Kluyveromyces marxianus strain for high-efficiency expression of biological enzymes and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a high-efficiency expression of a Kluyveromyces marxianus strain and application thereof. The Kluyveromyces marxianus strain is obtained by using CRISPR / Cas9 gene editing technology to knock out genes in a protein synthesis metabolic pathway, a secretion and transport pathway, a cytoskeleton synthesis or an intercellular information transmission pathway in a genome of the Kluyveromyces marxianus, and taking the Kluyveromyces marxianus FIM-1 in which a key gene URA3 of a uracil synthesis pathway is knocked out as a starting strain. A FIM-1 ura3Delta mnn11Delta is screened and obtained, and the alpha-galactosidase secretion expression level can be increased by more than 3 times. The Kluyveromyces marxianus strain can be applied to high-efficiency preparation of biological enzymes such as alpha-galactosidase, ferulic acid esterase, chitosanase, saccharifying enzyme, xylanase, mannanase, cellulase and beta-amylase, and has high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Kluyveromyces martensii strain that efficiently expresses biological enzymes and its applications. Background Technology

[0002] Yeast is a single-celled eukaryotic organism with many advantages, including high secretory capacity, high growth rate, a sophisticated internal membrane system for organelle division, a mature post-translational modification system, ease of cultivation in small dishes and large bioreactors, convenient product separation and purification, and lack of susceptibility to adverse factors such as bacteriophages. Saccharomyces cerevisiae is a yeast species that has been extensively studied, with numerous transcriptomic, proteomic, metabolomic, metabolic flux, and protein-protein interaction analyses conducted, making it one of the most widely used and frequently employed cell factories in the biotechnology field, holding a dominant position. However, S. cerevisiae also has disadvantages such as plasmid instability, low yield of target proteins, and low secretion efficiency due to excessive glycosylation of target proteins. [7] This limits its large-scale application.

[0003] Protein secretion is a crucial pathway in eukaryotic cells. In eukaryotes, approximately 10–20% of native proteins in fungi and 30–40% in mammals require post-translational modifications via the secretory pathway. This pathway involves translocation, folding, endoplasmic reticulum-associated protein degradation (ERAD), sorting, and various post-translational modifications (PTMs) involving several different organelles to ensure proper protein function. In *Saccharomyces cerevisiae*, approximately 200 proteins are involved in these functions through the secretory pathway. Early technological developments aimed to improve yeast protein production levels (titer, yield, and productivity) primarily focused on fine-tuning expression constructs, particularly optimizing promoters and regulatory sequences to enhance expression efficiency. The specific PTM modifications required for each secreted protein dictate the specific combination of processes needed for its production and secretion. This makes the secretion pathway a complex production line, making it difficult to elucidate its specific mechanisms. Therefore, it is necessary to study the energy and material costs of proteins passing through the secretion pathway, and how cells allocate energy and enzymes to process these proteins. This will contribute to a better understanding of the protein secretion process. In yeast, glycosylation is an important PTM modification in the secretion pathway, especially N-glycosylation. When yeast secretory proteins pass through the endoplasmic reticulum and Golgi apparatus, glycosylation enzymes located in these organelles perform complex modifications on the nascent peptide chains, producing high-mannose glycans. This process consumes a significant amount of cellular energy and material resources. Therefore, it can be inferred that reducing the degree of N-glycosylation modification can reduce the cost of cellular production of secretory proteins and improve protein production efficiency.

[0004] In unconventional yeast expression systems, *Kluyveromyces marxianus*, in addition to the advantages mentioned above, also possesses characteristics such as strong heat resistance, a broad carbon source substrate spectrum, high total protein content, and the ability to naturally secrete various hydrolytic enzymes (inulinase, lactase, and pectinase). Furthermore, due to its long-standing association with the safe use of dairy products, *Kluyveromyces marxianus* has obtained both the EU's QPS safety certification and the US GRAS certification. Therefore, *Kluyveromyces marxianus* is increasingly being widely used in agricultural, industrial, and medical fields, including recombinant protein production, aromatic hydrocarbon synthesis, polyketide compound processing, feed additives, and wastewater treatment.

