application also provides a recombinant expression vector combination, which comprises the recombinant expression vector as described above as a first recombinant expression vector, and a second recombinant expression vector comprising gene ptaI.

[0084] In some embodiments, the second recombinant expression vector further comprises gene pEX10. In the second recombinant expression vector, the gene pEX10 and gene ptal are each independently contained in different expression cassettes.

[0085] Referring to the above, the promoter is selected from P TEF1N , P GMP and P TEF and P FBA1N The terminator can be selected from the group consisting of XPR2 , T OCT1 and T LIP1 The group formed.

[0086] The second recombinant expression vector can be a linear plasmid or a circular plasmid.

[0087] The first recombinant expression vector and the second recombinant expression vector are each independently selected from pUTinX and pLTinX.

[0088] The present application also provides a method for preparing rheumatoid artemisia, the method comprising:

[0089] Cultivating the genetically engineered bacteria as described above in a fermentation medium to obtain a culture;

[0090] Isolation of rhein from the culture.

[0091] The fermentation medium contains: 5 g / L~25 g / L yeast extract, 10 g / L~30 g / L peptone and 30 g / L~80 g / L glucose.

[0092] In some embodiments, the fermentation medium uses an inorganic nitrogen source including ammonium sulfate or ammonium chloride as a nitrogen source, and glycerol, vegetable oil and acetate as a carbon source.

[0093] In some embodiments, the culture conditions include: a culture temperature of 28°C and a rotation speed of 180 rpm to 250 rpm.

[0094] With reference to the above content, the starting strain of the genetically engineered bacteria includes Yarrowia lipolytica, and the Yarrowia lipolytica can optionally be the Yarrowia lipolytica with the deposit number ATCC MYA-2613.

[0095] The present application also provides a method for preparing physaccharin methyl ether, the method comprising:

[0096] Cultivating the genetically engineered bacteria as described above in a fermentation medium to obtain a culture;

[0097] Isolation of physcion from culture.

[0098] In the method, the fermentation medium contains: 5 g / L~25 g / L yeast extract, 10 g / L~30 g / L peptone and 30 g / L~80 g / L glucose or glycerol.

[0099] In this method, the culture conditions include: a culture temperature of 28°C and a rotation speed of 180 rpm to 250 rpm.

[0100] With reference to the above, the starting strain of the genetically engineered bacteria includes Yarrowia lipolytica. The Yarrowia lipolytica strain may be, for example, the Yarrowia lipolytica with the deposit number ATCC MYA-2613.

[0101] The present application also provides a method for constructing a genetically engineered bacterium for producing rhamnosin, the method comprising: integrating at least one copy of the following gene into the genome of the starting strain:

[0102] The gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase also include at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, and also include at least one of the decarboxylase gene TpCK and the gene AfDC.

[0103] In some embodiments, the gene is integrated into the genome of the starting strain by transfecting the recombinant expression vector as described above into the starting strain.

[0104] The nucleic acid sequence of the gene npgA, the gene gedC, the gene HyTE, the gene AfDC, the gene gedB or the decarboxylase gene TpCK is as defined above.

[0105] The present application also provides a method for constructing a genetically engineered bacterium for producing physcion, the method comprising: integrating at least one copy of the following gene into the genome of the starting strain:

[0106] The gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding a polyketide synthase, at least one of the gene HyTE and the gene gedB encoding a β-lactamase type thioesterase, at least one of the decarboxylase gene TpCK and the gene AfDC, the gene ptaI, and optionally the gene pEX10.

[0107] In some embodiments, the gene is integrated into the genome of the starting strain by transfecting the recombinant expression vector combination described above into the starting strain.

[0108] Some examples are provided below.

[0109] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0110] Example 1

[0111] In the following, Y. lipolytica Po1f (ATCC MYA-2613) was used as the starting strain. Through the fermentation and expression optimization of heterologous genes, the biosynthetic pathway of rhamnosine and rhamnosine methyl ether was successfully constructed. The genetically recombinant strain can efficiently synthesize the target product.

