Recombinant pichia pastoris strain as well as construction method and application thereof

Abstract

The embodiment of the invention provides a recombinant pichia pastoris strain as well as a construction method and application thereof. The construction method comprises the following steps: by taking a pichia pastoris strain as a host strain, constructing a dual-synthesis path of phenylalanine ammonialyase and tyrosine ammonialyase in the host strain to obtain a first engineering strain; removing chorismic acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing an aromatic amino acid synthesis route of the second engineering strain to obtain a third engineering strain; knocking out a branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing precursor supply of the fourth engineering strain to obtain a recombinant pichia pastoris strain; according to the construction method provided by the embodiment of the invention, the synthesis path of p-coumaric acid can be systematically optimized, the recombinant pichia pastoris strain for efficiently producing p-coumaric acid by taking methanol as a carbon source is constructed, and compared with a host strain, the recombinant pichia pastoris strain has the advantage that the yield of p-coumaric acid is remarkably increased.

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: In a first aspect, the present invention provides a method for constructing a Pichia pastoris strain, the method comprising: 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; 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; The precursor supply of the fourth engineered strain was increased to obtain a recombinant Pichia pastoris strain.

[0007] As one embodiment of the present application, the dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain, comprising: The phenylalanine ammonia lyase gene AtPAL2 was integrated into the chromosome PNSI-2 site of the host strain, the cinnamate hydroxylase AtC4H and P450 reductase AtATR2 were integrated into the chromosome PNSII-4 site of the host strain, and the tyrosine ammonia lyase FjTAL was integrated into the chromosome PNSII-5 site of the host strain.

[0008] As one embodiment of the present application, the metabolic engineering-based removal of chorismate feedback inhibition in the first engineered strain includes: Chorismate mutase mutant Aro7 G141S Integrate into the chromosome PNSI-8 site of the first engineered strain, and use the PAOX1 promoter to control the chorismate mutase mutant Aro7 G141S expression.

[0009] As an embodiment of the present application, the chorismate mutase mutant Aro7 G141S It is produced by the G141S mutation of chorismate mutase Aro7.

[0010] As an embodiment of the present application, the optimization of the aromatic amino acid synthesis pathway of the second engineered strain includes: The prephenate dehydratase ScPHA2 was integrated into the chromosome PNSI-6 site of the second engineered strain, and the expression of the prephenate dehydratase ScPHA2 was controlled by the PAOX1 promoter.

[0011] As an embodiment of the present application, the knockout of the branch metabolic pathway of the third engineered strain includes: Gene editing technology was used to knock out the endogenous phenylpyruvate decarboxylase Aro10 in the third engineered strain.

[0012] As an embodiment of the present application, the gene editing technology is CRISPR / Cas9 gene editing technology.

[0013] As an embodiment of the present application, the increasing the precursor supply of the fourth engineered strain includes: Endogenous fructose-1,6-bisphosphatase was integrated into the chromosome PNSII-6 site of the fourth engineered strain, and the overexpression of endogenous fructose-1,6-bisphosphatase was controlled by the PAOX1 promoter.

[0014] In a second aspect, the embodiments of the present application provide a recombinant Pichia pastoris strain, which is prepared using the construction method described in the first aspect.

[0015] In a third aspect, the examples of the present application propose the use of the recombinant Pichia pastoris strain described in the second aspect in synthesizing p-coumaric acid.

[0016] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects: The embodiment of the present application conducts a targeted transformation of the host strain. First, a dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain to optimize the metabolic flow of aromatic amino acids in the Pichia pastoris strain; then, by relieving the feedback inhibition of chorismate, the limitation of the chorismate pathway is broken, ensuring that the pathway can operate continuously and efficiently, stably generating intermediates such as chorismate, and providing a 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 increased, sufficient substrate is provided for the synthesis of p-coumaric acid, and the production of p-coumaric acid is directly promoted; furthermore, by knocking out the branched metabolic pathway, the ineffective consumption of carbon sources and related energy is reduced, so that intermediates such as chorismate flow more to the tyrosine synthesis pathway, thereby improving the carbon flow allocation efficiency of p-coumaric acid synthesis; finally, increasing the precursor supply is conducive to transforming the upstream metabolic pathway, increasing the supply of PEP and E4P, and can improve the metabolic flux of the shikimic acid pathway from the source, providing more sufficient starting materials for chorismate synthesis, thereby increasing the entire metabolic flow of p-coumaric acid synthesis.

[0017] In summary, the construction method provided in the examples of the present application can systematically optimize the synthesis pathway of p-coumaric acid by optimizing metabolic flux, removing restrictions, strengthening direct precursors, reducing diversion, and increasing sources (precursor supply). Ultimately, a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source was constructed. Compared with the host strain, the recombinant Pichia pastoris strain has significantly improved p-coumaric acid production.

[0018] Additional aspects and advantages of the present application will be given in part in the following description, which will become apparent from the following description, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of metabolic engineering modification of a Pichia pastoris engineered strain for synthesizing p-coumaric acid is shown in this example; Figure 2 shows the liquid chromatograms of p-coumaric acid synthesis by different engineered strains; Figure 3 A schematic diagram showing the p-coumaric acid production results of engineered strains CA01-CA07 by shake flask fed-batch fermentation; Figure 4 Schematic diagram showing the results of fermentation production of p-coumaric acid by the engineered strain CA05 in Example 5 in a 15 L bioreactor. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only some of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0021] It will be understood that when used in this specification and the appended claims, the terms “comprises” and “comprising” indicate the presence of described features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0022] It should also be understood that the terms used in this 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, the singular forms "a", "an", and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0023] The recombinant Pichia pastoris strains, their construction methods, and uses according to the embodiments of the present application are described in detail below.

[0024] First, the construction method of the recombinant Pichia pastoris strain according to the first aspect of this embodiment is described.

[0025] Construction method In view of this, this embodiment provides a method for constructing a Pichia pastoris strain, through which a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source was successfully constructed.

[0026] Specifically, the construction method of this embodiment includes the following steps: S1. 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.

[0027] It is understandable that the host strain of this embodiment can adopt an existing known Pichia pastoris strain; for example, the recombinant Pichia pastoris S12 described in Chinese patent publication number CN118530863A is used as the host strain (hereinafter referred to as starting strain S12) for subsequent targeted transformation.

[0028] In step S1, a dual synthesis pathway of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) is simultaneously constructed in Pichia pastoris. This is achieved by synergistically utilizing two precursors (phenylalanine and tyrosine) to break through the metabolic bottleneck of a single pathway and maximize the production of p-coumaric acid.

[0029] Specifically, although Pichia pastoris can synthesize two aromatic amino acids, phenylalanine and tyrosine, the synthesis flux of the two may differ due to metabolic regulation (e.g., accumulation of one amino acid is more abundant). Currently, a single pathway (PAL or TAL) can only utilize one amino acid as a substrate, which may be limited by the supply of that amino acid, resulting in wasteful carbon flow.

