Engineered yeast for fermentation production of L-tryptophan by using methanol carbon source and application of engineered yeast

Abstract

The invention provides yeast engineering bacteria for producing L-tryptophan by utilizing methanol carbon source fermentation and application, and belongs to the technical field of biology. According to the engineered yeast, a methanol substrate is coupled with a methanol inducible promoter induction system, so that the gene expression intensity is dynamically matched with the methanol consumption rate, and the problem of regulation lag caused by separation of an inducer and a substrate in a traditional carbon source is solved; in addition, a gene recombination technology is adopted to enhance expression of key genes in the synthesis route of the L-tryptophan, and the biosynthesis efficiency of the L-tryptophan is remarkably improved by increasing the dosage of target genes, enhancing supply of precursor substances and knocking out genes related to catabolism of the tryptophan in chassis cells at the same time. The constructed yeast engineering bacterium has the characteristics of clear metabolic background and strong heterologous expression ability, can be used for synthesizing L-tryptophan from the beginning completely by taking methanol as a carbon source, effectively gets rid of the dependence of a traditional process on grain resources, reduces the production cost, and provides a brand new technical path for industrial production of L-tryptophan.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, specifically to engineered yeast strains that utilize methanol as a carbon source for the fermentation production of L-tryptophan and their applications. Background Technology

[0002] L-Tryptophanin, as a key essential amino acid, has irreplaceable application value in feed additives, pharmaceutical intermediates, and other fields. Currently, the industrial production of L-Tryptophanin mainly relies on microbial fermentation processes using glucose as a carbon source. The commonly used chassis cells are Escherichia coli or Corynebacterium glutamicum, and the fermentation level is usually 50-55 g / L, with a sugar-acid conversion rate of only 0.20-0.22 g / g (Liu S, Wang BB, Xu JZ, Zhang WG. Engineering of Shikimate Pathway and Terminal Branch for Efficient Production of L-Tryptophanin Escherichia coli. Int J Mol Sci. 2023;24(14):11866. Published 2023 Jul24.doi:10.3390 / ijms241411866). This process has significant drawbacks: on the one hand, glucose needs to be obtained through starch hydrolysis, consuming 3.5-4 tons of corn starch per ton of product, creating direct resource competition with the feed industry; on the other hand, the high cost of raw materials restricts the market competitiveness of the product, especially in the field of pharmaceutical-grade high value-added products (unit price reaches US$120 / kg), where cost control has become a key bottleneck for industrial upgrading.

[0003] To overcome these limitations, developing alternative carbon source technologies to replace grain has become a hot research topic in the industry. Methanol, as a potential carbon source, has significant advantages such as wide availability and independence from grain resources: it can be industrialized through natural gas reforming and coal gasification (with a significant cost advantage in coal-rich areas such as Xinjiang and Inner Mongolia), or it can be produced using photovoltaic hydrogen production coupled with CO2 capture technology. Furthermore, global methanol production capacity has exceeded 160 million tons per year, and a mature transportation network exists (e.g., sea freight costs between China and Southeast Asia are approximately US$30 per ton), which can fully guarantee the stability of raw material supply for industrialized production.

[0004] However, current research on the de novo synthesis of L-tryptophan using methanol as a carbon source still faces technological gaps, particularly the lack of efficient fermentation systems using yeast as a chassis cell. Yeast, as a eukaryotic microorganism, possesses advantages such as a clear genetic background, well-developed metabolic regulatory mechanisms, and good product compatibility, making it more suitable for the synthesis of complex metabolites compared to traditional prokaryotic chassis cells. However, the L-tryptophan synthesis pathway in yeast is long, involving multiple cellular compartments (such as the cytoplasm and mitochondria), and its regulatory network is more complex than that of prokaryotic microorganisms. Many regulatory details remain incompletely elucidated, increasing the difficulty of rationally designing high-yield strains for the de novo synthesis of L-tryptophan using methanol as a carbon source.

[0005] Therefore, constructing an engineered strain for the fermentation production of L-tryptophan using yeast as the chassis cell and methanol as the substrate, as well as a fermentation process for L-tryptophan, is of great significance for improving the biosynthetic yield of L-tryptophan and reducing its production cost. Summary of the Invention

[0006] In view of this, the present invention mainly constructs an engineered strain that uses yeast as a chassis cell and utilizes methanol as a carbon source to ferment and produce L-tryptophan through multi-dimensional metabolic engineering, and achieves efficient biosynthesis of L-tryptophan.

[0007] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides an engineered yeast strain for the fermentation production of L-tryptophan using methanol as a carbon source, including transformation of the yeast containing the gene... TRP1 (N-(5'-phosphoribosyl)anthranilate isomerase, N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase) TRP2 (Anthranilate synthase II) 、 TRP3 (Indole-3-glycerol-phosphate synthase) TRP4 (Anthranilate phosphoribosyltransferase) and TRP5 An overexpression cassette for Tryptophan synthase, wherein the overexpression cassette includes a methanol-inducible promoter.

[0008] Furthermore, to enhance the supply of the precursor shikimic acid via the shikimic acid pathway, the engineered yeast strain was further transformed with two key genes containing the shikimic acid pathway. ARO4(Phospho-2-dehydro-3-deoxyheptonate aldolase) and CRS The expression cassettes for chorismate synthase (ARO4) include a methanol-inducible promoter in each cassette. Preferably, the key gene ARO4 encodes a mutant ARO4. K229L The genes.

[0009] Furthermore, to optimize the extracellular transport efficiency of tryptophan, the engineered yeast strain was further transformed with a gene containing a tryptophan-specific transporter protein. yddG An overexpression cassette for (aromatic amino acid exporter), which includes a constitutive promoter.

[0010] Furthermore, to enhance the supply level of the cofactor NADPH, the engineered yeast strain is further converted to contain NADP. + Citrate-dependent dehydrogenase gene ScIDP2 The expression box includes a constitutive promoter.

[0011] Furthermore, to inhibit tryptophan degradation and reduce carbon diversion, the key gene for tryptophan degradation was knocked out in the engineered yeast strain. ARO9 (Aromatic aminotransferase I).