[0112] (1) Construction of the expression vector for the exogenous gene for synthesizing rhamnosine

[0113] like Figure 1As shown, in order to realize the synthesis of rhein in Yarrowia lipolytica, it is necessary to introduce the synthesis gene of rhein. The candidate genes for the synthesis of rhein include the polyketide synthase gedC gene (SEQ ID NO: 1), β-lactamase type thioesterase gedB (SEQ ID NO: 2) from the fungus A. terreus, and the HyTE gene (SEQ ID NO: 3) from Hypoxylon sp. EC38, and the decarboxylase genes TpCK (SEQ ID NO: 4) and AfDC (SEQ ID NO: 5) from Aspergillus fumigatus and Aspergillus fischeri NRRL 181, respectively. At the same time, it is necessary to express the gene npgA (SEQ ID NO: 6) encoding phosphopantetheinyl transferases (PPTase) from A. nidulans to activate the polyketide synthase.

[0114] Under the catalysis of polyketide synthase, acetyl-CoA and malonyl-CoA are used as substrates, and then decarboxylase is used to synthesize rhamnosin. Under the action of methyltransferase, rhamnosin methyl ether is finally formed. The synthetic pathways of rhamnosin and rhamnosin methyl ether can be found in Figure 1 .

[0115] The above genes were optimized according to the codon preference of Y. lipolytica and directly synthesized into the expression plasmid pLTinX (see Figure 8 ) HindIII / NdeI restriction site. The expression plasmid pLTinX carries the strong promoter P of Y. lipolytica TEF1N (translation elongation factor, including the first intron), the termination sequence is T xpr2(alkaline extracellular protease). As shown in Table 1, with pLTinX as the starting vector, the recombinant plasmids carrying the synthetic genes were named pLTinX-gedC, pLTinX-gedB, pLTinX-HyTE, pLTinX-TpcK, pLTinX-AfDC and pLTinX-npgA. Subsequently, the single gene expression cassettes consisting of the promoter, the functional gene and the termination sequence were connected in sequence until a tandem structure of four gene expression cassettes was formed. The constructed plasmids pEM15, pEM16, pEM17 and pEM18 have all the genes required for the synthesis of rhamnosin, and carry the leu2 gene, which can be used to transform the leucine synthesis-deficient Y. lipolytica strain. For example, plasmid pEM15 carries the Escherichia coli selection marker ampicillin resistance gene, the Y. lipolytica selection marker leu2 gene, and cloned four gene expression cassettes P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 ,P TEF1N- HyTE-T xpr2 and P TEF1N- AfDC-T xpr2 . Figure 2 A schematic diagram of the representative plasmid pEM15 is shown. The leu2 gene of pEM17 was replaced with the yeast ura3 selection marker to generate plasmid pEM17-ura3 (Table 1). Plasmid pEM17-ura3 can be used to transform Y. lipolytica strains deficient in uracil synthesis.

[0116] Table 1 Genetic recombinant plasmids constructed in this application

[0117]

[0118] Plasmid pEM15 carries the ampicillin resistance gene (Amp R ), the Yarrowia lipolytica selection marker leu2 gene, and four gene expression cassettes P TEF1N- npgA-T xpr2 , P TEF1N- gedC-T xpr2 , P TEF1N- HyTE-T xpr2 and P TEF1N- AfDC-T xpr2 , the schematic diagram of the spectrum can be seen in Figure 2 .

[0119] (2) Cloning and expression of exogenous genes for the synthesis of physaccharin methyl ether

[0120] like Figure 1 As shown, the synthesis of physaccharin methyl ether requires further methylation of physaccharin. This application introduces the methyltransferase encoding gene ptaI (SEQ ID NO: 7) from Pestalotiopsis fici to catalyze the methylation of physaccharin. The ptaI gene will be optimized according to the codon preference of Y. lipolytica and directly synthesized into the expression plasmid pUTinX (see Fig. 9 ) with HindIII / NdeI restriction sites, and the resulting plasmid was pUTinX-ptaI (Table 1). TEF1N Promoter and T xpr2 At the same time, pUTinX carries the yeast ura3 selection marker and can be used to transform yeast deficient in uracil synthesis. Since the synthesis of physaccharin and physaccharin methyl ether requires acetyl-CoA and malonyl-CoA as precursors ( Figure 1 ), an expression vector of the pEX10 gene (YALI1_C01416g) that can enhance fatty acid ß-oxidation was constructed. The plasmid pPEX10-ptaI was further obtained, on which the expression of genes pEX10 and ptaI was driven by independent promoters P TEF1N control (Table 1). Figure 3 The map diagram of plasmid pPEX10-ptaI is shown as an example.

