Recombinant pichia strain and its construction method and use

Abstract

The application provides a recombinant Pichia pastoris strain and a construction method and application thereof. The construction method comprises the following steps: taking a Pichia pastoris strain as a host strain, constructing a double synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase in the host strain to obtain a first engineering strain; relieving the branch acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing the aromatic amino acid synthesis pathway of the second engineering strain to obtain a third engineering strain; knocking out the branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing the precursor supply of the fourth engineering strain to obtain the recombinant Pichia pastoris strain. The construction method can systematically optimize the synthesis path of p-coumaric acid, and the recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as a carbon source is constructed. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of genetic engineering technology, and specifically to a recombinant Pichia pastoris strain, a construction method thereof, and uses thereof. Background Art

[0002] p-Coumaric acid (trans-4-hydroxycinnamic acid, also known as p-CA) is a natural aromatic compound found widely in plants. Its applications span a wide range of industries, including pharmaceuticals, food, daily necessities, feed, and chemicals. p-Coumaric acid exhibits diverse biological activities, including antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, cardiovascular, diabetes prevention and improvement, and neuroprotective effects. Currently, microbial synthesis of p-coumaric acid has garnered significant attention due to its advantages over traditional plant extraction and chemical synthesis, including shorter production cycles and higher conversion efficiencies.

[0003] Pichia pastoris ( Komagataella phaffii, Also known as Pichia pastoris ) has become an ideal platform for the production of high-value chemicals due to its efficient methanol conversion capability; in the methanol utilization pathway, Pichia pastoris can use the alcohol oxidase I (AOX1) promoter to regulate the metabolism of methanol; when methanol is used as the sole carbon source, it acts as both a carbon source and an inducer. Due to its strict regulation and strong inducibility, it is widely used to drive the expression of heterologous proteins.

[0004] Although various microbial cells have been constructed for the production of p-coumaric acid, significant challenges remain in achieving efficient microbial synthesis of p-coumaric acid. Pichia pastoris' ability to utilize methanol as a carbon and energy source allows it to convert inexpensive methanol into high-value-added compounds. Therefore, developing a Pichia pastoris strain that efficiently synthesizes p-coumaric acid from methanol, enabling it to convert inexpensive and readily available methanol into high-value-added p-coumaric acid, is of great significance for promoting the industrial production of coumaric acid. Summary of the Invention

[0005] The purpose of the embodiments of the present application is to provide a recombinant Pichia pastoris strain and its construction method and use.

[0006] To achieve the above objectives, the present application proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing a Pichia pastoris strain, the method comprising:

[0008] Using Pichia pastoris as a host strain, a dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase was constructed in the host strain to obtain the first engineered strain;

[0009] Eliminating chorismate feedback inhibition of the first engineered strain based on metabolic engineering to obtain a second engineered strain;

[0010] optimizing the aromatic amino acid synthesis pathway of the second engineered strain to obtain a third engineered strain;

[0011] knocking out the branched metabolic pathway of the third engineered strain to obtain a fourth engineered strain;

[0012] increasing the precursor supply of the fourth engineered strain to obtain a recombinant Pichia pastoris strain.

[0013] As an embodiment of the present application, the construction of the dual synthesis pathway of phenylalanine ammonia-lyase and tyrosine ammonia-lyase in the host strain comprises:

[0014] integrating the phenylalanine ammonia-lyase gene AtPAL2 into the chromosomal PNSI-2 site of the host strain, integrating the cinnamic acid hydroxylase AtC4H and the P450 reductase AtATR2 into the chromosomal PNSII-4 site of the host strain, and integrating the tyrosine ammonia-lyase FjTAL into the chromosomal PNSII-5 site of the host strain.

[0015] As an embodiment of the present application, the relieving of the feedback inhibition of the first engineered strain based on metabolic engineering comprises:

[0016] mutating the Aro7 G141S into the chromosomal PNSI-8 site of the first engineered strain, and using the PAOX1 promoter to control the expression of the Aro7 G141S mutant.

[0017] As an embodiment of the present application, the Aro7 G141S mutant is prepared after the G141S mutation of the Aro7

[0018] As an embodiment of the present application, the optimization of the aromatic amino acid synthesis pathway of the second engineered strain comprises:

[0019] integrating the prephenate dehydratase ScPHA2 into the chromosomal PNSI-6 site of the second engineered strain, and using the PAOX1 promoter to control the expression of the prephenate dehydratase ScPHA2.

[0020] As an embodiment of the present application, the knocking out of the branched metabolic pathway of the third engineered strain comprises:

[0021] knocking out the endogenous phenylpyruvate decarboxylase Aro10 of the third engineered strain by using a gene editing technology.

[0022] As an embodiment of the present application, the gene editing technology is the CRISPR / Cas9 gene editing technology.

[0023] As an embodiment of the present application, the increasing precursor supply of the fourth engineered strain comprises:

[0024] The endogenous fructose-1,6-bisphosphatase is integrated into the chromosome of the fourth engineered strain at the PNSII-6 site, and the overexpression of the endogenous fructose-1,6-bisphosphatase is controlled by the PAOX1 promoter.

[0025] In a second aspect, the embodiments of the present application provide a recombinant Pichia pastoris strain, which is prepared by the construction method of the first aspect.

[0026] In a third aspect, the embodiments of the present application provide the use of the recombinant Pichia pastoris strain of the second aspect in synthesis of p-coumaric acid.

[0027] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects:

[0028] The embodiments of the present application optimize the metabolic flow of aromatic amino acids in the Pichia pastoris strain by constructing a double synthetic pathway of phenylalanine ammonia-lyase and tyrosine ammonia-lyase in the host strain; then, by relieving the feedback inhibition of shikimic acid, the limitation of the shikimic acid pathway is broken, ensuring that the pathway can continuously and efficiently operate to stably generate intermediate products such as shikimic acid, thereby providing sufficient material basis for the subsequent synthesis of p-coumaric acid; secondly, by optimizing the synthesis pathway of aromatic amino acids, the yield of the direct precursor tyrosine is improved, which provides sufficient substrate for the synthesis of p-coumaric acid and directly promotes the generation of p-coumaric acid; thirdly, by knocking out the branched metabolic pathway, the invalid consumption of carbon source and related energy is reduced, so that intermediate products such as shikimic acid flow more to the tyrosine synthesis pathway, thereby improving the carbon flow distribution efficiency of p-coumaric acid synthesis; finally, increasing the precursor supply is conducive to the modification of the upstream metabolic pathway, thereby improving the supply amount of PEP and E4P, which can improve the metabolic flux of the shikimic acid pathway from the source, thereby providing more starting materials for the synthesis of shikimic acid, and further increasing the entire metabolic flow of p-coumaric acid synthesis.

[0029] In summary, the construction method provided by the embodiments of the present application can systematically optimize the synthesis path of p-coumaric acid by optimizing the metabolic flow, relieving the limitation, strengthening the direct precursor, reducing the diversion, increasing the source (precursor supply), and the like, and finally constructs a recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as the carbon source. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

[0030] Additional aspects and advantages of the application will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following description and the attached drawings. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1A schematic diagram of metabolic engineering of the Pichia pastoris engineering strain for constructing p-coumaric acid is shown.

[0032] Figure 2 A liquid chromatogram of different engineering strains for synthesizing p-coumaric acid is shown.

[0033] Figure 3 A schematic diagram of p-coumaric acid production results of the engineering strains CA01-CA07 in shake flask fed-batch fermentation is shown.

[0034] Figure 4 A schematic diagram of the results of the engineering strain CA05 in 15 L bioreactor fermentation for producing p-coumaric acid in Example 5 is shown. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments will be described clearly and completely below with reference to the embodiments of the present application and the drawings. Obviously, the embodiments described below are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without any creative effort fall within the scope of protection of the present application.

[0036] It should be understood that, when used in the specification and the appended claims, the terms “comprise” and “include” indicate the presence of described features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0037] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless otherwise clearly indicated by the context, the singular forms “a”, “an” and “the” are intended to include the plural forms.

[0038] The recombinant Pichia pastoris strain of the embodiments of the present application and the construction method and use thereof will be described in detail below.