[0012] Furthermore, to achieve precise and targeted branch blocking and reduce carbon diversion, the key gene for tryptophan degradation was also knocked out in the engineered yeast strain. ARO9 Aromatic amino acid branch point genes ARO7 (Chorismate mutase).

[0013] Secondly, the present invention provides a method for producing L-tryptophan by fermentation, comprising inoculating the above-mentioned engineered yeast culture into a culture medium and fermenting it at 25-37°C for 1-15 days to synthesize L-tryptophan; wherein the culture medium uses methanol as the sole carbon source.

[0014] To improve the yield of L-tryptophan by shake-flask fermentation, the method includes: adjusting the concentration of L-tryptophan to (1–10) × 10 7Yeast engineered strain seed culture at a concentration of CFU / mL was inoculated into an Erlenmeyer flask containing culture medium at pH 5.8–7.6 and fermented at 26–34°C for 2–5 days at a rotation speed of 150–250 r / min. 0.5%–2% (m / v) methanol was added to the culture medium every 12–36 h to further increase the yield of L-tryptophan produced by shake-flask fermentation. To increase the yield of L-tryptophan produced in the fermenter, the method includes: adding (1–5) × 10⁻⁶ CFU / mL yeast seed culture to the culture medium. 8 The engineered yeast seed culture (cfu / mL) was inoculated into a fermenter containing BMMY fermentation medium and fermented at 25–37°C for 3–10 days at a rotation speed of 200–1000 r / min.

[0015] To further increase the yield of L-tryptophan, methanol was added to the BMMY fermentation medium at a flow rate of 4–25 g / h during the fermentation process, and the dissolved oxygen (DO) in the fermenter was maintained above 30%.

[0016] Thirdly, the present invention provides an application of the above-mentioned engineered yeast strain in the production of L-tryptophan using methanol as a carbon source.

[0017] Fourthly, the present invention provides a kit for producing L-tryptophan, the kit comprising the aforementioned engineered yeast strain.

[0018] Therefore, the technical solution provided by this invention achieves a step-by-step increase in tryptophan production through a step-by-step metabolic engineering strategy of "core pathway enhancement → precursor supply improvement → product efflux optimization → cofactor synergy → branched carbon flow regulation." The synergistic effects of each modification step are significant, with the enhancement of the precursor pathway and the blocking of the branched pathway being particularly effective. Furthermore, for the first time, methanol substrate is coupled with a methanol-inducible promoter induction system, dynamically matching gene expression intensity with methanol consumption rate, thus solving the regulatory lag problem caused by "inducer-substrate separation" in traditional carbon sources. In addition, methanol is a liquid substrate, easy to sterilize and precisely control, and exhibits higher pH stability during fermentation; moreover, methanol is relatively inexpensive. Therefore, the technical solution provided by this invention offers an industrially feasible solution for the efficient microbial synthesis of tryptophan. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthetic pathway for L-tryptophan production from methanol metabolism used in this invention; Figure 2 This is a diagram showing the identification results of the fermentation products of the engineered strain provided in an embodiment of the present invention; Figure 3 The graph shows the L-tryptophan yield produced by shake-flask fermentation of different metabolically engineered strains provided in the embodiments of the present invention. Figure 4 This invention provides a diagram illustrating the yield of L-tryptophan produced by engineered yeast using methanol as a carbon source in a 5L reactor. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0021] the term In this invention, the "expression cassette" or "gene expression cassette" refers to a gene expression system containing all the necessary elements required to express a target polypeptide. Typically, it includes the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; additionally, it may optionally include a signal peptide encoding sequence, etc.; these elements are operatively linked.

[0022] In this invention, "operably linked" or "operationally linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is "operably linked" to the nucleic acid sequence.

[0023] In this invention, the term "expression construct" refers to a recombinant DNA molecule containing a desired nucleic acid coding sequence, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector.

[0024] In this invention, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, then the nucleic acid is exogenous to that host cell. A specific sequence is "exogenous" to the cell or organism into which it is inserted.

[0025] In the description of this invention, the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. It should be noted that, herein, relational terms such as "first," "second," etc., are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0026] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0027] In this invention, all terms used are common terms in the relevant field. Unless otherwise specified, the preparation processes and testing methods used in each embodiment are conventional methods well known to those skilled in the art, and the raw materials and equipment used can be obtained from publicly available commercial channels.

[0028] This invention primarily uses methanol as the sole carbon source substrate and yeast as the substrate to synthesize L-tryptophan via the following pathway: Figure 1 As shown, methanol is metabolized to produce branched acid, a precursor in the L-tryptophan synthesis pathway. This branched acid is then catalyzed by enzymes to synthesize L-tryptophan, which is finally transported extracellularly via the yddG protein. The entire reaction process is a de novo synthesis of the final product. Therefore, this invention constructs a yeast strain capable of efficiently synthesizing tryptophan through multi-dimensional metabolic engineering. Its innovation and technological advantages lie in the synergistic design of the methanol substrate and the metabolic regulatory system. Based on this, the main objective of this invention is to provide a genetically engineered strain that utilizes methanol as a carbon source for fermentation to produce L-tryptophan and its applications.

[0029] To achieve the above objectives, the specific embodiments of the present invention adopt the following solutions: In a first aspect, the present invention provides an engineered yeast strain for the fermentation production of L-tryptophan using methanol as a carbon source, including transformation of the yeast containing the gene... TRP1, TRP2, TRP3, TRP4 and TRP5 An overexpression cassette, wherein the overexpression cassette includes a methanol-inducible promoter. (Gene) TRP1, TRP2, TRP3, TRP4 and TRP5 All of these are genes involved in tryptophan biosynthesis.

[0030] Preferably, the engineered yeast strain is Pichia pastoris (Pichia pastoris). Pichia pastoris (More preferably, the engineered yeast strain is Pichia pastoris GS115 or Δku70.)

[0031] In one specific embodiment, the engineered yeast strain is transformed with overexpression cassettes containing the following genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4 and TRP5 This enhances the supply of the precursor shikimic acid via the pathway.