[0005] The global market size for biopharmaceutical proteins and industrial enzymes reaches hundreds of billions and tens of billions of US dollars annually, respectively. Efficient recombinant protein expression systems are crucial for the manufacture of pharmaceutical proteins. *Kluyveromyces martensii*, with its high growth rate and high biomass, offers significant advantages in reducing industrial production costs, making it ideal for producing edible, feed, and pharmaceutical proteins. However, while *Kluyveromyces martensii* exhibits high intraspecific diversity, its physiology and metabolism remain underdeveloped. Furthermore, as an unconventional yeast, its genetic background is not well-understood, and its metabolic pathways, signal transduction, and regulatory mechanisms are not fully understood. This invention will investigate the synthesis, metabolism, translation, modification, and transfer of *Kluyveromyces martensii* proteins to obtain recombinant *Kluyveromyces martensii* strains that efficiently express industrial enzyme proteins, laying the foundation for the application of *Kluyveromyces martensii* in pharmaceutical proteins and industrial enzymes. Summary of the Invention

[0006] The purpose of this invention is to provide a Max Kluyveromyces strain capable of efficiently secreting and expressing biological enzymes and its applications.

[0007] The Kluyveromycea marxianus strain for efficiently secreting and expressing biological enzymes provided by this invention is based on Kluyveromycea marxianus strain FIM-1, which has the key gene URA3 in the uracil synthesis pathway knocked out. FIM-1 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as *Kluyveromycea marxianus*, with accession number CGMCC No. 10621 and a deposit date of March 13, 2015. This gene-deleted mutant strain was obtained by seamlessly knocking out genes in protein synthesis and metabolism pathways, secretion and transport pathways, cytoskeleton synthesis, or intercellular communication pathways in the *Kluyveromycea marxianus* genome using CRISPR / Cas9 gene editing technology.

[0008] The knocked-out gene is selected from one or more of the following genes: GDS1, TRM1, OPT2, FLC1, PPM1, HUB1, MNN11, CWH41, UBX2, KEX1, OST5, NVJ3, ASE1, CAP1, YBL081W, YLR407W, and YGL242C.

[0009] Preferably, the gene knocked out is the MNN11 gene, and the resulting gene-deleted mutant strain is denoted as FIM-1ura3Δmnn11Δ strain.

[0010] The method for constructing a Kluyveromyces martensii mutant strain that efficiently secretes and expresses biological enzymes, provided by this invention, comprises the following steps:

[0011] (1) Key candidate genes regulating the secretory expression of Kluyveromyces martensii protein were screened through bioinformatics analysis;

[0012] (2) Using Kluyveromyces martensii FIM-1ura3Δ as the starting strain, genes related to protein synthesis metabolism, secretion and transport, cytoskeleton synthesis or intercellular communication pathways in the genome of Kluyveromyces martensii were knocked out without scarring using CRISPR / Cas9 gene editing technology to obtain gene deletion mutant strains.

[0013] Specifically, the target sequence 20 bp preceding NGG in the CDS of the gene to be edited was inserted into the CRISPR-Cas9 plasmid pARS1-CRISPR-2. Using the Kluyveromyces martensii genome as a template, fragments of approximately 500 bp each upstream and downstream of the ORF of the gene to be knocked out were amplified by PCR. Then, the upstream and downstream fragments were fused by overlap extension PCR to form a knockout DNA fragment of approximately 1 kb in length. The pARS1-CRISPR-2 containing the target sequence and the knockout DNA fragment were co-transformed into Kluyveromyces martensii strain FIM-1URA3Δ using lithium acetate chemical transformation. Positive clones were then screened on SC-Ura plates and verified by PCR to obtain mutant strains with the target gene knocked out.

[0014] The gene to be edited is selected from one or more of the following genes: GDS1, TRM1, OPT2, FLC1, PPM1, HUB1, MNN11, CWH41, UBX2, KEX1, OST5, NVJ3, ASE1, CAP1, YBL081W, YLR407W, and YGL242C.

