Recombinant strain for synthesizing homogentisic acid and construction method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and relates to a recombinant Yarrowia lipolytica for synthesizing homogentisic acid as well as a construction method and application thereof. The recombinant strain is obtained by genetic modification of a starting strain. Compared with the starting strain, the activity of phenylpyruvate decarboxylase ARO10 of the recombinant strain is weakened or inactivated. The construction method comprises: genetic modification of the starting strain to weaken or inactivate the activity of phenylpyruvate decarboxylase of the starting strain. The method for fermenting and synthesizing homogentisic acid comprises: inoculating the aforementioned recombinant strain into a fermentation medium added with L-tyrosine to perform fermentation; or, constructing the recombinant strain according to the aforementioned method, and inoculating the obtained recombinant strain into a fermentation medium added with L-tyrosine to perform fermentation. More phenylpyruvic acid in the recombinant strain flows to the homogentisic acid synthesis pathway, and the yield of homogentisic acid can be effectively improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a recombinant Yarrowia lipolytica for synthesizing high gentisic acid, a construction method and application thereof. BACKGROUND

[0002] Melanin is a brown or black chemical substance with physiological activity. Its unique chemical composition and physical and chemical properties endow melanin with various functions. The cell can secrete melanin to protect the cell from various environmental stresses: melanin can absorb ultraviolet rays, X-rays and gamma rays, and scavenge reactive oxygen species (ROS) and free radicals; meanwhile, melanin acts as an ion exchanger in the cell and plays an important role in the cell. Studies have shown that the synthesis of melanin in Yarrowia lipolytica helps to isolate metal ions and improve the tolerance of the strain to copper ions. Considering the excellent ultraviolet resistance and antioxidant function of melanin, melanin is often used in medical and cosmetic products; the excellent ion exchange capacity of melanin makes melanin be used to synthesize silver nanoparticles, which are extremely important coating additives due to their antifungal properties. In addition, melanin can also repair soil contaminated by metals. Although melanin has powerful functions and wide application fields, it is hoped that its yield can be further improved through synthetic biology means. High gentisic acid is one of the metabolic products of microorganisms, and high gentisic acid can be rapidly converted and polymerized into melanin under the participation of enzymes or oxides after being transported outside the cell. Increasing the yield of high gentisic acid in microorganisms can greatly increase the market supply of melanin.

[0003] The abundant acetyl-CoA and malonyl-CoA precursors in Yarrowia lipolytica can be used to synthesize plant secondary metabolites, including flavonoids, polyketides, polyunsaturated fatty acids, and isoprenaline. A large number of gene editing techniques (Gateway cloning, genome integration, CRISPR-9 / Cpf1 genome editing, transposon, auxotrophic marker, resistance screening marker and promoter library) greatly facilitate the metabolic engineering of Yarrowia lipolytica. Compared with Saccharomyces cerevisiae, Yarrowia lipolytica does not flow to ethanol production under high-sugar conditions, and is more suitable for high-density fermentation and process-controllable industrial production. At present, Yarrowia lipolytica has been widely used in the production of aromatic compounds, such as resveratrol and 2-phenylethanol. In addition, Yarrowia lipolytica is a natural melanin-producing strain, which can catalyze the synthesis of L-tyrosine to phenylpyruvic acid, and then obtain high gentisic acid under the catalysis of phenyl-4-hydroxyphenylpyruvic acid dioxygenase HPD. High gentisic acid can be oxidized and polymerized outside the cell to obtain melanin.

[0004] However, the ability of microorganisms including Yarrowia lipolytica to accumulate homogentisic acid still needs to be further improved for producing homogentisic acid products, so as to reduce the production cost of homogentisic acid products. ARO10 encodes phenylpyruvate decarboxylase, which can catalyze the reaction of phenylpyruvate to phenylacetaldehyde, and disperse the flow of phenylpyruvate to the phenylethanol synthesis pathway, thereby reducing the yield of homogentisic acid. By knocking out ARO10, the bypass phenylethanol synthesis pathway is knocked out, so that more phenylpyruvate flows to the homogentisic acid synthesis, thereby realizing the improvement of the yield of homogentisic acid. SUMMARY

[0005] The present application aims to overcome the problem that the ability of microorganisms to accumulate homogentisic acid still needs to be improved for producing homogentisic acid in the prior art, and provides a recombinant strain for synthesizing homogentisic acid from L-tyrosine as a precursor, a construction method thereof and a method for fermenting and synthesizing homogentisic acid from L-tyrosine as a precursor. In the recombinant strain, more phenylpyruvate flows to the homogentisic acid synthesis pathway, which can effectively improve the yield of homogentisic acid.

[0006] Based on the above purpose, the present application provides a recombinant strain for synthesizing homogentisic acid, which is obtained by knocking out the phenylpyruvate decarboxylase gene of a starting strain with a hygr gene of a hygromycin resistance gene. Compared with the starting strain, the activity of phenylpyruvate decarboxylase of the recombinant strain is weakened or inactivated.

[0007] Further, the amino acid sequence of the substituted phenylpyruvate decarboxylase gene of the starting strain is shown in SEQ ID NO. 1.

[0008] Further, the nucleotide sequence of the substituted phenylpyruvate decarboxylase gene of the starting strain is shown in SEQ ID NO. 2.