[0121] Plasmid pPEX10-ptaI carries the E. coli selection marker ampicillin resistance gene (Amp R ), Y. lipolytica selection marker ura3 gene, and two gene expression cassettes P TEF1N- ptaI-T xpr2 and P TEF1N- pEX10-T xpr2 .

[0122] (3) Construction of Y. lipolytica recombinant strains

[0123] Escherichia coli DH5α competent cells from Sangon were used for plasmid transformation and amplification. The plasmids were spread on LB solid plates (2% agar) containing 100 mg / mL ampicillin (Amp). After 12 h of culture, single colonies were picked and transferred to LB liquid medium containing 100 mg / mL ampicillin for 12 h to 16 h. After restriction digestion identification, the linearized plasmid was transferred into Yarrowia lipolytica. Yarrowia lipolytica Po1f was cultured in YPD liquid medium, and the Frozen-EZ Yeast Transformation II Kit (Zymo) was used for the preparation and transformation of yeast competent cells.

[0124] The construction process of the recombinant Yarrowia lipolytica for synthesizing rhamnocin and rhamnocin methyl ether is as follows Figure 4 As shown. The plasmids pEM15, pEM16, pEM17 and pEM18 in Table 1 were linearized by endonuclease BamHI (FastDigest Restriction Enzyme, Thermo Fisher Scientific), and the digestion products were recovered and transformed into Y.lipolytica Po1f. After static culture at 28°C for 96 hours, a single colony of the transformed recombinant yeast appeared on a leucine-deficient yeast basal nitrogen base (YNB) agar plate. The recombinant bacteria integrated with the linearized plasmids pEM15, pEM16, pEM17 and pEM18 were named Y. lipolytica GEN1-1, GEN1-2, GEN1-3 and GEN1-4, respectively. Y. lipolytica GEN1-1, GEN1-2, GEN1-3 and GEN1-4 are integrated with genes for the synthesis of rhamnosine. The linearized plasmid pEM17-ura3 was then introduced into Y. lipolytica GEN1-1, and yeast recombinants were screened in YNB solid medium lacking uracil to obtain strain Y. lipolytica GEN2. The recombinant strain Y. lipolytica GEN2 contained 2 copies of the rhamnosine synthesis gene. The pPEX10-ptaI plasmid DNA was integrated into the chromosome of Y. lipolytica GEN1-1 to obtain Y. lipolytica GEN3. Y. lipolytica GEN3 has integrated the ptaI gene for the synthesis of rhamnosine methyl ether.

[0125] Y. lipolytica GEN1-1, GEN1-2, GEN1-3 and GEN1-4 were developed by transforming strain Po1f with pEM15, pEM16, pEM17 and pEM18. pEM17-ura3 plasmid DNA was further integrated into the chromosome of Y. lipolytica GEN1-1 to obtain Y. lipolytica GEN2, and pPEX10-ptaI plasmid DNA was integrated into the chromosome of Y. lipolytica GEN1-1 to obtain Y. lipolytica GEN3. The construction process of the genetically recombinant Yarrowia lipolytica that synthesizes rhamnosin and rhamnosin methyl ether can be found in Figure 4 .