[0015] The gene to be edited is preferably MNN11, and the resulting mutant strain with the gene knocked out is the FIM-1ura3Δmnn11Δ strain. This mutant strain has the characteristic of being able to efficiently secrete and express biological enzymes.

[0016] In this invention, the nucleotide sequence of the MNN11 gene is SEQ ID No.1, and the amino acid sequence is SEQ ID No.2.

[0017] In this invention, the bioenzyme is one or more of the following: α-galactosidase, ferulic acid esterase, chitosanase, saccharifying enzyme, xylanase, mannanase, cellulase, and β-amylase. The *Kluyveromyces martensii* expression host strain constructed in this invention can efficiently secrete and express these bioenzymes, indicating that the *Kluyveromyces martensii* expression host strain constructed in this invention efficiently secretes and expresses bioenzymes and has versatility.

[0018] In this invention:

[0019] The α-galactosidase described has the nucleotide sequence of SEQ ID No. 3 and the amino acid sequence of SEQ ID No. 4.

[0020] The ferulic acid esterase described herein has the nucleotide sequence of SEQ ID No. 5 and the amino acid sequence of SEQ ID No. 6.

[0021] The chitosanase described has the nucleotide sequence of SEQ ID No. 7 and the amino acid sequence of SEQ ID No. 8.

[0022] The saccharifying enzyme described has the nucleotide sequence of SEQ ID No. 9 and the amino acid sequence of SEQ ID No. 10.

[0023] The xylanase described has the nucleotide sequence of SEQ ID No. 11 and the amino acid sequence of SEQ ID No. 12.

[0024] The mannanase described has the nucleotide sequence of SEQ ID No. 13 and the amino acid sequence of SEQ ID No. 14.

[0025] The cellulase described has the nucleotide sequence of SEQ ID No. 15 and the amino acid sequence of SEQ ID No. 16.

[0026] The β-amylase described has the nucleotide sequence of SEQ ID No. 17 and the amino acid sequence of SEQ ID No. 18.

[0027] The FIM-1ura3ΔMNN11Δ strain provided by this invention increases the secretion yield of α-galactosidase MEL1 by 182%. It also increases the secretion levels of other enzymes, such as ferulic acid esterase AnFaeA, chitosanase PJY15, saccharifying enzyme AaGlA, xylanase Xyn-CDBFV, mannanase Man330, cellulase RuCelA, and β-amylase Bce, by 30%, 452%, 76.3%, 28.6%, 137%, 37%, and 45%, respectively. Therefore, the Kluyveromyces martensii strain provided by this invention can serve as a universal host strain for efficient secretion and expression of various enzymes. Attached Figure Description

[0028] Figure 1 The effect of gene deletion in the protein secretion pathway of Kluyveromyces max-Kluyveromyces on the expression level and secretion efficiency of α-galactosidase.

[0029] Figure 2High-density fermentation of recombinant α-galactosidase expression strains FIM-1ura3Δ / MEL1 and FIM-1ura3ΔMNN11Δ / MEL1. (a) Enzyme activity of α-galactosidase in the fermentation supernatant of the strains; (b) Growth curve of the strains; (c) SDS-PAGE analysis of the fermentation supernatant of the recombinant strain FIM-1ura3Δ / MEL1; (d) SDS-PAGE analysis of the fermentation supernatant of FIM-1ura3ΔMNN11Δ / MEL1.

[0030] Figure 3 The effect of gene deletion in the protein secretion pathway of *Kluyveromyces martensii* on the secretory expression level of ferulic acid esterase AnFaeA.

[0031] Figure 4 The secretion and expression level of chitosanase PJY15 was increased in the FIM-1ura3ΔMNN11 strain.

[0032] Figure 5 The expression level of saccharifying enzyme AaGlA was increased in the FIM-1ura3ΔMNN11 strain.

[0033] Figure 6 The expression level of xylanase Xyn-CDBFV was increased in strain FIM-1ura3ΔMNN11.

[0034] Figure 7 The expression level of mannanase Man330 was increased in the FIM-1ura3ΔMNN11 strain.

