Saccharomyces cerevisiae engineering bacteria for improving protein content, and construction method and application thereof

Abstract

The present application relates to a kind of protein content of saccharomyces cerevisiae engineering bacteria and its construction method and application, belong to microbial technology field.The protein content of the present application is raised saccharomyces cerevisiae engineering bacteria with saccharomyces cerevisiae uracil deficiency haploid strain as starting strain, while overexpressing citrate synthase gene CIT1, isocitrate dehydrogenase gene IDH1, glutamate synthetase gene GLT1, glutamate dehydrogenase gene GDH1, glutamine synthetase gene GLN1, ribosome transcription regulatory factor gene IFH1, valyl-tRNA synthetase gene VAS1 And one or more of ribosomal protein genes;Haploid hybridization obtains diploid, and transcription factor gene SUT1 is also overexpressed in diploid.The technical scheme realizes the systematic reconstruction from metabolic basis to synthesis machine to cell function for the first time, provides innovative technical path and efficient strain resources for the industrialized production of microbial cell protein.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an engineered strain of Saccharomyces cerevisiae with increased protein content, its construction method, and its application. Background Technology

[0002] Microbial cell protein (MCP), also known as single-cell protein (SCP), is widely considered a strategic solution to address future protein supply crises due to its advantages such as high production efficiency, small footprint, immunity to climate changes, and availability of various inexpensive raw materials. Among these, fungal MCPs, represented by yeast, have broad application prospects in the food and feed industries due to their rich nutritional content and high safety. Saccharomyces cerevisiae is a long-established and generally recognized as safe (GRAS) microorganism in the food industry. Its clear genetic background, simple culture conditions, and mature fermentation technology make it an ideal substrate cell for MCP production. Yeast cells themselves are rich in protein, B vitamins, and various trace elements, and the presence of the cell wall acts as a natural microencapsulation, helping to protect nutrients. However, the inherent defects of wild-type or industrial wild-type Saccharomyces cerevisiae strains severely restrict the economic feasibility of their MCP production: their natural protein content is limited, typically accounting for 40%-50% of the cell dry weight. This inherent bottleneck results in low protein yield per unit of biomass, leading to high downstream processing costs and making it difficult to gain an advantage in competition with traditional protein sources (such as soybean meal and fishmeal) and emerging alternative proteins. The development of this technology has evolved from traditional single-cell protein production to modern synthetic biology techniques: In terms of technological evolution, early methods primarily involved simple cultivation of wild-type microbial strains, resulting in limited protein content and nutritional value. With the development of metabolic engineering, researchers began to optimize the protein synthesis capabilities of microorganisms through genetic modification. In recent years, the integration of systems biology and synthetic biology has brought new breakthroughs to microbial cell protein production, enabling the synergistic regulation of multiple genes and pathways. In terms of product characteristics, microbial cell proteins have significant advantages: 1. Protein content can reach 40%-70% of cell dry weight; 2. Balanced amino acid composition, rich in essential amino acids; 3. Production process is not affected by seasons or climate; 4. Sustainable production can be achieved using a variety of inexpensive raw materials. In terms of application areas, microbial cell proteins have expanded from traditional feed additives to human nutritional supplements, functional food ingredients, specialty feed proteins, and food processing aids. However, this field still faces significant technological bottlenecks: the protein content and synthesis efficiency of existing production strains are still insufficient to meet the economic requirements of industrial production, especially when using conventional carbon sources, as the inherent metabolic regulation mechanisms of the strains limit the full realization of their protein synthesis potential. This necessitates the urgent development of new engineering techniques to fundamentally overcome the natural limitations of microbial protein synthesis.