[0039] First, the construction method of the recombinant Pichia pastoris strain of the first aspect of the embodiments will be described.

[0040] Construction method

[0041] Therefore, the embodiments of the present application provide a construction method of a Pichia pastoris strain, by which a recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as a carbon source is successfully constructed.

[0042] Specifically, the construction method of the embodiments of the present application specifically includes the following steps:

[0043] S1, a Pichia pastoris strain is used as a host strain, and a double synthetic pathway of phenylalanine ammonia-lyase and tyrosine ammonia-lyase is constructed in the host strain to obtain a first engineering strain.

[0044] It can be understood that the host strain of the embodiment can use an existing known Pichia pastoris strain; for example, the recombinant Pichia pastoris S12 disclosed in the Chinese patent with the publication number CN118530863A is used as a host strain (hereinafter referred to as a starting strain S12) for subsequent directed modification.

[0045] In step S1, the double synthetic pathway of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) is constructed in Pichia pastoris, which is achieved by synergistically using two precursors (phenylalanine and tyrosine) to break through the metabolic bottleneck of a single pathway and maximize the yield of p-coumaric acid.

[0046] Specifically, although Pichia pastoris can synthesize both phenylalanine and tyrosine, the synthesis fluxes of the two aromatic amino acids may differ due to metabolic regulation (for example, one kind of amino acid is more abundant). At present, a single pathway (PAL or TAL) can only use one kind of amino acid as a substrate, which may be limited by the supply amount of the amino acid, resulting in carbon flow waste.

[0047] Firstly, the double pathway used in step S1 can simultaneously use phenylalanine and tyrosine, and direct the metabolic flow of the two kinds of amino acids to the synthesis of the target aromatic compound (p-coumaric acid), thereby maximizing the utilization of the intracellular aromatic amino acid reserves and improving the carbon source conversion efficiency.

[0048] Secondly, the double pathway can also improve the yield through metabolic complementation: for example, when the supply of tyrosine is insufficient, the cinnamic acid generated by PAL catalysis can be converted into p-coumaric acid by hydroxylase, thereby indirectly supplementing the synthesis flux of the target product and avoiding the bottleneck limitation of a single pathway.

[0049] Furthermore, in the metabolism of aromatic amino acids, the synthesis of phenylalanine and tyrosine is in a competitive relationship (for example, sharing upstream intermediates such as branch acid and prephenic acid). Excessive strengthening of a single pathway may lead to excessive accumulation of another kind of amino acid (for example, only strengthening TAL will lead to excess phenylalanine), causing feedback inhibition or byproduct generation; the double pathway used in step S1 can balance the consumption rate of the two kinds of amino acids through the synergistic action of the two enzymes, reduce the unreasonable accumulation of metabolic intermediates, maintain the homeostasis of the intracellular metabolic network, and reduce the adverse effects on the growth of the host strain.

[0050] Therefore, by constructing the PAL and TAL dual synthetic pathway in step S1, the metabolic flow of aromatic amino acids in the Pichia pastoris strain can be optimized through diversion utilization and complementary synergistic strategies, ultimately achieving the enhancement of the yield and synthesis stability of the target aromatic compound (p-coumaric acid), and laying a foundation for providing more efficient engineering strains in the subsequent.

[0051] S2, relieving the feedback inhibition of the chorismate pathway in the first engineering strain to obtain a second engineering strain.

[0052] It can be understood that the chorismate pathway is the core stage of generating chorismate (a key intermediate product of aromatic compound synthesis) in the shikimic acid pathway, and the key enzymes (such as chorismate synthase) of this pathway will be inhibited in activity by the downstream end products (such as tyrosine, phenylalanine and other aromatic amino acids) through the feedback inhibition mechanism (to avoid excessive accumulation of products).

[0053] The feedback inhibition of the chorismate pathway in step S2 is to purposefully genetically modify the metabolic network of the first engineering strain through molecular biology, genetic engineering and systems biology, etc., to achieve the goal of relieving the feedback inhibition of the chorismate node.

[0054] For example, through genetic engineering means (such as site-directed mutation of key enzyme genes), these enzymes are modified so that they are no longer inhibited by downstream end products.

[0055] Based on this, step S2 can break the limitation of the chorismate pathway by relieving the feedback inhibition of chorismate, ensuring that the pathway can run continuously and efficiently, and stably generating intermediate products such as chorismate, providing sufficient material basis for the subsequent synthesis of p-coumaric acid.

[0056] S3, optimizing the aromatic amino acid synthesis pathway of the second engineering strain to obtain a third engineering strain.

[0057] It should be noted that aromatic amino acids (especially tyrosine) are direct precursors of p-coumaric acid (p-coumaric acid is generated from tyrosine through TAL catalysis). The optimization of the aromatic amino acid synthesis pathway in step S3 means to enhance the efficiency of the pathway through genetic engineering means.

[0058] For example, by overexpressing the key enzyme of the pathway (such as prephenate dehydrogenase), prephenic acid can be catalyzed to generate p-hydroxyphenylpyruvic acid, and finally generate tyrosine; or by balancing the branch flow, the synthesis of tyrosine can be effectively avoided from being excessively competed by the tryptophan / phenylalanine branch.

[0059] Therefore, by optimizing the synthesis pathway of aromatic amino acids in step S3, the yield of the direct precursor tyrosine can be significantly improved (directly increasing the supply of p-coumaric acid precursors), providing sufficient substrate for the synthesis of p-coumaric acid, and directly promoting the generation of p-coumaric acid.

[0060] S4, knocking out the branched metabolic pathway of the third engineering strain to obtain a fourth engineering strain.

[0061] Shikimic acid, as the core intermediate of the shikimic acid pathway, will flow to other non-target products, such as the synthesis of tryptophan and phenylalanine, or the generation of quinolinic acid and homogentisic acid, in addition to the synthesis of tyrosine (target pathway).

[0062] The step S4 of knocking out the branched metabolic pathway is to block the flow of intermediates to non-target products by knocking out the key enzyme genes in these branched pathways (such as the anthranilate synthase gene for tryptophan synthesis).

[0063] Therefore, step S4 can reduce the inefficient consumption of carbon sources and related energy by knocking out the branched metabolic pathway, so that more intermediates such as shikimic acid flow to the tyrosine synthesis pathway, and the carbon flow distribution efficiency of coumaric acid synthesis is improved.

[0064] S5, increasing the precursor supply of the fourth engineering strain to obtain a recombinant Pichia pastoris strain.

[0065] The initiation of the shikimic acid pathway depends on the precursors provided by the upstream sugar metabolism, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), which are the substrates of DAHP synthase.

[0066] The step S5 of increasing the precursor supply is to improve the supply amount of PEP and E4P by modifying the upstream metabolic pathway (such as overexpressing phosphoenolpyruvate carboxykinase to reduce the conversion of PEP to pyruvate, or enhancing the key enzyme activity of the pentose phosphate pathway to increase the generation of E4P).

[0067] That is, increasing the precursor supply is beneficial to modify the upstream metabolic pathway, increase the supply amount of PEP and E4P, and improve the metabolic flux of the shikimic acid pathway from the source, so as to provide more sufficient starting materials for shikimic acid synthesis, and further increase the entire metabolic flow of coumaric acid synthesis.

[0068] In summary, the construction method provided in the embodiment can systematically optimize the synthesis path of coumaric acid by optimizing the metabolic flow, removing the limitation, strengthening the direct precursor, reducing the diversion, increasing the source (precursor supply), and the like. Ultimately, a recombinant Pichia pastoris strain for efficiently producing coumaric acid with methanol as the carbon source is constructed. Compared with the host strain, the yield of coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

[0069] In the following, the related steps of the above construction method will be further described.

[0070] In step S1, the double synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain, which includes:

[0071] The phenylalanine ammonia-lyase gene AtPAL2 is integrated into the chromosomal PNSI-2 site of the host strain, the cinnamic acid hydroxylase AtC4H and the P450 reductase AtATR2 are integrated into the chromosomal PNSII-4 site of the host strain, and the tyrosine ammonia-lyase FjTAL is integrated into the chromosomal PNSII-5 site of the host strain.