[0035] Figure 8 The expression level of cellulase RuCelA ​​was increased in the FIM-1ura3ΔMNN11 strain.

[0036] Figure 9 The expression level of β-amylase Bce was increased in the FIM-1ura3ΔMNN11 strain.

[0037] Figure 10 This is a schematic diagram of the structure of plasmid pARS1-CRISPR-2. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0039] Example 1: Knockout of genes involved in the protein secretion pathway in Kluyveromyces martensii

[0040] There are various strategies to enhance protein expression levels, such as optimizing transcription and enhancing translation, optimizing folding and secretion pathways, designing protein sequences, and optimizing cell proliferation and survival engineering. For host cells, high expression of exogenous proteins can lead to various stressors and excessive energy and material demands, thereby adversely affecting the levels of secretory proteins and membrane proteins in the endoplasmic reticulum (ER), particularly high levels of protein synthesis. Therefore, optimizing folding and secretion pathways is an important approach to improving protein secretion expression levels.

[0041] This invention screened 17 genes from the genome of *Kluyveromyces martensii* that may affect protein secretion and expression. The specific gene function annotations are shown in Table 1. The selected genes were divided into four categories based on their function: the first category consists of six genes involved in metabolic pathways: GDS1, TRM1, OPT2, FLC1, PPM1, and HUB1; the second category consists of five genes involved in protein modification and signal peptide cleavage: MNN11, CWH41, UBX2, KEX1, and OST5; ​​the third category consists of three genes that form the cytoskeleton or contribute to intercellular signal transduction: ASE1, CAP1, and NVJ3; and the last category includes three genes with unknown functions: YBL081W, YLR407W, and YGL242C.

[0042] Table 1: Annotation of genes involved in protein secretion in Kluwer max.

[0043]

[0044]

[0045] The Kluyveromyces macrocarpa gene knockout was performed using the CRISPR-Cas9 gene editing method. First, the target sequence, 20 bp preceding the NGG sequence in the CDS of the gene to be edited, was inserted into the CRISPR-Cas9 plasmid pARS1-CRISPR-2. Using the Kluyveromyces macrocarpa genome as a template, fragments approximately 500 bp upstream and downstream of the ORF of the gene to be knocked out were amplified using PCR. Then, overlap extension PCR was used to fuse the upstream and downstream fragments to form a knockout DNA fragment approximately 1 kb in length.

[0046] A lithium acetate chemical transformation method was used to co-transform *Kluyveromyces martensii* strain FIM-1ura3Δ with the pARS1-CRISPR-2 gene containing the target sequence and the knockout DNA fragment. Positive clones were then screened on SC-Ura plates and verified by PCR to obtain mutant strains with the target gene knocked out. Further, the mutant strains were cultured in YPD liquid medium, and then streaked on YPD+5-FOA (5-fluoroorotic acid) plates for reverse screening to obtain strains with the pARS1-CRISPR-2 plasmid deletion. PCR verification yielded a *Kluyveromyces martensii* mutant strain with the deletion of the protein secretion pathway gene, which was then used as a host strain for expressing the exogenous protein.

[0047] Example 2: Screening strains with high expression levels of α-galactosidase were obtained from 17 Kluyveromyces martensii protein secretion pathway gene deletion mutants.