[0003] In the field of microbial cell protein production, conventional strategies for increasing protein content mainly include random mutagenesis and fermentation process optimization. Studies have shown that strains with improved protein content can be obtained through adaptive evolution combined with high-throughput screening. However, the effectiveness of these methods is often limited by the inherent metabolic capacity and protein synthesis potential of natural strains. In recent years, the development of synthetic biology and systems metabolic engineering has provided new technical pathways for the systematic modification of Saccharomyces cerevisiae. With the support of genome editing tools such as CRISPR / Cas9, researchers have developed multi-level engineering strategies, including metabolic pathway reconstruction, key enzyme engineering, and optimization of protein synthesis mechanisms. Representative works reported in the existing literature include: using a stepwise engineering strategy to block ethanol synthesis and gluconeogenesis pathways while optimizing the fermentation medium composition, resulting in a significant increase in the protein content of engineered strains; and successfully increasing protein content by modifying the synthesis pathways of cell wall structural components through cell wall engineering strategies. At the level of basic biological research, there is ample evidence that protein biosynthesis is a highly coordinated and resource-intensive core cellular process, the efficiency of which is precisely regulated at multiple levels. This process encompasses multiple stages, including transcription, translation, and post-translational modifications. Its throughput efficiency primarily depends on the following key factors: the supply level of substrates such as amino acids, the number and activity state of ribosomes, translation initiation efficiency, tRNA gene copy number and aminoacyl-tRNA synthetase activity, as well as an adequate supply of ATP and reducing power. However, there is currently no strategy for systematically modifying and studying the protein synthesis and intracellular protein content of Saccharomyces cerevisiae.

[0004] Traditional methods employ physicochemical mutagenesis combined with high-throughput screening to obtain mutant strains with increased protein content. However, this method, based on random mutations, struggles to achieve targeted optimization of metabolic networks. In metabolic engineering, existing technologies include single-pathway modification strategies such as enhancing specific carbon metabolic pathways, optimizing culture media, and knocking out byproduct synthesis pathways. The closest prior art discloses a stepwise metabolic engineering method that increases protein yield by sequentially blocking ethanol synthesis, weakening gluconeogenesis, and combining it with culture media optimization. However, this technology still has significant limitations: it primarily focuses on carbon metabolism modification without achieving synergistic carbon-nitrogen regulation, fails to address the systematic enhancement of protein synthesis machinery, and is limited to haploid strains, neglecting the stability requirements of industrial production. Other related technologies include methods that redirect carbon flux to protein synthesis through cell wall engineering strategies. Overall, existing technologies generally suffer from a lack of multi-level system optimization, simplistic modification strategies, insufficient industrial stability, and a ceiling on protein content enhancement—the core technical problems that this patent application aims to solve. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing microbial cell protein production technologies, such as single-dimensional modification, insufficient system coordination, and poor industrial adaptability. By constructing a multi-level synergistic engineering strategy that includes a precursor supply layer, a translation machine layer, and a cell physiological layer, a brewer's yeast engineered strain with both high protein content and excellent industrial adaptability is developed. This effectively solves the difficulty in increasing protein content caused by single-gene modification, breaks through the technical bottleneck of difficulty in increasing protein content, and achieves the goal of maintaining stable and high performance in industrial production in bioreactors of 5 liters or more.

[0006] To address the technical problems of existing microbial cell protein production technologies, such as limited modification dimensions, insufficient system coordination, and poor industrial adaptability, this invention provides an engineered Saccharomyces cerevisiae strain with increased protein content, its construction method, and its applications. The core of this invention lies in the systematic three-tiered engineering strategy for full-chain optimization of Saccharomyces cerevisiae: at the precursor supply layer, by coordinating and strengthening key genes in central carbon and nitrogen metabolism, the carbon skeleton supply and nitrogen assimilation efficiency for amino acid synthesis are improved. This optimization of the precursor supply layer includes overexpressing at least one of the following genes: citrate synthase gene CIT1, isocitrate dehydrogenase gene IDH1, glutamate synthase gene GLT1, glutamate dehydrogenase gene GDH1, and glutamine synthase gene GLN1; at the translation machine layer, by enhancing ribosomal biosynthesis-related transcription factors and key ribosomal proteins... The expression of genes simultaneously enhances the capacity and efficiency of the translation machine. Optimization of the translation machine layer includes overexpression of at least one of the following genes: ribosomal transcription factor gene IFH1, valine-tRNA synthetase gene VAS1, and single-copy ribosomal protein genes RPS31, RPS15, RPL28, and RPL29. At the cellular physiological layer, by constructing a diploid industrial chassis and integrating key transcriptional regulatory factors, the environmental adaptability and protein accumulation capacity of the strain are significantly improved. Optimization of the cellular physiological layer includes converting high-protein-producing haploid strains into diploid strains and overexpressing the transcription factor gene SUT1. This technical solution achieves, for the first time, a systematic reconstruction from metabolic basis to synthetic machinery and then to cellular function, providing an innovative technical path and efficient strain resources for the industrial production of microbial cell proteins.

