Engineering bacterium capable of producing trans-aconitic acid at high yield as well as construction method and application of engineering bacterium

Abstract

The invention relates to a high-yield trans-aconitic acid engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of gene engineering. In the scheme of the invention, the inventor introduces mitochondrial localization signal peptide to be combined with the aconitic acid isomerase to form a gene element, and an expression cassette containing the gene element is introduced into the strain by adopting a genetic engineering technical means, so that the aconitic acid isomerase expressed in the constructed recombinant strain can be localized into mitochondria, and the expression efficiency of the recombinant strain is improved. Metabolic pathways related to trans-aconitic acid synthesis are located in the same zone, so that the capacity of fermenting and producing trans-aconitic acid by the recombinant strain is improved. The technical scheme disclosed by the invention not only has a good application prospect and production economic value, but also provides a referential new thought for transformation of strains related to other fermentation processes.

Description

Technical Field

[0001] The present application relates to an engineering bacterium with high production of trans-aconitic acid and a construction method and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Trans-aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid that can be used as a food additive. It contains unsaturated double bonds and abundant hydroxyl groups. Trans-aconitic acid can be used as a raw material to develop a new type of bio-based plasticizer of trans-aconitic acid esters. At the same time, trans-aconitic acid also has a good nematode control effect, and has the potential to become a new generation of efficient and non-toxic nematode killing preparations. Trans-aconitic acid is mainly produced by chemical synthesis, but the process is complicated, there are many by-products, the cost is high, and large-scale production has not been formed. In order to develop a greener and more efficient trans-aconitic acid production process, the inventors used Aspergillus terreus as the starting strain, and blocked the cis-aconitic acid decarboxylase gene (cadA) that catalyzes the decarboxylation of cis-aconitic acid to produce itaconic acid in Aspergillus terreus, thereby obtaining an Aspergillus terreus engineered strain At-ΔcadA that can produce aconitic acid (see patent CN110527637A for details). Furthermore, based on the At-ΔcadA strain, the inventors also made improvements by overexpressing aconitate isomerase Adi1 / TbrA, and obtained several engineered strains of Aspergillus terreus with high production of trans-aconitate (see patents CN112011469A and CN112029671A for details).

[0003] The inventors have found through research that cis-aconitic acid plays a very important role in the synthesis of trans-aconitic acid. However, the supply of cis-aconitic acid needs to be produced by the mitochondrial tricarboxylic acid cycle, and can reach the cytoplasm after a one-step transmembrane transport for the synthesis of trans-aconitic acid. If cis-aconitic acid cannot be transported out in time for the synthesis of trans-aconitic acid, it will be rapidly converted into isocitrate and used for growth metabolism. Therefore, in order to improve the synthesis efficiency of trans-aconitic acid from the source, it is necessary to provide a solution that can further improve the production efficiency of trans-aconitic acid. Summary of the invention

[0004] In order to solve the above problems, an engineered bacterium with high trans-aconitic acid production and a construction method and application thereof are provided. A mitochondrial signal peptide is combined with aconitate isomerase to form a gene expression element, and a gene expression cassette containing the element is introduced into the strain by means of genetic engineering technology, so that the aconitate isomerase expressed in the constructed recombinant strain can be localized in the mitochondria, so that the two-step reaction of trans-aconitate synthesis is carried out in the same compartment, avoiding excessive precursor cis-aconitate from being used for growth metabolism, which is more conducive to the production of trans-aconitate, thereby improving the efficiency of the recombinant strain in producing trans-aconitate by fermentation.

[0005] According to one aspect of the present application, a method for constructing a high-yield trans-aconitate strain is provided, the construction method comprising the step of using gene editing technology to target aconitate isomerase to the mitochondria.

[0006] In the present application, the inventors found, based on the aforementioned research contents, that there is still room for improvement in the trans-aconitic acid production efficiency of the modified strain. By localizing the successfully expressed aconitate isomerase into the mitochondria, the metabolic pathway for the production of trans-aconitic acid is located in the same compartment, thereby inhibiting the metabolic pathway for the conversion of cis-aconitic acid to citric acid, and allowing the cis-aconitic acid produced in the mitochondria to be rapidly converted into trans-aconitic acid under the catalysis of aconitate isomerase, thereby significantly improving the production efficiency of trans-aconitic acid.

[0007] It can be understood by those skilled in the art that the use of gene editing technology referred to in the present invention to target the aconitate isomerase to the mitochondria can adopt the technical means commonly used in the art, such as using fusion PCR technology or gene synthesis technology to add a mitochondrial localization signal peptide sequence in front of the gene encoding aconitate isomerase, and introduce it into the strain through PEG-mediated transformation or electroporation. The translated signal peptide will guide the aconitate isomerase sequence into the mitochondria, fold and perform its function in the mitochondria. It should be noted that the same technical means commonly used in the art that can achieve the targeted localization of aconitate isomerase to the mitochondria should also be included in the scope of the present invention.

[0008] Optionally, the construction method enables the strain to express aconitate isomerase with a mitochondrial localization signal peptide fused to the N-terminus, and the expressed mitochondrial localization signal peptide can localize the aconitate isomerase amino acid sequence into the mitochondria, and the aconitate isomerase is refolded into a normally functioning aconitate isomerase.

[0009] Optionally, the amino acid sequence of the mitochondrial localization signal peptide contains the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; and / or, the aconitate isomerase is Adi1 or TbrA.

[0010] It should be noted that other amino acid sequences with the same function obtained by replacing one or more amino acids on the basis of the above-mentioned mitochondrial localization signal peptide without other significant changes in effect should also be included in the scope of the present invention. In addition, the aconitate isomerase being Adi1 or TbrA does not constitute a limitation on the present application. It is known to those skilled in the art that other genes with aconitate isomerase function can also achieve the effect achieved by the present application after being expressed and entering the mitochondria. Other proteins encoded by other sequences but having no obvious functional differences from aconitate isomerase should also be included in the scope of the present invention.

[0011] According to another aspect of the present application, an engineered bacterium with high production of trans-aconitic acid is provided, wherein the engineered bacterium expresses aconitate isomerase with a mitochondrial localization signal peptide fused to its N-terminus.

[0012] Optionally, the amino acid sequence of the mitochondrial localization signal peptide contains the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; and / or, the aconitate isomerase is Adi1 or TbrA.

[0013] It should be noted that other amino acid sequences with the same function obtained by replacing one or more amino acids on the basis of the above-mentioned mitochondrial localization signal peptide without other significant changes in effect should also be included in the scope of the present invention. In addition, the aconitate isomerase being Adi1 or TbrA does not constitute a limitation on the present application. It is known to those skilled in the art that other genes with aconitate isomerase function can also achieve the effect achieved by the present application after being expressed and entering the mitochondria. Other proteins encoded by other sequences but having no obvious functional differences from aconitate isomerase should also be included in the scope of the present invention.

[0014] Optionally, the starting strain of the engineered bacteria is Aspergillus.

[0015] Optionally, the Aspergillus is selected from Aspergillus terreus or Aspergillus niger.

[0016] Optionally, the Aspergillus niger is selected from one or more of Aspergillus niger MEFC1501, Aspergillus niger ATCC 1015, Aspergillus niger NRRL 41873, Aspergillus niger NRRL 27809, Aspergillus niger NRRL 31821, Aspergillus niger NRRL 6276, Aspergillus niger Co827, Aspergillus niger 5016, Aspergillus niger 3008, Aspergillus niger GCB75, Aspergillus niger ATCC 9142, and Aspergillus niger MTCC 282; and / or,

[0017] The Aspergillus terreus is selected from one or more of Aspergillus terreus CICC40205, Aspergillus terreus NRRL1960, Aspergillus terreus DSM23081, Aspergillus terreus TN484, and Aspergillus terreus TN484-M1.

[0018] Optionally, when the starting strain is Aspergillus terreus, in order to prevent the influence of itaconic acid pathway on the synthesis of trans-aconitic acid, the cadA gene of aconitate decarboxylase is also knocked out to improve the production efficiency of trans-aconitic acid.