[0030] First, the dual pathway used in step S1 can simultaneously utilize phenylalanine and tyrosine, directing the metabolic flow of both amino acids toward the synthesis of the target aromatic compound (p-coumaric acid), maximizing the utilization of aromatic amino acid reserves within the cell and improving carbon source conversion efficiency.

[0031] Secondly, the dual pathway can also increase production through metabolic complementarity: for example, when tyrosine supply is insufficient, cinnamic acid catalyzed by PAL can be converted into p-coumaric acid by hydroxylase, indirectly supplementing the synthesis flux of the target product and avoiding the bottleneck limitation of a single pathway.

[0032] Furthermore, in aromatic amino acid metabolism, the synthesis of phenylalanine and tyrosine compete (e.g., they share upstream intermediates such as chorismate and prephenic acid). Over-enhancing a single pathway can lead to excessive accumulation of the other amino acid (e.g., enhancing only TAL can result in excess phenylalanine), triggering feedback inhibition or byproduct formation. The dual pathway employed in step S1 balances the consumption rates of the two amino acids through the synergistic action of the two enzymes, reducing the irrational accumulation of metabolic intermediates, maintaining homeostasis in the intracellular metabolic network, and minimizing adverse effects on host strain growth.

[0033] Therefore, by constructing the dual synthesis pathways of PAL and TAL in step S1, the metabolic flow of aromatic amino acids in the Pichia pastoris strain can be optimized through the strategies of diversion utilization and complementary enhancement, ultimately achieving an increase in the yield of the target aromatic compound (p-coumaric acid) and enhanced synthetic stability, laying the foundation for the subsequent provision of more efficient engineered strains.

[0034] S2. Based on metabolic engineering, the feedback inhibition of chorismate in the first engineered strain is relieved to obtain a second engineered strain.

[0035] It is understandable that the chorismate pathway is the core stage in the shikimate pathway for producing chorismate (a key intermediate in the synthesis of aromatic compounds). The key enzymes in this pathway (such as chorismate synthase) will be inhibited by downstream end products (such as aromatic amino acids such as tyrosine and phenylalanine) through a feedback inhibition mechanism (to avoid excessive accumulation of products).

[0036] The release of feedback inhibition of the chorismate pathway in step S2 is to perform targeted genetic modification on the metabolic network of the first engineered strain through tools such as molecular biology, genetic engineering and systems biology to achieve the goal of releasing feedback inhibition of the chorismate node.

[0037] For example, these enzymes can be modified through genetic engineering methods (such as site-directed mutagenesis of key enzyme genes) so that they are no longer inhibited by downstream end products.

[0038] Based on this, step S2 can break the limitation of the chorismic acid pathway by relieving the feedback inhibition of chorismic acid, ensuring that the pathway can continue to operate efficiently and stably generate intermediates such as chorismic acid, and provide sufficient material basis for the subsequent synthesis of p-coumaric acid.

[0039] S3. Optimize the aromatic amino acid synthesis pathway of the second engineered strain to obtain a third engineered strain.

[0040] It should be noted that aromatic amino acids (especially tyrosine) are direct precursors for the synthesis of p-coumaric acid (p-coumaric acid is produced from tyrosine via TAL catalysis). Optimizing the aromatic amino acid synthesis pathway in step S3 refers to enhancing the efficiency of this pathway through genetic engineering.

[0041] For example, by overexpressing key enzymes in the pathway (such as prephenate dehydrogenase), prephenate can be catalyzed to produce p-hydroxyphenylpyruvate and ultimately tyrosine; alternatively, by balancing branch flow, excessive competition for tyrosine synthesis by the tryptophan / phenylalanine branch can be effectively avoided.

[0042] Therefore, by optimizing the synthesis pathway of aromatic amino acids in step S3, the yield of the direct precursor tyrosine can be significantly increased (directly increasing the supply of p-coumaric acid precursor), providing sufficient substrate for the synthesis of p-coumaric acid, and directly promoting the formation of p-coumaric acid.

[0043] S4. Knock out the branch metabolic pathway of the third engineered strain to obtain the fourth engineered strain.

[0044] As the core intermediate product of the shikimic acid pathway, chorismic acid not only flows to tyrosine synthesis (target pathway), but also diverts to other non-target products through branched metabolic pathways, such as the synthesis of tryptophan and phenylalanine, or the production of by-products such as quinolinic acid and homogentisate.

[0045] The knockout of branch metabolic pathways in step S4 is to block the flow of intermediate products to non-target products by knocking out key enzyme genes in these branch pathways (such as the anthranilate synthase gene for tryptophan synthesis).

[0046] Therefore, step S4 can reduce the ineffective consumption of carbon sources and related energy by knocking out the branched metabolic pathway, so that intermediates such as branched acid flow more to the tyrosine synthesis pathway, thereby improving the carbon flow allocation efficiency of p-coumaric acid synthesis.

[0047] S5. Increase the precursor supply of the fourth engineered strain to obtain a recombinant Pichia pastoris strain.

[0048] Among them, the initiation of the shikimate pathway depends on the precursors provided by upstream sugar metabolism, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), both of which are substrates of DAHP synthase.

[0049] Increasing the precursor supply in step S5 is to increase the supply 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 activity of key enzymes in the pentose phosphate pathway to increase E4P production).

[0050] That is, increasing the supply of precursors is beneficial to the transformation of upstream metabolic pathways, increasing the supply of PEP and E4P, and improving the metabolic flux of the shikimic acid pathway from the source, providing more sufficient starting materials for the synthesis of chorismic acid, thereby increasing the entire metabolic flow of p-coumaric acid synthesis.

[0051] In summary, the construction method provided in this example can systematically optimize the synthesis pathway of p-coumaric acid by optimizing metabolic flux, removing restrictions, strengthening direct precursors, reducing diversion, and increasing the source (precursor supply). Ultimately, a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source was constructed. Compared with the host strain, the recombinant Pichia pastoris strain has significantly increased p-coumaric acid production.

[0052] The following further describes the relevant steps of the above construction method.

[0053] In step S1, a dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain, comprising: The phenylalanine ammonia lyase gene AtPAL2 was integrated into the chromosome PNSI-2 site of the host strain, the cinnamate hydroxylase AtC4H and P450 reductase AtATR2 were integrated into the chromosome PNSII-4 site of the host strain, and the tyrosine ammonia lyase FjTAL was integrated into the chromosome PNSII-5 site of the host strain.

[0054] It can be understood that the chromosomal PNSI-2 site, PNSII-4 site and PNSII-5 site are neutral integration sites screened through gene editing technology, which can be used for the stable insertion and efficient expression of exogenous genes. At the same time, the insertion of exogenous genes does not affect the basic growth and methanol utilization ability of Pichia pastoris (Pichia pastoris uses methanol as a carbon source and inducer).

[0055] This example integrates the phenylalanine ammonia-lyase gene, AtPAL2, into the host strain's chromosome at the PNSI-2 locus. PNSI-2 is a neutral locus with high expression potential in Pichia pastoris, making it suitable for driving overexpression of key rate-limiting enzymes. Integrating AtPAL2 into this locus significantly increases its expression via a strong promoter (such as PAOX1), enhancing the conversion flux of phenylalanine to cinnamic acid and providing ample substrate for downstream reactions.