[0007] This invention is achieved through the following technical solution:

[0008] The first objective of this invention is to provide an engineered Saccharomyces cerevisiae strain with increased protein content. This engineered strain uses a uracil-deficient haploid strain of Saccharomyces cerevisiae as the starting strain and simultaneously overexpresses one or more of the following genes: citrate synthase CIT1, isocitrate dehydrogenase IDH1, glutamate synthase GLT1, glutamate dehydrogenase GDH1, glutamine synthase GLN1, ribosomal transcription factor gene IFH1, valine-tRNA synthase gene VAS1, and ribosomal protein genes. The haploid is then hybridized to obtain a diploid strain, in which the transcription factor gene SUT1 is overexpressed.

[0009] In one embodiment of the present invention, the ribosomal protein gene is one or more of the following: ribosomal protein gene RPS31, ribosomal protein gene RPS15, ribosomal protein gene RPL28, and ribosomal protein gene RPL29.

[0010] The sequence of the ribosomal protein gene RPS31 is shown in SEQ ID NO.8; the sequence of the ribosomal protein gene RPS15 is shown in SEQ ID NO.9; the sequence of the ribosomal protein gene RPL28 is shown in SEQ ID NO.10; and the sequence of the ribosomal protein gene RPL29 is shown in SEQ ID NO.11.

[0011] In one embodiment of the present invention, the NCBI ID of the citrate synthase is 855732;

[0012] The NCBI ID of the isocitrate dehydrogenase is 855691;

[0013] The NCBI ID of the glutamate synthase is 851383;

[0014] The NCBI ID of the glutamate dehydrogenase is 854557;

[0015] The NCBI ID of the glutamine synthase is 856147;

[0016] The NCBI ID of the valine-tRNA synthetase is 852986.

[0017] In one embodiment of the present invention, the sequence of the citrate synthase gene CIT1 is shown in SEQ ID NO.1;

[0018] The sequence of the isocitrate dehydrogenase gene IDH1 is shown in SEQ ID NO.2;

[0019] The sequence of the glutamate synthase gene GLT1 is shown in SEQ ID NO.3;

[0020] The sequence of the glutamate dehydrogenase gene GDH1 is shown in SEQ ID NO.4;

[0021] The sequence of the glutamine synthase gene GLN1 is shown in SEQ ID NO.5;

[0022] The sequence of the ribosomal transcription regulatory factor gene IFH1 is shown in SEQ ID NO.6;

[0023] The sequence of the valine-tRNA synthetase gene VAS1 is shown in SEQ ID NO.7;

[0024] The sequence of the transcription factor gene SUT1 is shown in SEQ ID NO.12.

[0025] The second objective of this invention is to provide a method for constructing the engineered brewer's yeast, comprising the following steps:

[0026] (1) Using a uracil-deficient strain of Saccharomyces cerevisiae as the starting strain, the glutamate synthase gene GLT1, the glutamate dehydrogenase gene GDH1, and the glutamine synthase gene GLN1 were co-expressed.

[0027] (2) Co-expression of citrate synthase gene CIT1 and isocitrate dehydrogenase gene IDH1;

[0028] (3) Overexpression of the valine-tRNA synthetase gene VAS1;

[0029] (4) Integrating ribosomal transcription regulatory factor gene IFH1 and ribosomal protein genes RPS31, RPS15, RPL28, and RPL29;

[0030] (5) Re-fuse the haploid engineered strain obtained in step (4) into a diploid strain;

[0031] (6) Overexpress the transcription factor gene SUT1 in the diploid strain obtained in step (5) to obtain the engineered Saccharomyces cerevisiae.

[0032] A third objective of this invention is to provide the application of the engineered Saccharomyces cerevisiae in increasing the protein content of microbial cells.