[0019] Optionally, the method of knocking out the aconitate decarboxylase cadA gene is to use the gene expression cassette to replace the nucleotide sequence of the Aspergillus terreus cadA gene with the nucleotide sequence of the mitochondrial localization signal peptide and aconitate isomerase.

[0020] It should be noted that the present invention does not limit the specific method of knocking out the cadA gene. For example, a single gene knockout element can be used to knock out the cadA gene, and an additional gene expression element can be used to target aconitate isomerase to the mitochondria. This method can achieve the construction of expressing aconitate isomerase that targets the inside of mitochondria while knocking out the cadA gene, which can reduce the operating procedures and difficulty of operation in strain transformation.

[0021] Optionally, when the starting strain is Aspergillus niger, a gene expression cassette containing a promoter, an aconitate isomerase gene expression element fused to a mitochondrial localization signal peptide at the N-terminus and a terminator sequence is used to insert the genome of the starting strain by transformation. It should be noted that the insertion method can be selected to randomly insert and screen out the modified strain that does not affect the normal life activities of the strain in the later stage, or it can be directed to insert, and the insertion method is not specifically limited in the present invention. It should also be noted that it is known to those skilled in the art that the genome of the above-mentioned insertion starting strain is not necessary, and it can also be present on a plasmid in a free state, as long as it can achieve the targeted localization of the successfully expressed aconitate isomerase to the mitochondria.

[0022] It should be noted that since there is a synthetic pathway in Aspergillus terreus for decarboxylation of the precursor cis-aconitic acid to form itaconic acid, in order to further improve the synthesis efficiency of trans-aconitic acid, the key gene cadA in the itaconic acid synthesis pathway is knocked out; Aspergillus niger does not have the key gene for the synthesis of itaconic acid, so it is sufficient to directly introduce aconitate isomerase that can be localized in the mitochondria.

[0023] According to another aspect of the present application, an engineered bacterium obtained by any of the above-mentioned construction methods is provided.

[0024] According to another aspect of the present application, there is provided a use of any of the above-mentioned engineered bacteria in the production of trans-aconitic acid, products containing trans-aconitic acid, or products containing a trans-aconitic acid structure.

[0025] According to another aspect of the present application, the following biomaterial is provided, wherein the biomaterial is any one of A) to C):

[0026] A) a mitochondrial localization signal peptide, the amino acid sequence of which is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;

[0027] B) an aconitate isomerase gene expression element with a mitochondrial localization signal peptide fused to the N-terminus, the gene expression element comprising a mitochondrial localization signal peptide sequence and an aconitate isomerase sequence, the mitochondrial localization signal peptide sequence is selected from the mitochondrial localization signal peptide described in A), and the aconitate isomerase is adi1 or tbrA;

[0028] C) a gene expression cassette, which contains, from 5' to 3', the upstream homology arm of the cadA gene, B) the aconitate isomerase gene expression element with the N-terminal fused mitochondrial localization signal peptide, a terminator and the downstream homology arm of the cadA gene; or,

[0029] The gene expression cassette contains a promoter, the aconitate isomerase gene expression element with the N-terminal fused mitochondrial localization signal peptide as described in B) and a terminator in sequence from 5' to 3'.

[0030] Optionally, a person skilled in the art may add a resistance gene to the gene expression box to facilitate the screening of successfully transformed strains. It is also known to a person skilled in the art that a resistance gene is not necessary, and other methods commonly used in the art such as gene sequence detection may be used to determine and screen successfully transformed strains.

[0031] Optionally, the promoter is PicdA, PgpdA, PacoA, PcitA, PglaA, PcadA, PmfsA or Pgas; and the terminator is Tpgk or TtrpC.

[0032] Optionally, the nucleotide sequence of the upstream homology arm of the cadA gene is shown as SEQ ID NO.52; the nucleotide sequence of the downstream homology arm of the cadA gene is shown as SEQ ID NO.53.

[0033] The beneficial effects of this application include but are not limited to:

[0034] 1. According to the high-yield trans-aconitic acid strain of the present application, the mitochondrial localization signal peptide is used to locate aconitate isomerase to enter the mitochondria to play its role, so that the cis-aconitic acid in the mitochondria can be efficiently converted into trans-aconitic acid under the catalysis of aconitate isomerase, thereby improving the efficiency of the strain in producing trans-aconitic acid, and providing a new engineered strain for the efficient production of trans-aconitic acid.

[0035] 2. According to the high-yield trans-aconitic acid strain of the present application, the problem in the prior art that the trans-aconitic acid synthesis pathway is in two compartments, resulting in the consumption and insufficient supply of the precursor cis-aconitic acid, is solved. The aconitate isomerase located in the mitochondria can efficiently catalyze the cis-aconitic acid reactants produced in the mitochondria, thereby improving the production efficiency of trans-aconitic acid and having great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0037] Figure 1 The genomic PCR verification results of the green fluorescent protein StayGold recombinant engineering strain mediated by different mitochondrial localization signal peptides constructed in Example 1 of the present application (M is a DNA molecular weight marker, and primers YZ-ku80-F / YZ-ku80-R are used for PCR verification, and C is the control strain At-Δku80);

[0038] Figure 2 This is a fluorescence result diagram of hyphae fused with different mitochondrial targeting sequences (MTS) observed by laser confocal microscopy (LSCM) in the characterization of the mitochondrial localization effect of the signal peptides MTSacoAt, MTScitAt, and MTSicdAn involved in Example 1 of the present application;

[0039] Figure 3 The PCR verification result of the genome of the transformant of the strain expressing mitochondrial aconitate isomerase involved in Example 2 of the present application (M is a DNA molecular weight marker, primers C-cadA-F / C-cadA-R are used for PCR verification, and wt is the control strain At-Δku80);

[0040] Figure 4 This is a graph showing the results of shake flask fermentation to evaluate the trans-aconitic acid production and conversion rate of an engineered strain of Aspergillus terreus;

[0041] Figure 5 The PCR verification results of the genome of the An::PicdAn-MTSicdAn-tbrA Aspergillus niger engineered strain constructed in Example 3 of the present application (M is a DNA molecular weight marker, C is a control strain MEFC 1501, and 1-3 are the results of PCR verification using primers PicdAn-F / TtrpC-R);

[0042] Figure 6This is a graph showing the results of shake flask fermentation evaluation of the trans-aconitic acid production of the MTSicdAn-tbrA Aspergillus niger engineered strain. DETAILED DESCRIPTION

[0043] The present application is described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are purchased through commercial channels.

[0044] Trans-Aconitic acid (CAS: 4023-65-8) is an unsaturated tricarboxylic acid that can be used as a food additive. It contains unsaturated double bonds and abundant hydroxyl groups. Trans-Aconitic acid can be used as a raw material to develop new bio-based plasticizers. At the same time, trans-Aconitic acid also has a good nematode control effect and has great potential to become a new generation of highly effective and non-toxic nematicide. Trans-Aconitic acid is mainly produced by chemical synthesis, but the process is complicated, there are many by-products, the cost is high, and it has not formed large-scale production. In order to develop a greener and more efficient trans-aconitic acid production process, the inventors have obtained an engineered strain of Aspergillus terreus that can produce aconitic acid, At-ΔcadA (see patent CN110527637A for details), by blocking the aconitate decarboxylase gene (cadA) that catalyzes the decarboxylation of aconitate to produce itaconic acid in Aspergillus terreus in previous studies. Furthermore, based on the At-ΔcadA strain, the inventors also made improvements by overexpressing aconitate isomerase Adi1 / TbrA. Further, several engineered strains of Aspergillus terreus with high production of trans-aconitic acid were obtained (see patents CN112011469A and CN112029671A for details). The above technical means uses Aspergillus terreus as the starting strain, knocks out the aconitate decarboxylase cadA gene in the starting strain to block the pathway of itaconic acid production, and overexpresses the aconitate isomerase Adi1 / TbrA to produce trans-aconitate, thereby obtaining a recombinant strain with high production of trans-aconitate.