[0072] It can be understood that the chromosomal PNSI-2 site, the PNSII-4 site and the PNSII-5 site are neutral integration sites screened by gene editing technology, which can be used for stable insertion and efficient expression of exogenous genes, and meanwhile, the basic growth and methanol utilization ability of Pichia pastoris (Pichia pastoris uses methanol as a carbon source and an inducer) are not affected after the insertion of the exogenous genes.

[0073] In this embodiment, the phenylalanine ammonia-lyase gene AtPAL2 is integrated into the chromosomal PNSI-2 site of the host strain, and the PNSI-2 site is a neutral site in Pichia pastoris with high expression potential, which is suitable for driving the overexpression of key rate-limiting enzymes. By integrating AtPAL2 into the PNSI-2 site, the expression amount of AtPAL2 can be significantly improved by using a strong promoter (such as PAOX1), and the conversion flux of phenylalanine to cinnamic acid can be enhanced, thereby providing sufficient substrate for downstream reactions.

[0074] Secondly, AtC4H (derived from Arabidopsis thaliana) is a cytochrome P450 enzyme, which catalyzes the para-hydroxylation of cinnamic acid to produce p-coumaric acid, and is a key enzyme for connecting cinnamic acid and p-coumaric acid; AtATR2 (derived from Arabidopsis thaliana) is a P450 reductase, which provides electrons for AtC4H, and both of them need to work together to efficiently catalyze the reaction. In this embodiment, the PNSII-4 site is suitable for the integration of multiple genes in series, and the co-expression of AtC4H and AtATR2 can be realized by using the same promoter, so as to ensure the proportional coordination of the two enzymes and avoid metabolic bottlenecks (such as cinnamic acid accumulation toxicity) caused by the lack of a certain enzyme.

[0075] Furthermore, FjTAL catalyzes the direct deamination of tyrosine to produce p-coumaric acid, which is the second independent pathway for the synthesis of p-coumaric acid (not dependent on the intermediate of cinnamic acid). The PNSII-5 site has good genetic stability and metabolic compatibility, and is suitable for introducing key enzymes of independent pathways. In this embodiment, FjTAL is integrated into the PNSII-5 site, which can avoid the expression competition with the first two genes, and meanwhile, the small influence of the PNSII-5 site on the growth of the host can ensure the stable operation of the second pathway.

[0076] Therefore, the present embodiment is based on the above-mentioned means to construct a double-pathway synthesis of p-coumaric acid, so as to maximize the carbon source utilization rate; the two pathways respectively utilize the phenylalanine and tyrosine synthesized by Pichia pastoris itself as substrates, so as to avoid the limitation of insufficient single amino acid supply, guide the metabolic flow of the two kinds of aromatic amino acids to the target product, and significantly improve the conversion efficiency of the carbon source to p-coumaric acid.

[0077] Meanwhile, it is known to those skilled in the art that, if only AtPAL2 is expressed and AtC4H / AtATR2 is lacking, cinnamic acid will accumulate (which is cytotoxic); if only FjTAL is expressed, it may be limited by the synthesis flux of tyrosine (there is competition between the synthesis of tyrosine and phenylalanine in Pichia pastoris). The present embodiment is based on the synergistic expression of the above-mentioned three, so as to realize the efficient flow of substrates-intermediates-products: the cinnamic acid generated by AtPAL2 is rapidly converted by AtC4H / AtATR2, and FjTAL splits the tyrosine metabolism, so as to avoid the overload of a single pathway and reduce the risk of toxicity.

[0078] The present embodiment can also utilize the site characteristics to optimize the expression intensity and improve the product yield; wherein, the high expression characteristics of PNSI-2 strengthen the activity of AtPAL2, so as to ensure the sufficiency of the cinnamic acid precursor; the multi-gene co-expression ability of PNSII-4 guarantees the synergistic catalytic efficiency of AtC4H and AtATR2; the stability of PNSII-5 supports the sustained expression of FjTAL, so as to maintain the flux of the second pathway. The matching design of site-gene in the present embodiment can avoid the metabolic bottleneck caused by the imbalance of expression intensity, and finally realize the synergistic improvement of the yield of p-coumaric acid.

[0079] Therefore, the present embodiment is based on the above-mentioned means to construct a double-pathway synthesis of p-coumaric acid, so as to maximize the carbon source utilization rate; the two pathways respectively utilize the phenylalanine and tyrosine synthesized by Pichia pastoris itself as substrates, so as to avoid the limitation of insufficient single amino acid supply, guide the metabolic flow of the two kinds of aromatic amino acids to the target product, and significantly improve the conversion efficiency of the carbon source to p-coumaric acid.

[0080] In step S2, the branch acid feedback inhibition of the first engineering strain is relieved based on metabolic engineering, which includes:

[0081] The branch acid mutase Aro7 G141S is integrated into the PNSI-8 site of the chromosome of the first engineering strain, and the expression of the branch acid mutase Aro7 G141S is controlled by the PAOX1 promoter.

[0082] The function of shikimate mutase (Aro7, from Saccharomyces cerevisiae) is to catalyze the intramolecular rearrangement of shikimate (the core intermediate of the shikimic acid pathway) to prephenate, which is the direct precursor of phenylalanine and tyrosine synthesis. Among them, the activity of wild-type Aro7 is strongly inhibited by the downstream end product tyrosine (feedback inhibition), resulting in limited synthesis of prephenate.

[0083] It can be understood that the Aro7 G141S of the present embodiment is a mutant obtained by site-directed mutagenesis, in which the glycine (G) at position 141 is replaced by serine (S), thereby eliminating the feedback inhibition of tyrosine on it. Even if tyrosine accumulates in the cell, it can still maintain high catalytic activity and continuously promote the conversion of shikimate to prephenate.

[0084] The selected site PNSI-8 in the present embodiment belongs to the neutral integration site of Pichia pastoris. This site is located in the non-essential region of the genome, and after insertion of the exogenous gene, it does not interfere with the basic metabolism and growth of the host strain itself. At the same time, it has good genetic stability, which can ensure that the integrated Aro7 G141S gene is stably inherited with the host chromosome, avoiding the common problem of gene loss in plasmid expression systems. At the same time, this site is compatible with other PNS family sites (such as PNSI-2, PNSII-4), which can support the coordinated integration of multiple genes (for example, with AtPAL2, FjTAL, etc. to form a metabolic pathway).

[0085] As mentioned earlier, shikimate is a key branch point of the shikimic acid pathway: in addition to flowing to prephenate (aromatic amino acid synthesis), it may also branch to the tryptophan synthesis pathway or other byproduct metabolism. The efficient expression of Aro7 G141S can competitively enhance the flow of shikimate to prephenate, reduce the waste of carbon sources to non-target pathways, and increase the carbon flow proportion of aromatic amino acid synthesis, indirectly providing more abundant substrates for downstream products such as p-coumaric acid. Specifically, by inducing regulation through the PAOX1 promoter, Aro7 G141S can be overexpressed during the rapid growth phase of P. pastoris (at this time the cell needs to preferentially allocate resources for proliferation), reducing the inhibition of host growth.

[0086] The neutral nature of the PNSI-8 site allows it to work synergistically with other integrated genes (AtPAL2, AtC4H / AtATR2, FjTAL) at other sites (PNSI-2, PNSII-4, PNSII-5): Aro7 G141S provides a pathway that can more efficiently convert it to p-coumaric acid, forming an integrated metabolic network of upstream strengthening-midstream conversion-downstream synthesis, ultimately significantly improving the yield of target products.

[0087] In summary, step S2 introduces a shikimate mutase mutant Aro7G141S The expression of the branchinase mutant Aro7 is controlled by the PAOX1 promoter integrated into the PNSI-8 site of the chromosome of the first engineered strain, the core purpose of which is to strengthen the conversion flux of chorismate to prephenate, increase the supply of phenylalanine and tyrosine, and provide sufficient precursors for the synthesis of p-coumaric acid downstream, while balancing the growth of the host and the synthesis of the product. G141S The core purpose of the present embodiment is to strengthen the conversion flux of chorismate to prephenate, increase the supply of phenylalanine and tyrosine, and provide sufficient precursors for the synthesis of p-coumaric acid downstream, while balancing the growth of the host and the synthesis of the product.