[0009] The starting strain of the present application is Yarrowia lipolytica in which the gene Ku70 is knocked out and the 4-hydroxyphenylpyruvate dioxygenase encoding gene ylHPD is overexpressed. The nucleotide sequence of the gene Ku70 is shown in SEQ ID NO. 3, and the encoding gene ylHPD is shown in SEQ ID NO. 4.

[0010] Before knocking out the phenylpyruvate decarboxylase gene of the starting strain with the hygr gene of the hygromycin resistance gene, a recombinant vector containing the hygr gene of the hygromycin resistance gene needs to be constructed, and then the hygr gene of the recombinant vector is used to replace the phenylpyruvate decarboxylase gene in the starting strain through homologous recombination.

[0011] Further, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO. 5, and the nucleotide sequence of the hygr gene of the hygromycin resistance gene is shown in SEQ ID NO. 6.

[0012] The application further provides application of the recombinant strain in synthesis of homogentisic acid.

[0013] Specifically, the method for synthesizing homogentisic acid by using the recombinant strain comprises inoculating the recombinant strain into a fermentation medium added with L-tyrosine for fermentation culture to obtain a fermentation liquor containing homogentisic acid.

[0014] Further, the fermentation culture conditions comprise that the inoculation amount of the recombinant strain is 0.5-2% of the volume of the fermentation medium, the culture temperature is 25-35 DEG C, the rotation speed is 180-250 rpm, and the culture time is 80-160 h.

[0015] Further, the fermentation medium contains at least one of carbon source, nitrogen source, sulfate, adenine and precursor L-tyrosine.

[0016] Further, the fermentation medium contains carbon source, nitrogen source, sulfate, L-tyrosine, adenine, L-arginine, L-aspartate, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan and L-valine.

[0017] Most preferably, the fermentation medium contains glucose 30-50 g / L, ammonium sulfate 0.8-1.3 g / L, YNB (amino-free yeast nitrogen base) 1.5-2 g / L, precursor L-tyrosine 8 g / L, adenine 0.01-0.02 g / L, L-arginine 0.02-0.08 g / L, L-aspartate 0.05-1 g / L, L-histidine 0.01-0.03 g / L, L-isoleucine 0.02-0.08 g / L, L-lysine 0.02-0.08 g / L, L-methionine 0.01-0.03 g / L, L-phenylalanine 0.02-0.08 g / L, L-threonine 0.05-0.15 g / L, L-tryptophan 0.02-0.08 g / L, and L-valine 0.05-0.25 g / L.

[0018] Compared with the prior art, the application has the following beneficial effects or advantages:

[0019] (1) The recombinant strain provided by the application knocks out phenylacetaldehyde decarboxylase gene, knocks out phenylethanol pathway, reduces the flux from phenylpyruvic acid to phenylethanol, makes more phenylpyruvic acid flow to homogentisic acid synthesis pathway, promotes the synthesis and accumulation of microbial homogentisic acid, effectively improves the yield of homogentisic acid, lays a foundation for metabolic engineering of efficient production of homogentisic acid, and provides a more optimal potential selection for microbial fermentation production of homogentisic acid.

[0020] (2) The application uses Yarrowia lipolytica with Ku70 knocked out and ylHPD overexpressed as a starting strain, and the concentration of homogentisic acid in the fermentation liquor of the recombinant Yarrowia lipolytica strain can reach 2.59 g / L, and the accumulation amount of homogentisic acid is increased by 331.66% compared with the starting strain. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a structural schematic diagram of the recombinant plasmid pYLXP-hygr-loxP-ARO10 in Example 1.

[0022] Figure 2 is a column chart of the yield of homogentisic acid synthesized by L-tyrosine as a precursor in Example 4 and Comparative Example 1. DETAILED DESCRIPTION

[0023] The specific embodiments of the application are described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and explanatory in nature and are not intended to limit the application.

[0024] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are understood to be open-ended. Each range endpoint is thus provided as a separate point value, and each point value is provided as a separate range endpoint. The disclosure is therefore understood to encompass all values and ranges between the low and high values of the ranges and the point values.

[0025] The terms "increase" or "enhance" are used herein generally to mean a statistically significant increase. However, for the avoidance of doubt, the terms "increase" or "enhance" mean an increase of at least 5%, for example, an increase of at least about 10%, or more; the terms "weaken" or "inactivate" mean a decrease in the ability of an enzyme to catalyze a reaction of at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%, or a complete loss of the ability to catalyze a reaction.

[0026] The first aspect of the application provides a recombinant strain for synthesizing homogentisic acid from L-tyrosine as a substrate, which is obtained by genetic modification of a starting strain, and the activity of phenylpyruvate decarboxylase in the recombinant strain is weakened or inactivated compared with the starting strain.

[0027] According to the present application, the starting strain can be any strain capable of synthesizing homogentisic acid from L-tyrosine. Preferably, the starting strain is Yarrowia lipolytica. More preferably, the starting strain is Yarrowia lipolytica in which the gene Ku70 is knocked out and ylHPD is overexpressed, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO. 3. The Yarrowia lipolytica in which the gene Ku70 is knocked out can be prepared by referring to the method disclosed in the prior art, for example, see the document Gao S, Tong Y, Zhu L, et al. Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production [J]. Metabolic Engineering, 2017: 192. Based on the original strain MYA2613 (purchased from the American Type Culture Collection, ATCC), after knocking out the gene Ku70 responsible for non-homologous recombination, and introducing the expression plasmid pYLXP'-ylHPD, Yarrowia lipolytica Po1f Δku70 ylHPD is obtained as the starting strain in the present application.