[0056] Secondly, AtC4H (from Arabidopsis thaliana) is a cytochrome P450 enzyme that catalyzes the para-hydroxylation of cinnamic acid to form p-coumaric acid, serving as a key enzyme in the linking of cinnamic and p-coumaric acids. AtATR2 (from Arabidopsis thaliana) is a P450 reductase that provides electrons to AtC4H. The two require synergistic action for efficient catalytic reaction. In this example, the selected PNSII-4 locus is suitable for tandem multi-gene integration, enabling co-expression of AtC4H and AtATR2 via the same promoter. This ensures a balanced ratio of the two enzymes and avoids metabolic bottlenecks caused by insufficient expression of either enzyme (e.g., cinnamic acid accumulation and toxicity).

[0057] Furthermore, FjTAL catalyzes the direct deamination of tyrosine to p-coumaric acid, providing a second independent pathway for the synthesis of p-coumaric acid (independent of the cinnamic acid intermediate). The PNSII-5 locus offers excellent genetic stability and metabolic compatibility, making it suitable for introducing key enzymes of this independent pathway. In this example, integrating FjTAL at this locus avoids competition with the expression of the first two genes while leveraging its minimal impact on host growth to ensure stable operation of the second pathway.

[0058] Therefore, this example constructs a dual pathway for synthesizing p-coumaric acid based on the above-mentioned means to maximize carbon source utilization; the two pathways utilize phenylalanine and tyrosine synthesized by Pichia pastoris as substrates, respectively, avoiding the limitation of insufficient supply of a single amino acid, and directing the metabolic flow of both aromatic amino acids to the target product, significantly improving the conversion efficiency of the carbon source to p-coumaric acid.

[0059] At the same time, those skilled in the art will recognize that expressing only AtPAL2 without AtC4H / AtATR2 can lead to cinnamic acid accumulation (which is cytotoxic); expressing only FjTAL can be limited by tyrosine synthesis flux (tyrosine and phenylalanine synthesis compete in Pichia pastoris). This implementation, based on the coordinated expression of these three components, achieves efficient substrate-intermediate-product flow: cinnamic acid generated by AtPAL2 is rapidly converted by AtC4H / AtATR2, while FjTAL diverts tyrosine metabolism, avoiding overloading of a single pathway and reducing the risk of toxicity.

[0060] This example also leverages site-specific expression to optimize expression intensity and increase product yield. Specifically, the high expression of PNSII-2 enhances AtPAL2 activity, ensuring sufficient cinnamic acid precursors. The multigene coexpression capability of PNSII-4 ensures the synergistic catalytic efficiency of AtC4H and AtATR2. The stability of PNSII-5 supports sustained expression of FjTAL, maintaining flux in the second pathway. This example employs a site-gene matching design to avoid metabolic bottlenecks caused by imbalanced expression intensity, ultimately achieving a synergistic increase in p-coumaric acid production.

[0061] Therefore, this step should use the strategy of site-by-site integration + dual-pathway collaboration to accurately assign the key enzyme genes for p-coumaric acid synthesis to the optimized sites of Pichia pastoris, which not only utilizes the functional complementarity of each gene, but also plays the expression advantages of different sites, and ultimately achieves the maximization of p-coumaric acid synthesis flux and the balance of the metabolic network, laying the foundation for the construction of Pichia pastoris engineered strains with high p-coumaric acid production.

[0062] In step S2, the chorismate feedback inhibition of the first engineered strain is released based on metabolic engineering, comprising: Chorismate mutase mutant Aro7 G141S Integrate into the chromosome PNSI-8 site of the first engineered strain, and use the PAOX1 promoter to control the chorismate mutase mutant Aro7 G141S expression.

[0063] Chorisate mutase (Aro7, from Saccharomyces cerevisiae) catalyzes the intramolecular rearrangement of chorismate (a core intermediate in the shikimate pathway) to produce prephenate, a direct precursor for the synthesis of phenylalanine and tyrosine. Wild-type Aro7 activity is strongly inhibited (feedback inhibition) by the downstream end product tyrosine, limiting prephenate synthesis.

[0064] It is understandable that Aro7 of this embodiment G141SIt is a mutant obtained by site-directed mutagenesis. After the glycine (G) at position 141 was replaced by serine (S), the feedback inhibition of tyrosine on it was released. Even if tyrosine accumulates in the cell, it can still maintain efficient catalytic activity and continue to promote the conversion of chorismate to prephenic acid.

[0065] The site PNSI-8 selected in this example belongs to the neutral integration site of Pichia pastoris. This site is located in the non-essential region of the genome. After the exogenous gene is inserted, it does not interfere with the basal metabolism and growth of the host strain itself. At the same time, it has good genetic stability, which can ensure the integration of Aro7. G141S The gene is stably inherited along the host chromosome, avoiding the common gene loss problem in plasmid expression systems; at the same time, this site is compatible with other PNS family sites (such as PNSI-2 and PNSII-4) and can support the coordinated integration of multiple genes (for example, forming metabolic pathways with genes such as AtPAL2 and FjTAL).

[0066] As mentioned above, chorismate is a key branch point in the shikimate pathway: in addition to flowing to prephenate (aromatic amino acid synthesis), it may also be diverted to the tryptophan synthesis pathway or other byproduct metabolism. G141S The efficient expression of PAOX1 can competitively enhance the flow of branched acid to prephenic acid, reduce the waste of carbon sources to non-target pathways, increase the carbon flow ratio of aromatic amino acid synthesis, and indirectly provide more sufficient substrates for downstream products such as p-coumaric acid. Specifically, this example can avoid Aro7 by inducing the regulation of PAOX1 promoter. G141S Overexpression during the rapid growth phase of Pichia pastoris (when cells need to prioritize resource allocation for proliferation) reduces inhibition of host growth.

[0067] The neutrality of the PNSI-8 site allows it to synergize with genes (AtPAL2, AtC4H / AtATR2, FjTAL) integrated into other sites (PNSI-2, PNSII-4, PNSII-5): Aro7 G141S The provided pathway can more efficiently convert it into p-coumaric acid, forming a complete metabolic network of upstream enhancement-midstream transformation-downstream synthesis, and ultimately significantly increasing the yield of the target product.

[0068] In summary, step S2 involves converting the chorismate mutase mutant Aro7 into G141S Integrate into the chromosome PNSI-8 site of the first engineered strain, and use the PAOX1 promoter to control the chorismate mutase mutant Aro7 G141S The core purpose of the expression of chorismate is to enhance the conversion flux of chorismate to prephenic acid, increase the supply of phenylalanine and tyrosine, provide sufficient precursors for the synthesis of downstream p-coumaric acid, and take into account the balance between host growth and product synthesis.

[0069] As an implementation of this example, the chorismate mutase mutant Aro7 G141S It is produced by the G141S mutation of chorismate mutase Aro7.