[0032] In one specific embodiment, the engineered yeast strain is transformed with overexpression cassettes containing the following genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4 , TRP5 and yddG This can further optimize the efficiency of extracellular transport of tryptophan.

[0033] In one specific embodiment, the engineered yeast strain is transformed with overexpression cassettes containing the following genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4 , TRP5, yddG and ScIDP2 This can further improve the supply level of the cofactor NADPH.

[0034] In one specific embodiment, the engineered yeast strain is transformed with expression cassettes containing the following genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4, TRP5, yddG and ScIDP2 And the strain was knocked out ARO9 Genes; this can further inhibit tryptophan degradation and reduce carbon flow diversion.

[0035] In one specific embodiment, the engineered yeast strain is transformed with expression cassettes containing the following genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4, TRP5, yddG and ScIDP2 And the strain was knocked out ARO9 , ARO7 Genes; this allows for precise, targeted branch blocking, further reducing carbon diversion.

[0036] Among them, the gene described in this invention ARO4, CRS, TRP1, TRP2, TRP3, TRP4 and TRP5 The expression cassettes all use methanol-inducible promoters, such as P whose expression is strictly dependent on methanol concentration. AOX1 Promoters; genes described in this invention yddG and ScIDP2 The expression boxes all use constitutive promoters, such as GAP Promoter.

[0037] In one or more embodiments, the ARO4 gene is a mutant. ARO4 K229L It has the nucleotide sequence shown in SEQ ID NO: 1, or its degenerate sequence, or the nucleotide coding sequence of a functional protein that has more than 70% homology with the sequence of SEQ ID NO: 1; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0038] In one or more embodiments, the CRS gene has the nucleotide sequence shown in SEQ ID NO: 2, or its degenerate sequence, or the nucleotide coding sequence of a functional protein that has more than 70% homology with the sequence of SEQ ID NO: 1; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0039] In one or more embodiments, the TRP1 gene has the nucleotide sequence shown in SEQ ID NO: 3, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 3; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0040] In one or more embodiments, the TPR2 gene has the nucleotide sequence shown in SEQ ID NO: 4, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 4; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0041] In one or more embodiments, the TRP3 gene has the nucleotide sequence shown in SEQ ID NO: 5, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 5; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0042] In one or more embodiments, the TRP4 gene has the nucleotide sequence shown in SEQ ID NO: 6, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 6; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0043] In one or more embodiments, the TRP5 gene has the nucleotide sequence shown in SEQ ID NO: 7, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 7; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0044] In one or more embodiments, the yddG gene has the nucleotide sequence shown in SEQ ID NO: 8, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 8; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; and more preferably more than 97%.

[0045] In one or more embodiments, the ScIDP2 gene has the nucleotide sequence shown in SEQ ID NO: 9, or its degenerate sequence, or the nucleotide coding sequence of a functional protein having more than 70% homology with the sequence of SEQ ID NO: 9; wherein the homology is preferably more than 80%; more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%.

[0046] ARO9 , ARO7 , PRK The gene is the name of the knockout gene, and its core conserved sequence needs to be knocked out. ARO9, ARO7, and PRK can be found in the Genebank database with IDs of PAS_chr4_0147, PAS_chr4_0050, and PAS_chr2-1_0769, respectively.

[0047] Genes and their expression systems In this invention, L-tryptophan is produced in engineered yeast strains by introducing exogenous genes and utilizing the intracellular system of yeast cells, thereby utilizing methanol as a carbon source. In a preferred embodiment, Pichia pastoris is used as the substrate cell, and through metabolic engineering of the strain, the yeast cells are fermented to synthesize L-tryptophan using methanol as a substrate.

[0048] In this invention, the aforementioned genes can be naturally occurring, for example, they can be isolated or purified from autotrophic plants or microorganisms. Furthermore, the genes can also be artificially prepared, for example, by using conventional genetic engineering recombination techniques or by artificial synthesis methods.

[0049] The nucleotide sequences of the aforementioned genes may be identical to the corresponding sequences shown in SEQ ID NO: 1–9, or they may be degenerate variants of them. As used herein, “degenerate variant” refers to a nucleic acid sequence that encodes a protein having the same function but differs from the corresponding sequences shown in SEQ ID NO: 1–9.

[0050] The gene may include: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0051] The present invention also relates to variants of the aforementioned gene that encode a polypeptide that differs from its corresponding wild-type polypeptide in amino acid sequence, and is a fragment, analogue, or derivative of the wild-type polypeptide. This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a form of polynucleotide substitution, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0052] In this invention, "strict conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the similarity between the two sequences is at least 90%, preferably more than 95%. Furthermore, the hybridizable polynucleotide-encoded polypeptide has the same biological function and activity as the corresponding wild-type polypeptide.

[0053] It should be understood that although the genes of this invention are preferably obtained from *Saccharomyces cerevisiae* and *Pichia pastoris*, other genes obtained from other microorganisms that are highly homologous to the corresponding genes in *Saccharomyces cerevisiae* and *Pichia pastoris*, such as those with more than 70%, such as 80%, 90%, 95%, or even 98% sequence identity, are also within the scope of this invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0054] The full-length sequences or fragments of the genes of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications can be performed, and then the fragments amplified from each amplification can be spliced ​​together in the correct order.

[0055] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell generated by genetic engineering using the aforementioned vector.

[0056] In this invention, the sequences of each gene can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0057] The sequences of each gene can be inserted separately into a recombinant expression vector, and multiple recombinant expression vectors can be co-transformed into host cells; expression cassettes of multiple genes can also be inserted in tandem into the same recombinant expression vector and transformed into host cells. The recombinant expression vector may also contain expression regulatory sequences operatively linked to the gene sequences to facilitate protein expression. It should be understood that those skilled in the art, after understanding the technical content of this invention, can easily construct recombinant expression vectors. The obtained recombinant expression vectors are also included in this invention.

[0058] In expression regulatory sequences or expression cassettes, inducible or constitutive promoters can be applied according to different needs. Inducible promoters can achieve more controllable protein expression and compound production, which is beneficial for industrial applications.

[0059] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0060] The obtained transformants can be cultured using conventional methods, and the culture medium used can be a yeast culture medium well-known in the art. Culture should be carried out under conditions suitable for yeast cell growth.