[0048] Using *Kluyveromyces martensii* FIM-1ura3Δ as a control strain and 17 strains with deleted genes involved in protein secretion as expression hosts, recombinant strains secreting α-galactosidase were constructed. The specific construction process is as follows: The *Saccharomyces cerevisiae* α-galactosidase MEL1 gene (SEQ ID No. 3) was inserted between the Sma I and Spe I sites of the *Kluyveromyces martensii* expression vector pUKDN132 (patent application number CN201810153537.X) to construct the recombinant expression vector pUKDN132 / MEL1. The recombinant expression vector pUKDN132 / MEL1 was transformed into FIM-1ura3Δ and the 17 mutant strains with deleted genes related to protein secretion using lithium acetate chemical transformation. The transformants were plated on SC-Ura plates, and the resulting clones were verified by PCR. Verified clones were picked and cultured in 50 mL of YP medium (2% Yeast extract, 4% glucose) at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged and the α-galactosidase activity was measured. The detection method was as follows: 1) Enzyme activity was measured using a 96-well plate. Each well contained 20 μL of 9.9 mM pNPαG (4-Nitrophenylα-D-galactopyranoside), 70 μL of phosphate buffer (pH 6.5), and 10 μL of fermentation supernatant; 2) Immediately after aliquoting, the plates were placed in a 37°C incubator for 30 min; 3) The reaction was terminated by adding 200 μL of Borate Buffer (pH 9.8) to each well; 4) The absorbance was measured at 405 nm using a microplate reader; 5) Enzyme activity was defined as the amount of enzyme required to convert 1 μmol of substrate PNPG or release 1 μmol of chromogenic group pNP per minute at 37°C and pH 6.5. One enzyme activity unit (U) was defined as the amount of enzyme required to convert 1 μmol of substrate PNPG or release 1 μmol of chromogenic group pNP per minute.

[0049] The results of enzyme activity assay in culture supernatant are as follows: Figure 1 As shown, knockout of GDS1, TRM1, MNN11, and CWH41 among 17 candidate Max Kluyvei genes significantly enhanced the secretory expression efficiency of α-galactosidase MEL1, increasing it by 1.7, 0.8, 3.0, and 0.7-fold, respectively. Furthermore, the effect of these gene deletions on protein secretion efficiency was analyzed by detecting intracellular and extracellular α-galactosidase MEL1 retention. The results showed that the deletion of GDS1, TRM1, and MNN11 significantly improved the secretion efficiency of MEL1 protein. Most significantly, the secretion efficiency of α-galactosidase MEL1 in the MNN11 gene-deleted strain increased from 70.2% to 92.4%.

[0050] Furthermore, the recombinant Kluyveromyces martensii strain FIM-1ura3ΔMNN11Δ / MEL1, which highly expressed α-galactosidase and was obtained through shake-flask screening, was subjected to high-density fermentation in a fermenter to verify the performance of this strain in producing α-galactosidase under industrial production conditions. The specific process is as follows: (1) The strain FIM-1ura3ΔMNN11Δ / MEL1, which was stored in a glycerol tube at -80℃, was streaked into YPD medium and cultured statically at 30℃ for 48 hours; (2) Single clones were picked and inoculated into 150 mL of seed medium and cultured on a shaker at 30℃ and 220 rpm for 16-20 hours; 1.5 L of medium (2% Yeast Extract, 4% glucose) for preparing the fermenter bottom material was poured into a 5 L fermenter, and then mixed with feed liquid (13% Yeast Extract, 4% glucose, 4% Yeast Extract ... (3) Sterilize the extract (65% glucose) and other accessories such as feed pipe at 115℃ for 15min; (4) Inoculate all the fermentation seed liquid (10% inoculation amount) into a 5L fermenter containing 1.5L of base culture medium. The fermenter parameters are set as follows: temperature 30℃, aeration rate 3.0L / min, initial speed 200rpm. Dissolved oxygen is controlled at about 30% by linkage with the speed of the stirring paddle. The stirring speed can reach up to 850rpm; (5) When the glucose in the base is basically exhausted, start adding sugar for continuous fermentation. Use ammonia water to control the pH value at about 5.5. Take samples during the fermentation process to test all the indicators to be tested.