[0070] Among them, the chorismate mutase mutant Aro7 G141S It is obtained by mutation in which the glycine (G) at position 141 of chorismate mutase Aro7 is replaced by serine (S). This design relieves the feedback inhibition of phenylalanine. Even if tyrosine accumulates in the cell, it can still maintain efficient catalytic activity and continuously promote the conversion of chorismate to prephenylate.

[0071] In step S3, the aromatic amino acid synthesis pathway of the second engineered strain is optimized, including: The prephenate dehydratase ScPHA2 was integrated into the chromosome PNSI-6 site of the second engineered strain, and the expression of the prephenate dehydratase ScPHA2 was controlled by the PAOX1 promoter.

[0072] Understandably, the prephenate dehydratase ScPHA2 is a key, rate-limiting enzyme in the phenylalanine biosynthesis pathway, catalyzing the dehydration and decarboxylation of prephenate to phenylpyruvate (the immediate precursor of phenylalanine, which is subsequently converted to phenylalanine via transaminases). In Pichia pastoris, endogenous enzyme activity is low or subject to strong feedback inhibition, creating a natural bottleneck for phenylalanine biosynthesis.

[0073] The PNSI-6 selected in this example is a neutral integration site in Pichia pastoris. Insertion of exogenous genes into this site does not interfere with host metabolism and growth, and exhibits good transcriptional activity, making it suitable for stable expression of exogenous genes. Furthermore, it can collaborate with other neutral sites (such as the aforementioned PNSI-2 and PNSII-4) to carry multiple gene metabolic pathways, avoiding expression competition caused by single-site integration.

[0074] PAOX1 is a highly inducible promoter commonly used in Pichia pastoris. Its activity is highly induced by methanol (expression levels after induction are significantly higher than those of constitutive promoters). Controlling ScPHA2 expression through PAOX1 enables controlled, high expression of the enzyme. ScPHA2 activity can be precisely enhanced by methanol induction during specific stages of fermentation, such as the late logarithmic growth phase.

[0075] Therefore, this example, incorporating a mutation that relieves feedback inhibition and driving ScPHA2 overexpression via PAOX1, not only improves the conversion efficiency of prephenate to phenylpyruvate, driving more chorismate into the phenylalanine biosynthesis pathway, but also counteracts the negative effects of feedback inhibition, allowing phenylalanine to accumulate in the cell and provide ample substrate for downstream p-coumaric acid synthesis (catalyzed by PAL). Furthermore, overexpression of ScPHA2 specifically enhances carbon flow through the phenylalanine branch, reducing the diversion of intermediates to other non-target pathways and improving carbon source utilization efficiency.

[0076] At the same time, step S3 can also synergize with other genes already integrated into the Pichia pastoris strain in steps S1 and S2, such as AtPAL2, AtC4H / AtATR2, and other genes. The enhanced expression of ScPHA2 can form an upstream donor-downstream conversion synergy with these pathways. In particular, upstream ScPHA2 increases the supply of phenylalanine, and downstream enzymes such as AtPAL2 efficiently convert phenylalanine into p-coumaric acid, ultimately forming a complete and efficient pathway from phenylalanine synthesis to p-coumaric acid conversion, significantly increasing the yield of the target product p-coumaric acid.

[0077] In step S4, the branch metabolic pathway of the third engineered strain is knocked out, including: Gene editing technology was used to knock out the endogenous phenylpyruvate decarboxylase Aro10 in the third engineered strain.

[0078] For example, the endogenous phenylpyruvate decarboxylase Aro10-1 of the third engineered strain is knocked out by gene editing technology; and / or, the endogenous phenylpyruvate decarboxylase Aro10-2 of the third engineered strain is knocked out by gene editing technology.

[0079] In step S4, the core function of knocking out the endogenous phenylpyruvate decarboxylases Aro10-1 and Aro10-2 in Pichia pastoris is to block the non-target metabolic diversion of phenylpyruvate and enhance the synthesis flux of phenylalanine and p-coumaric acid.

[0080] Phenylpyruvate decarboxylase (Aro10 family) is a key enzyme involved in the degradation of aromatic amino acids in Pichia pastoris. Its core function is to catalyze the decarboxylation of phenylpyruvate to phenylacetaldehyde (which can be further converted into byproducts such as phenylethanol). Phenylpyruvate is both a direct precursor for phenylalanine synthesis and an intermediate in phenylalanine degradation (phenylalanine can be reversed to phenylpyruvate by transaminases). Aro10-1 and Aro10-2 are the primary homologous enzymes catalyzing this reaction in Pichia pastoris. Their functional redundancy allows them to jointly catalyze the degradation and diversion of phenylpyruvate.

[0081] Understandably, in the intact strain, a futile cycle of phenylpyruvate synthesis and degradation occurs. On the one hand, enzymes like ScPHA2 catalyze the production of phenylpyruvate for phenylalanine synthesis; on the other hand, Aro10-1 and Aro10-2 decarboxylate phenylpyruvate to phenylacetaldehyde, resulting in the precursor being consumed in the synthesis of byproducts (such as phenylethanol). Knocking out these two genes in this example completely blocks the degradation shunt of phenylpyruvate, allowing more phenylpyruvate to be used for phenylalanine synthesis. This directly increases the precursor reserve in the target metabolic pathway, provides more substrate for enzymes like PAL, and promotes the efficient synthesis of the target product, p-coumaric acid.

[0082] Therefore, the purpose of knocking out Pichia pastoris Aro10-1 and Aro10-2 in step S4 is to maximize the synthesis and accumulation of phenylalanine by blocking the degradation shunt of phenylpyruvate, providing sufficient precursors for the downstream product p-coumaric acid, while reducing by-product consumption and toxicity, and ultimately synergizing with other metabolic engineering modifications to significantly improve the yield and production efficiency of the target product p-coumaric acid.

[0083] As an implementation method of this embodiment, the gene editing technology is CRISPR / Cas9 gene editing technology.

[0084] Among them, CRISPR / Cas9 gene editing technology boasts high targeting accuracy, ease of use, and high efficiency. Through the complementary pairing of artificially designed guide RNA (sgRNA) with the target gene sequence, CRISPR / Cas9 precisely directs the Cas9 protein to cleave specific DNA sites. Off-target effects can be significantly reduced through optimization, ensuring editing specificity. Furthermore, compared to earlier technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 eliminates the need for complex protein design and modification, requiring only the synthesis of specific sgRNAs for editing. This streamlined construction process and short experimental cycles significantly lower the technical barriers to entry for gene editing.

[0085] The CRISPR / Cas9 gene editing technology used in this embodiment is a conventional technology known in the art.

[0086] In step S5, increasing the precursor supply of the fourth engineered strain includes: Endogenous fructose-1,6-bisphosphatase was integrated into the chromosome PNSII-6 site of the fourth engineered strain, and the overexpression of endogenous fructose-1,6-bisphosphatase was controlled by the PAOX1 promoter.

[0087] It should be noted that step S5 integrates endogenous fructose-1,6-bisphosphatase (FBPase) into the PNSII-6 site of the Pichia pastoris chromosome and controls its overexpression through the PAOX1 promoter. This is a precise design for optimizing carbon metabolic flow. Its core function is to strengthen the gluconeogenesis pathway and increase the supply of precursors for p-coumaric acid synthesis.