[0088] As an embodiment of the present embodiment, the branchinase mutant Aro7 G141S is prepared after the G141S mutation of the branchinase Aro7.

[0089] The branchinase mutant Aro7 G141S is obtained after the glycine (G) at position 141 of the branchinase Aro7 is replaced by serine (S). This design eliminates the feedback inhibition of phenylalanine, and even if tyrosine accumulates in the cell, it can still maintain high catalytic activity and continuously promote the conversion of chorismate to prephenate.

[0090] In step S3, the aromatic amino acid synthesis pathway of the second engineered strain is optimized, including:

[0091] The prephenate dehydratase ScPHA2 is integrated into the PNSI-6 site of the chromosome of the second engineered strain, and the expression of the prephenate dehydratase ScPHA2 is controlled by the PAOX1 promoter.

[0092] It can be understood that the prephenate dehydratase ScPHA2 is a key rate-limiting enzyme in the phenylalanine synthesis pathway, which catalyzes the dehydration and decarboxylation of prephenate to generate phenylpyruvic acid (the direct precursor of phenylalanine, which is subsequently generated by transaminase to generate phenylalanine). In Pichia pastoris strains, the endogenous enzyme activity is low or strongly inhibited by feedback, becoming a natural bottleneck for phenylalanine synthesis.

[0093] The PNSI-6 selected in the present embodiment belongs to the neutral integration site of Pichia pastoris strains, which does not interfere with the host's own metabolism and growth after inserting exogenous genes, and has good transcriptional activity, suitable for stable expression of exogenous genes; at the same time, it can cooperate with other neutral sites (such as the aforementioned PNSI-2, PNSII-4, etc.) to carry multiple gene metabolic pathways, avoiding the expression competition caused by single site integration.

[0094] PAOX1 is a commonly used strong inducible promoter in Pichia pastoris, and its activity can be efficiently induced by methanol (the expression after induction is significantly higher than that of the constitutive promoter). By controlling the expression of ScPHA2 through PAOX1, controllable high expression of the enzyme can be realized, and the enzyme activity of ScPHA2 can be precisely enhanced through methanol induction at a specific stage (such as the late logarithmic growth phase) of fermentation.

[0095] Therefore, the present embodiment, in combination with the mutation of relieving feedback inhibition, can not only improve the conversion efficiency of prebenzoic acid to phenylpyruvic acid, promote more shikimic acid to flow to the phenylalanine synthesis pathway, but also offset the negative impact of feedback inhibition, so as to accumulate phenylalanine in the cell, provide sufficient substrate for the synthesis of p-coumaric acid (catalyzed by PAL), and at the same time, the overexpression of ScPHA2 can specifically enhance the carbon flow of the phenylalanine branch, reduce the diversion of intermediates to other non-target pathways, and improve the carbon source utilization efficiency.

[0096] At the same time, step S3 can also cooperate with other genes integrated in the Pichia pastoris strain in steps S1 and S2, such as AtPAL2, AtC4H / AtATR2, etc. The enhanced expression of ScPHA2 can form a synergy with these pathways to form an upstream donor-downstream conversion; wherein the upstream ScPHA2 increases the supply of phenylalanine, and the downstream AtPAL2 and other enzymes efficiently convert phenylalanine to p-coumaric acid, ultimately forming a complete and efficient pathway of phenylalanine synthesis-p-coumaric acid conversion, which significantly improves the yield of the target product p-coumaric acid.

[0097] In step S4, the shunt metabolic pathway of the third engineering strain is knocked out, including:

[0098] The endogenous phenylpyruvic acid decarboxylase Aro10 of the third engineering strain is knocked out by using gene editing technology.

[0099] For example, the endogenous phenylpyruvic acid decarboxylase Aro10-1 of the third engineering strain is knocked out by using gene editing technology; and / or, the endogenous phenylpyruvic acid decarboxylase Aro10-2 of the third engineering strain is knocked out by using gene editing technology.

[0100] In step S4, the core role of knocking out the endogenous phenylpyruvic acid decarboxylases Aro10-1 and Aro10-2 of Pichia pastoris is to block the non-target metabolic diversion of phenylpyruvic acid and to strengthen the synthesis flux of phenylalanine and p-coumaric acid.

[0101] Among them, phenylpyruvic acid decarboxylase (Aro10 family) is a key enzyme involved in the degradation of aromatic amino acids in Pichia pastoris, and its core function is to catalyze the decarboxylation of phenylpyruvic acid to produce phenylacetaldehyde (which can be further converted into byproducts such as phenylethanol). Phenylpyruvic acid is a direct precursor of phenylalanine synthesis and an intermediate product of phenylalanine degradation (phenylalanine can be reversibly converted into phenylpyruvic acid by transaminase). Aro10-1 and Aro10-2 are the main homologous enzymes in Pichia pastoris that catalyze this reaction, and they are functionally redundant, together responsible for the degradation and diversion of phenylpyruvic acid.

[0102] It can be understood that in the unknocked-out strain, there is an ineffective cycle of synthesis-degradation of phenylpyruvic acid; on the one hand, ScPHA2 and other enzymes catalyze the generation of phenylpyruvic acid for the synthesis of phenylalanine; on the other hand, Aro10-1 and Aro10-2 decarboxylate phenylpyruvic acid to benzene acetaldehyde, resulting in the consumption of the precursor for the synthesis of by-products (benzene ethanol, etc.). The present embodiment can completely block the degradation diversion of phenylpyruvic acid after knocking out the two genes, so that more phenylpyruvic acid is used for the synthesis of phenylalanine, directly increasing the precursor reserve of the target metabolic flow, providing more sufficient substrate for PAL and other enzymes, and promoting the efficient synthesis of the target product p-coumaric acid.

[0103] Therefore, the purpose of step S4 is to maximize the synthesis and accumulation of phenylalanine by blocking the degradation diversion of phenylpyruvic acid, to provide sufficient precursors for downstream product p-coumaric acid, while reducing by-product consumption and toxicity, and finally to significantly improve the yield and production efficiency of the target product p-coumaric acid in cooperation with other metabolic engineering modifications.

[0104] As an embodiment of the present embodiment, the gene editing technology is CRISPR / Cas9 gene editing technology.

[0105] Among them, the CRISPR / Cas9 gene editing technology has the advantages of high targeting accuracy and simple and efficient operation; the CRISPR / Cas9 gene editing technology can precisely guide the Cas9 protein to cut the specific DNA site through the complementary pairing of the artificially designed guide RNA (sgRNA) and the target gene sequence, and the off-target effect can be significantly reduced after optimization, ensuring the specificity of the editing. At the same time, compared with the early zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and other technologies, CRISPR / Cas9 does not need complex protein design and modification, only needs to synthesize a specific sgRNA to realize editing, the construction process is simple, the experimental period is short, and the technical threshold of gene editing is greatly reduced.

[0106] The CRISPR / Cas9 gene editing technology used in the present embodiment is a conventional technology known at present.

[0107] In step S5, the precursor supply of the fourth engineering strain is increased, including:

[0108] The endogenous fructose-1,6-bisphosphatase is integrated into the chromosome PNSII-6 site of the fourth engineering strain, and the overexpression of the endogenous fructose-1,6-bisphosphatase is controlled by using the PAOX1 promoter.

[0109] It should be noted that step S5 integrates the endogenous fructose-1,6-bisphosphatase (FBPase) into the Pichia pastoris chromosome PNSII-6 site and controls its overexpression by the PAOX1 promoter, which is a precise design for carbon metabolic flow optimization. The core role is to strengthen the gluconeogenesis pathway and increase the supply of precursors for the synthesis of p-coumaric acid.

[0110] The Pichia pastoris strain can use methanol as a carbon source in fermentation. Methanol is metabolized to generate energy and intermediates (such as dihydroxyacetone phosphate), which need to be converted into sugar metabolism intermediates (such as fructose-6-phosphate, F6P) through gluconeogenesis. By overexpressing FBPase, the conversion of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) can be accelerated, breaking the rate-limiting bottleneck of gluconeogenesis, and more non-sugar carbon sources can flow to F6P accumulation.

[0111] F6P is the starting substrate of the pentose phosphate pathway (PPP), which can be further converted into erythrose-4-phosphate (E4P). E4P is a key precursor of the shikimic acid pathway, which requires E4P to combine with phosphoenolpyruvate (PEP) to generate DAHP.