[0045] In the scheme of the present invention, the inventors continue to analyze according to the biosynthetic pathway and find that the synthesis of trans-aconitic acid involves a two-step transmembrane transport process: in the first step, cis-aconitic acid is synthesized in the mitochondria and transported to the cytoplasm across the mitochondrial membrane, and trans-aconitic acid is formed in the cytoplasm through catalysis, and in the second step, it is transported to the extracellular space across the cell membrane. During the two-step transmembrane transport, cis-aconitic acid plays a very important role in the synthesis of trans-aconitic acid. However, the supply of cis-aconitic acid needs to be produced by the mitochondrial tricarboxylic acid cycle and can reach the cytoplasm after a one-step transmembrane transport for the synthesis of trans-aconitic acid. If cis-aconitic acid cannot be transported out in time for the synthesis of trans-aconitic acid, it will be rapidly converted into isocitrate and used for growth metabolism. Based on the inventor's continued exploration and research on the recombinant strains constructed in previous patents, the inventors continue to provide a scheme that can further improve the efficiency of trans-aconitic acid synthesis in the present invention to solve the technical problem that the precursor cis-aconitic acid is insufficiently supplied due to the compartmentalization of the synthesis pathway, thereby affecting the yield of trans-aconitic acid.

[0046] In the previous research results of the inventors, a recombinant strain with high trans-aconitate production was obtained by knocking out the cadA gene of aconitate decarboxylase in the starting strain and overexpressing aconitate isomerase Adi1 / TbrA, which has made great technical progress in increasing the production of trans-aconitate. In the present application, the inventors analyzed the pathway of trans-aconitate synthesis in cells in the prior art, proposed a feasible optimization strategy, and verified through experiments that aconitate isomerase can be localized in mitochondria by combining a mitochondrial localization signal peptide with aconitate isomerase, and the relocated aconitate isomerase can still function normally, and finally a fermentation strain with higher trans-aconitate production was obtained.

[0047] Specifically, although the inventors have greatly improved the production of trans-aconitic acid in previous studies, the inventors did not stop the research. After further analyzing and studying the production pathway of trans-aconitic acid in the modified strains obtained in previous studies, the inventors found that the fermentation production link of trans-aconitic acid still has room for optimization, and proposed a solution to locate aconitate isomerase to mitochondria. Through experiments, at least the following facts were successfully verified: first, the mitochondrial localization signal peptide provided in the present application has the ability to locate the target sequence to the mitochondria; second, aconitate isomerase can be located to the mitochondria by using the mitochondrial localization signal peptide; third, the aconitate isomerase located in the mitochondria can still function normally; fourth, after the aconitate isomerase is located to the mitochondria, the production of trans-aconitic acid can indeed be greatly increased.

[0048] In the present application, in order to achieve the above-mentioned successful localization of aconitate isomerase to mitochondria and increase the production of trans-aconitate, the inventors have gone through the steps of selecting mitochondrial signal peptides, verifying the localization effect of mitochondrial localization signal peptides, constructing recombinant strains and verifying the fermentation of trans-aconitate produced by the recombinant strains. Finally, not only did they successfully obtain a high-yield trans-aconitate fermentation strain, but they also obtained the mitochondrial localization signal peptides and the gene expression elements used in constructing the high-yield trans-aconitate fermentation strain. The trans-aconitate high-yield strain obtained in the present application greatly improves the production efficiency of trans-aconitate, has huge economic value and market application prospects, and the present application has important theoretical and practical significance for the use of mitochondrial localization signal peptides to artificially intervene in metabolic pathways.

[0049] It should be noted that the experimental materials used in the specific embodiments of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the specific embodiments of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd Edition), Science Press, 2002; DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or the conditions recommended by the manufacturer.

[0050] It should be noted that the concept of gene expression cassette in the present application can be understood as a nucleotide sequence composed of biological elements with biological functions such as promoters, protein-coding genes, and terminators, which can stably guide cells to produce corresponding mRNA and proteins when transformed into cells. It can also be understood as a sequence with the target expression gene that has the ability to express by replacing the original gene fragment and utilizing the original promoter and terminator, but it is not limited to having a promoter or terminator. For example, when Aspergillus terreus is genetically recombined in the present application, the nucleotide sequence of the Aspergillus terreus cadA gene is replaced by the nucleotide sequence of the mitochondrial localization signal peptide and aconitate isomerase, and then the promoter of the Aspergillus terreus cadA gene is used to achieve expression. Therefore, the concept of gene expression cassette conventionally understood in the prior art does not constitute a limitation on the gene expression cassette described in the present application.

[0051] The scheme of the present invention is specifically described below through specific embodiments.

[0052] Example 1: Characterization of the mitochondrial localization effects of signal peptides MTSacoAt, MTScitAt, and MTSicdAn

[0053] 1) Prediction of mitochondrial localization signal peptides of acoA and citA genes from Aspergillus terreus and icdA genes from Aspergillus niger

[0054] The amino acid sequences of acoAt (Genbank accession number: EAU36599.1), citAt (Genbank accession number: EAU31163.1) and icdAn (Genbank accession number: BAA19073.1) genes were predicted using the MITOPROTⅡ program (MGClaros et al., 1996). The probability of localizing to mitochondria was 90.54%, 95.91% and 99.55%, respectively. Therefore, it was confirmed that the signal peptides contained therein were most likely targeting sequences localized to mitochondria. Finally, three mitochondrial localization signal peptide sequences were obtained as shown below.

[0055] MTSacoAt (SEQ ID NO. 1):

[0056] MISTRLARMGALAPKSRLLLGTRGLATVADSP

[0057] MTScitAt (SEQ ID NO. 2):

[0058] MAASLRLGTSALRSTTLAGKPVVQSAAFNGLRCY

[0059] MTSicdAn (SEQ ID NO. 3):

[0060] MSSVRFSSALARRSFAVASPPLSAPLSSSARRFLSSSSSISSSSSSVSTRSPRSLTSASSLLSSRTASARWTGLSSLNLTQSRT

[0061] 2) Construction of fluorescent protein gene expression cassette fused with mitochondrial signal peptide

[0062] Although three mitochondrial localization signal peptides were predicted, their mitochondrial localization ability still needs to be experimentally verified. The inventors here use fluorescent protein genes as the directed target proteins for mitochondrial localization testing, which is convenient for verification by fluorescence detection.

[0063] KU80: This site is a DNA repair-related protein, Genbank accession number EAU32303.1.

[0064] StayGold: a highly green fluorescent protein, Genbank accession number LC601652. The coding nucleotide fragment of StayGold was optimized and synthesized according to the codon preference of Aspergillus and is shown in SEQ ID NO.4.

[0065] Primers were designed and synthesized based on the gene sequence information. The specific primer sequences are shown below.

[0066] Plasmid pXH106 (recorded in CN110527637A, a kind of aconitic acid-producing Aspergillus terreus strain and its construction method and application, and available to the public through the Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences) was used as a template, DNA polymerase (Nanjing Novozyme Medical Technology Co., Ltd., 2×Phanta Max Master Mix) was used for PCR amplification, and primers pXH106-XF / pXH106-XR were used to amplify a pXH106 plasmid backbone fragment of about 6.6 kb containing the downstream homology arm D-ku80 of the ku80 gene, the plasmid backbone fragment of pUC57 (containing the ampicillin resistance gene amp), the upstream homology arm U-ku80 of the ku80 gene, and the orotidine-5'-decarboxylase gene pyrG.

[0067] Using Aspergillus terreus At-Δku80 (Aspergillus terreus At-Δku80 is an engineered strain for knocking out the ku80 gene in Aspergillus terreus CICC40205, see CN104894165A A method and application for improving the efficiency of gene targeting technology in Aspergillus terreus) genomic DNA as a template, primers PgpdAt-F (PyrG) / PgpdAt-R, PgpdAt-F (PyrG) / PgpdAt-R (MTScitA t), PgpdAt-F(PyrG) / PgpdAt-R(MTSacoAt), PgpdAt-F(PyrG) / PgpdAt-R(MTSicdAt) were amplified in sequence by PCR to obtain the Aspergillus terreus constitutive promoter PgpdAt containing the downstream partial sequence, namely, PgpdAt, PgpdAt(MTScitAt), PgpdAt(MTSacoAt) and PgpdAt(MTSicdAn) fragments.