[0028] According to the present application, in order to improve the yield of homogentisic acid synthesized by the recombinant strain from L-tyrosine, preferably, at least part of the phenylpyruvate decarboxylase gene in the recombinant strain is knocked out. More preferably, the phenylpyruvate decarboxylase gene knocked out encodes an amino acid sequence shown in SEQ ID NO. 1. Further preferably, the nucleotide sequence of the phenylpyruvate decarboxylase gene (ARO10 gene) knocked out is shown in SEQ ID NO. 2, which can be specifically referred to Gene ID: 2910787 in the GeneBank database of NCBI. The inventors of the present application found that, starting from the Yarrowia lipolytica Po1f Δku70 ylHPD strain, knocking out the phenylpyruvate decarboxylase gene with the nucleotide sequence shown in SEQ ID NO. 2 can more significantly improve the yield of homogentisic acid in Yarrowia lipolytica.

[0029] In the present application, the knock-out refers to a technology of site-specific integration of an exogenous gene into a certain site on the genome of a target cell by homologous recombination, so as to achieve the purpose of site-specific modification of a certain gene on the chromosome. Further preferably, at least part of the phenylpyruvate decarboxylase gene in the recombinant strain is knocked out, so that the knocked-out phenylpyruvate decarboxylase gene in the recombinant strain is replaced by the gene of the hygromycin resistance gene hygr.

[0030] The second aspect of the present application provides a method for constructing a recombinant strain for synthesizing homogentisic acid, which comprises: genetically modifying a starting strain so that the activity of phenylpyruvate decarboxylase of the starting strain is weakened or inactivated.

[0031] According to the present application, preferably, the starting strain is Yarrowia lipolytica. More preferably, the starting strain is Yarrowia lipolytica with the gene Ku70 knocked out and the coding gene ylHPD of 4-hydroxyphenylpyruvate dioxygenase overexpressed, wherein the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO. 3, and the coding gene ylHPD is shown in SEQ ID NO. 4.

[0032] According to the present application, preferably, the activity of phenylpyruvate decarboxylase of the starting strain is weakened or inactivated by gene knockout. More preferably, the amino acid sequence encoded by the knocked-out phenylpyruvate decarboxylase gene is shown in SEQ ID NO. 1. Further preferably, the nucleotide sequence of the knocked-out phenylpyruvate decarboxylase gene is shown in SEQ ID NO. 2.

[0033] In the present application, the specific open reading frame sequence or promoter sequence in the genome of Yarrowia lipolytica is preferably knocked out by gene knockout.

[0034] In the present application, the homologous sequence fragment used for the gene knockout can be obtained by: artificially synthesizing the upstream and downstream fragment sequences of the target gene (for example, the phenylpyruvate decarboxylase gene Gene ID: 2910787) in Yarrowia lipolytica according to the sequences disclosed in the database (for example, GenBank database, https: / / www.ncbi.nlm.nih.gov / genbank / ) known in the art as the homologous arms; or amplifying the upstream and downstream fragment sequences of the target gene from the genome of the starting strain (for example, Yarrowia lipolytica) as the homologous arms by the method of PCR, so as to obtain the initial homologous sequence fragment of the target gene, but the present application is not limited thereto. Part or all of the initial homologous sequence of the target gene refers to the sequence containing the above-mentioned target gene.

[0035] In the present application, a recombinant vector can be first constructed, which can knock out the phenylpyruvate decarboxylase gene of the starting strain (e.g., Yarrowia lipolytica). Various methods for constructing a recombinant vector are known in the art to link a target gene fragment to a vector to prepare a gene knockout recombinant vector, such as but not limited to, a classic "restriction-ligation" method, a Gateway cloning system developed by Invitrogen, a Creator cloning system developed by Clon tech, a Univector cloning system developed by Stephen Elledge laboratory, and a Golden Gate cloning method based on type II restriction enzymes.

[0036] For example, the recombinant vector of the present application can be constructed by a recombinase method: based on the genome of the starting strain (e.g., Yarrowia lipolytica), the upstream and downstream homologous arm sequences of the target insertion site are amplified by PCR; the target gene sequence, the upstream and downstream homologous arm sequences, and the resistance gene expression cassette are connected in series to obtain the recombinant vector, but the present application is not limited thereto.

[0037] Subsequently, the recombinant vector can be introduced into the starting strain (e.g., Yarrowia lipolytica) by conventional methods in the art, such as but not limited to microinjection, gene gun, transformation (e.g., electroporation), infection, or transfection. The above-mentioned microinjection, gene gun, transformation, infection, or transfection are all conventional operations in the art. For example, transformation refers to treating cells by using some known methods in molecular biology and genetic engineering, so that the treated cells are in a competent state, and thus come into contact with foreign DNA, so that the foreign DNA enters the cells in a competent state. Commonly used transformation methods include protoplast transformation, chemical transformation, and electroporation transformation. Infection refers to using artificially modified bacteriophage live viruses as carriers, recombining the carrier with the target DNA sequence, and then using the coat protein of the bacteriophage or virus to package the recombinant DNA into a live bacteriophage or virus in vitro, so that the recombinant DNA enters the host cell in an infected manner. Transfection refers to treating cells into a competent state by CaCl2, electroporation, etc., and then making the competent cells receive the recombinant bacteriophage DNA.