[0112] In unmodified Pichia pastoris, the metabolic flow of non-sugar carbon sources such as methanol may flow more to energy generation (such as the TCA cycle) rather than to the synthesis of sugar metabolism intermediates required for the synthesis of target products. Overexpression of FBPase can redirect carbon flow, allowing more carbon sources to be used for synthesis in the target pathway, thereby improving the conversion efficiency of carbon sources to target products.

[0113] Therefore, the core purpose of step S5 is to overexpress the endogenous FBPase at the PNSII-6 site driven by the PAOX1 promoter, strengthen the gluconeogenesis pathway of Pichia pastoris, increase the supply of key precursors (E4P) for the synthesis of p-coumaric acid, and ultimately improve the yield of target product p-coumaric acid and the carbon source utilization efficiency in cooperation with other metabolic engineering strategies, providing an efficient cell factory basis for industrial production.

[0114] Next, the recombinant Pichia pastoris strain of the second aspect of the present embodiment will be described.

[0115] Recombinant Pichia pastoris strain

[0116] The recombinant Pichia pastoris strain provided in the present embodiment is prepared by the construction method of the first aspect. As described in the first aspect, the present embodiment can systematically optimize the synthesis pathway of p-coumaric acid through the combined action of optimizing metabolic flow, removing limitations, strengthening direct precursors, reducing shunts, and increasing sources (precursor supply), etc. Finally, a recombinant Pichia pastoris strain for efficient production of p-coumaric acid using methanol as a carbon source is constructed. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

[0117] The following will illustrate the use of the recombinant Pichia pastoris strain of the third aspect of the present embodiment in the synthesis of p-coumaric acid.

[0118] Use

[0119] It can be understood that the recombinant Pichia pastoris strain provided in the present embodiment can realize the efficient, low-cost and high-purity production of p-coumaric acid by systematically optimizing the synthesis pathway of p-coumaric acid. Therefore, the recombinant Pichia pastoris strain of the present embodiment can be used for the efficient synthesis of p-coumaric acid.

[0120] The present application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the present application and are not used to limit the scope of the present application.

[0121] In the following examples, the materials, reagents and instruments used can be obtained from commercial channels if not specifically stated.

[0122] Figures 1-4 The following will illustrate the use of the recombinant Pichia pastoris strain of the third aspect of the present embodiment in the synthesis of p-coumaric acid. Figure 1 Figure 2 shows the metabolic engineering modification schematic diagram of the Pichia pastoris engineering strain for synthesizing p-coumaric acid in the present embodiment; Figure 2 Figure 3 shows the liquid chromatogram of the synthesis of p-coumaric acid by different engineering strains (including strain CA01); Figure 3 Figure 4 shows the schematic diagram of the p-coumaric acid yield results of the fed-batch fermentation of engineering strains CA01-CA07 in a shake flask; Figure 4 Figure 5 shows the results of the fermentation of engineering strain CA05 in a 15 L bioreactor for producing p-coumaric acid in Example 5.

[0123] In the following examples, engineering strain CA01 is a strain obtained by integrating phenylalanine ammonia lyase gene AtPAL2 (Gene ID: 824493) into the chromosome PNSI-2 site of the starting strain S12, and integrating cinnamic acid hydroxylase AtC4H (Gene ID: 817599) and P450 reductase AtATR2 (Gene ID: 829144) into the chromosome PNSII-4 site of the starting strain S12; engineering strain CA02 is a strain obtained by integrating tyrosine ammonia lyase FjTAL into the chromosome PNSII-5 site of engineering strain CA01; engineering strain CA03 is a strain obtained by integrating DNA fragment P AOX1 -ScAro7 G141S -T AOX1Strain CA02 is obtained by integrating the gene of prephenate dehydratase from S. cerevisiae into the PNSI-8 site in the chromosome of the engineering strain CA01; engineering strain CA04 is obtained by integrating the gene of prephenate dehydratase from S. cerevisiae into the PNSI-6 site in the chromosome of the engineering strain CA03, and using P AOX1 Strain CA05 is obtained by knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) in the engineering strain CA04 using the CRISPR / Cas9 technology; strain CA06 is obtained by knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) and the endogenous phenylpyruvate decarboxylase Aro10-2 (Gene ID: 8197764) in the engineering strain CA04 using the CRISPR / Cas9 technology; strain CA07 is obtained by integrating the endogenous fructose-1, 6-bisphosphatase FBP (Gene ID: 8199670) into the PNSII-6 site in the chromosome of the engineering strain CA05, and using P AOX1 Strain CA08 is obtained by overexpressing the above-mentioned genes under the control of the promoter.

[0124] As shown in the following examples, the p-coumaric acid production strain, i.e., the recombinant Pichia pastoris strain, is constructed by the method shown in the following. Figure 1 Figure 1 As shown in the following examples, the p-coumaric acid production strain, i.e., the recombinant Pichia pastoris strain, is constructed by the method shown in the following.

[0125] In this example, the recombinant Pichia pastoris engineering strain for efficiently producing p-coumaric acid with methanol as the carbon source is successfully constructed by introducing the phenylalanine deamination pathway and / or the tyrosine deamination pathway into Pichia pastoris S12, realizing the combination of rational metabolic engineering strategies such as biosynthesis of p-coumaric acid, relieving feedback inhibition of the shikimic acid pathway, optimizing biosynthesis of aromatic amino acids, knocking out competitive metabolic pathways, and increasing precursor supply, etc. The yield of the recombinant Pichia pastoris engineering strain is significantly improved compared with the starting strain.

[0126] The initial strain (starting strain S12) used in the following examples is the existing recombinant Pichia pastoris S12 (genotype his4 - , P GAP -PpPSA-T AOX1 ,P AOX1 -PpPET2-T AOX1 , which is derived from the strain described in patent No. CN118530863A.

[0127] ​The modification methods in the following examples are all carried out by CRISPR-Cas9 technology (the strain construction method is carried out according to Cai et al. Nucleic Acids Res. 2021; 49(13): 7791-7805).

[0128] In the following, the related raw materials used in this embodiment will be described.

[0129] In the recombinant Pichia pastoris engineering strain constructed in this embodiment for efficiently synthesizing p-coumaric acid from methanol, the phenylalanine deamination pathway enzyme genes include phenylalanine deaminase AtPAL2, cinnamic acid hydroxylase AtC4H, and cytochrome P450 reductase AtATR2, all of which are derived from Arabidopsis thaliana, and are introduced into the starting strain after codon optimization and gene synthesis of Pichia pastoris, so that L-Phe in Pichia pastoris can be used to synthesize trans-cinnamic acid and p-coumaric acid.

[0130] Tyrosine deaminase FjTAL, derived from Flavobacterium johnsoniae, is introduced into the starting strain after codon optimization and gene synthesis of Pichia pastoris, so that L-Tyr (L-tyrosine) in Pichia pastoris can be used to synthesize p-coumaric acid; Aro7 G141S , derived from Saccharomyces cerevisiae, the 141st glycine is mutated to serine, which eliminates the feedback inhibition of L-Tyr on the enzyme, and the enzyme can effectively increase the yield of p-coumaric acid precursors.

[0131] Prephenate dehydratase ScPHA2, derived from Saccharomyces cerevisiae, encodes the first enzyme for tyrosine synthesis, which can effectively increase the amount of L-Tyr, the precursor of p-coumaric acid; knock out the endogenous phenylpyruvate decarboxylase Aro10 of Pichia pastoris, which can catalyze the decarboxylation of the precursor of L-Phe, phenylpyruvic acid, to benzaldehyde, and the deletion of this gene can increase the accumulation of L-Phe; Fructose-1,6-bisphosphatase FBP, a key regulatory enzyme in the anaplerotic pathway, which participates in the assimilation of formaldehyde and xylulose monophosphate pathway, overexpression of this enzyme can increase the metabolic flux of the shikimic acid pathway.

[0132] It should be noted that the key enzymes of the above pathways are controlled by the PAOX1 promoter to express the genes.

[0133] Example 1: Construction of p-coumaric acid synthesis pathway.