[0068] Using the genomic DNA of Aspergillus terreus At-△ku80 as a template, the primers MTScitAt-F / MTScitAt-R and MTSacoAt / MTSacoAt-R were used to amplify the mitochondrial localization signal peptide MTS in sequence to obtain the MTScitAt and MTSacoAt fragments. Using the genomic DNA of Aspergillus niger MEFC1501 (deposited in the China General Microbiological Culture Collection Center, the culture collection number is: CGMCCNO.40614, the storage time is: April 27, 2023, and the storage location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) as a template, the primers MTSicdAn-F / MTSicdAn-R were used to amplify the mitochondrial localization signal peptide MTS to obtain the MTSicdAt fragment.

[0069] Using pUC-staygold-Kan (the plasmid carrying the StayGold gene synthesized by Jinweizhi, the public can synthesize it by themselves through the nucleotide sequence of the StayGold gene) as a template, StayGold(PgpdAt)-F / StayGold-R(Ku80-D), StayGold(MTScitAt)-F / StayGold-R(Ku80-D), StayGold(MTSacoAt)-F / StayGold-R(Ku80-D) and StayGold(MTSicdAn)-F / StayGold-R(Ku80-D) were PCR amplified in sequence to obtain the green fluorescent protein StayGold fragments containing the upstream partial sequence, namely, StayGold, StayGold(MTScitAt), StayGold(MTSacoAt) and StayGold(MTSicdAn) fragments.

[0070] All PCR products were detected by 0.8% agarose gel electrophoresis and purified by gel excision. One-step cloning kit (purchased from Nanjing Novozyme Medical Technology Co., Ltd., ClonExpress Ultra One Step Cloning The plasmids pXH106-PgdpAt-StayGold, pXH106-PgdpAt-MTScitAt-StayGold, pXH106-PgdpAt-MTScitAt-StayGold, pXH106-PgdpAt-MTSicd ... coli transformation, amplification culture and plasmid extraction to obtain a plasmid with a high concentration. Using D-ku80-F / U-ku80-R as primers to amplify the gene targeting elements ku80::PgpdAt-staygold-pyrG, ku80::PgpdAt-MTScitAt-staygold-pyrG, ku80::PgpdAt-MTSacoAt-staygold-pyrG and ku80::PgpdAt-MTSicdAn-staygold-pyrG with a size of about 5.5 kb, the gene targeting elements constructed above can be used to construct a genetic engineering strain that expresses a fluorescent protein located in mitochondria using a PgpdAt promoter at the ku80 site.

[0071] 3) Construction of an engineered strain of Aspergillus terreus that heterologously expresses fluorescent proteins localized to mitochondria

[0072] At-Δku80-ΔpyrG is an engineered strain with uracil auxotrophy obtained by knocking out the pyrG gene based on the At-Δku80 strain (see patent CN104894165A, a method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus). The spores of the engineered strain At-Δku80-ΔpyrG were inoculated into 55 mL of IPM-FU (IPM with 5 mM 5-fluoroorotic acid and 10 mM uridine) liquid culture medium (wherein the preparation method of IPM is 100 g / L glucose, 2 g / L NH4NO3, 0.2 g / L (NH4)2HPO4, 20 mg / L FeSO4, 0.4 g / L MgSO4, 40 mg / L ZnSO4, 40 mg / L CuSO4, adjusted to pH 3.5 with sulfuric acid, and sterilized at 115°C for 30 min) to obtain a spore concentration of about 10 7 / mL, culture at 220rpm and 37℃ for 36h. Collect the grown mycelium by filtering with a sterile single layer of 500 mesh nylon cloth, rinse three times with a sterilized 0.6M MgSO4 solution, squeeze dry and place in a sterile 50mL conical flask, add an appropriate amount of enzymatic solution according to the weight of the mycelium (add 10mL of enzymatic solution for every 1g of mycelium), and treat at 30℃ and 130rpm for 1 to 3h. Filter the above enzymatically hydrolyzed mixture with a 300 mesh nylon cloth or lens paper, and collect the filtrate. Collect the protoplasts by centrifugation at 4℃ and 4000rpm, wash once with a precooled 1.2M sorbitol solution, and then wash once with a precooled STC (STC consists of 1.2M sorbitol, 50mM Tris-HCl (pH=8.0), 50mM CaCl2), and finally resuspend the protoplasts in precooled STC, and adjust the protoplast concentration to 5×10 7 / mL to obtain a protoplast suspension. Add 10 μL of the DNA fragments (about 5 μg) of the targeting elements ku80::PgpdAt-staygold-pyrG, ku80::PgpdAt-MTScitAt-staygold-pyrG, ku80::PgpdAt-MTSacoAt-staygold-pyrG and ku80::PgpdAt-MTSicdAn-staygold-pyrG to 150 μL of the protoplast suspension. Add 50 μL of PSTC in an ice bath (PSTC composition is 40% PEG4000, 1.2M sorbitol, 50mM Tris-HCl (pH=8.0), 50mM CaCl2), mix gently, and place on ice for 30 minutes. Add 1mL of PSTC at room temperature, mix well, and place at room temperature for 20 minutes. Then, it was mixed with 30 mL of PDBS top agar and poured onto 10 PDAS plates for regeneration screening and culture, and cultured at 30°C in the dark for 5 to 7 days.

[0073] Transformants with good growth conditions were picked from the transformation screening plate and transferred to the PDA plate, and cultured at 30°C for 5 days for subculture purification. The spores of stable subculture transformants were inoculated into IPM liquid culture medium, cultured at 30°C and 200rpm for 48 hours, the mycelium was collected to extract genomic DNA, and PCR verification was performed with primers YZ-ku80-F / YZ-ku80-R. At the same time, the genome of the At-Δku80-ΔpyrG strain was used as a control. The positive transformants can amplify a band of approximately 3.0-3.5kb in size, and the control can amplify a band of approximately 2.0kb in size. Five positive transformants were selected for single spore isolation and purification, 3 single spores were verified for each transformant, and the primers YZ-ku80-F / YZ-ku80-R were used again for genomic PCR verification. The results are as follows Figure 1 As shown, homozygous transformants with the green fluorescent protein StayGold element mediated by different mitochondrial localization signal peptides integrated at the ku80 site were successfully obtained, namely, the genetically engineered strains used to evaluate the mitochondrial localization effect of the signal peptide, which were marked as At-Δku80::PgpdAt-staygold, At-Δku80::PgpdAt-MTScitAt-staygold, At-Δku80::PgpdAt-MTSacoAt-staygold and At-Δku80::PgpdAt-MTSicdAt-staygold, respectively.

[0074] 4) Fluorescence co-localization to evaluate the mitochondrial localization effect of signal peptides

[0075] The positive transformant strains of At-Δku80::PgpdAt-MTScitAt-staygold, At-Δku80::PgpdAt-MTSacoAt-staygold and At-Δku80::PgpdAt-MTSicdAn-staygold were selected, and At-Δku80::PgpdAt-staygold was used as the control strain. They were inoculated into IPM medium at a final concentration of 5×10 5 spores / mL, culture at 37°C, 220 rpm for 24 h. Take 1 ml of the bacterial solution and wash it twice with sterile water. Collect the bacteria by centrifugation, remove the supernatant, and use 300 nM Mito Resuspend the cells with Red CM-H2XRos reagent (Invitrogen, Cat. no. M7513) and incubate at 37°C for 30 min in the dark. After staining, collect the cells by centrifugation and resuspend them in fresh culture medium preheated at 37°C. Observe the hyphae fluorescence co-localization results under a confocal microscope (FluoView FV1000).