[0088] Pichia pastoris strains can use methanol as a carbon source during fermentation. Methanol is metabolized to generate energy and intermediates (such as dihydroxyacetone phosphate), which then need to be converted to sugar metabolic intermediates (such as fructose-6-phosphate, F6P) through gluconeogenesis. Overexpressing FBPase can accelerate the conversion of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P), overcoming the rate-limiting bottleneck of gluconeogenesis and allowing more non-sugar carbon sources to accumulate in F6P.

[0089] F6P is the starting substrate of the pentose phosphate pathway (PPP) and can be further converted into erythrose 4-phosphate (E4P). E4P is a key precursor of the shikimate pathway. The shikimate pathway requires E4P to combine with phosphoenolpyruvate (PEP) to produce DAHP.

[0090] In unmodified Pichia pastoris, metabolic flux from non-sugar carbon sources, such as methanol, may be directed toward energy production (e.g., the TCA cycle) rather than toward the production of sugar metabolic intermediates required for the synthesis of target products. Overexpression of FBPase can redirect this carbon flux, allowing more carbon sources to be used for the synthesis of target pathways and improving the efficiency of carbon source conversion to target products.

[0091] Therefore, the core purpose of step S5 is to drive the overexpression of endogenous FBPase at the PNSII-6 site through the PAOX1 promoter, strengthen the gluconeogenesis pathway of Pichia pastoris, increase the supply of the key precursor (E4P) for p-coumaric acid synthesis, and ultimately collaborate with other metabolic engineering strategies to improve the yield of the target product p-coumaric acid and the carbon source utilization efficiency, providing an efficient cell factory foundation for industrial production.

[0092] Next, the recombinant Pichia pastoris strain according to the second aspect of this embodiment will be described.

[0093] Recombinant Pichia pastoris strains The recombinant Pichia pastoris strain provided in this embodiment is prepared using the construction method described in the first aspect. As described in the first aspect, this embodiment can systematically optimize the synthesis pathway of p-coumaric acid by optimizing metabolic flux, removing restrictions, strengthening direct precursors, reducing diversion, and increasing sources (precursor supply). Ultimately, a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source is constructed. Compared with the host strain, the recombinant Pichia pastoris strain has significantly increased p-coumaric acid production.

[0094] Hereinafter, the use of the recombinant Pichia pastoris strain according to the third aspect of this embodiment in synthesizing p-coumaric acid will be described.

[0095] use It is understood that the recombinant Pichia pastoris strain provided in this example, by systematically optimizing the synthesis pathway of p-coumaric acid, can achieve efficient, low-cost, and high-purity production of p-coumaric acid. Therefore, the recombinant Pichia pastoris strain of this example can be used to efficiently synthesize p-coumaric acid.

[0096] The present application will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate / explain the present application and are not intended to limit the scope of the present application.

[0097] In the following examples, all materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0098] Figure 1-Figure 4 1 is a diagram illustrating the present embodiment; wherein, Figure 1 A schematic diagram of metabolic engineering modification of a Pichia pastoris engineered strain for synthesizing p-coumaric acid is shown in this example; Figure 2 shows the liquid chromatograms of p-coumaric acid synthesis by different engineered strains (including strain CA01); Figure 3 A schematic diagram showing the p-coumaric acid production results of engineered strains CA01-CA07 by shake flask fed-batch fermentation; Figure 4 Schematic diagram showing the results of fermentation production of p-coumaric acid by the engineered strain CA05 in Example 5 in a 15 L bioreactor.

[0099] In the following examples, the engineered strain CA01 was obtained by integrating the phenylalanine ammonia lyase gene AtPAL2 (Gene ID: 824493) into the chromosome PNS1-2 site of the starting strain S12, and integrating the cinnamate hydroxylase AtC4H (Gene ID: 817599) and P450 reductase AtATR2 (Gene ID: 829144) into the chromosome PNSII-4 site of the starting strain S12; the engineered strain CA02 was obtained by integrating the tyrosine ammonia lyase FjTAL into the chromosome PNSII-5 site of the engineered strain CA01; and the engineered strain CA03 was obtained by integrating the DNA fragment P AOX1 -ScAro7 G141S -T AOX1 The engineered strain CA04 was obtained by integrating the prephenate dehydratase ScPHA2 (Gene ID: 855400) from Saccharomyces cerevisiae into the PNSI-6 site of the engineered strain CA03, and using PAOX1 The strains were obtained by controlling the expression of the above genes with the promoter; the engineered strain CA05 was obtained by knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) of the engineered strain CA04 using CRISPR / Cas9 technology; the engineered strain CA06 was obtained by simultaneously knocking out the endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) and endogenous phenylpyruvate decarboxylase Aro10-2 (Gene ID: 8197764) of the engineered strain CA04 using CRISPR / Cas9 technology; the engineered strain CA07 was obtained by integrating the endogenous fructose-1,6-bisphosphatase FBP (Gene ID: 8199670) into the chromosome PNSII-6 site of the engineered strain CA05, and using P AOX1 The strain obtained by overexpressing the above genes controlled by the promoter.

[0100] like Figure 1 As shown, in the following embodiments, Figure 1 The method shown is used to construct a p-coumaric acid-producing strain, namely a recombinant Pichia pastoris strain.

[0101] In this example, a combination of rational metabolic engineering strategies, including introducing the phenylalanine ammonia-lysis pathway and / or the tyrosine ammonia-lysis pathway into Pichia pastoris S12 to achieve p-coumaric acid biosynthesis, relieve feedback inhibition of the shikimic acid pathway, optimize aromatic amino acid biosynthesis, knock out competing metabolic pathways, and increase precursor supply, was successfully constructed. A recombinant Pichia pastoris engineered strain that efficiently produces p-coumaric acid using methanol as a carbon source was successfully constructed. The yield of this recombinant Pichia pastoris engineered strain was significantly improved compared to the starting strain.

[0102] The initial strain (starting strain S12) used in the following examples is an existing recombinant Pichia pastoris S12 (genotype his4 - , P GAP -PpPSA-T AOX1 ,P AOX1 -PpPET2-T AOX1 , the strain is derived from the record in patent number CN118530863A).

[0103] The transformation methods in the following examples all use CRISPR-Cas9 technology (the strain construction method is carried out according to Cai et al. Nucleic Acids Res. 2021; 49(13): 7791-7805) for directed transformation.

[0104] Next, the relevant raw materials used in this embodiment will be described.

[0105] In the recombinant Pichia pastoris engineered strain constructed in this example for efficiently synthesizing p-coumaric acid using methanol, the phenylalanine ammonia-lysis pathway enzyme genes include phenylalanine ammonia-lyase AtPAL2, cinnamate hydroxylase AtC4H, and cytochrome P450 reductase AtATR2, all of which are derived from Arabidopsis thaliana. After Pichia codon optimization and gene synthesis, they are introduced into the starting strain and can utilize the endogenous L-Phe of Pichia pastoris to synthesize trans-cinnamic acid and p-coumaric acid.