[0033] A fourth objective of this invention is to provide a method for increasing the protein content of microbial cells, including the step of fermentation production using the engineered strain of *Saccharomyces cerevisiae*.

[0034] In one embodiment of the present invention, the fermentation temperature is 28℃-32℃; and the culture is carried out at 300 rpm-1000 rpm.

[0035] In one embodiment of the present invention, the fermentation includes shake-flask fermentation or fed-batch fermentation.

[0036] In one embodiment of the present invention, the fermentation includes the following steps: inoculating the engineered Saccharomyces cerevisiae into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation.

[0037] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0038] (1) This invention provides an engineered strain of *Saccharomyces cerevisiae* with increased protein content, its construction method, and its application. This invention uses a uracil-deficient strain of *Saccharomyces cerevisiae* as the starting strain, and simultaneously overexpresses one or more of the following genes: citrate synthase CIT1, isocitrate dehydrogenase IDH1, glutamate synthase GLT1, glutamate dehydrogenase GDH1, glutamine synthase GLN1, ribosomal transcription factor gene IFH1, valine-tRNA synthase gene VAS1, and ribosomal protein genes. The resulting haploid engineered strain is then re-fused into a diploid strain and overexpressed with the transcription factor gene SUT1. The resulting engineered *Saccharomyces cerevisiae* strain has a protein content ≥70 g / 100g DCW, which is significantly higher than the protein content of *Saccharomyces cerevisiae* in the prior art.

[0039] (2) This invention effectively solves the common problem of insufficient amino acid supply in conventional modification by synergistically strengthening key genes of central nitrogen metabolism (GLT1, GDH1, GLN1) and core node genes of central carbon metabolism (CIT1, IDH1). Experiments have shown that this strategy increases the intracellular α-ketoglutarate (α-KG) level to 120.2%, which drives the overall expansion of the total amino acid library, thereby providing sufficient carbon backbone and amino acid donors for protein synthesis and achieving simultaneous growth in protein content and yield.

[0040] (3) This invention optimizes the matching between the "translation machine" and "translation efficiency," eliminating the rate-limiting link in protein synthesis. This invention not only enhances the quantity and function of ribosomes as "synthetic factories" by overexpressing the ribosomal transcription regulator IFH1 and specific single-copy ribosomal protein genes (such as RPS15 and RPL28), but also removes the substrate activation bottleneck in the translation process by precisely upregulating aminoacyl-tRNA synthetases (such as VAS1). This multi-level synergistic strategy of "substrate supply - synthetic machine - translation efficiency" enables the protein content of haploid strains to leapfrog to 57.31 g / 100g DCW.

[0041] (4) This invention demonstrates industrial potential and high-density fermentation stability. In a 5 L fermenter fed-batch fermentation verification, the engineered strain D3 constructed in this invention exhibited extremely strong growth and proliferation capabilities and metabolic homeostasis. Its intracellular protein content steadily increased with the fermentation process, reaching a peak of 75.16 g / 100g DCW, and it could still maintain highly efficient protein synthesis activity in the presence of high concentrations of ethanol. This data is significantly better than the protein content levels of Saccharomyces cerevisiae reported in current literature, and has extremely high industrial application value.

[0042] (5) This invention provides a systematic protein synthesis optimization model and a universal target library, which not only obtained specific superior strains, but also verified a series of effective targets across metabolic levels (involving carbon metabolism, nitrogen metabolism, and translation machines), providing a universal technical reference solution for constructing other high-yield protein microbial cell factories. Attached Figure Description

[0043] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0044] Figure 1 This invention relates to computer prediction and experimental verification of protein synthesis targets; wherein, a) is the protein synthesis optimization target predicted based on a genomic metabolic model; b) is the protein content of the strain verifying the upregulation of the target; c) is the protein yield and cell dry weight of the strain verifying the upregulation of the target; d) is the protein content of the strain verifying the knockout of the target; e) is the protein yield and cell dry weight of the strain verifying the knockout of the target.

[0045] Figure 2 This invention relates to the effect of combined nitrogen assimilation engineering on protein biosynthesis; where a is the growth curve of the combined enhanced strain; b is the nitrogen source consumption curve of the combined enhanced strain; c is the protein content of the combined enhanced strain; and d is the protein yield and cell dry weight graph of the combined enhanced strain.