[0076] Mitochondrial localization results Figure 2 As shown, in Figure 2 The wild-type strain (WT) without the introduction of StayGold fluorescent protein was used as a blank control, the StayGold fluorescent protein without MTS sequence was expressed in the cytoplasm (cStayGold) as a negative control, and the mStayGOLD fused with the N-terminal MTS sequence of citAt, acoAt and icdAn genes was expressed in the mitochondria as an experimental group. At the same time, in order to show the location of mitochondria, the hyphae were stained with mitotracker Red. Figure 2 The results show that the At-Δku80::PgpdAt-staygold strain without a signal peptide sequence will cause the entire hyphae to be filled with green fluorescence. In contrast, when any of the signal peptides MTScitAt, MTSacoAt and MTSicdAn is combined with Staygold, the green fluorescence will be distributed in a dotted manner in the hyphae. At the same time, when the mitochondrial staining reagent MitoTracker is used for co-localization with Staygold, it is found that the green fluorescence stimulated by the At-Δku80::PgpdAt-MTScitAt-staygold, At-Δku80::PgpdAt-MTSacoAt-staygold and At-Δku80::PgpdAt-MTSicdAn-staygold strains fused with signal peptides highly overlaps with the position of mitochondria stained by the MitoTracker reagent. According to the above results, three signal peptides MTScitAt, MTSacoAt and MTSicdAn that can be localized to mitochondria have been successfully obtained.

[0077] Here, the inventors obtained three signal peptides with mitochondrial localization function. However, when applying them to the process of constructing a trans-aconitate strain, it is still necessary to determine whether the introduction of aconitate isomerase into the mitochondria works. Therefore, the inventors continued to construct and verify the recombinant Aspergillus terreus strain of mitochondrial localized aconitate isomerase.

[0078] Example 2: Construction of a recombinant Aspergillus terreus strain producing mitochondrial-localized aconitate isomerase

[0079] 1) Construction of gene targeting element for knocking out cadA and overexpressing mitochondrial aconitate isomerase adi1 / tbrA

[0080] The amino acid sequence of aconitate isomerase Adi1 of Ustilago maydis (as shown in SEQ ID NO.27, see CN 112011469 A, a recombinant Aspergillus terreus strain with high trans-aconitate production and its construction method and application):

[0081] The amino acid sequence of aconitate isomerase TbrA of Bacillus thuringiensis (as shown in SEQ ID NO.28, see CN 112029671 A for details, a recombinant Aspergillus terreus strain with high trans-aconitate production and its preparation method and application):

[0082] Primers were designed and synthesized based on the gene sequence information. The specific primer sequences are shown below.

[0083] Using Aspergillus terreus At-Δku80 (Aspergillus terreus At-Δku80 is an engineering strain for knocking out the ku80 gene in Aspergillus terreus CICC40205, see patent CN104894165A, a method and application for improving the application efficiency of gene targeting technology in Aspergillus terreus) genomic DNA as a template, primers U-cadA-F / U-cadA-R (MTScitAt) and U-cadA-F / U-cadA-R (MTSacoAt) can be used to amplify the upstream homologous arms U-cadA (MTScitAt) and U-cadA (MTSacoAt) fragments of the cadA gene with a size of about 1.2 kb, and primers D-cadA-F (pyrG) / D-cadA-R can be used to amplify the downstream homologous arm D-cadA fragment of the cadA gene with a size of about 1.3 kb. The U-cadA nucleotide sequence is shown in SEQ ID NO.51, and the D-cadA nucleotide sequence is shown in SEQ ID NO.52.

[0084] Using the genomic DNA of Aspergillus terreus At-△ku80 as a template, the primers MTScitAt-F / MTScitAt-R and MTSacoAt / MTSacoAt-R were used to amplify the mitochondrial localization signal peptide MTS in sequence to obtain the MTScitAt and MTSacoAt fragments.

[0085] Using the synthetic adi1 gene plasmid as a template, primers adi1-F (MTScitAt) / adi1-R and adi1-F (MTSacoAt) / adi1-R were used to sequentially amplify aconitate isomerase adi1 to obtain adi1 (MTScitAt) and adi1 (MTSacoAt) fragments. Using the synthetic tbrA gene plasmid as a template, primers tbrA-F (MTScitAt) / tbrA-R and tbrA-F (MTSacoAt) / tbrA-R were used to sequentially amplify aconitate isomerase tbrA to obtain tbrA (MTScitAt) and tbrA (MTSacoAt) fragments.

[0086] Using plasmid pXH2-1 as a template, the terminator TtrpC was amplified sequentially with primers TtrpC-F(adi1) / TtrpC-R and TtrpC-F(tbrA) / TtrpC-R to obtain TtrpC(adi1) and TtrpC(tbrA) fragments.

[0087] The nucleotide sequence of TtrpC is shown in SEQ ID NO.53.

[0088] Plasmid pXH-106 was used as a template and primers pyrG-F (TtrpC) / pyrG-R were used to amplify orotidine-5'-decarboxylase gene pyrG to obtain the pyrG fragment.

[0089] The nucleotide sequence of PyrG is shown in SEQ ID NO.54.

[0090] All PCR products were detected by 0.8% agarose gel electrophoresis and purified by gel excision. The "U-cadA (MTScitAt) fragment, MTScitAt fragment, adi1 (MTScitAt) fragment, TtrpC (adi1) fragment, pyrG fragment and D-cadA fragment", "U-cadA (MTScitAt) fragment, MTScitAt fragment, tbrA (MTScitAt) fragment, TtrpC (tbrA) fragment, pyrG fragment and D-cadA fragment", "U-cadA (MTSacoAt) fragment, MTSacoAt fragment, adi1 (MTSacoAt) fragment, TtrpC (adi1) fragment, pyrG fragment and D-cadA fragment", "U-cadA (MTSacoAt) fragment, MTSacoAt fragment, adi1 (MTSacoAt) fragment, TtrpC (adi1) fragment, pyrG fragment and D-cadA fragment", "U-cadA (MTSacoAt) fragment, MTSacoAt fragment, The fusion PCR product was used as a template and C-cadA-F / C-cadA-R were used as primers to amplify the gene targeting elements of about 6.1 kb, namely ΔcadA::MTScitAt-adi1, ΔcadA::MTScitAt-tbrA, ΔcadA::MTSacoAt-adi1 and ΔcadA::MTSacoAt-tbrA, which can be used to site-directedly overexpress the mitochondrial-localized aconitate isomerase adi1 / tbrA gene at the cadA site using the PcadA promoter.

[0091] 2) Construction of an engineered strain of Aspergillus terreus that heterologously overexpresses the mitochondrial-localized aconitate isomerase adi1 / tbrA at the cadA locus

[0092] The specific operation of transforming the gene into the strain is detailed in Example 1, 3) Construction of an engineered strain of Aspergillus terreus heterologously expressing a fluorescent protein localized to mitochondria.

[0093] Transformants with good growth conditions were picked from the transformation screening plate and transferred to the PDA plate, and cultured at 30°C for 5 days for subculture purification. The spores of the stable subculture transformants were inoculated into IPM liquid culture medium, cultured at 30°C and 200rpm for 48 hours, the mycelium was collected to extract genomic DNA, and PCR verification was performed with primers C-cadA-F / C-cadA-R. At the same time, the genome of the At-Δku80-ΔpyrG strain was used as a control. The positive transformants can amplify a band of approximately 6.2kb in size, and the control can amplify a band of approximately 4.5kb in size. Five positive transformants were selected for single spore isolation and purification, 3 single spores were verified for each transformant, and genomic PCR verification was performed again with primers C-cadA-F / C-cadA-R. The results are as follows Figure 3 As shown, homozygous transformants that heterologously overexpressed the mitochondrial-localized aconitate isomerase adi1 / tbrA at the cadA site were successfully obtained, i.e., genetically engineered strains with higher production of trans-aconitate, and were marked as At-ΔcadA::MTScitAt-adi1, At-ΔcadA::MTScitAt-tbrA, At-ΔcadA::MTSacoAt-adi1 and At-ΔcadA::MTSacoAt-tbrA, respectively.

[0094] 3) Shake flask analysis of fermentation of trans-aconitate by an engineered strain of Aspergillus terreus overexpressing the mitochondrial-localized aconitate isomerase adi1 / tbrA

[0095] Three positive transformant strains of At-ΔcadA::MTScitAt-adi1, At-ΔcadA::MTScitAt-tbrA, At-ΔcadA::MTSacoAt-adi1 and At-ΔcadA::MTSacoAt-tbrA were selected, and At-ΔcadA was used as a control strain. They were inoculated into PDA medium and cultured at 30°C for 7 days to obtain mature spores. The spores on the plates were then inoculated into IPM medium (500 mL of triangular flasks containing 55 mL of medium), with an inoculum size of about 5x10 5spores / mL, 3 shake flasks were set up as parallel for each transformant, and fermented at 37°C and 220rpm for 72h. The supernatant was filtered through a 0.45μm filter and appropriately diluted, and the organic acid content was detected by high performance liquid chromatography (HPLC), wherein the chromatographic column was Bio-rad Aminex HPX-87H, 300mm x 7.8mm, the mobile phase was 5mM sulfuric acid, the flow rate was 0.5mL / min, the column temperature was 55°C, the inspection temperature was 35°C, and the UV detector was 210nm.