[0126] (4) Synthesis of rhamnosine by recombinant strains

[0127] The recombinant strains Y. lipolytica GEN1-1, GEN1-2, GEN1-3, GEN1-4 and GEN2 ( Figure 4 ) were cultured on YPD plates (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 18 g / L agar) at 28°C. After 48 hours of static culture, single colonies were obtained on the plates. Single colonies were picked and inoculated into YPD liquid medium for shaking culture at 28°C and the shaking speed was set to 200 rpm. After 48 hours of culture, they were inoculated into medium containing 50 g / L glucose, which also contained 10 g / L yeast extract and 20 g / L peptone. After 120 hours of culture in a shaker, 100 μL of the culture was taken and centrifuged at 12000 rpm for 5 minutes, and the supernatant was discarded. 500 μL methanol and 500 μL dichloromethane were added to the obtained bacteria, mixed and ultrasonicated for 30 min, and then centrifuged again at 12000 rpm for 5 min. The organic phase was aspirated and filtered through a 0.22 μm organic phase filter membrane for high performance liquid chromatography (HPLC) detection. The results can be found in Figure 7 . HPLC analysis was performed using a Shimadzu LC-2050C instrument, using a C18 (4.6 mmx250 mm, 5 μm) chromatographic column. The HPLC mobile phase was 0.1% phosphoric acid in pump A, methanol in pump B, and a flow ratio of 15:85 in pump AB. The flow rate of the mobile image was 1 mL / min, the column temperature was set at 30°C, the injection volume was 10 μL, and the detection wavelength was 440 nm. A standard curve was prepared using rhubarb standard, and quantitative analysis was performed based on the standard curve. Figure 5As shown, the recombinant strains Y. lipolytica GEN1-1, GEN1-2, GEN1-3, and GEN1-4 can synthesize rhein, while the product rhein was not detected in the culture of Y. lipolytica Po1f. Among them, the rhein production of Y. lipolytica GEN1-1 and GEN1-3 was relatively high, reaching 453 mg / L and 440 mg / L respectively. The rhein titer of GEN2, which has an increased copy number of the synthetic gene, is higher than 800 mg / L, which can be seen in Figure 5 This data is much higher than the reported production of rhamnocin by recombinant Saccharomyces cerevisiae, which is 528.4 mg / L (Lei Sun, Guiyou Liu, Ya Li, Dayong Jiang, Wenfeng Guo, Hui Xu, Ruoting Zhan. Metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin. Metabolic Engineering. 2019; 54:212-221).

[0128] The culture medium consisted of 10 g / L yeast extract, 20 g / L peptone and 50 g / L glucose. The culture was incubated for 120 hours at 28°C and the shaking speed was set at 200 rpm.

[0129] (5) Synthesis of physcion methyl ether by recombinant strains

[0130] The recombinant strain Y. lipolytica GEN3 ( Figure 4 ) was cultured on a YPD plate (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 18 g / L) at 28°C. After 48 hours of static culture, a single colony was obtained on the plate. A single colony was picked and inoculated into YPD liquid culture medium for shaking culture at 28°C and a shaking speed of 200 rpm. After 48 hours of culture, they were inoculated into a culture medium containing 50 g / L glucose or 50 g / L glycerol. After 120 hours of culture in a shaker, the culture was taken and centrifuged to obtain the bacterial cells. After treatment with organic solvents methanol and dichloromethane, HPLC analysis was performed. The results can be seen in Figure 7 The standard curve was established using rhamnosin and rhamnosin methyl ether standards, and quantitative analysis was performed based on the standard curve. Figure 6Y. lipolytica GEN3 can synthesize rhamnosin methyl ether from glucose and glycerol, with yields of 675 mg / L and 713 mg / L, respectively, which is comparable to that of genetically engineered Aspergillus terreus under similar conditions (Lü Xuefeng, Qi Feifei, Huang Xuenian, Xue Yingying, Zhang Wei. An engineered strain accumulating rhamnosin-8-methyl ether, its construction method and application. CN: CN110964649A, 2020-04-07). Another major advantage of Y. lipolytica GEN3 is that the accumulation of rhamnosin is as low as about 20 mg / L, see Figure 6 Combined with the various excellent properties of Y. lipolytica, this application provides key technical support for the development of the biomanufacturing industry of rhamnosin and rhamnosin methyl ether.

[0131] The culture medium consists of 10 g / L yeast extract, 20 g / L peptone and 50 g / L glucose or glycerol. The culture is carried out for 120 hours at 28°C and the shaking speed is set at 200 rpm.

[0132] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A genetically engineered bacterium, characterized in that: It carries foreign genes; the foreign genes include the gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase, and also include at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, and also include at least one of the decarboxylase gene TpCK and the gene AfDC; Optionally: The exogenous genes include gene npgA, gene gedC, gene HyTE and gene AfDC; The exogenous genes include gene npgA, gene gedC, gene HyTE and decarboxylase gene TpCK; The exogenous genes include gene npgA, gene gedC, gene gedB and gene AfDC; or, The exogenous genes include gene npgA, gene gedC, gene gedB and decarboxylase gene TpCK.