[0134] P-coumaric acid is synthesized by phenylalanine deaminase and tyrosine deaminase double pathway.

[0135] Firstly, the donor expression cassette for single expression of the codon-optimized whole gene-synthesized phenylalanine ammonia-lyase gene AtPAL2 (Gene ID: 824493), cinnamic acid carboxylase gene AtC4H (Gene ID: 817599), cell P450 reductase AtATR2 (Gene ID: 829144), and tyrosine ammonia-lyase FjTAL (Sequence ID: WP_012023194.1) was constructed; the donor DNA fragment included 750 bp of homologous arms upstream and downstream of the PNSI-2 or PNSII-4 or PNSII-5 site amplified from the Pichia pastoris GS115 genome (reference Yu et al. Synth Syst Biotechnol 2021, 6:63-68), the promoter P AOX1 , the terminator T AOX1 , and the structural gene.

[0136] The CRISPR / Cas9 gene editing method used in this example refers to the literature (Cai P, Duan X, Wu X, Gao L, Ye M, Zhou YJ. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Res. 2021 Jul 21;49(13):7791-7805. doi: 10.1093 / nar / gkab535. PMID: 34197615; PMCID: PMC8287956).

[0137] Specifically, the donor DNA and gRNA were transformed into the starting strain S12 or GS115 by electroporation, plated on YPD plates containing G418 antibiotic, and incubated at 30°C for 3-4 days for screening. The correct ones were verified by colony PCR and subcultured in YPD liquid medium for plasmid loss. The obtained engineering strains were fermented, extracted and detected, and it was confirmed that the integration of AtPAL2, AtC4H and AtATR2 could obtain coumaric acid (engineering strain CA01) with a yield of 1.54 g / L. On the basis of CA01, the integration of FjTAL obtained the engineering strain CA02, which had a coumaric acid yield of 1.79 g / L (see Figure 3 ).

[0138] The specific experimental steps are as follows:

[0139] Refer to the amplification primers in Table 2 to amplify the phenylalanine ammonia lyase gene AtPAL2, cinnamate carboxylase gene AtC4H, cytochrome P450 reductase gene AtATR2, and tyrosine ammonia lyase gene FjTAL from the vectors pPICZA-AtPAL2, pPICZA-AtC4H, pPICZA-AtATR2, or pPICZA-FjTAL to obtain structural gene fragments.

[0140] First, vectors pPICZA-AtPAL2 or pPICZA-AtC4H or pPICZA-AtATR2 or pPICZA-FjTAL are all obtained by synthesizing the entire gene of AtPAL2 after codon optimization and inserting it between EcoR I and Not I of the pPICZA plasmid to construct the structural gene.

[0141] Next, using the Pichia pastoris genome GS115 as a template, primers PNS*-up-F / PNS*-S-up-R and PNS*-down-F / PNS*-S-down-R (where * represents different neutral sites on the Pichia pastoris genome) were used. For specific primers, please refer to the primers shown in Table 2. Then, the neutral site on the chromosome was amplified using TOYOBO's KOD FX high-fidelity polymerase kit (neutral site reference Cai et al. Nucleic Acids Res. 2021; 49(13): 7791-7805) to obtain 750 bp homology arm fragments upstream and downstream of PNSI-2 or PNSII-4 or PNSII-5 and promoter P AOX1 With terminator T AOX1 The pPICZA vector was used to construct a promoter containing upstream and downstream homology arms of the neutral site and promoter P. AOX1 With terminator T AOX1 structural gene expression vector.

[0142] The structural gene expression vector was then double-enzyme digested, and homologous recombination was carried out with the structural gene fragment obtained above and slowly transformed into Escherichia coli TOP10 competent cells. The expression vector of the donor DNA was obtained by sequencing identification. After sequencing identification, the plasmid was double-enzyme digested to obtain the donor DNA fragment (PNS neutral site-UP-P AOX1 -Structural gene-T AOX1 -PNS neutral site-DOWN).

[0143] Preparation of competent yeast cells:

[0144] (1) Pichia pastoris strain S12 or GS115 stored at -80°C was streaked onto a YPD plate and cultured in a 30°C incubator for 3 days;

[0145] (2) Pick a single colony with a sterilized gun tip and inoculate into 10 mL YPD medium, and place it in a shaker, set the conditions as 30 ℃, 250 rpm, and culture for 20-24 h;

[0146] (3) Measure the OD600 of the initial seed liquid, and transfer it to 50 mL YPD medium with an initial OD600 of 0.5-0.6, and culture for 4-5 h until the OD600 is 1.2-1.8;

[0147] (4) In the super-clean bench, transfer the bacterial liquid to a sterile 50 mL centrifuge tube, and centrifuge in a refrigerated benchtop centrifuge, set the conditions as 4000 rpm, 5 min and 4 ℃;

[0148] (5) In the super-clean bench, remove the supernatant, add 40 mL of filtered and sterilized LDST solution to the centrifuge tube, and resuspend the bacteria thoroughly, then place it in a 30 ℃ incubator for 30 min;

[0149] (6) Centrifuge in a high-speed refrigerated benchtop centrifuge, set the conditions as 4000 rpm, 5 min, 4 ℃, and remove the supernatant in the super-clean bench. Then, transfer the bacteria in the 50 mL centrifuge tube to a sterilized 1.5 mL centrifuge tube using pre-cooled 1 mL 1 M sorbitol, resuspend the precipitate. 4 ℃, 4000 rpm, 1 min, remove the supernatant, and repeat the washing three times;

[0150] (7) Add 400 μL of ice-bath 1M sorbitol, resuspend the bacteria thoroughly, and aliquot 50 or 80 μL per tube in a 1.5 mL sterile centrifuge tube, and store it in a -80 ℃ refrigerator for standby.

[0151] The yeast electroporation method specifically includes:

[0152] (1) Linearize the successfully constructed PNSI-2-AtPAL2, PNSII-4-AtC4H+AtATR2 or PNSII-5-FjTAL plasmid using enzymes PacI and AvrII, and incubate at 37 ℃ for 2 h; specifically, the linearization system is (50 μL): 10×FastDigest Buffer 5 μL, 2.5 μL of each enzyme, 2.0 μg-3.0 μg of plasmid, ddH2O to 50 μL. After the reaction is completed, use the PCR product recovery kit to recover the linearization system;

[0153] (2) After mixing the linearized fragment with 1.5 μg-2.0 μg of each neutral site corresponding Cas9 plasmid, the mixture was added to the Pichia pastoris competent cells, mixed gently, and then transferred to the ice-bath shock cup, and placed on ice for 5 min;

[0154] (3) The shock instrument parameters were set to Fungi mode, the outside of the shock cup was wiped dry, and then 1 mL of ice-bath 1 M sorbitol was added;

[0155] (4) The mixture in the shock cup was completely aspirated and transferred to a 1.5 mL centrifuge tube. It was placed in a 30 ℃ incubator and incubated for 1.0-1.5 h. Then it was centrifuged at 5000 rpm for 2 min;

[0156] (5) 800-900 μL of supernatant was removed, the remaining supernatant was used to resuspend the bacteria, and the whole was aspirated and plated on YPD (YPDG) plates containing 200-250 μg / mL G418 antibiotic, and incubated at 30 ℃ in an incubator for 2-3 days.

[0157] With the aid of the CRISPR / Cas9 system, the above obtained donor DNA fragment and PNS neutral site-Cas9 expression plasmid (the construction method of the PNS neutral site-Cas9 expression vector (containing gRNA) is referred to Cai et al. Nucleic Acids Res. 2021; 49(13): 7791-7805) were each 3000 ng transformed into Pichia pastoris competent cells by electroporation method, the strain was plated on G418 antibiotic YPD (YPDG) selection plate, and incubated at 30 ℃ for 3-4 days for screening. The correct strain was verified by colony PCR, and the verified correct strain was inoculated in YPD antibiotic-free medium for subculture to lose plasmid. After plasmid loss, the process strain was obtained.