[0096] The fermentation yield results are as follows Figure 4 As shown, Figure 4 The left side shows the aconitic acid production, and the right side shows the ratio of trans-aconitic acid to aconitic acid. The trans-aconitic acid production of strain At-ΔcadA::MTSacoAt-adi1 is 37.67 g / L, and the conversion rate of trans-aconitic acid is 88.63%. The trans-aconitic acid production of strain At-ΔcadA::MTScitAt-adi1 is 33.72 g / L, and the conversion rate of trans-aconitic acid is 88.77%. The trans-aconitic acid production of TSacoAt-tbrA was 11.18 g / L, and the conversion rate of trans-aconitic acid was 68.43%; the trans-aconitic acid production of At-ΔcadA::MTScitAt-tbrA was 7.16 g / L, and the conversion rate of trans-aconitic acid was 62.09%; the trans-aconitic acid production of the starting strain At-ΔcadA was 7.08 g / L, and the conversion rate of trans-aconitic acid was 43.78%. The Error bar in the figure represents the SEM of three parallel experiments (significance: *p≤0.05; **p<0.01; ***p<0.001).

[0097] Statistical comparative analysis of the fermentation yields of the engineered strains showed that after 72 hours of shake flask fermentation, the trans-aconitic acid production of the strains At-ΔcadA::MTSacoAt-adi1 (37.67 g / L), At-ΔcadA::MTScitAt-adi1 (33.72 g / L) and At-ΔcadA::MTSacoAt-tbrA (11.18 g / L) was significantly higher than that of the starting strain At-ΔcadA (7.08 g / L). At the same time, although the trans-aconitic acid production of the other strain At-ΔcadA::MTScitAt-tbrA (7.16 g / L) was not significantly increased compared with that of the starting strain At-ΔcadA, its trans-aconitic acid conversion rate reached 62.09%, which was much higher than 43.78% of the starting strain At-ΔcadA, indicating that localizing aconitate isomerase to mitochondria can significantly improve the production and conversion rate of trans-aconitic acid.

[0098] The inventors speculate that due to the specificity of the enzyme, the Adi1 strains (At-ΔcadA::MTScitAt-adi1 and At-ΔcadA::MTSacoAt-adi1) mediated by the two signal peptides MTScitAt and MTSacoAt are significantly higher than the TbrA strains (At-ΔcadA::MTScitA-tbrA and At-ΔcadA::MTSacoA-tbrA). At the same time, the trans-aconitic acid production of these two strains is significantly improved compared with the trans-aconitic acid production of 24.65g / L (168h) (202010866757.4) and 15.34g / L (72h) (202010866728.8) in other patents. Among them, the strain targeting the Adi1 isomerase to the mitochondria by the MTSacoAt signal peptide is the best, which can significantly improve the yield and conversion rate of trans-aconitic acid.

[0099] According to the above experimental verification results, compared with the trans-aconitate production and conversion rate of the starting strain At-ΔcadA, it can be confirmed that localizing aconitate isomerase to the mitochondria can significantly increase the production and conversion rate of trans-aconitate. The improved strategy based on the high-yield trans-aconitate strain constructed by the inventors in the past can be successfully implemented, and also achieved very obvious improvement results.

[0100] Example 3 Construction of a recombinant Aspergillus niger strain producing mitochondrial localized aconitate isomerase

[0101] In order to further verify that the above strategy has universal applicability and that the construction idea of ​​localizing trans-aconitate-related enzymes to mitochondria by introducing mitochondrial localization signal peptides can be applied to other strains, the inventors continued to transform the Aspergillus niger strain based on the above construction strategy.

[0102] 1) Construction of a targeting element for overexpressing aconitate isomerase tbrA gene containing the mitochondrial localization signal peptide MTSicdAn

[0103] The recombinant Aspergillus niger includes a promoter, which drives the expression of aconitate isomerase, and the promoter is a PicdA promoter, wherein the PicdA promoter sequence is shown in SEQ ID NO. 55. However, the selection of the promoter here is not limited to the PicdA promoter, and other promoters conventionally used in the art can be used, for example, it can be a PgpdAt promoter, whose nucleotide sequence is shown in SEQ ID NO. 56, a PgpdAn promoter, whose nucleotide sequence is shown in SEQ ID NO. 57, a PacoA promoter, whose nucleotide sequence is shown in SEQ ID NO. 58, a PcitA promoter, whose nucleotide sequence is shown in SEQ ID NO. 59, a PglaA promoter, whose nucleotide sequence is shown in SEQ ID NO. 60, a PcadA promoter, whose nucleotide sequence is shown in SEQ ID NO. 61, a PmfsA promoter, whose nucleotide sequence is shown in SEQ ID NO. 62, and a Pgas promoter, whose nucleotide sequence is shown in SEQ ID NO. 63.

[0104] The recombinant Aspergillus niger also includes a terminator, which is used to terminate the expression of aconitate isomerase, and the terminator is a TtrpC terminator, and the sequence of the TtrpC terminator is shown in SEQ ID NO. 53. It should be noted that the choice of the terminator here is not limited to this, and those skilled in the art can select other terminators according to routine, such as a Tpgk terminator, and the sequence of the Tpgk terminator is shown in SEQ ID NO. 64.

[0105] Primers were designed and synthesized based on the gene sequence information. The specific primer sequences are shown below.

[0106] Using the genome of Aspergillus niger MEFC1501 as a template, primers PicdAn-F / PicdAn-R were used to amplify the promoter PicdAn (PicdA is a general term for promoters of the same gene, PicdAn is a promoter obtained in Aspergillus niger, and PicdAt is a corresponding promoter obtained in Aspergillus terreus. For other promoters, those skilled in the art can know their expression significance according to common knowledge in the art or the above-mentioned numbering rules) to obtain the PicdAn fragment.

[0107] Similarly, the genome of Aspergillus niger MEFC1501 was used as a template and primers MTSicdAn(PicdAn)-F / MTSicdAn(tbrA)-R were used to amplify the mitochondrial localization signal peptide to obtain the MTSicdAn fragment.

[0108] Using plasmid puc57-tbrA as a template, primers tbrA-F / tbrA-F were used to amplify tbrA to obtain the tbrA fragment.

[0109] Using plasmid pXH2-1 as a template, primers TtrpC-F(TbrA) / TtrpC-R were used to amplify the terminator TtrpC to obtain the TtrpC(tbrA) fragment.

[0110] All PCR products were detected by 0.8% agarose gel electrophoresis and purified by gel excision. The "PicdAn fragment, MTSicdAn fragment, tbrA fragment and TtrpC (tbrA) fragment" were fused by fusion PCR, and the product of the fusion PCR was used as a template and PicdAn-F / TtrpC-R was used as primers to amplify the obtained PicdA-MTSicdA-tbrA-TtrpC gene targeting element, which can be used for subsequent random insertion and overexpression of aconitate isomerase tbrA strain containing mitochondrial localization signal peptide MTSicdAn.

[0111] Plasmid pSGF957 (obtained from Seoul National University, the plasmid is recorded in Kim, JG, Choi, YD, Chang, YJ, Kim, SU, Genetic transformation of Monascus purpureus DSM1379, Biotechnology Letters, 2003, 25, 1509-1514) was used as a template to perform PCR amplification of the PtrpC-hph-TtrpC fragment, where PtrpC is the promoter of the Aspergillus nidulans tryptophan synthase gene, hph is the hygromycin phosphotransferase gene, and TtrpC is the terminator of the Aspergillus nidulans tryptophan synthase. The PCR product was detected by 0.8% agarose gel electrophoresis and purified by gel excision recovery to obtain the hygromycin B-resistant hph gene expression cassette PtrpC-hph-TtrpC, which can be used to randomly insert and overexpress aconitate isomerase tbrA containing the mitochondrial localization signal peptide MTSicdAn to construct a strain.