[0046] Figure 3 This invention enhances the effect of the tricarboxylic acid cycle on protein biosynthesis; where a is the growth curve of the enhanced strain; b is the nitrogen source consumption curve of the enhanced strain; c is the protein content of the enhanced strain; d is the protein yield and cell dry weight of the enhanced strain; e is the relative amino acid content of the enhanced strain; and f is the relative α-ketoglutarate content of the enhanced strain.

[0047] Figure 4This invention enhances the effect of ribosome synthesis on protein production; where a represents the protein content of the strain with enhanced transcription factor IFH1; b represents the protein yield and cell dry weight of the strain with enhanced transcription factor IFH1; c represents the protein content of the strain with enhanced 40S subunit single gene encoding ribosomal protein; d represents the protein content of the strain with enhanced 60S subunit single gene encoding ribosomal protein; e represents the protein content of the combined enhanced strain; and f represents the protein yield and cell dry weight of the combined enhanced strain.

[0048] Figure 5 This invention describes the construction of a diploid yeast strain and the enhancement of transcription factor SUT1 to improve protein synthesis; wherein, a is a schematic diagram of the construction of the diploid strain; b is a gel electrophoresis diagram of the diploid strain verification; c is the protein content of the diploid yeast strain; d is the protein yield and cell dry weight of the diploid yeast strain; e is the protein content of the strain with the introduced mutation site; f is a graph showing the protein yield and cell dry weight of the strain with the introduced mutation site.

[0049] Figure 6 This invention describes the performance of the high-protein yeast strain D3 in a 5-L bioreactor fermentation. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0052] Example 1: Validation of protein synthesis targets predicted by a genome-scale model

[0053] This embodiment aims to verify the gene manipulation targets for increasing the protein content of Saccharomyces cerevisiae, predicted by a genome-scale metabolic model.

[0054] The ura 3Δ strain of Saccharomyces cerevisiae, Y1 (CEN. PK 113-7D, https: / / doi.org / 10.1016 / S0141-0229(00)00162-9, derived strain), preserved in the laboratory, was used as the starting strain. Using CRISPR-Cas9 technology, overexpression strains targeting predicted upregulated targets (aminoacyl-tRNA synthetase encoding genes ALA1, NCBI ID 854513; VAS1, NCBI ID 852986; GUS1, NCBI ID 852606; KRS1, NCBI ID 851607; ILS1, NCBI ID 852202) and gene knockout strains targeting predicted knockout targets (OAC1, NCBI ID 853739; TPO5, NCBI ID 853680; MDH3, NCBI ID 851481; RPE1, NCBI ID 853322) were constructed. The MET22 knockout strain (obtained by knocking out the MET22 gene solely in strain Y1) was not included in subsequent analyses due to severely impaired growth. All genetic modifications were validated by colony PCR and / or DNA sequencing, and CRISPR plasmids were removed using 5-fluoroorotic acid plates.

[0055] The constructed bacterial strains and control strain Y1 were cultured in synthetic medium via shake-flask fermentation (30°C, 220 rpm). After fermentation, the cells were collected by centrifugation, freeze-dried to constant weight, and the cell dry weight was determined. The total protein content was determined using the Kjeldahl method, and the protein yield (g / L) and protein content (g / 100 g DCW) were calculated. The results are as follows: Figure 1 As shown, compared with the starting strain Y1 (protein yield 0.8 g / L, protein content ~48 g / 100 g DCW), strains overexpressing aminoacyl-tRNA synthetase (such as Y1-ILS1 and Y1-GUS1) showed a slight increase in protein yield, reaching 0.9 g / L, but their protein content did not increase significantly, and even decreased in some knockout strains. This indicates that while enhancing aminoacyl-tRNA supply during translation can slightly increase total protein yield, it cannot effectively increase the steady-state concentration of intracellular protein due to bottlenecks such as precursor supply.

[0056] Example 2: Effects of nitrogen metabolism combinatorial modification on protein synthesis

[0057] To enhance nitrogen assimilation efficiency, this embodiment modifies key genes of the nitrogen metabolism pathway based on strain Y1.