[0106] Tyrosine ammonia lyase FjTAL, derived from Flavobacterium johnsonii, was introduced into the starting strain after codon optimization and gene synthesis in Pichia pastoris, and can use the endogenous L-Tyr (L-tyrosine) of Pichia pastoris to synthesize p-coumaric acid; chorismate mutase gene ScAro7 G141S , derived from Saccharomyces cerevisiae, mutates glycine at position 141 into serine, which relieves the feedback inhibition of L-Tyr on the enzyme. The enzyme can effectively increase the production of p-coumaric acid precursors.

[0107] Prephenate dehydratase ScPHA2, derived from Saccharomyces cerevisiae, encodes the first enzyme in tyrosine synthesis, which can effectively increase the amount of L-Tyr, a precursor of p-coumaric acid; knocking out the endogenous phenylpyruvate decarboxylase Aro10 in Pichia pastoris, which catalyzes the decarboxylation of phenylpyruvate, the precursor of L-Phe, to phenylacetaldehyde, and deleting this gene can increase the accumulation of L-Phe; fructose-1,6-bisphosphatase FBP, a key regulatory enzyme in the gluconeogenesis pathway, is involved in formaldehyde assimilation and the xylulose monophosphate pathway. Overexpression of this enzyme can increase the metabolic flux of the shikimate pathway.

[0108] It should be noted that the key enzymes in the above pathways all control gene expression through the PAOX1 promoter.

[0109] Example 1: Construction of a p-coumaric acid synthesis pathway.

[0110] p-Coumaric acid is synthesized by a dual pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase.

[0111] First, a donor expression cassette for the independent expression of the phenylalanine ammonia lyase gene AtPAL2 (Gene ID: 824493), cinnamate carboxylase gene AtC4H (Gene ID: 817599), cellular P450 reductase AtATR2 (Gene ID: 829144), and tyrosine ammonia lyase FjTAL (Sequence ID: WP_012023194.1) synthesized after codon optimization was constructed; the donor DNA fragment included 750 bp of upstream and downstream homology arms of the PNSI-2 or PNSII-4 or PNSII-5 sites amplified from the Pichia pastoris GS115 genome (gene integration sites refer to Yu et al. Synth Syst Biotechnol 2021, 6: 63-68), the promoter P AOX1 , terminator T AOX1 and structural genes.

[0112] 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).

[0113] Specifically, the donor DNA and gRNA were transformed into the starting strain S12 or GS115 by electroporation, plated onto YPD plates containing G418 antibiotics, and incubated at 30°C for 3-4 days for screening. Colony PCR was used to verify the correctness of the strain, and the strain was subcultured in YPD liquid medium to eliminate the plasmid. The resulting engineered strain was fermented, and after extraction and testing, it was confirmed that the integration of AtPAL2, AtC4H, and AtATR2 could produce p-coumaric acid (engineered strain CA01) with a yield of 1.54 g / L. Based on CA01, FjTAL was integrated to obtain the engineered strain CA02, which produced 1.79 g / L of p-coumaric acid (see Figure 3 ).

[0114] The specific experimental steps are: 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.

[0115] 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.

[0116] 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.

[0117] 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).

[0118] Preparation of competent yeast cells: (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; (2) Use a sterilized pipette tip to pick up a single colony and inoculate it into 10 mL of YPD medium. Place it on a shaker at 30°C and 250 rpm for 20-24 hours. (3) Measure the OD600 of the initial seed solution and transfer it to 50 mL YPD medium with an initial OD600 of 0.5-0.6. Cultivate for 4-5 hours until the OD600 reaches 1.2-1.8. (4) In a clean bench, transfer the entire bacterial solution to a sterile 50 mL centrifuge tube and centrifuge in a refrigerated benchtop centrifuge at 4000 rpm, 5 min, and 4°C. (5) In a clean bench, remove the supernatant and add 40 mL of filtered and sterilized LDST solution to the centrifuge tube. After thoroughly resuspending the bacteria, place them in a 30°C incubator for 30 min. (6) Centrifuge in a high-speed refrigerated benchtop centrifuge at 4000 rpm for 5 min at 4°C. Remove the supernatant in a clean bench. Then, transfer the bacteria from the 50 mL centrifuge tube to a sterilized 1.5 mL centrifuge tube using 1 mL of pre-chilled 1 M sorbitol and resuspend the pellet. Centrifuge at 4°C at 4000 rpm for 1 min. Remove the supernatant and repeat the washing three times. (7) Add 400 μL of 1M sorbitol in an ice bath to thoroughly resuspend the cells. Aliquot 50 or 80 μL into 1.5 mL sterile centrifuge tubes and store in a -80 °C refrigerator for later use.

[0119] Yeast electroporation transformation method specifically includes: (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°C for 2 h. Specifically, the linearization system (50 μL) is as follows: 5 μL of 10× FastDigest Buffer, 2.5 μL of each enzyme, 2.0 μg-3.0 μg of plasmid, and ddH2O to 50 μL. After the reaction, the linearization system is recovered using a PCR product recovery kit. (2) Mix the linearized fragment with 1.5 μg-2.0 μg of the Cas9 plasmid corresponding to each neutral site, add it to the Pichia competent cells, mix it gently, transfer the mixture to the electroporation cup in an ice bath, and place it on ice for 5 minutes; (3) Set the electroshock instrument parameters to Fungi mode, wipe the water outside the electroshock cup, perform the electroshock, and then add 1 mL of 1 M sorbitol in an ice bath; (4) Aspirate all the mixed solution in the electroporation cup and transfer it to a 1.5 mL centrifuge tube. Place it in a 30°C incubator and incubate it for 1.0-1.5 hours. Then centrifuge it at 5000 rpm for 2 minutes. (5) Remove 800-900 μL of supernatant, resuspend the bacteria with the remaining supernatant, and spread all of it on YPD (YPDG) plates containing 200-250 μg / mL G418 antibiotics. Incubate the plates in an inverted incubator at a constant temperature of 30°C for 2-3 days.

[0120] With the help of the CRISPR / Cas9 system, 3000 ng of each of the donor DNA fragment obtained above and the PNS neutral site-Cas9 expression plasmid (the construction method of the PNS neutral site-Cas9 expression vector (containing gRNA) vector refers to Cai et al. Nucleic Acids Res. 2021; 49(13): 7791-7805) were transformed into Pichia pastoris competent cells by electroporation, and the strain was spread on the YPD (YPDG) screening plate with G418 antibiotic and cultured at 30°C for 3 to 4 days for screening. The correctness was verified by colony PCR, and the verified correct strain was inoculated and subcultured in YPD antibiotic-free culture medium for plasmid loss. After the plasmid was lost, the process strain was obtained.