[0038] After the recombinant vector is introduced into the starting strain (e.g., Yarrowia lipolytica), positive clones can be screened by a screening marker (e.g., a resistance gene), and verified by genome PCR or by sequencing the genomic DNA, so as to obtain the recombinant strain for synthesizing homogentisic acid with L-tyrosine as a precursor.

[0039] According to the present application, preferably, the gene knockout process comprises: first constructing a recombinant vector containing a hygromycin resistance gene hygr, and then replacing the phenylpyruvate decarboxylase gene in the original strain with the hygromycin resistance gene hygr in the recombinant vector through homologous recombination. More preferably, the recombinant vector is pYLXP-hygr-loxP-ARO10, the nucleotide sequence of which is shown in SEQ ID NO. 5, and the nucleotide sequence of the hygromycin resistance gene hygr is shown in SEQ ID NO. 6.

[0040] The third aspect of the present application provides the use of the aforementioned recombinant strain or the aforementioned method in the synthesis of homogentisic acid with L-tyrosine as a precursor.

[0041] The fourth aspect of the present application provides a method for fermentatively synthesizing homogentisic acid with L-tyrosine as a precursor, which comprises: inoculating the aforementioned recombinant strain into a fermentation medium added with L-tyrosine to perform fermentation.

[0042] Alternatively, a recombinant strain is constructed according to the aforementioned method, and the obtained recombinant strain is inoculated into a fermentation medium added with L-tyrosine to perform fermentation.

[0043] According to the present application, preferably, the recombinant strain is first prepared into a seed liquid, and then the seed liquid is inoculated into a fermentation medium added with L-tyrosine to perform fermentation, so as to obtain a fermentation liquid.

[0044] According to the present application, preferably, the preparation method of the seed liquid comprises: picking a single colony of the recombinant strain and inoculating it into a seed culture medium to perform seed culture, so as to obtain the seed liquid.

[0045] The method for seed culture is not particularly limited in the present application, as long as the method can activate and proliferate the recombinant strain. Preferably, the inoculation amount of the recombinant strain in the seed culture medium is 0.5-2% by volume; if the recombinant strain preserved in a glycerol tube is inoculated, the strain in each glycerol tube is inoculated into 100 mL of the seed culture medium. The temperature, pH, rotation speed, time and other parameters used in the seed culture can be the conventional settings in the art. Preferably, the conditions for the seed culture comprise: a temperature of 25-35°C, a rotation speed of 180-250 rpm, and a time of 40-60 h.

[0046] In the present application, the single colony of the recombinant strain can be selected from the freshly prepared recombinant strain or the low-temperature preserved recombinant strain (for example, the recombinant strain for synthesizing homogentisic acid with L-tyrosine as a precursor, which is preserved in a glycerol preservation tube in a -80°C refrigerator).

[0047] In the present application, the seed culture medium is not particularly limited and can be a seed culture medium commonly used in the art. Preferably, when the starting strain is Yarrowia lipolytica, the seed culture medium contains a carbon source, a nitrogen source, a sulfate, adenine and at least one amino acid. More preferably, the seed culture medium contains a carbon source, a nitrogen source, a sulfate, adenine, L-arginine, L-aspartate, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan and L-valine.

[0048] Further preferably, the seed culture medium contains 15-25 g / L of glucose, 4-6 g / L of ammonium sulfate, 1.5-2 g / L of yeast nitrogen base (YNB) without amino acids, 0.01-0.02 g / L of adenine, 0.02-0.08 g / L of L-arginine, 0.05-1 g / L of L-aspartate, 0.01-0.03 g / L of L-histidine, 0.02-0.08 g / L of L-isoleucine, 0.02-0.08 g / L of L-lysine, 0.01-0.03 g / L of L-methionine, 0.02-0.08 g / L of L-phenylalanine, 0.05-0.15 g / L of L-threonine, 0.02-0.08 g / L of L-tryptophan and 0.05-0.25 g / L of L-valine.

[0049] In the present application, the method of fermentation is not particularly limited and can be a method of fermenting to synthesize homogentisic acid commonly used in the art, for example, the seed liquid is inoculated into the fermentation medium containing L-tyrosine (e.g. a shake flask or a fermenter containing the fermentation medium) to perform fermentation culture to obtain a fermentation broth.

[0050] In the present application, in order to improve the yield of homogentisic acid, preferably, the inoculation amount of the seed liquid is 0.5-2 parts by volume relative to 100 parts by volume of the fermentation medium.

[0051] In the present application, in order to improve the yield of homogentisic acid, preferably, the fermentation conditions include a temperature of 25-35°C, a rotation speed of 180-250 rpm and a time of 80-160 h.

[0052] According to the present application, preferably, the fermentation medium contains a carbon source, a nitrogen source, a sulfate, adenine, L-tyrosine and at least one amino acid.