[0058] Combination strains overexpressing key genes of the glutamate / glutamine synthesis network were constructed: Y4 (co-overexpressing GLT1 and GDH1), Y5 (co-overexpressing GLT1 and GLN1), and Y6 (co-overexpressing GLT1, GDH1, and GLN1). Shake-flask fermentation was performed according to the method described in Example 1, and OD was measured periodically. 600 To monitor growth, the concentration of ammonium ions in the fermentation supernatant was determined using Nessler's reagent method to assess nitrogen source consumption. After fermentation, cell dry weight, protein content, and yield were measured.

[0059] The results are as follows Figure 2 As shown. The growth of the combined modified strains was not significantly affected ( Figure 2 (a) Among them, the three-gene overexpression strain Y6 showed the fastest ammonium consumption rate ( Figure 2 (b) and the highest biomass (DCW increased by 10.8% compared to Y1) Figure 2 (d in the text). However, the protein content of all combined strains was not significantly increased compared to Y1 (~48.4 g / 100 g DCW). Figure 2 (c) This result indicates that enhanced nitrogen metabolic flux primarily promoted cell proliferation and total protein production, but failed to break through the plateau in protein content, suggesting that carbon skeleton supply has become a new limiting factor.

[0060] Example 3: Enhancing the TCA cycle to improve the effect of carbon skeleton supply on protein synthesis

[0061] This embodiment further enhances the TCA cycle based on the nitrogen metabolism-optimized strain Y6, aiming to achieve synergistic carbon and nitrogen metabolic flows.

[0062] Using strain Y6 as a chassis, strain Y7 overexpressing the citrate synthase gene CIT1, strain Y8 overexpressing the isocitrate dehydrogenase gene IDH1, and strain Y9 co-overexpressing CIT1 and IDH1 were constructed. Shake-flask fermentation, growth monitoring, nitrogen consumption analysis, and protein content determination were performed according to the methods described in Examples 1 and 2. Simultaneously, the fermentation cells were collected, broken up with glass beads, and proteins were removed. Intracellular α-ketoglutarate and amino acid content were determined using high-performance liquid chromatography (HPLC) and an amino acid analyzer, respectively.

[0063] The results are as follows Figure 3 As shown. The double-overexpression strain Y9 exhibited the best performance: its growth rate and ammonium consumption rate were further improved ( Figure 3 (a and b in the text); protein content was significantly increased to 50.3 g / 100 g DCW, and protein yield reached 0.9 g / L ( Figure 3(c and d in the text). Metabolite analysis revealed its mechanism: compared with strain Y6, which only optimized nitrogen metabolism, the intracellular α-KG level of Y9 was increased to 120.2%, and the total amino acid pool was also increased to 106.8% (c and d in the text). Figure 3 (e and f in the text). This confirms that enhancing the TCA cycle, particularly the carbon metabolic flux to α-KG, can effectively supply the carbon skeleton required for nitrogen assimilation, thereby synergistically enhancing protein synthesis capacity. Based on Y9, the five aminoacyl-tRNA synthases in Example 1 were re-evaluated, and it was found that overexpression of VAS1 (strain Y10) could further increase the protein content to 52.3 g / 100 g DCW, indicating that after removing the precursor restriction, a specific aminoacyl-tRNA synthase becomes a new rate-limiting step.

[0064] Example 4: The effect of enhancing the ribosome synthesis pathway on protein synthesis

[0065] To enhance the efficiency of the protein synthesis machinery itself, this embodiment focuses on the modification of the ribosome synthesis pathway.

[0066] First, strain Y15 was obtained by overexpressing the key transcriptional regulator IFH1 of ribosomal protein genes in strain Y1. Shake-flask fermentation results showed that the protein content of Y15 was increased by 6.1% compared to Y1, while the biomass was unaffected. Figure 4 (a and b in the text). This indicates that enhancing ribosome synthesis capacity can directly increase protein synthesis throughput.