[0112] 2) Construction of an engineered strain of Aspergillus niger with randomly inserted aconitate isomerase tbrA and overexpressed mitochondrial localization signal peptide MTSicdAn

[0113] The specific operation of transforming the gene into the strain is detailed in Example 1, 3) Construction of an engineered strain of Aspergillus terreus heterologously expressing a fluorescent protein localized to mitochondria.

[0114] Pick the recombinant Aspergillus niger transformants obtained above and inoculate them on PDA-H plates to culture spores, then inoculate the spores into liquid culture medium for culture, collect the mycelium to extract the genome, and perform PCR verification with PicdA-F / TtrpC-R as primers, and use the genome of Aspergillus niger MEFC1501 as a control. The positive transformants can amplify a band of about 3.0 kb, while the control cannot amplify the target band. The transformants with correct genotype verification were transferred to PDA-H plates for subculture, and subcultured 3 times. Then collect the spores separately and make appropriate gradient dilutions with physiological saline, take 100 μL and spread it on the PDA-H plate to make it grow an independent single colony, which is single spore isolation. Take spores from the single colony and perform single spore isolation again, and perform single spore separation and subculture a total of 4 times. The recombinant strain after single spore isolation was genotyped and verified again, and the results are as follows. Figure 5 As shown, a genetically engineered strain with high production of trans-aconitic acid was successfully obtained and marked as An::PicdA-MTSicdA-tbrA.

[0115] 3) Shake flask analysis of fermentation of trans-aconitic acid by an engineered strain of Aspergillus niger overexpressing the mitochondrial-localized aconitate isomerase tbrA

[0116] The An::PicdA-MTSicdA-tbrA engineered strain and the starting strain Aspergillus niger MEFC1501 were selected and inoculated into the Aspergillus niger spore production medium and cultured at 32°C for 6 days to obtain mature spores. Then, the spores cultured to maturity were inoculated into the trans-aconitate fermentation medium IPM, and the inoculation amount was about 5×10 7 spores / mL, and 3 shake flasks were set up as parallel for each transformant, and fermented at 34°C and 220rpm for 170h. The supernatant was filtered through a 0.45μm filter and appropriately diluted, and the organic acid content was detected by high performance liquid chromatography (HPLC), wherein the chromatographic column was Bio-rad Aminex HPX-87H, 300mmx7.8 mm, the mobile phase was 5mM sulfuric acid, the flow rate was 0.5mL / min, the column temperature was 55°C, the inspection temperature was 35°C, and the wavelength of the ultraviolet detector was 210nm.

[0117] The results of trans-aconitic acid production are as follows Figure 6 As shown, Figure 6The white in the middle is cis-aconitic acid, the left oblique pattern is trans-aconitic acid, WT is the control strain MEFC 1501, MTSicdAn-tbrA is the engineered strain of A. niger containing the mitochondrial localization signal peptide MTSicdAn, and the Error bar in the figure represents the SEM of three parallel experiments (significance: *p≤0.05; **p<0.01; ***p<0.001). Figure 6 The results show that the trans-aconitic acid yield (17.19 g / L) of strain An::PicdA-MTSicdA-tbrA is much higher than that of the starting strain MEFC1501 (1.02 g / L), and the yield is increased by 1685%. The inventors also used different transformation methods, such as co-transformation with a screening tag or constructing the expression cassette of the target gene and the expression cassette of the screening tag together for transformation, and obtained similar results. It can be seen that locating the isomerase tbrA to the mitochondria can significantly increase the trans-aconitic acid yield of Aspergillus niger.

[0118] Based on the above embodiments and verification results, it can be seen that the mitochondrial localization signal peptides MTSacoAt, MTScitAt, and MTSicdAn can accurately locate the target protein in the mitochondria, and the mitochondrial localization signal peptides can indeed locate aconitate isomerase in the mitochondria of Aspergillus terreus and Aspergillus niger. Based on the above research results, those skilled in the art can reasonably expect that the strategy of using mitochondrial localization signal peptides to locate aconitate isomerase in the mitochondria can successfully affect the metabolic pathway of trans-aconitate, and significantly improve the trans-aconitate yield and conversion rate of Aspergillus terreus and Aspergillus niger.

[0119] The primers used in the specific implementation mode of this application are listed as follows:

[0120] pXH106-XF:5'-cccggagttaggtagataga-3' (SEQ ID NO.5);

[0121] pXH106-XR:5'-gcatcaaatcgtcgtaccgca-3' (SEQ ID NO.6);

[0122] PgpdAt-F(PyrG):5'-gcggtacgacgatttgatgcttacactctgggaggatccagg-3'(SEQID NO.7);

[0123] PgpdAt-R: 5'-gttgtgatgattgatgagtt-3' (SEQ ID NO. 8);

[0124] PgpdAt-R(MTScitAt):5’-tctgagagaggcagccatgttgtgatgattgatgagttgttgg-3’(SEQ ID NO.9);

[0125] PgpdAt-R(MTSacoAt):5’-gcaaggcgggtggagatcatgttgtgatgattgatgagtt-3’(SEQID NO.10);

[0126] PgpdAt-R(MTSicdAn):5’-ctgaaccggacagacgacatgttgtgatgattgatgagtt-3’(SEQID NO.11);

[0127] MTScitAt-F:5’-atggctgcctctctcagac-3’(SEQ ID NO.12);

[0128] MTScitAt-R:5’-gtagcagcgcaagccattga-3’(SEQ ID NO.13);

[0129] MTSacoAt-F:5’-atgatctccacccgccttg-3’(SEQ ID NO.14);

[0130] MTSacoAt-R:5’-ataggggaatcagcaacggtg-3’(SEQ ID NO.15);

[0131] MTSicdAn-F:5’-atgtcgtctgtccggttcag-3’(SEQ ID NO.16);

[0132] MTSicdAn-R:5’-ggtgcgggattgagtaaggt-3’(SEQ ID NO.17);

[0133] StayGold(PgpdAt)-F:

[0134] 5’-ctcatcaatcatcacaacatggcctccacccccttcaagt-3’(SEQ ID NO.18);

[0135] StayGold(MTScitAt)-F:

[0136] 5'-aatggcttgcgctgctacatggcctccacccccttc-3'(SEQ ID NO.19);

[0137] StayGold(MTSacoAt)-F:

[0138] 5'-accgtgctgattcccctatggcctccacccccttc-3'(SEQ ID NO.20);

[0139] StayGold(MTSicdAn)-F:

[0140] 5'-acctactcaatcccgcaccatggcctccacccccttcaa-3'(SEQ ID NO.21);

[0141] StayGold-R(Ku80-D):

[0142] 5'-attctatctacctaactccgggttacaggtgggcctccagg-3'(SEQ ID NO.22);

[0143] D-ku80-F:5'-gggtttctagaagtcacatcagc-3'(SEQ ID NO.23);

[0144] U-ku80-R:5'-atcaccgaccctacgctgtg-3'(SEQ ID NO.24);

[0145] YZ-ku80-F:5'-atataccactccacactaaccg-3'(SEQ ID NO.25);

[0146] YZ-ku80-F:5'-gccatagagttgctcttgatc-3'(SEQ ID NO.26);

[0147] U-cadA-F:5'-gcgataaatgttgaacgaggc-3'(SEQ ID NO.29);

[0148] U-cadA-R(MTScitAt):