[0053] More preferably, the fermentation medium contains a carbon source, a nitrogen source, a sulfate, L-tyrosine, adenine, L-arginine, L-aspartate, L-histidine, L-isoleucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan and L-valine.

[0054] Further preferably, the fermentation medium contains: glucose 30-50 g / L, ammonium sulfate 0.8-1.3 g / L, YNB (amino acid-free yeast nitrogen base) 1.5-2 g / L, L-tyrosine 8 g / L, adenine 0.01-0.02 g / L, L-arginine 0.02-0.08 g / L, L-aspartate 0.05-1 g / L, L-histidine 0.01-0.03 g / L, L-isoleucine 0.02-0.08 g / L, L-lysine 0.02-0.08 g / L, L-methionine 0.01-0.03 g / L, L-phenylalanine 0.02-0.08 g / L, L-threonine 0.05-0.15 g / L, L-tryptophan 0.02-0.08 g / L, L-valine 0.05-0.25 g / L.

[0055] In the present application, the homogentisic acid in the obtained fermentation liquor can be isolated by known methods. For example, the fermentation liquor is first subjected to solid-liquid separation to obtain the homogentisic acid in the fermentation liquor.

[0056] In the present application, the homogentisic acid in the fermentation liquor or the homogentisic acid isolated from the fermentation liquor can also be detected by known methods. For example, the content of homogentisic acid can be detected by high-performance liquid chromatography and the like.

[0057] According to a particularly preferred embodiment of the present application, the method for fermenting and synthesizing homogentisic acid by using L-tyrosine as a precursor by a recombinant strain comprises the following steps: picking a single colony of the recombinant strain and inoculating it into a seed culture medium, and then performing seed culture at a temperature of 25-35°C and a rotation speed of 180-250 rpm for 40-60 h to obtain a seed liquor; inoculating the seed liquor into the fermentation medium with L-tyrosine added thereto at a volume ratio of 0.5-2% (v / v), and then performing fermentation culture at a temperature of 25-35°C and a rotation speed of 180-250 rpm for 80-160 h to obtain a fermentation liquor; and subjecting the fermentation liquor to solid-liquid separation to obtain the homogentisic acid in the fermentation liquor.

[0058] The seed culture medium contains glucose 15-25 g / L, ammonium sulfate 4-6 g / L, amino acid-free yeast nitrogen base (YNB) 1.5-2 g / L, adenine 0.01-0.02 g / L, L-arginine 0.02-0.08 g / L, L-aspartate 0.05-1 g / L, L-histidine 0.01-0.03 g / L, L-isoleucine 0.02-0.08 g / L, L-lysine 0.02-0.08 g / L, L-methionine 0.01-0.03 g / L, L-phenylalanine 0.02-0.08 g / L, L-threonine 0.05-0.15 g / L, L-tryptophan 0.02-0.08 g / L, and L-valine 0.05-0.25 g / L.

[0059] The fermentation medium contains glucose 30-50 g / L, ammonium sulfate 0.8-1.3 g / L, YNB (yeast nitrogen base without amino) 1.5-2 g / L, L-tyrosine 8 g / L, adenine 0.01-0.02 g / L, L-arginine 0.02-0.08 g / L, L-aspartate 0.05-1 g / L, L-histidine 0.01-0.03 g / L, L-isoleucine 0.02-0.08 g / L, L-lysine 0.02-0.08 g / L, L-methionine 0.01-0.03 g / L, L-phenylalanine 0.02-0.08 g / L, L-threonine 0.05-0.15 g / L, L-tryptophan 0.02-0.08 g / L, L-valine 0.05-0.25 g / L.

[0060] The present application will be described in detail by way of examples below. However, the examples are only used to illustrate the present application and not intended to limit the scope of the present application. In the following examples, unless otherwise specified, the experimental methods used are conventional methods known to those skilled in the art.

[0061] In the following examples, Yarrowia lipolytica po1f is a derivative strain of Yarrowia lipolytica ATCC20460, which is directly provided by Professor Peng Xu of the University of Maryland and purchased from Yeastern Biotech Company in Taiwan, China; the plasmids pYLXP'-URA-loxP, pYLXP' are constructed by ourselves; unless otherwise specified, the reagents and media used are commercially available products, and the methods used are conventional methods.

[0062] 1. Medium and reagents

[0063] Formulation of hygromycin resistance plate: hygromycin 50 ug / mL, yeast extract 10 g / L, proteose peptone 20 g / L, glucose 20 g / L, agar 20 g / L;

[0064] Uracil-deficient plate: glucose 20 g / L, ammonium sulfate 5 g / L, YNB (yeast nitrogen base without amino) 1.7 g / L, adenine hemisulfate 10 mg / L, L-arginine 0.05 g / L, L-aspartate 0.08 g / L, L-histidine 0.02 g / L, L-isoleucine 0.05 g / L, L-lysine 0.05 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-threonine 0.1 g / L, L-tryptophan 0.05 g / L, L-tyrosine 0.05 g / L, L-valine 0.14 g / L, L-leucine 0.05 g / L, agar 20 g / L;

[0065] Leucine-deficient plate: glucose 20 g / L, ammonium sulfate 5 g / L, amino acid-free yeast nitrogen base (YNB) 1.7 g / L, adenine hemisulfate 10 mg / L, L-arginine 0.05 g / L, L-aspartic acid 0.08 g / L, L-histidine 0.02 g / L, L-isoleucine 0.05 g / L, L-lysine 0.05 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-threonine 0.1 g / L, L-tryptophan 0.05 g / L, L-tyrosine 0.05 g / L, L-valine 0.14 g / L, agar 20 g / L;