2. The genetically engineered bacterium according to claim 1, characterized in that The exogenous gene satisfies one or more of the following conditions (A1) to (A6): (A1) The gene gedC is derived from Aspergillus terreus, and optionally, the nucleic acid sequence is shown in SEQ ID NO: 1; (A2) The gene gedB is derived from Aspergillus terreus, and optionally, the nucleic acid sequence is shown in SEQ ID NO: 2; (A3) The gene HyTE is derived from Hypoxylon sp. EC38; optionally, the nucleic acid sequence is shown in SEQ ID NO: 3; (A4) the decarboxylase gene TpCK is derived from Aspergillus fumigatus, and optionally, the nucleic acid sequence is shown in SEQ ID NO: 4; (A5) the gene AfDC is derived from Aspergillus fischeri NRRL 181, and optionally, the nucleic acid sequence is shown in SEQ ID NO: 5; and, (A6) The gene npgA is derived from Aspergillus nidulans (A. nidulans), and optionally, the nucleic acid sequence is shown in SEQ ID NO:

6.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: The exogenous gene contained in the genetically engineered bacteria satisfies one or two of the following conditions (B1) to (B3): (B1) The exogenous gene is integrated into the genome of the genetically engineered bacteria; optionally, the exogenous gene is integrated into the genome by linear plasmid transformation; further optionally, the exogenous genes are independently contained in different expression cassettes of the same linear plasmid; (B2) the exogenous gene is contained in a recombinant expression vector; and, (B3) The genetically engineered bacteria contain multiple copies of foreign genes; Optionally, the expression cassette comprises a promoter and a terminator; Wherein, the promoter is optionally selected from P TEF1N , P GMP , P TEF and P FBA1N The terminator is optionally selected from T XPR2 , T OCT1 and T LIP1 The group formed.

4. The genetically engineered bacterium according to claim 1 or 2, characterized in that: The genetically engineered bacteria further include one or more of the gene leu2, the gene ura3, the gene ptaI encoding a methyltransferase, and the gene pEX10; Optionally, the nucleic acid sequence of the gene ptaI is shown in SEQ ID NO: 7; Optionally, the gene pEX10 and the gene ptaI contained in the genetically engineered bacteria satisfy one or two of the following conditions (C1) to (C2): (C1) the gene pEX10 and the gene ptaI are integrated into the genome of the genetically engineered bacteria; optionally, the gene pEX10 and the gene ptaI are integrated into the genome by linear plasmid transformation; further optionally, the gene pEX10 and the gene ptaI are each independently contained in different expression cassettes of the same linear plasmid; the expression cassette is optionally as defined in claim 2; (C2) The gene pEX10 and the gene ptaI are contained in a recombinant expression vector.

5. The genetically engineered bacterium according to claim 1, characterized in that The starting strain of the genetically engineered bacteria includes Yarrowia lipolytica; Optionally, the Yarrowia lipolytica is the Yarrowia lipolytica with the deposit number ATCC MYA-2613.

6. A recombinant expression vector, characterized in that: The method comprises the following genes: a gene npgA encoding phosphopantetheinyl transferase and a gene gedC encoding a polyketide synthase, and also comprises at least one of a gene HyTE and a gene gedB encoding a β-lactamase type thioesterase, and also comprises at least one of a decarboxylase gene TpCK and a gene AfDC; Optionally, The recombinant expression vector comprises gene npgA, gene gedC, gene HyTE and gene AfDC; The recombinant expression vector comprises gene npgA, gene gedC, gene HyTE and decarboxylase gene TpCK; The recombinant expression vector comprises gene npgA, gene gedC, gene gedB and gene AfDC; or, The recombinant expression vector comprises gene npgA, gene gedC, gene gedB and decarboxylase gene TpCK; Optionally, the nucleic acid sequence of the gene npgA, the gene gedC, the gene HyTE, the gene AfDC, the gene gedB or the decarboxylase gene TpCK is as defined in claim 1; Optionally, the recombinant expression vector satisfies one or more of the following conditions (D1) to (D3): (D1) The genes in the recombinant expression vector are each independently contained in different expression cassettes; optionally, the expression cassette comprises a promoter and a terminator; wherein the promoter is optionally selected from P TEF1N , P GMP and P TEF and P FBA1N The terminator is selected from the group consisting of XPR2 , T OCT1 and T LIP1 The group formed; (D2) the recombinant expression vector is a linear plasmid; and, (D3) The recombinant expression vector further comprises one or more of the gene leu2 and the gene ura3.