[0149] 5'-agtctgagaggcagccatggtcaatttaataggacaatttc-3'(SEQ ID NO.30); <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> U-cadA-R(MTSacoAt):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> 5'-aggcgggtggagatcatggtcaatttaataggacaattttc-3'(SEQ ID NO.31);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0152] <h2 style=";text-align:left;direction:ltr"> MTScitAt-F:5'-atggctgcctctctcagact-3'(SEQ ID NO.32);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0153] <h2 style=";text-align:left;direction:ltr"> MTScitAt-R:5'-gtagcagcgcaagccattga-3'(SEQ ID NO.33);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0154] <h2 style=";text-align:left;direction:ltr"> MTSacoAt-F:5'-atgatctccacccgccttg-3'(SEQ ID NO.34);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0155] <h2 style=";text-align:left;direction:ltr"> MTSacoAt-R:5'-ataggggaatcagcaacggtg-3'(SEQ ID NO.35);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0156] <h2 style=";text-align:left;direction:ltr"> adi1-F(MTScitAt):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0157] <h2 style=";text-align:left;direction:ltr"> 5'-ttcaatggcttgcgctgctacatgctgcaccccatcgacac-3'(SEQ ID NO.36);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0158] <h2 style=";text-align:left;direction:ltr"> adi1-F(MTSacoAt):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0159] <h2 style=";text-align:left;direction:ltr"> 5'-gccaccgttgctgattcccctatgctgcaccccatcgacac-3'(SEQ ID NO.37);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0160] <h2 style=";text-align:left;direction:ltr"> adi1-R:5'-ttaggacaggctacggtcgctag-3'(SEQ ID NO.38);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0161] <h2 style=";text-align:left;direction:ltr"> tbrA-F(MTScitAt):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0162] <h2 style=";text-align:left;direction:ltr"> 5'-caatggcttgcgctgctacatgaagatcccctgcttcgtc-3'(SEQ ID NO.39);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0163] <h2 style=";text-align:left;direction:ltr"> tbrA-F(MTSacoAt):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0164] <h2 style=";text-align:left;direction:ltr">5'-aattgtcctattaaattgaccatgatctccacccgccttg-3'(SEQ ID NO.40);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0165] <h2 style=";text-align:left;direction:ltr"> tbrA-R:5'-ttaggggatgatcagctcgc-3'(SEQ ID NO.41);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0166] <h2 style=";text-align:left;direction:ltr"> TtrpC-F(adi1):5'-gcgaccgtagcctgtcctaaggatccacttaacgttactg-3'(SEQ IDNO.42);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0167] <h2 style=";text-align:left;direction:ltr"> TtrpC-F(tbrA):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0168] <h2 style=";text-align:left;direction:ltr"> 5'-gcgagctgatcatcccctaaggatccacttaacgttactg-3'(SEQ ID NO.43);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0169] <h2 style=";text-align:left;direction:ltr"> TtrpC-R:5'-aagaaggttacctctctaaacaag-3'(SEQ ID NO.44);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0170] <h2 style=";text-align:left;direction:ltr"> pyrG-F(TtrpC):5'-cttgtttagaggtaaccttctttaagggagatggtgattgaactag-3'(SEQ ID NO.45);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0171] <h2 style=";text-align:left;direction:ltr"> pyrG-R:5'-gcatcaaatcgtcgtaccgca-3'(SEQ ID NO.46);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0172] <h2 style=";text-align:left;direction:ltr"> D-cadA-F(pyrG):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0173] <h2 style=";text-align:left;direction:ltr"> 5'-tgcggtacgacgatttgatgcatgggaagcgatatggaaac-3'(SEQ ID NO.47);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0174] <h2 style=";text-align:left;direction:ltr"> D-cadA-R:5'-cattgcagggaagtatatgcttc-3'(SEQ ID NO.48);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0175] <h2 style=";text-align:left;direction:ltr"> C-cadA-F:5'-tgtggttcctaccaaggtggc-3'(SEQ ID NO.49);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0176] <h2 style=";text-align:left;direction:ltr"> C-cadA-R:5'-cgactatagctggattgatcac-3'(SEQ ID NO.50);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0177] <h2 style=";text-align:left;direction:ltr"> PicdAn-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0178] <h2 style=";text-align:left;direction:ltr"> 5'-CTTTAACGTTGCAGATACAGGGATGCG-3'(SEQ ID NO.65);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0179] <h2 style=";text-align:left;direction:ltr"> PicdAn-R:5'-TTCGACAGTGAAGCGTGGCC-3'(SEQ ID NO.66);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0180] <h2 style=";text-align:left;direction:ltr"> MTSicdAn(PicdAn)-F:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0181] <h2 style=";text-align:left;direction:ltr"> 5'-GGCCACGCTTCACTGTCGAAATGTCGTCTTGTCCGGTTCAG-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0182] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO.67);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0183] <h2 style=";text-align:left;direction:ltr"> MTSicdAn(tbrA)-R:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0184] <h2 style=";text-align:left;direction:ltr"> 5'-gacgaagcaggggatcttcatGGTGCGGGATTGAGGAAGGTTC-3'(SEQ ID NO.68);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0185] <h2 style=";text-align:left;direction:ltr"> tbrA-F:5'-atgaagatcccctgcttcgtc-3'(SEQ ID NO.69);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0186] <h2 style=";text-align:left;direction:ltr"> tbrA-R:5'-ttaggggatgatcagctcgcc-3'(SEQ ID NO.70);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0187] <h2 style=";text-align:left;direction:ltr"> TtrpC-F(TbrA):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0188] <h2 style=";text-align:left;direction:ltr"> 5'-ggcgagctgatcatcccctaaggatccacttaacgttactgaaatcatc-3'(SEQ IDNO.71);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0189] <h2 style=";text-align:left;direction:ltr"> TtrpC-R:5'-GAATCAGAGCTCCATCATGTCTTG-3'(SEQ ID NO.72);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0190] <h2 style=";text-align:left;direction:ltr"> hph-F:5'-ttcgggatcgcaagcgtaaag-3'(SEQ ID NO.73);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0191] <h2 style=";text-align:left;direction:ltr"> hph-R:5'-caattatctttgcgaacccagg-3'(SEQ ID NO.74)。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0192] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a high-yield trans-aconitic acid strain, characterized in that: The construction method includes the step of using gene editing technology to target aconitate isomerase to the mitochondria.

2. The construction method according to claim 1, characterized in that: The construction method enables the strain to express aconitate isomerase with a mitochondrial localization signal peptide fused to the N-terminus.

3. The construction method according to claim 2, characterized in that: The amino acid sequence of the mitochondrial localization signal peptide contains the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; and / or, The aconitate isomerase is Adi1 or TbrA.

4. An engineered bacterium for high production of trans-aconitic acid, characterized in that: The engineered bacteria express aconitate isomerase with a mitochondrial localization signal peptide fused to the N-terminus.

5. The engineered bacteria according to claim 4, characterized in that The amino acid sequence of the mitochondrial localization signal peptide contains the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; and / or, The aconitate isomerase is Adi1 or TbrA.

6. The engineered bacteria according to claim 4, characterized in that The starting strain of the engineering bacteria is Aspergillus; Preferably, the Aspergillus is selected from Aspergillus terreus or Aspergillus niger.

7. The engineered bacteria according to claim 6, characterized in that When the starting strain is Aspergillus terreus, the aconitate decarboxylase cadA gene is also knocked out.

8. An engineered bacterium obtained by the construction method according to any one of claims 1 to 3.

9. Use of the engineered bacteria according to any one of claims 4 to 7 or the engineered bacteria according to claim 8 in the production of trans-aconitic acid, products containing trans-aconitic acid, or products containing a trans-aconitic acid structure.

10. Biomaterial, characterized in that The biological material is any one of A) to C): A) a mitochondrial localization signal peptide, the amino acid sequence of which is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; B) an aconitate isomerase gene expression element with a mitochondrial localization signal peptide fused to the N-terminus, the gene expression element comprising a mitochondrial localization signal peptide sequence and an aconitate isomerase sequence, the mitochondrial localization signal peptide sequence is selected from the mitochondrial localization signal peptide described in A), and the aconitate isomerase is adi1 or tbrA; C) a gene expression cassette, wherein the gene expression cassette comprises, from 5' to 3', an upstream homology arm of the cadA gene, B) the aconitate isomerase gene expression element with a mitochondrial localization signal peptide fused to the N-terminus, a terminator, and a downstream homology arm of the cadA gene; or, The gene expression cassette contains a promoter, the aconitate isomerase gene expression element with the N-terminal fused mitochondrial localization signal peptide as described in B) and a terminator in sequence from 5' to 3'.

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