[0066] YPD medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L;

[0067] Fermentation medium: glucose 40 g / L, YNB (amino acid-free yeast nitrogen base) 1.7 g / L, ammonium sulfate 1.1 g / L, L-tyrosine 8 g / L, adenine 0.01 g / L, L-arginine 0.05 g / L, L-aspartic acid 0.08 g / L, L-histidine 0.02 g / L, L-isoleucine 0.05 g / L, L-lysine 0.05 g / L, L-methionine 0.02 g / L, L-phenylalanine 0.05 g / L, L-threonine 0.1 g / L, L-tryptophan 0.05 g / L, L-valine 0.14 g / L.

[0068] 2. Detection method

[0069] (1) High gentisic acid determination method: centrifuge the fermentation supernatant, dilute 10 times, and perform high performance liquid chromatography analysis;

[0070] High performance liquid chromatography (HPLC) detection parameters: Agilent 1200, VWD detector, ZORBAX Eclipse Plus C18 chromatographic column (4.6x100 mm, 3.5 μm, Agilent), mobile phase: 20% (v / v) methanol aqueous solution, flow rate: 0.6 mL / min, detection spectrum: 225 nm, column temperature: 35 °C, sample injection volume: 10 μL, retention time of high gentisic acid: about 4.501 min.

[0071] Preparation Example 1

[0072] pYLXP'-URA-loxP-ku70 knockout plasmid and Ku70 gene knockout strain Yarrowia lipolytica Po1fΔku70 construction.

[0073] (1) using AvrII and SalI to linearize plasmid pYLXP'-URA-loxP (nucleotide sequence as shown in SEQ ID NO. 7, containing URA gene with nucleotide sequence as shown in SEQ ID NO. 15), then using primers Ku70_Up_F / Ku70_Up_R (nucleotide sequences as shown in SEQ ID NO. 8 and SEQ ID NO. 9) and Ku70_Dw_F / Ku70_Dw_R (nucleotide sequences as shown in SEQ ID NO. 10 and SEQ ID NO. 11) to amplify Ku70 Upstream Arm Ku70_Up and Downstream Arm Ku70_Dw (nucleotide sequences as shown in SEQ ID NO. 12 and SEQ ID NO. 13, each 1000 bp) from Yarrowia lipolytica genome template, respectively, and then using Gibson assembly to assemble linearized pYLXP'-URA-loxP, Ku70_Up and Ku70_Dw to obtain recombinant plasmid pYLXP'-URA-loxP-ku70 (nucleotide sequence as shown in SEQ ID NO. 14), which is sequenced by Shanghai Biosciences to verify the correctness before being used for gene knockout;

[0074] (2) using Yarrowia lipolytica po1f as the original strain, culturing the po1f strain in 2 mL of YPD medium to the exponential growth phase (16-24 h), collecting po1f cells in 1 mL of fermentation broth, centrifuging and discarding the supernatant, adding 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2M), 5 μL of single-stranded DNA (salmon sperm) and 5 μL of recombinant plasmid pYLXP'-URA-loxP-ku70 obtained in step (1), mixing, incubating at 39°C for 1 h, then plating on uracil-deficient plates, and screening to obtain a recombinant strain in which the Ku70 gene (nucleotide sequence as shown in SEQ ID NO. 3) is replaced by the uracil selection marker URA (nucleotide sequence as shown in SEQ ID NO. 15);

[0075] (3) Pick single colony on the uracil-deficient plate in step (2) for colony PCR verification, use primer pair Ku70_ChkUp_F / TEF_R (nucleotide sequences are shown as SEQ ID NO. 16 and SEQ ID NO. 17) and Ku70_ChkDw_R / XPR_F (nucleotide sequences are shown as SEQ ID NO. 18 and SEQ ID NO. 19) to verify the upstream and downstream sites of the recombinant plasmid pYLXP'-URA-loxP-Ku70 integration, both sites are correct to prove the upstream and downstream sites of the recombinant plasmid pYLXP'-URA-loxP-Ku70 knock-out box integration, both sites are correct to prove the integration of the recombinant plasmid pYLXP'-URA-loxP-Ku70 to the knock-out site of the po1f cell body, and the obtained recombinant engineering bacteria is Yarrowia lipolytica Po1f Δku70 strain with Ku70 gene knockout.

[0076] Preparation Example 2

[0077] pYLXP'-ylHPD expression plasmid and control strain Yarrowia lipolytica Po1f Δku70 ylHPD construction.

[0078] (1) Use KpnI and SnaBI to linearize the plasmid pYLXP'-(nucleotide sequence is shown as SEQ ID NO. 20), then use primer HPD_F / HPD_R (nucleotide sequences are shown as SEQ ID NO. 21 and SEQ ID NO. 22) to amplify ylHPD (nucleotide sequence is shown as SEQ ID NO. 4) from Yarrowia lipolytica genome, and then use Gibson assembly method to assemble linearized pYLXP'- and ylHPD to obtain recombinant plasmid pYLXP'-ylHPD (nucleotide sequence is shown as SEQ ID NO. 23), and then use the recombinant plasmid pYLXP'-ylHPD for cell expression after sequencing verification by Shanghai Generay Biotech Co., Ltd.