[0067] Secondly, to identify potential rate-limiting ribosomal proteins, 19 single-copy ribosomal protein genes (9 40S subunits and 10 60S subunits) were systematically overexpressed and screened in strain Y1. Using the empty vector control strain P0 (protein content 47.9 g / 100 g DCW) as a baseline, strains overexpressing RPS31 (P7), RPS15 (P8), RPL28 (P10), and RPL29 (P19) all showed protein contents exceeding 50.0 g / 100 g DCW. Among them, strain P8, which overexpressed RPS15, had the highest protein content, reaching 50.4 g / 100 g DCW. Figure 4 (c and d in the text).

[0068] Finally, the best-performing strain Y10 (VAS1 overexpression) was selected as the chassis, and IFH1 and the four key ribosomal protein genes (RPS31, RPS15, RPL28, RPL29) were integrated to construct the hybrid strain Y16. Shake-flask fermentation results showed that Y16 achieved a breakthrough increase in protein content, reaching 57.3 g / 100 g DCW, an increase of more than 18.4% compared to Y1. However, Y16 exhibited significant growth defects, with DCW decreasing to 1.3 g / L, and the total protein yield also decreasing to 0.7 g / L. Figure 4 (e and f in the text). This indicates that extremely enhanced protein synthesis machinery places a huge metabolic burden on cells, thereby affecting growth.

[0069] Example 5: Construction of diploid strains and the effect of enhanced transcription factor SUT1 on protein accumulation

[0070] To improve the industrial applicability and protein accumulation capacity of the strain, this embodiment converts the haploid strain into a diploid strain and further introduces transcriptional regulation modification.

[0071] The optimal haploid strain Y16 (MATa) was converted to MATα using a CRISPR / Cas9-mediated mating-type conversion method. This MATα strain was then hybridized with the retained MATa strain to obtain the diploid strain D2. Simultaneously, a control diploid D1 was constructed from the starting strain Y1 using the same method. The diploid genotype was verified by triple primer PCR (simultaneously amplifying 544 bp MATa and 404 bp MATα bands). Figure 5 (b) Shake-flask fermentation results showed that diploidization significantly increased protein content, with D2 reaching 63.1 g / 100 g DCW, significantly higher than its haploid ancestor Y16 and the control diploid D1 ( Figure 5 (c and d in the text).

[0072] Based on the diploid strain D2, the transcription factor SUT1 was further overexpressed to construct strain D3. Shake-flask fermentation results showed that the protein content of D3 was further increased to 66.5 g / 100 g DCW, a 5.4% increase compared to D2, with a slight increase in biomass, while the protein yield remained at 0.8 g / L. Figure 5 (e and f in the text). This suggests that SUT1 may promote protein accumulation by regulating pathways such as cell cycle and nitrogen metabolism.

[0073] Example 6: Performance evaluation of production at a 5 L bioreactor scale

[0074] To evaluate the scale-up production potential of the engineered strain, this embodiment describes fed-batch fermentation of the optimal strain D3 in a controlled 5 L bioreactor.

[0075] Single colonies of D3 were activated by inoculation in YPD medium (20 g / L casein peptone, 10 g / L yeast extract, 20 g / L glucose). After two stages of seed culture, the culture was transferred to a 5 L reactor containing 1 L of initial fermentation medium. Fermentation was maintained at 30°C, with pH 5.0 maintained by automatic addition of 15% ammonia. Dissolved oxygen (DO) was maintained above 30% by adjusting the stirring speed (300-1000 rpm) and aeration rate (1-3 vvm). Two feed solutions were used to supplement the carbon source (50% w / v glucose) and nitrogen / phosphorus source (20% w / v (NH4)2SO4 and 10.0% w / v NH4H2PO4), respectively. OD was measured periodically. 600 Protein content and ethanol concentration.

[0076] The results are as follows Figure 6 As shown. Strain D3 grew well in the reactor, OD... 600 The protein content increased from 1.3 to 30.6 within 32 hours. Notably, the protein content continued to increase during fermentation, peaking at 75.2 g / 100 g DCW at 26 hours, a 50.3% increase compared to the starting strain Y1 (50.0 g / 100 g DCW). In the later stages of fermentation, the protein content decreased slightly, accompanied by a continuous accumulation of ethanol (up to a maximum of 18.2 g / L). These results demonstrate that strain D3 has extremely high protein accumulation potential under controlled fermentation conditions, but further optimization is needed to address the accumulation of metabolic byproducts and potential metabolic burden in the later stages.