[0061] Secondly, the present invention provides a method for producing L-tryptophan by fermentation, comprising inoculating the above-mentioned engineered yeast strain into a culture medium and fermenting it at 25–37°C for 1–15 days to synthesize L-tryptophan; wherein the culture medium uses methanol as the sole carbon source. The pH value of the culture medium is preferably 4.0–7.6, such as 4.0, 4.5, 5.0, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, etc. The culture temperature of the engineered yeast strain is limited to 25–37°C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37°C, etc., preferably, the fermentation culture temperature is 30±2°C.

[0062] Because excessively high methanol concentrations inhibit the growth of the engineered yeast and slow down its growth rate, in order to shorten the fermentation cycle and improve the cell viability of the engineered yeast, in the fermentation process using methanol as a substrate, glucose is first used as the carbon source to rapidly grow the engineered yeast strain, and then the process is switched to methanol as the carbon source for product synthesis. Genes related to the tryptophan synthesis pathway are used... AOX1 Promoter expression does not occur without methanol, and no product is synthesized. Gene expression and product synthesis only begin when the methanol carbon source is switched. Therefore, before fermentation, the engineered yeast strain is first seed cultured using a glucose-based medium, such as YPD medium; then the seed culture is inoculated into shake flask or fermenter medium for fermentation.

[0063] The culture media used for culturing engineered yeast strains include, but are not limited to, YPM, YPD, YPG, and YPE media. YPD or YEPD (Yeast Extract Peptone Dextrose Medium) is also called yeast extract peptone dextrose medium; when agar is added, it is called yeast extract peptone dextrose (YPD) agar medium. YPG medium is a commonly used yeast culture medium, whose main components include yeast extract, peptone, and glycerol. YPE medium is a commonly used yeast culture medium, whose main components include yeast extract, peptone, and ethanol. YPM medium is a commonly used yeast culture medium, whose main components include yeast extract, peptone, and methanol, such as BMMY medium.

[0064] The method for producing methanol-based yeast by shake-flask fermentation includes: inoculating the above-mentioned engineered yeast strain into liquid YPD medium and culturing it at 28–32°C and 150–250 r / min to obtain a concentration of (1–10) × 10⁻⁶. 7 A seed culture of cfu / mL was prepared; this seed culture was inoculated into a 250 mL Erlenmeyer flask containing 40–60 mL of BMMY liquid medium and fermented at 26–34 °C for 2–4 days. The BMMY medium contained a phosphate buffer pair with a pH of 5.8–7.6, preferably 5.8–6.5.

[0065] To further increase the yield of L-tryptophan, during the shake-flask fermentation process, 0.5%–2% (w / v) methanol is added to the BMMY liquid medium every 12–36 hours, such as 12, 15, 18, 20, 22, 24, 28, 32, and 36 hours. More preferably, 0.5%–2% (m / v) methanol is added every 16–30 hours. Preferably, during the shake-flask fermentation process, the shaking speed is 150–250 r / min, such as 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, and 250 r / min; more preferably, it is 180–220 r / min.

[0066] The method for producing methanol by reactor fermentation using methanol as a substrate includes the following steps: Seed culture: The engineered yeast strain was inoculated into a shake flask containing YPD medium and cultured to obtain (1–5) × 10⁻⁶ samples. 8 Seed culture with cfu / mL; Fermentation in a fermenter: The seed culture is transferred to a fermenter containing BMMY fermentation medium and fermented at 25–37°C for 3–10 days, preferably 3–7 days, such as 3, 4, 5, 6, or 7 days. Specifically, BMMY fermentation medium is added to the fermenter, and glucose is initially added at a flow rate of 20–40 g / h. When the wet weight of the engineered yeast cells reaches 180–220 g / L, glucose feeding is stopped, and the cells are starved for 1.5–2.5 h. Then, methanol is added at a flow rate of 4–6 g / h, and the methanol utilization capacity is monitored and evaluated to prevent excessive methanol accumulation. Subsequently, the methanol flow rate is gradually increased, with a maximum flow rate of 25 g / h. During the batch addition stage of the culture medium, the pH value in the fermenter was maintained at 4.5, and during the feeding stage, it was adjusted to 5.5-6.5 by automatically controlling the addition of NH4OH (50%, v / v); the dissolved oxygen (DO) was maintained above 30% by automatically adjusting the stirring speed between 200-1000 rpm.

[0067] To maintain the dissolved oxygen in the fermenter at a level above 30%, the stirring speed is limited to 200–1000 rpm, such as 200, 300, 400, 500, 600, 700, 800, 900, or 1000 rpm; the liquid volume of the fermenter is preferably 40%–75% (volume fraction), such as 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

[0068] Thirdly, the present invention provides the application of the described engineered yeast strain in the production of L-tryptophan using methanol as a carbon source.

[0069] This invention provides an application of a gene combination, wherein the gene combination comprises the following group of genes: ARO4, CRS, TRP1, TRP2, TRP3, TRP4, TRP5, yddG and ScIDP2 ; and knockout of ARO9, ARO7, and PRK genes; this gene combination is used to transform engineered yeast strains to produce L-tryptophan using methanol as a substrate.

[0070] Fourthly, the present invention provides a kit for producing L-tryptophan, the kit comprising the aforementioned engineered yeast strain. Preferably, the culture medium used for culturing the engineered yeast strain includes, but is not limited to, YPM, YPD, YPG, YPE, etc.

[0071] The recombinant Pichia pastoris strain obtained by the present invention through gene recombination technology has the characteristics of low metabolic background, strong heterologous expression ability, de novo synthesis of end products using methanol, easy separation of end products and few by-products, which will largely solve the problems existing in traditional biological and chemical synthesis methods and provide a new idea for the industrial production of L-tryptophan.

[0072] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] Material The plasmid construction method used was Novizan Biosciences' Seamless Cloning Kit.

[0074] All enzymes used were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), and the specific reaction conditions and methods were as per the product instructions. Unless otherwise specified, all promoters used in the examples were commercially available products.