[0079] (2) Take Yarrowia lipolytica Po1f Δku70 as the original strain, cultivate po1f Δku70 strain in 2 mL of YPD medium to the exponential growth phase (16-24 h), collect 1 mL of po1f Δku70 bacteria in the fermentation broth, centrifuge and discard the supernatant, then add 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2M), 5 μL of single-stranded DNA (salmon sperm) and 5 μL of the recombinant plasmid pYLXP'-ylHPD obtained in step (2), mix well, then incubate at 39°C for 1 h, then plate on a leucine-deficient plate, and screen to obtain a recombinant strain carrying the leucine marker pYLXP'-ylHPD plasmid (nucleotide sequence as shown in SEQ ID NO. 23) single colony; the obtained recombinant engineering bacteria are Yarrowia lipolytica Po1f Δku70 ylHPD strain with Ku70 gene knockout and overexpression of ylHPD.

[0080] Example 1

[0081] Recombinant plasmid construction.

[0082] The plasmid pYLXP-hygr-loxP (nucleotide sequence as shown in SEQ ID NO. 24, containing the hygr gene with the nucleotide sequence as shown in SEQ ID NO. 6) is linearized by enzyme digestion using AvrII and SalI, and then primers ARO10_Up_F / ARO10_Up_R (nucleotide sequences as shown in SEQ ID NO. 25 and SEQ ID NO. 26) and ARO10_Dw_F / ARO10_Dw_R (nucleotide sequences as shown in SEQ ID NO. 27 and SEQ ID NO. 28) are used to amplify the upstream arm ARO10_Up and the downstream arm ARO10_Dw (nucleotide sequences as shown in SEQ ID NO. 29 and SEQ ID NO. 30, each 1000 bp) of ARO10 from the Yarrowia lipolytica genome, respectively. Then, the linearized pYLXP-hygr-loxP, ARO10_Up and ARO10_Dw are assembled by Gibson assembly to obtain the recombinant plasmid pYLXP-hygr-loxP-ARO10 (structure as shown in SEQ ID NO. 5, nucleotide sequence as shown in SEQ ID NO. 5), and the recombinant plasmid pYLXP-hygr-loxP-ARO10 is sequenced by Shanghai Biosciences to verify its correctness, and then used in Example 2 for gene knockout. Figure 1

[0083] Example 2

[0084] (1) Strain homologous recombination

[0085] ​The knockout frame ARO10_Up-hygr-loxP-ARO10_Dw (as shown in SEQ ID NO. 31) was amplified using the recombinant plasmid pYLXP-hygr-loxP-ARO10 obtained in Example 1 as a template and primers ARO10_Up_F / ARO10_Dw. The Yarrowia lipolytica Po1fΔku70 obtained in Preparation 1 was used as a starting strain. The Yarrowia lipolytica Po1fΔku70 strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h), and 1 mL of Yarrowia lipolytica Po1fΔku70 cells in the fermentation broth was collected by centrifugation. After the supernatant was discarded, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2M), 5 μL of single-stranded DNA (salmon sperm), and the obtained knockout frame ARO10_Up-hygr-loxP-ARO10_Dw were added. After mixing and incubation at 39°C for 1 h, the mixture was plated on a hygromycin-resistant plate, and a recombinant strain single colony in which the hygromycin resistance gene (nucleotide sequence as shown in SEQ ID NO. 6) replaced the phenylpyruvate decarboxylase ARO10 gene (nucleotide sequence as shown in SEQ ID NO. 2, amino acid sequence as shown in SEQ ID NO. 1) was screened.

[0086] (2) Verification of the recombinant strain

[0087] Four single colonies (numbered as single colony 1, single colony 2, single colony 3, and single colony 4) randomly selected from the hygromycin-resistant plate in step (1) were subjected to colony PCR verification using primer pairs ARO10_ChkUp_F / TEF_R (nucleotide sequences as shown in SEQ ID NO. 32 and SEQ ID NO. 17) and ARO10_ChkDw_R / XPR_F (nucleotide sequences as shown in SEQ ID NO. 33 and SEQ ID NO. 19). The upstream and downstream sites of the knockout frame ARO10_Up-hygr-loxP-ARO10_Dw were verified using primer pairs ARO10_ChkUp_F / TEF_R and ARO10_ChkDw_R / XPR_F. If both sites are correct, it indicates that the knockout frame is correctly integrated into the knockout site of the Yarrowia lipolytica Po1fΔku70 strain. The recombinant strain of single colony 2 is the ARO10 gene-knocked-out Yarrowia lipolytica strain po1fk△ARO10, which was used as a recombinant Yarrowia lipolytica to synthesize homologancic acid from L-tyrosine as a precursor.