7. A recombinant expression vector combination, characterized in that: It comprises the recombinant expression vector as claimed in claim 6 as a first recombinant expression vector, and a second recombinant expression vector; The second recombinant expression vector comprises a gene ptaI; the nucleic acid sequence of the gene ptaI may be as shown in SEQ ID NO:7; Optionally, the second recombinant expression vector satisfies one or more of the following conditions (E1) to (E2): (E1) The second recombinant expression vector further comprises gene pEX10; optionally, the gene pEX10 and gene ptaI are each independently contained in different expression cassettes; the expression cassette optionally comprises a promoter and a terminator; wherein the promoter is optionally selected from P TEF1N , P GMP and P TEF and P FBA1N The terminator is selected from the group consisting of XPR2 , T OCT1 and T LIP1 The group formed; and (E2) The second recombinant expression vector is a linear plasmid.

8. A method for preparing rheumatoid arthritis, characterized in that: The method comprises: Cultivating the genetically engineered bacteria according to any one of claims 1 to 5 in a fermentation medium to obtain a culture; Isolation of rhein from culture; Optionally, the fermentation medium comprises: 5 g / L-25 g / L yeast extract, 10 g / L-30 g / L peptone and 30 g / L-80 g / L glucose; Optionally, the fermentation medium uses an inorganic nitrogen source including ammonium sulfate or ammonium chloride as a nitrogen source, and glycerol, vegetable oil and acetate as a carbon source; Optionally, the culture conditions include: a culture temperature of 28°C and a rotation speed of 180 rpm to 250 rpm; Optionally, the starting strain of the genetically engineered bacteria includes Yarrowia lipolytica, and the Yarrowia lipolytica may be Yarrowia lipolytica with the deposit number ATCC MYA-2613.

9. A method for preparing physaccharin methyl ether, characterized in that: The method comprises: Cultivating the genetically engineered bacteria as claimed in claim 4 or 5 in a fermentation medium to obtain a culture; Isolation of physaccharin methyl ether from culture; Optionally, the fermentation medium comprises: 5 g / L-25 g / L yeast extract, 10 g / L-30 g / L peptone and 30 g / L-80 g / L glucose or glycerol; Optionally, the culture conditions include: a culture temperature of 28°C and a rotation speed of 180 rpm to 250 rpm; Optionally, the starting strain of the genetically engineered bacteria includes Yarrowia lipolytica, and the Yarrowia lipolytica may be Yarrowia lipolytica with the deposit number ATCC MYA-2613.

10. A method for constructing a genetically engineered bacterium for producing rhamnosin, characterized in that: The method comprises integrating at least one copy of the following gene into the genome of the starting strain: The gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding polyketide synthase, and at least one of the gene HyTE and the gene gedB encoding β-lactamase type thioesterase, and at least one of the decarboxylase gene TpCK and the gene AfDC; Optionally, the gene is integrated into the genome of the starting strain by transfecting the recombinant expression vector as claimed in claim 6 into the starting strain; Optionally, the nucleic acid sequence of the gene npgA, the gene gedC, the gene HyTE, the gene AfDC, the gene gedB or the decarboxylase gene TpCK is as defined in claim 1; Optionally, the starting strain includes Yarrowia lipolytica; further optionally, the Yarrowia lipolytica is the Yarrowia lipolytica with the deposit number ATCC MYA-2613.

11. A method for constructing a genetically engineered bacterium for producing physaccharin methyl ether, characterized in that: The method comprises integrating at least one copy of the following gene into the genome of the starting strain: at least one of the gene npgA encoding phosphopantetheinyl transferase and the gene gedC encoding a polyketide synthase, the gene HyTE and the gene gedB encoding a beta-lactamase type thioesterase, at least one of the decarboxylase gene TpCK and the gene AfDC, the gene ptaI, and optionally the gene pEX10; Optionally, the gene is integrated into the genome of the starting strain by transfecting the recombinant expression vector combination as claimed in claim 7 into the starting strain; Optionally, the nucleic acid sequence of the gene npgA, the gene gedC, the gene HyTE, the gene AfDC, the gene gedB or the decarboxylase gene TpCK is as defined in claim 1; the nucleic acid sequence of the gene ptaI is as defined in claim 3; Optionally, the starting strain includes Yarrowia lipolytica; further optionally, the Yarrowia lipolytica is the Yarrowia lipolytica with the deposit number ATCC MYA-2613.

Images