[0075] The following commercially available plasmids and strains were used for gene cloning and protein expression: *E. coli* Top10 strains, all purchased from Qingke Biotechnology Co., Ltd. The yddG gene was synthesized by Qingke Biotechnology Co., Ltd. and constructed into the PpicZB vector, resulting in the plasmid PpicZB-yddG. Δ ku70 See Liu Q, Shi X, Song L, Liu H, Zhou X, Wang Q, Zhang Y, Cai M. CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris . Microbial Cell Factories 2019, 18:144.

[0076] The exogenous gene sequences introduced into the constructed strains are shown in Table 1, the primer sequences used are shown in Table 2, and the gRNA sequences used for the insertion sites are shown in Table 3. Among these, the exogenous gene... yddG It is a synthetically produced Pichia pastoris codon-optimized DNA sequence, gene ARO4 , TRP1 , TRP2 , TRP3 , TRP4 , TRP5 and ScIDP2 All are derived from Saccharomyces cerevisiae, gene CRS It is derived from Pichia pastoris.

[0077] culture medium YPD liquid medium: glucose 20.0 g / L, peptone 20.0 g / L, yeast extract 10.0 g / L; YPD solid medium: glucose 20.0 g / L, peptone 20.0 g / L, yeast extract 10.0 g / L, agar 20.0 g / L; BMMY: Peptone 20.0 g / L, Yeast extract 10.0 g / L, 0.67% YNB, 0.575 g / L dipotassium hydrogen phosphate, 2.95 g / L potassium dihydrogen phosphate, methanol 10 g / L; When preparing the above culture media, glucose should be autoclaved at 115°C for 20 min, and methanol should be added before use. Other components should be autoclaved at 121°C for 20 min.

[0078] Table 1. Nucleotide sequences of exogenous genes used in the embodiments of the present invention. Gene name DNA sequence number Gene name DNA sequence number Gene name DNA sequence number SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 7 CRS SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 8 SEQ ID NO: 3 SEQ ID NO: 6 SEQ ID NO: 9 Table 2. Nucleotide sequences of primers used in the embodiments of the present invention Serial Number Primer name Serial Number Primer name Serial Number Primer name SEQ ID NO: 10 int12-UP-F SEQ ID NO:32 TRP3-F SEQ ID NO:54 int11-pG-R SEQ ID NO:11 int12-pA-R SEQ ID NO:33 TRP3-R SEQ ID NO:55 Int11-tA-F SEQ ID NO:12 pAOX1-F SEQ ID NO:34 TRP4-F SEQ ID NO:56 Int11-DO-R SEQ ID NO:13 pAOX1-R SEQ ID NO:35 TRP4-R SEQ ID NO:57 ScIDP2-F SEQ ID NO:14 TRP2-F SEQ ID NO:36 IntE16-tA-F SEQ ID NO:58 ScIDP2-R SEQ ID NO:15 TRP2-TB-R SEQ ID NO:37 IntE16-DO-R SEQ ID NO:59 int1-UP-F SEQ ID NO:16 TRP2-TB-F SEQ ID NO:38 int15-UP-F SEQ ID NO:60 int1-pG-R SEQ ID NO:17 TRP2-R SEQ ID NO:39 int15-pA-R SEQ ID NO:61 Int1-tA-F SEQ ID NO:18 AOXTT-F SEQ ID NO:40 ARO4-F SEQ ID NO:62 Int1-DO-R SEQ ID NO:19 AOXTT-R SEQ ID NO:41 ARO4-TB-R SEQ ID NO:63 ARO9-UP-F SEQ ID NO:20 int12-tA-F SEQ ID NO:42 ARO4-TB-F SEQ ID NO:64 ARO9-UP-R SEQ ID NO:21 int12-DO-R SEQ ID NO:43 ARO4-R SEQ ID NO:65 ARO9-DO-F SEQ ID NO:22 int18-UP-F SEQ ID NO:44 pAOX2-F2 SEQ ID NO:66 ARO9-DO-R SEQ ID NO:23 int18-pA-R SEQ ID NO:45 CRS-F SEQ ID NO:67 ARO7-UP-F SEQ ID NO:24 TRP1-F SEQ ID NO:46 CRS-R SEQ ID NO:68 ARO7-UP-R SEQ ID NO:25 TRP1-R SEQ ID NO:47 CYC1TT-F SEQ ID NO:69 ARO7-DO-F SEQ ID NO:26 TRP5-F SEQ ID NO:48 CYC1TT-R SEQ ID NO:70 ARO7-DO-R SEQ ID NO:27 TRP5-R SEQ ID NO:49 pAOX2-F SEQ ID NO:71 pyk-UP-F SEQ ID NO:28 int18-tA-F SEQ ID NO:50 pAOX2-R SEQ ID NO:72 pyk-UP-R SEQ ID NO:29 int18-DO-R SEQ ID NO:51 Int15-tA-F SEQ ID NO:73 pyk-DO-F SEQ ID NO:30 intE16-UP-F SEQ ID NO:52 Int15-DO-R SEQ ID NO:74 pyk-DO-R SEQ ID NO:31 intE16-pA-R SEQ ID NO:53 int11-UP-F SEQ ID NO:84 pGAP-F SEQ ID NO:85 pGAP-R Table 3. Nucleotide sequences of gRNA targets used in the embodiments of the present invention. Serial Number gRNA Serial Number gRNA Serial Number gRNA SEQ ID NO:75 Int1 SEQ ID NO:78 Int15 SEQ ID NO:81 ARO9 SEQ ID NO:76 Int11 SEQ ID NO:79 IntE16 SEQ ID NO:82 ARO7 SEQ ID NO:77 Int12 SEQ ID NO:80 Int18 SEQ ID NO:83 pyk Methanol is metabolized to produce branched acid, a precursor in the L-tryptophan synthesis pathway. The branched acid is then synthesized into L-tryptophan by enzyme catalysis. Finally, the generated L-tryptophan is transported to the extracellular space via the yddG protein. The entire reaction process is a de novo synthesis of the final product.