[0158] According to the above CRISPR / Cas9 gene editing method, AtPAL2 was integrated into Pichia pastoris S12 PNSI-2 neutral site, and the promoter P AOX1 controlled the expression of the structural gene. The engineering strain after colony PCR verification and plasmid loss was named SP. The above experimental operation was repeated, and AtC4H and AtATR2 were sequentially integrated into the engineering strain SP PNSII-4 neutral site. After verification and plasmid loss, the engineering strain CA01 was obtained, which was a Pichia pastoris cell factory for de novo synthesis of p-coumaric acid in phenylalanine ammonia lyase pathway; please refer to Figure 2 , Figure 2 The liquid chromatograms of different engineering strains synthesizing p-coumaric acid are shown. The engineering strain CA01 can specifically synthesize p-coumaric acid, and has higher yield.

[0159] Then, according to the above method, FjTAL was integrated into the PNSII-5 site on the basis of the engineering strain CA01, and the engineering strain CA02 was obtained after verification and plasmid loss, which was a Pichia pastoris cell factory for constructing phenylalanine deamination and tyrosine deamination double pathways to synthesize p-coumaric acid from scratch.

[0160] The fermentation extraction and detection steps in this example include: activating the engineering strain (engineering strain CA01 or CA02) after plasmid loss in BMGY medium, 30 °C, 220 rpm for 24 h, then transferring to 25 mL BMMY medium / 250 mL flask, initial OD 600 =1.0, under the condition of 30 °C, 220 rpm for 120 h, and determining the biomass and yield.

[0161] Among them, the extraction and detection method of cinnamic acid and p-coumaric acid is: take 200 μL of fermentation broth, vortex extraction with 800 μL of methanol, 12000 rpm, 10 minutes, take the supernatant, filter sterilization with 0.22 μm organic filter membrane, and then place it in the liquid phase detection sample bottle. Use (Synergi™ 4 μm Hydo-RP 80 Å, Kromasil, Sweden, 250 mm×4.6 mm×5 μm, Aphenomenex 100-spherical silica), the injection amount is 10 μL, the best detection wavelength is 310 nm, the mobile phase is respectively: A phase, 0.1% (v / v) trifluoroacetic acid or formic acid; B phase, 100% acetonitrile, the flow rate is 1 mL / min, and the column temperature is 30 °C). The specific gradient elution program is shown in Table 1:

[0162] Table 1: HPLC detection gradient elution program

[0163]

[0164] The fermentation broth was extracted by methanol and detected by high performance liquid chromatography to quantitatively synthesize the precursors-trans-cinnamic acid or p-coumaric acid. The yield of cinnamic acid in the engineering strain SP was 288.1 mg / L; the yield of p-coumaric acid in the engineering strain CA01 was 1.54 g / L. The yield of p-coumaric acid in the engineering strain CA02 was 1.79 g / L.

[0165] Among them, the engineering strain CA02 is used for the modification of subsequent examples.

[0166] Table 2: Amplification primers involved in the construction of phenylalanine deaminase and tyrosine deaminase double pathways

[0167]

[0168] In Table 2, the primers TEST-AtPAL2-R, TEST-AtC4H-R, TEST-AtATR2-R and TEST-FjTAL-R are used to verify whether the target gene is inserted into the target site, and the rest of the primers are used to amplify the corresponding gene fragments.

[0169] The construction of the engineering strains and the transformation, fermentation conditions in the following examples are the same as those in Example 1, and the expression and overexpression of the structural genes in the engineering strains are all controlled by the promoter P AOX1 Control.

[0170] Example 2: Release of shikimic acid pathway feedback inhibition.

[0171] In order to further increase the synthesis of coumaric acid, the key enzymes of the shikimic acid pathway in the engineering strain CA02 were released from feedback inhibition in this embodiment. According to the steps described in Example 1, the branchmutase mutant Aro7 G141S was integrated into the PNSI-8 site of the chromosome of the engineering strain CA02, and the PAOX1 promoter was used to overexpress the shikimic acid pathway branchmutase ScAro7 G141S .

[0172] The primers involved in this embodiment are shown in Table 3.

[0173] According to the steps described in Example 1, the branchmutase ScAro7 G141S (Gene ID: 856173) gene mutant nucleotide sequence at position 421 to 423 is TCG) from Saccharomyces cerevisiae was overexpressed at the PNSI-8 site of the engineering strain CA02, and the coumaric acid yield was detected after fermentation extraction.

[0174] Among them, the branchmutase ScAro7 G141S The p-CA yield of the engineering strain CA03-1 overexpressing the branchmutase was increased, and the strain grew well, with a p-CA yield of 2.19 g / L. The strain was named CA03 for subsequent modification.

[0175] Table 3: Primers involved in this embodiment

[0176]

[0177] Example 3: Overexpression of key genes in aromatic amino acid synthesis pathway.

[0178] On the basis of engineering strain CA03, the aromatic amino acid synthesis pathway was further optimized in this embodiment. Referring to the steps described in Example 1, a donor expression cassette of prephenate dehydratase ScPHA2 (Gene ID: 855400) from Saccharomyces cerevisiae was constructed by means of CRISPR / Cas9 system, and was integrated into the genome PNSI-6 site together with PNSI-6-Cas9 by electroporation. The P AOX1 The promoter controls the overexpression of the above-mentioned genes, and the specific steps of the related gene expression cassette construction and genome integration are the same as those in Example 1. After double-pathway integration, feedback inhibition removal, and optimization of the aromatic amino acid synthesis pathway, the engineering strain CA04 was obtained, and the coumaric acid yield of the engineering strain was 2.33 g / L.

[0179] Table 4: Primers involved in this embodiment

[0180]

[0181] Similarly, in Table 4, primers TEST-ScPHA2-F and TEST-ScPHA2-R are used to verify whether the target gene is inserted into the target site, and primers ScPHA2-F and ScPHA2-R are used to amplify the target gene fragment.

[0182] Example 4: Knocking out the branched pathway and increasing the precursor supply to improve the coumaric acid yield.

[0183] Referring to the primers described in Table 5, this embodiment aims to illustrate the steps of knocking out the Aro10 gene of the genome phenylpyruvate decarboxylase. The specific steps are as follows:

[0184] (1) Using Pichia pastoris GS115 as a template, the upper and lower homologous arms were obtained by KODneo enzyme PCR amplification with △Aro10-F / △Aro10-S-F and △Aro10-X-F / △Aro10-R, respectively. Then, the donor △Aro10 gene knockout fragment was obtained by KODneo enzyme overlap PCR using the above-mentioned homologous arms as templates. The gene integration fragment was composed of the Aro10 upper and lower homologous arms.

[0185] (2) Using the StgRNA-F / gRNA-Aro10-R primer pair, a DNA fragment containing the target sequence was constructed by PCR annealing program using the pPIC-Cas9 plasmid as a template skeleton, and a circular PCR product was obtained. After product recovery, it was transformed into Top10 competent cells, and positive transformants were selected. The plasmid was sent for sequencing, and the plasmid with correct sequencing was named pPIC-Aro10-Cas9.

[0186] The obtained donor △Aro10 gene knockout fragment was electroporated into the competent strain CA04, and after identification, plasmid loss, fermentation extraction and detection, the yield of p-coumaric acid reached 2.49 g / L (△Aro10-1 (Gene ID: 8199377)) and 2.7 g / L (△Aro10-2 (Gene ID: 8197764)). The strain was named CA05 (knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377)).

[0187] Similarly, referring to the above method, the strain CA06 (knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) and the endogenous phenylpyruvate decarboxylase Aro10-2 (Gene ID: 8197764)) can be prepared.

[0188] Then, on the basis of the engineering strain CA05, the supply of precursor substances PEP and E4P in the strain was strengthened. Referring to the primers (FBP-F and FBP-R) described in Table 5, referring to the steps described in Example 1, the donor DNA expression vector of the endogenous fructose-1, 6-bisphosphatase FBP (a key regulatory enzyme in the glucose production pathway, Gene ID: 8199670) of Pichia pastoris was constructed by means of CRISPR / Cas9 system, and the gene fragment was obtained by PCR amplification from the genome of Pichia pastoris GS115, and was integrated into the corresponding site of the genome by electroporation method.