[0121] According to the above CRISPR / Cas9 gene editing method, AtPAL2 was integrated into the neutral site of Pichia pastoris S12 PNSI-2 and expressed through the promoter P AOX1 The engineered strain, after control of the expression of the structural gene and verification by colony PCR and plasmid loss, was named SP. Repeating the above experimental procedures, AtC4H and AtATR2 were sequentially integrated into the neutral site of PNSII-4 of the engineered strain SP. After verification and plasmid loss, the engineered strain CA01 was obtained, which is the Pichia pastoris cell factory for de novo synthesis of p-coumaric acid from the phenylalanine ammonia decomposition pathway; please refer to Figure 2 , Figure 2 The liquid chromatograms of the synthesis of p-coumaric acid by different engineered strains are shown; among them, the engineered strain CA01 can specifically synthesize p-coumaric acid and has a higher yield.

[0122] Next, according to the above method, based on the engineered strain CA01, FjTAL was integrated into the PNSII-5 site. After verification and plasmid loss, the engineered strain CA02 was obtained, which is a Pichia pastoris cell factory for the de novo synthesis of p-coumaric acid through both phenylalanine ammonialysis and tyrosine ammonialysis pathways.

[0123] The fermentation extraction and detection steps in this embodiment include: activating the engineered strain (engineered strain CA01 or CA02) after plasmid loss in BMGY medium, culturing at 30°C and 220 rpm for 24 h, and then transferring to 25 mL BMMY medium / 250 mL shake flask, with an initial OD 600 =1.0, and the culture was fermented at 30 °C and 220 rpm for 120 h, and the biomass and yield were measured.

[0124] The extraction and detection method for cinnamic acid and p-coumaric acid is as follows: 200 μL of fermentation broth was extracted with 800 μL of methanol by vortex extraction at 12,000 rpm for 10 minutes. The supernatant was sterilized by filtration through a 0.22 μm organic filter and then placed into a liquid chromatography injection vial. A Synergi™ 4 μm Hydo-RP 80 Å, Kromasil, Sweden, 250 mm × 4.6 mm × 5 μm, Aphenomenex 100 spherical silica was used. The injection volume was 10 μL, and the optimal detection wavelength was 310 nm. The mobile phases were: Phase A, 0.1% (v / v) trifluoroacetic acid or formic acid; Phase B, 100% acetonitrile. The flow rate was 1 mL / min, and the column temperature was 30°C. The specific gradient elution program is shown in Table 1: Table 1: HPLC detection gradient elution program

[0125] Methanol extraction of the fermentation broth and high-performance liquid chromatography analysis revealed the quantification of trans-cinnamic acid or p-coumaric acid, precursors of p-coumaric acid synthesis. The engineered strain SP produced 288.1 mg / L of cinnamic acid, while the engineered strain CA01 produced 1.54 g / L and the engineered strain CA02 produced 1.79 g / L.

[0126] Among them, the engineered strain CA02 was used for the transformation of subsequent examples.

[0127] Table 2: Amplification primers involved in the construction of the dual pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase

[0128] In Table 2 , 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 remaining primers are used to amplify the corresponding target gene fragments.

[0129] Unless otherwise specified, the construction, transformation, and fermentation conditions of the engineering strains in the following examples were the same as those in Example 1. The structural genes expressed and overexpressed in the engineering strains were all expressed through the promoter P AOX1 control.

[0130] Example 2: Relief of feedback inhibition of the shikimate pathway.

[0131] In order to further increase the synthesis of p-coumaric acid, this example, based on Example 1, removes the feedback inhibition of the key enzymes of the shikimate pathway of the engineered strain CA02; referring to the steps described in Example 1, the chorismate mutase mutant Aro7 is transformed into G141S The gene was integrated into the chromosomal PNSI-8 locus of the engineered strain CA02 and the PAOX1 promoter was used to regulate the chorismate mutase ScAro7 of the shikimate pathway. G141S Overexpressed.

[0132] The primers involved in this example are shown in Table 3.

[0133] Referring to the steps described in Example 1, the chorismate mutase ScAro7 from Saccharomyces cerevisiae was overexpressed at the PNSI-8 site of the engineered strain CA02. G141S (The nucleotide sequence from positions 421 to 423 after the mutation of the ScAro7 (Gene ID: 856173) gene is TCG), and the product was extracted through fermentation to detect the production of p-coumaric acid.

[0134] Among them, chorismate mutase ScAro7 G141S The p-CA production in the overexpressed engineered strain CA03-1 increased and the strain grew well, with a p-CA production of 2.19 g / L. The strain was named CA03 for subsequent modification.

[0135] Table 3: Primers involved in this implementation

[0136] Example 3: Overexpression of key genes in the aromatic amino acid synthesis pathway.

[0137] Based on the engineered strain CA03, this example further optimized the aromatic amino acid synthesis pathway. Referring to the steps described in Example 1, a donor expression cassette of the prephenate dehydratase ScPHA2 (GeneID: 855400) from Saccharomyces cerevisiae was constructed using the CRISPR / Cas9 system and electroporated together with PNSI-6-Cas9 into the genomic PNSI-6 locus using P AOX1The promoter controlled the overexpression of the above-mentioned gene. The specific steps of the construction of the relevant gene expression cassette and genome integration were the same as those in Example 1 above. After dual-pathway integration, release of feedback inhibition, and optimization of the aromatic amino acid synthesis pathway, the engineered strain CA04 was obtained. The engineered strain had a p-coumaric acid production of 2.33 g / L.

[0138] Table 4: Primers involved in this implementation

[0139] 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.

[0140] Example 4: Knocking out a branched metabolic pathway and increasing the precursor supply to increase p-coumaric acid production.

[0141] Please refer to the primers recorded in Table 5. This example is intended to illustrate the steps for knocking out the genomic phenylpyruvate decarboxylase Aro10 gene. The specific steps are as follows: (1) Using Pichia pastoris GS115 as a template, △Aro10-F / △Aro10-SF and △Aro10-XF / △Aro10-R were amplified by KODneo enzyme PCR to obtain the upstream and downstream homology arms, and then the donor △Aro10 gene knockout fragment was obtained by KODneo enzyme overlapping PCR using these as templates. The gene integration fragment consisted of the upstream and downstream homology arms of Aro10.

[0142] (2) Using the StgRNA-F / gRNA-Aro10-R primer pair, the pPIC-Cas9 plasmid was used as the template skeleton to construct the DNA fragment containing the target sequence through the PCR annealing procedure to obtain a circular PCR product. After the product was recovered, it was transformed into Top10 transfection competent cells, and positive transformants were screened. The plasmid was extracted and sent for sequencing. The plasmid with correct sequencing was named pPIC-Aro10-Cas9.

[0143] The obtained donor △Aro10 gene knockout fragment and the pPIC-Aro10-Cas9 plasmid were electroporated into the competent engineered strain CA04. After identification, plasmid loss, and fermentation extraction testing, the production 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 (endogenous phenylpyruvate decarboxylase Aro10-1 knockout (Gene ID: 8199377)).

[0144] Similarly, the strain CA06 (endogenous phenylpyruvate decarboxylase Aro10-1 (Gene ID: 8199377) and endogenous phenylpyruvate decarboxylase Aro10-2 (Gene ID: 8197764) knocked out) can be prepared by referring to the above method.