[0079] I. Construction of Donor Fragments 1. Construction of int12-TRP2 donor fragment Using the Pichia pastoris genome as a template, the int12-UP fragment was amplified using primers int12-UP-F / int12-pA-R; the P fragment was amplified using primers pAOX1-F / pAOX1-R using the Pichia pastoris genome as a template. AOX1 Promoter fragment; using the *Saccharomyces cerevisiae* genome as a template, the mutant TRP2 gene was amplified using primers TRP2-F / TRP2-TB-R and TRP2-TB-F / TRP2-R; using the *Pichia pastoris* genome as a template, the AOXTT terminator fragment was amplified using primers AOXTT-F / AOXTT-R; using the *Pichia pastoris* genome as a template, the int12-DO fragment was amplified using primers int12-tA-F / int12-DO-R; then, the above fragments were ligated by overlap PCR to obtain the int12-TRP2 donor fragment.

[0080] 2. Construction of int18-TRP1TRP5 donor fragments Using the Pichia pastoris genome as a template, primers int18-UP-F / int18-pA-R were used to amplify the int18-UP fragment; using the Pichia pastoris genome as a template, primers pAOX1-F / pAOX1-R were used to amplify the P fragment. AOX1 Promoter fragments; using *Saccharomyces cerevisiae* genome as a template, the TRP1 and TRP5 genes were amplified using primers TRP1-F / TRP1-R and TRP5-F / TRP5-R, respectively; using *Pichia pastoris* genome as a template, the AOXTT terminator fragment was amplified using primers AOXTT-F / AOXTT-R; using *Pichia pastoris* genome as a template, the CYC1 terminator fragment was amplified using primers CYC1TT-F / CYC1TT-R; using *Pichia pastoris* genome as a template, the P gene was amplified using primers pAOX2-F / pAOX2-R. AOX2 Promoter fragment; using the Pichia pastoris genome as a template, the int18-DO fragment was amplified using primers int18-tA-F / int18-DO-R; then, the above fragments were ligated by overlap PCR to obtain the int18-TRP1TRP5 donor fragment.

[0081] 3. Construction of intE16-TRP3TRP4 donor fragment Using the Pichia pastoris genome as a template, the primers intE16-UP-F / intE16-pA-R were used to amplify the intE16-UP fragment; using the Pichia pastoris genome as a template, the primers pAOX1-F / pAOX1-R were used to amplify the P fragment. AOX1 Promoter fragments were obtained; TRP3 and TRP4 genes were amplified using primers TRP3-F / TRP3-R and TRP4-F / TRP4-R, respectively, using *Saccharomyces cerevisiae* genome as a template; AOXTT terminator fragments were amplified using primers AOXTT-F / AOXTT-R, using *Pichia pastoris* genome as a template; CYC1 terminator fragments were amplified using primers CYC1TT-F / CYC1TT-R, using *Pichia pastoris* genome as a template; pAOX2 promoter fragments were amplified using primers pAOX2-F / pAOX2-R, using *Pichia pastoris* genome as a template; and intE16-DO fragments were amplified using primers intE16-tA-F / intE16-DO-R, using *Pichia pastoris* genome as a template. Then, the above fragments were ligated by overlap PCR to obtain the intE16-TRP3TRP4 donor fragment.

[0082] 4. Construction of int15-ARO4-CRS donor fragment Using the Pichia pastoris genome as a template, primers int15-UP-F / int15-pA-R were used to amplify the int15-UP fragment; using the Pichia pastoris genome as a template, primers pAOX1-F / pAOX1-R were used to amplify the P fragment. AOX1 Promoter fragments were obtained; the ARO4 gene was amplified using primers ARO4-F / ARO4-TB-R and ARO4-TB-F / ARO4-R as a template; the AOXTT terminator fragment was amplified using primers AOXTT-F / AOXTT-R as a template; the CYC1 terminator fragment was amplified using primers CYC1TT-F / CYC1TT-R as a template; the pAOX2 promoter fragment was amplified using primers pAOX2-F2 / pAOX2-R as a template; the int15-DO fragment was amplified using primers int15-tA-F / int15-DO-R as a template; and then, the above fragments were ligated by overlap PCR to obtain the int15-ARO4-CRS donor fragment.

[0083] 5. Construction of int11-yddG donor fragment The yddG gene was codon-optimized by Qingke Biotechnology and constructed into the pGAPZB vector. The yddG expression cassette was amplified using primers pGAP-F / AOXTT-R. The int11-UP fragment was amplified using int11-UP-F / int11-pG-R as a template. The int11-DO fragment was amplified using primers int11-tA-F / int11-DO-R. Then, the above fragments were ligated by overlap PCR to obtain the int11-yddG donor fragment.

[0084] 6. Construction of int1-ScIDP2 donor fragment Using the Pichia pastoris genome as a template, the int1-UP fragment was amplified using primers int1-UP-F / int1-pG-R; using the Pichia pastoris genome as a template, the P fragment was amplified using primers pGAP-F / pGAP-R. GAP Promoter fragment; using Saccharomyces cerevisiae genome as template, the ScIDP2 gene was amplified using primers ScIDP2-F / ScIDP2-R; using Pichia pastoris genome as template, the AOXTT terminator fragment was amplified using primers AOXTT-F / AOXTT-R; using Pichia pastoris genome as template, the int1-DO fragment was amplified using primers int1-tA-F / int1-DO-R; then, the above fragments were ligated by overlap PCR to obtain the int1-ScIDP2 donor fragment.

[0085] 7. Construction of ΔARO9 donor fragment Using the Pichia pastoris genome as a template, the ARO9-UP fragment was amplified using primers ARO9-UP-F / ARO9-UP-R; the ARO9-DO fragment was amplified using primers ARO9-DO-F / ARO9-DO-R; then, the above fragments were ligated by overlap PCR to obtain the ΔARO9 donor fragment.

[0086] 8. Construction of the ΔARO7 donor fragment Using the Pichia pastoris genome as a template, the ARO7-UP fragment was amplified using primers ARO7-UP-F / ARO7-UP-R; the ARO7-DO fragment was amplified using primers ARO7-DO-F / ARO7-DO-R; then, the above fragments were ligated by overlap PCR to obtain the ΔARO7 donor fragment.