[0077] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of engineered brewer's yeast with increased protein content, characterized in that, The engineered Saccharomyces cerevisiae strain uses a uracil-deficient haploid strain of Saccharomyces cerevisiae as the starting strain, and simultaneously overexpresses one or more of the following genes: citrate synthase CIT1, isocitrate dehydrogenase IDH1, glutamate synthase GLT1, glutamate dehydrogenase GDH1, glutamine synthase GLN1, ribosomal transcription factor gene IFH1, valine-tRNA synthase gene VAS1, and ribosomal protein gene; the haploid is hybridized to obtain a diploid, and the transcription factor gene SUT1 is overexpressed in the diploid.

2. The engineered brewer's yeast according to claim 1, characterized in that, The ribosomal protein gene is one or more of the following: ribosomal protein gene RPS31, ribosomal protein gene RPS15, ribosomal protein gene RPL28, and ribosomal protein gene RPL29. The sequence of the ribosomal protein gene RPS31 is shown in SEQ ID NO.8; the sequence of the ribosomal protein gene RPS15 is shown in SEQ ID NO.9; the sequence of the ribosomal protein gene RPL28 is shown in SEQ ID NO.10; and the sequence of the ribosomal protein gene RPL29 is shown in SEQ ID NO.

11.

3. The engineered brewer's yeast according to claim 1, characterized in that, The NCBI ID of the citrate synthase is 855732; The NCBI ID of the isocitrate dehydrogenase is 855691; The NCBI ID of the glutamate synthase is 851383; The NCBI ID of the glutamate dehydrogenase is 854557; The NCBI ID of the glutamine synthase is 856147; The NCBI ID of the valine-tRNA synthetase is 852986.

4. The engineered brewer's yeast according to claim 1, characterized in that, The sequence of the citrate synthase gene CIT1 is shown in SEQ ID NO.1; The sequence of the isocitrate dehydrogenase gene IDH1 is shown in SEQ ID NO.2; The sequence of the glutamate synthase gene GLT1 is shown in SEQ ID NO.3; The sequence of the glutamate dehydrogenase gene GDH1 is shown in SEQ ID NO.4; The sequence of the glutamine synthase gene GLN1 is shown in SEQ ID NO.5; The sequence of the ribosomal transcription regulatory factor gene IFH1 is shown in SEQ ID NO.6; The sequence of the valine-tRNA synthetase gene VAS1 is shown in SEQ ID NO.7; The sequence of the transcription factor gene SUT1 is shown in SEQ ID NO.

12.

5. The method for constructing engineered Saccharomyces cerevisiae according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Using a uracil-deficient strain of Saccharomyces cerevisiae as the starting strain, the glutamate synthase gene GLT1, the glutamate dehydrogenase gene GDH1, and the glutamine synthase gene GLN1 were co-expressed. (2) Co-expression of citrate synthase gene CIT1 and isocitrate dehydrogenase gene IDH1; (3) Overexpression of the valine-tRNA synthetase gene VAS1; (4) Integrating ribosomal transcription regulatory factor gene IFH1 and ribosomal protein genes RPS31, RPS15, RPL28, and RPL29; (5) Re-fuse the haploid engineered strain obtained in step (4) into a diploid strain; (6) Overexpress the transcription factor gene SUT1 in the diploid strain obtained in step (5) to obtain the engineered Saccharomyces cerevisiae.

6. The use of the engineered Saccharomyces cerevisiae according to any one of claims 1-4 in increasing the protein content of microbial cells.

7. A method for increasing the protein content of microbial cells, characterized in that, The step includes fermentation production using engineered brewer's yeast as described in any one of claims 1-4.

8. The method according to claim 7, characterized in that, The fermentation temperature was 28℃-32℃; and the culture was carried out at 300 rpm-1000 rpm.

9. The method according to claim 7, characterized in that, The fermentation includes shake-flask fermentation or fed-batch fermentation.

10. The method according to claim 7, characterized in that, The fermentation includes the following steps: inoculating the engineered Saccharomyces cerevisiae into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation.

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