[0145] Next, based on the engineered strain CA05, the supply of the precursor substances PEP and E4P in the strain was enhanced. Referring to the primers (FBP-F and FBP-R) listed in Table 5 and referring to the steps described in Example 1, a donor DNA expression vector for the endogenous fructose-1,6-bisphosphatase FBP (a key regulatory enzyme in the glucose biosynthesis pathway, Gene ID: 8199670) from Pichia pastoris was constructed using the CRISPR / Cas9 system. The gene fragment was amplified from the Pichia pastoris GS115 genome by PCR and integrated into the corresponding genomic locus by electroporation.

[0146] After fermentation, extraction and testing, the integration and expression of the FBP gene was found to be beneficial for the synthesis of p-coumaric acid. The engineered strain CA07 was obtained, which integrated the dual pathways of p-coumaric acid, relieved the feedback inhibition of shikimic acid, strengthened the aromatic amino acid synthesis pathway, knocked out the aromatic amino acid synthesis branch metabolic pathway, and enhanced the supply of PEP and E4P, with a yield of 2.84 g / L ( Figure 3 ).

[0147] See Figure 3 , Figure 3 A schematic diagram of the p-coumaric acid production results of the engineered strains CA01-CA07 by shake flask fed-batch fermentation is shown. The production of the engineered strains CA01-CA07 increases sequentially, indicating that the targeted modification in this example can work together from multiple aspects to systematically optimize the synthesis pathway of p-coumaric acid, and ultimately construct a recombinant Pichia pastoris strain CA07 that efficiently produces p-coumaric acid using methanol as a carbon source.

[0148] Table 5: Primers involved in this implementation

[0149] Note: The underlined gRNA sequence is the gRNA sequence.

[0150] Example 5: Batch fed-batch fermentation of engineered strain CA05.

[0151] In order to test the high-density fermentation ability of the engineered strain, a 15 L fermentor fed-batch fermentation experiment was carried out; the engineered strain CA05 obtained above was used as an exemplary strain for fed-batch fermentation in a 15 L fermentor.

[0152] The above test steps are as follows: The engineered strain CA05 was streaked onto YPD plates for activation and incubated at 30°C for 3 days. The strain was then inoculated into 10 mL / 50 mL YPD liquid medium in a triangular flask and incubated at 30°C, 220 rpm, for 24 hours to obtain a primary seed. A 4% inoculum was then transferred to 200 mL / 500 mL YPD liquid medium in a triangular flask and incubated at 30°C, 220 rpm, for 20-24 hours to obtain a secondary seed solution. Batch fermentation was performed using a 15 L Shanghai Bailun bioreactor system with a 6 L batch volume (15 L fermentor). The seed solution was inoculated into the fermentor at an 8% inoculum volume.

[0153] Fed-batch fermentation was performed using an inorganic salt-based medium (BSM with yeast extract at a concentration of 5 g / L and peptone at 10 g / L) at a pH of 5.5. Once glycerol was consumed, dissolved oxygen was maintained above 20%. Once a rebound in dissolved oxygen occurred, the next stage could be entered. 50% glycerol was added to achieve the desired induced OD (200-300), with the addition rate controlled at 6-11 g / L / h to maintain dissolved oxygen above 20%. 600 When the desired induction value is reached, a starvation phase begins. Supplementation with 50% glycerol is stopped for 30–60 minutes to deplete the remaining carbon source in the culture medium. During this period, the temperature is automatically adjusted to 25°C and the pH to 6.0. The methanol induction phase then begins. The methanol flow rate is controlled at 4–6 g / L / h, the dissolved oxygen content is maintained at no less than 20%, and the methanol induction time is limited to 120 hours. During this period, the fermentation broth is sampled every 12 hours for methanol extraction and determination of p-coumaric acid production.

[0154] Figure 4 Schematic diagram showing the results of fermentation of p-coumaric acid by the engineered strain CA05 in a 15 L bioreactor in Example 5; Figure 4 The first stage shown: after about 44 hours of carbon source consumption period, OD 600 When the OD value reaches 38.8, 50% glycerol is added to the fermenter to maintain rapid cell growth. The flow rate is controlled at 6~11g / L / h. When the glycerol supply is completed, the OD value 600 Around 194.

[0155] The 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 gradually accumulated. After 84 h of induction, OD 600 The titer of p-CA reached 202.9, the p-CA production reached 18.55±3.48 g / L (344.37 mg / gDCW), and the productivity was 662.605.30 mg / L / d (220.83 mgL / h), which is the highest p-CA titer so far.

[0156] In summary, the construction method provided in this example integrates the dual pathways for p-coumaric acid, relieves feedback inhibition of shikimic acid, strengthens the aromatic amino acid synthesis pathway, knocks out the aromatic amino acid synthesis branch metabolic pathway, and enhances the supply of PEP and E4P to obtain an engineered strain, which can systematically optimize the synthesis pathway of p-coumaric acid. Ultimately, a recombinant Pichia pastoris strain that efficiently produces p-coumaric acid using methanol as a carbon source was constructed. Compared with the host strain, the recombinant Pichia pastoris strain has a significantly improved p-coumaric acid yield.

[0157] The above is a detailed introduction to the technical solutions provided in the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above 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, there may be changes in the specific implementation methods and application scope. In summary, the contents of this specification 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: 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; 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; The precursor supply of the fourth engineered strain was increased to obtain a recombinant Pichia pastoris strain.

2. The construction method according to claim 1, characterized in that The dual synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase is constructed in the host strain, comprising: The phenylalanine ammonia lyase gene AtPAL2 was integrated into the chromosome PNSI-2 site of the host strain, the cinnamate hydroxylase AtC4H and P450 reductase AtATR2 were integrated into the chromosome PNSII-4 site of the host strain, and the tyrosine ammonia lyase FjTAL was integrated into the chromosome PNSII-5 site of the host strain.

3. The construction method according to claim 1, characterized in that The method of removing chorismate feedback inhibition of the first engineered strain based on metabolic engineering comprises: Chorismate mutase mutant Aro7 G141S Integrate into the chromosome PNSI-8 site of the first engineered strain using P AOX1 Promoter-controlled chorismate mutase mutant Aro7 G141S expression.

4. The construction method according to claim 3, characterized in that The chorismate mutase mutant Aro7 G141S It is produced by the G141S mutation of chorismate mutase Aro7.

5. The construction method according to claim 1, characterized in that The optimization of the aromatic amino acid synthesis pathway of the second engineered strain comprises: 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, a prephenate dehydratase.

6. The construction method according to claim 1, characterized in that The knockout of the branched metabolic pathway of the third engineered strain comprises: Gene editing technology was used to knock out the endogenous phenylpyruvate decarboxylase Aro10 in the third engineered strain.

7. The construction method according to claim 6, characterized in that: The gene editing technology is CRISPR / Cas9 gene editing technology.

8. The construction method according to claim 1, wherein: The method of increasing the precursor supply of the fourth engineered strain comprises: The endogenous fructose-1,6-bisphosphatase 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.

9. 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 to 8.

10. Use of the recombinant Pichia pastoris strain according to claim 9 in synthesizing p-coumaric acid.

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