[0087] 9. Construction of Δpyk donor fragments Using the Pichia pastoris genome as a template, the pyk-UP fragment was amplified using primers pyk-UP-F / pyk-UP-R; the pyk-DO fragment was amplified using primers pyk-DO-F / pyk-DO-R; then, the above fragments were ligated by overlap PCR to obtain the Δpyk donor fragment.

[0088] II. Electroporation of Pichia pastoris and Construction of Engineered Strains 1. Construction of TRP1, TRP2, and TRP3 engineered strains The int12-TRP2 donor fragment was converted together with int12-gRNA to Δ ku70 Transformants were screened on YPD+ZeocinR plates. Transformants with correctly integrated genes were obtained as strain TRP1. The int18-TRP1TRP5 donor fragment was transformed into TRP1 along with the int18-gRNA plasmid. Transformants were screened on YPD+ZeocinR plates again. Transformants with correctly integrated genes were obtained as strain TRP2. The intE16-TRP3TRP4 donor fragment was transformed into TRP2 along with the intE16-gRNA plasmid. Transformants were screened on YPD+ZeocinR plates again. Transformants with correctly integrated genes were obtained as strain TRP3.

[0089] 2. Construction of TRP4 engineered strains The int15-ARO4-CRS donor fragment was transformed into TRP3 along with int15-gRNA. The transformed cells were screened on YPD+ZeocinR plates. The TRP4 strain was obtained by screening for the correct transformed cells with gene integration.

[0090] 3. Construction of TRP5 engineered strains The int11-yddG donor fragment was transformed into TRP4 along with int11-gRNA. Transformants were screened on YPD+ZeocinR plates. The TRP5 strain was obtained by screening for the correct transformants with gene integration.

[0091] 4. Construction of TRP6 engineered strains The int1-ScIDP2 donor fragment was transformed into TRP5 along with int1-gRNA. The transformed cells were then screened on YPD+ZeocinR plates. The TRP6 strain was obtained by screening for the correct transformed cells with gene integration.

[0092] 5. Construction of TRP7 engineered strains The ΔARO9 donor fragment was transformed into TRP6 along with ARO9-gRNA. The transformed strains were then screened on YPD+ZeocinR plates. The TRP7 strain was obtained by screening for the correct transformed strain with gene integration.

[0093] 6. Construction of TRP8 engineered strains The ΔARO7 donor fragment was transformed into TRP7 along with ARO7-gRNA. The transformed strains were then screened on YPD+ZeocinR plates. The TRP8 strain was obtained by screening for the correct transformed strain with gene integration.

[0094] 7. Construction of TRP9 engineered strains The Δpyk donor fragment was transformed into TRP8 along with pyk-gRNA, and the transformants were screened on YPD+ZeocinR plates. The TRP9 strain was obtained by screening for the correct transformants with gene integration.

[0095] III. Fermentation and Product Identification of Recombinant Engineered Bacteria 1. Recombinant engineered bacteria shake-flask fermentation process Each TRP engineered strain constructed in "II. Construction of Pichia pastoris by Electroporation and Engineered Strains" was inoculated into liquid YPD medium at a 1% inoculum and cultured at 30℃ and 200 r / min until the recombinant bacteria reached the logarithmic growth phase. The bacterial resuspended was then inoculated into a 250 mL Erlenmeyer flask containing 50 mL of BMMY liquid medium with a pH of 6.0 phosphate buffer. The flask was cultured at 30℃ and 200 r / min for 3 days. 0.5% (w / v) methanol was added every 24 h. 1 OD is approximately 5 × 10⁻⁶. 7 One yeast cell (OD value is yeast cell concentration unit, OD value is measured by ultraviolet spectrophotometer at a wavelength of 600 nm).

[0096] 2. Identification of fermentation products from recombinant strains After fermentation, 1 mL of the fermentation broth was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for high performance liquid chromatography (HPLC) analysis.

[0097] The HPLC method used an Agilent C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was 0.3 g / L KH2PO4:methanol = 90:10; the flow rate was 1 mL / min; the detection wavelength was 280 nm; and the temperature was 39℃.

[0098] (1) The HPLC identification results of the fermentation broth of engineered strain TRP9 are as follows: Figure 2 As shown, from Figure 2 As can be seen from this, the engineered strain TRP9 can directly synthesize L-tryptophan after shaking flask fermentation with methanol as the sole carbon source.

[0099] (2) Product yield of each strain in shake flask fermentation The yields of L-tryptophan produced by engineered strains TRP1–TRP9 using methanol as a substrate in shake-flask fermentation are as follows: Figure 3 As shown.

[0100] from Figure 3 It can be seen from this: 1) Strain TRP3 showed a 14% increase in yield compared to wild-type WT, indicating that the methanol substrate and P... AOX1 The synergistic effect of the induction system can overcome the bottleneck of natural metabolism; 2) Based on TRP3, the engineered strain TRP4 expresses two key genes of the shikimic acid pathway, ARO4 (mutant ARO4), through a methanol-inducible promoter. K229L (Relief of feedback inhibition) and CRS construction; the tryptophan yield of the engineered strain TRP4 was increased by 37% compared with TRP3, which is significantly higher than the increase of single gene modification in the existing technology, proving that a leapfrog increase in product synthesis can be achieved by synergistically enhancing the precursor pathway. This combined modification strategy has a significant effect on improving product synthesis. It also proves that the carbon flow partition of methanol substrate is more suitable for tryptophan precursor synthesis; 3) The engineered strain TRP5 was constructed by overexpressing the tryptophan-specific transporter gene yddG in TRP4. The extracellular tryptophan production of TRP5 increased by about 10% compared with TRP4. This is the first demonstration of the promoting effect of yddG on tryptophan efflux in the methanol yeast system, providing a new technical means to relieve product inhibition. 4) Engineered strain TRP6 introduces NADP derived from Saccharomyces cerevisiae into TRP5. + Citrate-dependent dehydrogenase gene Sc IDP2, by enhancing NADP + The improved NADPH conversion efficiency and increased cofactor supply resulted in a 10% increase in yield compared to TRP5. This cofactor optimization strategy, combined with the precursor pathway, forms a metabolic synergy, further overcoming synthetic limitations. 5) Knocking out the key tryptophan degradation gene ARO9 in strain TRP6 to construct TRP7 resulted in a 24% increase in yield compared to TRP6. Further knocking out the aromatic amino acid branching gene ARO7 (blocking tyrosine / phenylalanine synthesis) to construct TRP8 resulted in an 11% increase in yield compared to TRP7. However, knocking out the PEP consumption gene pyk (blocking the ineffective shunting of PEP to pyruvate) did not show a significant change in yield compared to TRP8 in TRP9. This demonstrates that the combined knockout of ARO9 and ARO7 specifically reduces carbon flux loss. This precise, targeted branching blocking strategy is significantly superior to existing methods that blindly knock out multiple genes, showcasing the innovation of metabolic network regulation.