[0088] Example 3

[0089] The po1fkΔARO10 strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h), 1 mL of po1fkΔARO10 strain in the fermentation broth was collected, centrifuged and the supernatant was discarded, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2M), 5 μL of single-stranded DNA (salmon sperm) and 5 μL of the recombinant plasmid pYLXP'-ylHPD obtained in Preparation Example 2 were added, mixed, incubated at 39°C for 1 h, and then plated on a leucine-deficient plate to screen for a recombinant strain carrying the pYLXP'-ylHPD plasmid (nucleotide sequence as shown in SEQ ID NO. 23) with a leucine marker; the obtained recombinant engineering strain was Yarrowia lipolytica po1fkΔARO10 ylHPD strain with Ku70 gene knockout, ARO10 gene knockout and overexpression of ylHPD.

[0090] Example 4

[0091] Recombinant strain for fermentation synthesis of homogentisic acid with L-tyrosine as precursor

[0092] (1) The recombinant Yarrowia lipolytica po1fkΔARO10 ylHPD obtained in Example 3 was inoculated in a seed culture medium and cultured at a temperature of 30°C and a rotation speed of 220 rpm for 48 h to obtain a seed liquid of the recombinant Yarrowia lipolytica;

[0093] (2) The seed liquid of the recombinant Yarrowia lipolytica obtained in step (1) was inoculated in a fermentation culture medium with L-tyrosine at an inoculation amount of 1% by volume and fermented at a temperature of 30°C and a rotation speed of 220 rpm for 120 h to obtain a fermentation broth; after the fermentation, the content of homogentisic acid in the fermentation broth was detected by the homogentisic acid detection method and was 2.59 g / L, as shown in Table 1.

[0094] Comparative Example 1

[0095] Homogentisic acid was synthesized by fermentation with L-tyrosine as precursor according to the method of Example 3, except that the recombinant Yarrowia lipolytica obtained in Example 2 was replaced by the Yarrowia lipolytica strain Yarrowia lipolytica Po1fΔku70 ylHPD obtained in Preparation Example 2.

[0096] After the fermentation, the content of homogentisic acid in the fermentation broth was detected by the homogentisic acid detection method and was 0.6 g / L, as shown in Table 1.

[0097] Based on the above examples and comparative examples, from the data in Table 1, it can be seen that the recombinant Yarrowia lipolytica obtained in Example 3 accumulates 2.59 g / L of homogentisic acid in the fermentation broth obtained by fermentation, which is 331.66% higher than the starting strain Yarrowia lipolytica Po1fΔku70 ylHPD, achieving the improvement of the yield of homogentisic acid produced by L-tyrosine as a precursor in recombinant Yarrowia lipolytica.

[0098] Table 1 Yield of homogentisic acid under different conditions

[0099]

[0100] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A recombinant strain for synthesizing homogentisic acid, characterized in that, The phenylpyruvate decarboxylase gene of the starting strain is replaced by the hygr gene of hygromycin resistance; The starting strain is Yarrowia lipolytica with a knocked-out gene Ku70 and overexpressed 4-hydroxyphenylpyruvate dioxygenase gene ylHPD, the nucleotide sequence of the gene Ku70 is shown in SEQ ID NO. 3, and the nucleotide sequence of the ylHPD is shown in SEQ ID NO.

4. The amino acid sequence of the phenylpyruvate decarboxylase gene of the starting strain is shown in SEQ ID NO. 1, and the starting strain is Yarrowia lipolytica.

2. The recombinant strain for synthesis of homogentisic acid according to claim 1, characterized in that, The nucleotide sequence of the knocked-out phenylpyruvate decarboxylase gene of the starting strain is shown in SEQ ID NO.

2.

3. The method for constructing a recombinant strain for synthesis of homogentisic acid according to any one of claims 1-2, characterized in that, The recombinant vector containing the hygr gene of hygromycin resistance is constructed, and the phenylpyruvate decarboxylase gene in the starting strain is replaced by the hygr gene of hygromycin resistance in the recombinant vector through homologous recombination. The nucleotide sequence of the recombinant vector is shown in SEQ ID NO. 5, and the nucleotide sequence of the hygr gene of hygromycin resistance is shown in SEQ ID NO.

6.

4. The method for constructing a recombinant strain for synthesizing homogentisic acid according to claim 3, characterized in that, The recombinant strain is used to synthesize homogentisic acid, which comprises inoculating the recombinant strain into a fermentation medium added with L-tyrosine for fermentation culture to obtain a fermentation broth containing homogentisic acid.

5. Use of the recombinant strain according to any one of claims 1 to 2 for the synthesis of homogentisic acid, characterized in that, The fermentation culture conditions comprise that the inoculation amount of the recombinant strain is 0.5-2% of the volume of the fermentation medium, the culture temperature is 25-35°C, the rotation speed is 180-250 rpm, and the culture time is 80-160 h.

6. Use according to claim 5, characterized in that, The fermentation medium contains glucose 30-50 g / L, ammonium sulfate 0.8-1.3 g / L, YNB 1.5-2 g / L, precursor L-tyrosine 8 g / L, adenine 0.01-0.02 g / L, L-arginine 0.02-0.08 g / L, L-aspartic acid 0.05-1 g / L, L-histidine 0.01-0.03 g / L, L-isoleucine-0.02-0.08 g / L, L-lysine 0.02-0.08 g / L, L-methionine 0.01-0.03 g / L, L-phenylalanine 0.02-0.08 g / L, L-threonine 0.05-0.15 g / L, L-tryptophan 0.02-0.08 g / L, and L-valine 0.05-0.25 g / L.

7. Use according to claim 5, characterized in that, ​

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