[0189] After fermentation extraction and detection, the integration of FBP gene is beneficial to the synthesis of p-coumaric acid; the engineering strain CA07 which integrates the double pathways of p-coumaric acid, releases the feedback inhibition of shikimic acid, strengthens the aromatic amino acid synthesis pathway, knocks out the branch metabolic pathway of aromatic amino acid synthesis and enhances the supply of PEP and E4P, has a yield of 2.84 g / L (p-coumaric acid). Figure 3 )。

[0190] Please see Figure 3 , Figure 3 The results of the shake flask fed-batch fermentation of p-coumaric acid by the engineering strains CA01-CA07 are shown in the schematic diagram, and the yield of the engineering strains CA01-CA07 increases in turn, indicating that the directional modification in this embodiment can jointly act from multiple aspects to systematically optimize the synthesis pathway of p-coumaric acid, and finally construct the recombinant Pichia pastoris strain CA07 for efficiently producing p-coumaric acid with methanol as the carbon source.

[0191] Table 5: Primers involved in this embodiment

[0192]

[0193] Note: The underlined part is the gRNA sequence.

[0194] Example 5: Batch fed fermentation of engineered strain CA05.

[0195] To test the high-density fermentation ability of the engineered strain, a fed-batch fermentation experiment was performed in a 15 L fermenter; the above-obtained engineered strain CA05 was taken as an exemplary strain for the fed-batch fermentation in a 15 L fermenter.

[0196] The above test steps are specifically as follows:

[0197] The engineered strain CA05 was streaked on a YPD plate for activation and incubated at 30°C for 3 days; then inoculated in 10 mL / 50 mL of a triangular flask YPD liquid medium for culture, 30°C, 220 rpm, 24 h, to obtain a primary seed; then inoculated in 200 mL / 500 mL of a triangular flask YPD liquid medium at a 4% inoculation amount, 30°C, 220 rpm, 20-24 h, to obtain a secondary seed liquid. Batch fed fermentation was performed using a 15 L Shanghai Bailun bioreactor system, and the batch fed fermentation volume was 6 L (15 L fermenter); the seed liquid was inoculated in the fermenter at a 8% inoculation volume.

[0198] The batch fed fermentation used an inorganic salt basic medium (BSM with the addition of 5 g / L of yeast powder and 10 g / L of proteose peptone), and the pH was 5.5. In the batch fed fermentation, glycerol was consumed, the dissolved oxygen was controlled to be above 20%, and the next stage was entered when the dissolved oxygen rebounded; 50% glycerol was added to reach the desired OD induction period (200-300), the addition speed was controlled to be 6-11 g / L / h, and the dissolved oxygen was controlled to be above 20%. OD 600 When the desired induction value was reached, the starvation period was entered, the addition of 50% glycerol was stopped for 30-60 min, the remaining carbon source in the medium was consumed, and during this period, the temperature was automatically adjusted to 25°C, and the pH was 6.0; then the methanol induction period was entered, the methanol addition speed was controlled to be 4-6 g / L / h, the dissolved oxygen was controlled to be no less than 20%, and the methanol induction time was controlled to be 120 h. During this period, the fermentation broth was sampled every 12 h for methanol extraction, and the p-coumaric acid yield was detected.

[0199] Figure 4 shows the results of the fermentation of the engineered strain CA05 in Example 5 in a 15 L bioreactor to produce p-coumaric acid; as shown in Figure 4 the first stage: after about 44 h of carbon source consumption period, OD 600 reached 38.8, 50% glycerol was added to the fermenter to maintain rapid cell growth, and the flow rate was controlled to be 6-11 g / L / h. When the glycerol supply was completed, OD 600 was about 194.

[0200] Second stage: methanol was added as the only carbon source, and the flow rate was controlled at 3-6 g / L / h. The biomass continued to increase, and p-CA was also gradually accumulated. After 84 h of induction, OD 600 reached 202.9, and the p-CA yield reached 18.55±3.48 g / L (344.37 mg / g DCW), with a yield of 662.605.30 mg / L / d (220.83 mgL / h), which was the highest p-CA titer so far.

[0201] In summary, the construction method provided in the embodiment can systematically optimize the synthesis pathway of p-coumaric acid by integrating the p-coumaric acid double pathway, relieving the feedback inhibition of shikimic acid, strengthening the aromatic amino acid synthesis pathway, knocking out the branch metabolic pathway of aromatic amino acid synthesis, and enhancing the supply of PEP and E4P. Finally, a recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as the carbon source is constructed. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

[0202] The technical solutions provided by the embodiments of the present application are described in detail above, and the principles and implementation modes of the embodiments of the present application are described by applying specific examples. The above description of the embodiments is only applicable to help understand the principles of the embodiments of the present application; at the same time, for those skilled in the art, according to the embodiments of the present application, the specific implementation modes and application ranges will be changed, and the above description of the embodiments should not be understood as limiting the present application.

Claims

1. A method for constructing a recombinant Pichia pastoris strain, characterized in that: The construction method comprises: The Pichia pastoris strain was used as the host strain, and a dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase was constructed in the host strain to obtain the first engineered strain; the Pichia pastoris strain was a recombinant Pichia pastoris strain S12, and the genotype of the recombinant Pichia pastoris strain S12 was his4 - , P GAP -PpPSA-T AOX1 , P AOX1 -PpPET2-T AOX1 ; Eliminating chorismate feedback inhibition of the first engineered strain based on metabolic engineering to obtain a second engineered strain; Optimizing the aromatic amino acid synthesis pathway of the second engineered strain to obtain a third engineered strain; knocking out the branch metabolic pathway of the third engineered strain to obtain a fourth engineered strain; increasing the precursor supply of the fourth engineered strain to obtain a recombinant Pichia pastoris strain; The dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain, comprising: The gene encoding phenylalanine ammonia lyase AtPAL2 was integrated into the host strain's chromosome PNS1-2 site, the gene encoding cinnamate hydroxylase AtC4H and the gene encoding P450 reductase AtATR2 were integrated into the host strain's chromosome PNSII-4 site, and the gene encoding tyrosine ammonia lyase FjTAL was integrated into the host strain's chromosome PNSII-5 site; The Gene ID of the gene encoding the phenylalanine ammonia lyase AtPAL2 is 824493; the Gene ID of the gene encoding the cinnamate hydroxylase AtC4H is 817599; the Gene ID of the gene encoding the P450 reductase AtATR2 is 829144; the Sequence ID of the gene encoding the tyrosine ammonia lyase FjTAL is WP_012023194.1; The method of removing chorismate feedback inhibition of the first engineered strain based on metabolic engineering comprises: Chorismate mutase mutant Aro7 G141S The coding gene was integrated into the chromosome PNSI-8 site of the first engineering strain using P AOX1 Promoter-controlled chorismate mutase mutant Aro7 G141S expression; The chorismate mutase mutant Aro7 G141S It is prepared by subjecting chorismate mutase Aro7 to a G141S mutation; the gene encoding the chorismate mutase Aro7 has a Gene ID of 856173; The optimization of the aromatic amino acid synthesis pathway of the second engineered strain comprises: The gene encoding the prephenate dehydratase ScPHA2 was integrated into the chromosome PNSI-6 site of the second engineered strain using P AOX1 The promoter controls the expression of ScPHA2, wherein the gene encoding the ScPHA2 has a GeneID of 855400. The knockout of the branched metabolic pathway of the third engineered strain comprises: The endogenous phenylpyruvate decarboxylase Aro10-1 encoding gene of the third engineered strain is knocked out using gene editing technology; the Gene ID of the endogenous phenylpyruvate decarboxylase Aro10-1 encoding gene is 8199377; The method of increasing the precursor supply of the fourth engineered strain comprises: The endogenous fructose-1,6-bisphosphatase encoding gene was integrated into the chromosome PNSII-6 site of the fourth engineered strain using P AOX1 The promoter controls the overexpression of endogenous fructose-1,6-bisphosphatase; the Gene ID of the gene encoding the endogenous fructose-1,6-bisphosphatase is 8199670.

2. The construction method according to claim 1, characterized in that The gene editing technology is CRISPR / Cas9 gene editing technology.

3. A recombinant Pichia pastoris strain, characterized in that: The recombinant Pichia pastoris strain is prepared by the construction method according to any one of claims 1-2.

4. Use of the recombinant Pichia pastoris strain according to claim 3 in synthesizing p-coumaric acid.

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