[0101] IV. Product yield of engineered strains using methanol as a substrate in reactor fermentation Using the TRP9 engineered strain as the object and methanol as the substrate, the reactor fermentation method includes: Seed culture: The TRP9 engineered strain was incubated at a 1% inoculum in a 1 L shake flask containing 200 mL of YPD medium for 12–16 hours to obtain OD. 600 =6 seed liquid; Fermentation in a fermenter: The seed culture was transferred to a 5 L fermenter containing 3 L of BMMY fermentation medium. During the batch stage, the pH was maintained at 4.5, and during the fed-batch stage, it was adjusted to 6.0 by automatically controlled addition of NH4OH (50%, v / v). Dissolved oxygen (DO) was maintained above 30% by automatically adjusting the stirring speed between 200 and 1000 rpm. The fermentation stage involved first culturing TRP9 engineered bacteria using glucose as the carbon source, and then synthesizing L-tryptophan using methanol as the carbon source. Specifically, the initial culture used glucose as the substrate, with a feed rate of 30 g / h. When the cell wet weight reached 200 g / L, glucose feeding was stopped, and the cells were starved for 2 hours. Then, methanol was initially fed at a flow rate of 5 g / h, with feeding interrupted hourly to assess methanol utilization and prevent excessive methanol accumulation. The methanol feed rate was gradually increased, with a maximum flow rate of 20 g / h. Tryptophan synthesis in the fermenter culture was as follows... Figure 4 As shown, the tryptophan yield reached its maximum of 10 g / L after 140 h of fermentation.

[0102] In summary, this invention achieves a step-by-step increase in tryptophan production through a step-by-step metabolic engineering strategy of "core pathway enhancement → precursor supply improvement → product efflux optimization → cofactor synergy → branching carbon flow regulation." The synergistic effects of each modification step are significant, with the enhancement of the precursor pathway (37%) and the blocking of the branching pathway (ARO9 knockout 24%) being particularly outstanding. This invention is the first to combine methanol substrate with P... AOX1The induction system coupling dynamically matches gene expression intensity with methanol consumption rate, solving the regulatory lag problem caused by the separation of inducer and substrate in traditional carbon sources. Furthermore, methanol costs only one-third that of glucose and can be produced from natural gas or industrial waste gas, making it widely available. As a liquid substrate, methanol is easy to sterilize and precisely control, resulting in higher pH stability during fermentation and reducing byproduct (such as ethanol and acetic acid) content by more than 60% compared to the glucose system, simplifying downstream separation processes. This invention provides an industrially feasible technical solution for the efficient microbial synthesis of tryptophan.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A yeast strain that utilizes methanol as a carbon source to ferment and produce L-tryptophan, characterized in that, Including the transformation of genes TRP1, TRP2, TRP3, TRP4 and TRP5 An overexpression cassette, wherein the overexpression cassette includes a methanol-inducible promoter.

2. The engineered yeast strain according to claim 1, characterized in that, The basal cells of the engineered yeast strain are Pichia pastoris engineered strains. GS115 Or a strain lacking KU70.

3. The engineered yeast strain according to claim 1 or 2, characterized in that, The engineered yeast strain was further transformed with two key genes containing the shikimic acid pathway. ARO4 and CRS The expression cassettes include methanol-inducible promoters.

4. The engineered yeast strain according to claim 3, characterized in that, Furthermore, it was transformed to contain a gene containing a tryptophan-specific transporter protein. yddG An overexpression cassette, which includes a constitutive promoter.

5. The engineered yeast strain according to claim 4, characterized in that, It also further converts NADP-containing + Citrate-dependent dehydrogenase gene ScIDP2 The expression box includes a constitutive promoter.

6. The engineered yeast strain according to claim 5, characterized in that, They also knocked out key genes for tryptophan degradation. ARO9 .

7. The engineered yeast strain according to claim 5, characterized in that, They also knocked out key genes for tryptophan degradation. ARO9 Aromatic amino acid branch point genes ARO7 .

8. A method for the fermentation production of L-tryptophan, comprising inoculating an engineered yeast culture medium according to any one of claims 1 to 7 into a culture medium, and fermenting it at 25 to 37°C for 1 to 15 days to synthesize L-tryptophan; wherein, The culture medium uses methanol as the sole carbon source.

9. The fermentation production method according to claim 8, characterized in that, Concentration (1~10) ×10 7 The engineered yeast culture at cfu / mL was inoculated into an Erlenmeyer flask containing culture medium at pH 5.8–7.6 and fermented at 26–34°C for 2–5 days at a rotation speed of 150–250 r / min; and 0.5%–2% methanol was added to the culture medium every 12–36 h.

10. The fermentation production method according to claim 8 or 9, characterized in that, Concentration (1~5)×10 8 The engineered yeast culture at cfu / mL was inoculated into a fermenter containing BMMY fermentation medium and fermented at 25–37°C for 3–10 days at a rotation speed of 200–1000 r / min.

11. The fermentation production method according to claim 10, characterized in that, Methanol was added to the BMMY fermentation medium at a flow rate of 4–25 g / h, and the dissolved oxygen in the fermenter was maintained above 30%.

12. The use of the yeast engineered strain according to any one of claims 1 to 7 in the production of L-tryptophan using methanol as a carbon source.

13. A kit for producing L-tryptophan, characterized in that, It includes the engineered yeast strains as described in any one of claims 1 to 7.

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