[0051] The growth and α-galactosidase production of recombinant strain FIM-1ura3ΔMNN11Δ / MEL1 during high-density fermentation are shown in the following results. Figure 2As shown, the expression level of α-galactosidase based on MEL1 in the MNN11Δ strain was significantly higher than that in the original strain FIM-1ura3Δ. However, compared with the FIM-1ura3Δ / MEL1 strain, the growth rate of the FIM-1ura3ΔMNN11Δ / MEL1 strain was significantly lower. Meanwhile, the secreted expression of α-galactosidase MEL1 was significantly higher than that of the FIM-1 / MEL1 strain. After 72 h of fermentation, the MEL1 expression levels in the FIM-1 / MEL1 and FIM-1-MNN11Δ / MEL1 strains were 1.52 U / mL and 4.29 U / mL, respectively, indicating that the recombinant strain FIM-1ura3ΔMNN11Δ / MEL1 increased the α-galactosidase production by 1.82 times. SDS-PAGE electrophoresis of the fermentation broth supernatant also showed that the MEL1 protein band in the FIM-1-MNN11Δ / MEL1 strain was significantly higher than that in the FIM-1 / MEL1 strain.

[0052] Example 3: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses ferulic acid esterase AnFaeA

[0053] Furthermore, the secretory expression of ferulic acid esterase AnFaeA was tested in 17 mutant strains with deletions of genes involved in protein secretion. First, a recombinant ferulic acid esterase secretion expression vector was constructed by inserting the *Aspergillus niger* ferulic acid esterase AnFaeA gene (SEQ ID No. 5) into the Sma I and Spe I sites of the *Kluyveromyces martensii* expression vector pUKDN132, resulting in the recombinant expression vector pUKDN132 / AnFaeA. The expression vector pUKDN132 / AnFaeA was then transformed into FIM-1ura3Δ and 17 mutant strains with deletions of genes involved in protein secretion using lithium acetate chemical transformation. Transformants were then screened on SC-Ura plates and verified by PCR. Successfully verified clones were picked and cultured in 50 mL of YP medium (2% Yeast extract, 4% glucose) at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the ferulic acid esterase activity was detected. The detection method is as follows: 1) Enzyme activity was detected using a 96-well plate. Each well contained 10 μL of CNPF (10 mM), 170 μL of PBST (pH = 6.4), and 20 μL of fermentation supernatant; 2) After dispensing, the plates were immediately placed in a 37°C incubator for 20 min; 3) The absorbance was measured at 410 nm using a microplate reader; 4) Enzyme activity was defined as the amount of enzyme required to convert 1 nmol of substrate CNPF or release 1 nmol of chromogenic group CNP per minute under the conditions of 37°C and pH = 7.4. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 nmol of substrate CNPF or release 1 nmol of chromogenic group CNP per minute.

[0054] Among 17 mutant strains with deletions of genes involved in protein secretion, knockout of HUB1, MNN11, CWH41, UBX2, and CAP1, as well as three genes of unknown function, significantly increased the expression level of the ferulic acid esterase AnFaeA in *Kluyveromyces martensii* (e.g., ...). Figure 3 Among the strains, the MNN11 gene deletion showed the best effect in increasing AnFaeA production, reaching approximately 30%. Although the results of these gene deletions on AnFaeA secretion efficiency showed that only the OST5 deletion significantly improved AnFaeA protein secretion efficiency, the extracellular AnFaeA production was lower in the MNN11 gene-deleted strain. This indicates that the effects of genes involved in protein secretion vary on the expression of different proteins, but the MNN11 gene deletion has a general effect on increasing the expression of different proteins.

[0055] Example 4: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses chitosanase PJY15

[0056] To further test the universality of high-secretion expression of different enzymes by the *Kluyveromyces martensii* strain FIM-1ura3ΔMNN11Δ, the chitosanase PJY15 gene (SEQ ID No. 7) was inserted into the Sma I and Spe I sites of the *Kluyveromyces martensii* expression vector pUKDN132, constructing the recombinant expression vector pUKDN132 / PJY15. The expression vector pUKDN132 / PJY15 was transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains, respectively, using lithium acetate chemical transformation. Transformants were then screened on SC-Ura plates and verified by PCR, constructing the recombinant chitosanase PJY15 expression strains FIM-1ura3Δ / PJY15 and FIM-1ura3ΔMNN11Δ / PJY15. Verified clones were picked and cultured in 50 mL of YP medium at 30°C and 220 rpm for 72 h, followed by centrifugation to detect chitosanase activity. The detection method is as follows: 1) Detect enzyme activity using 1.5 mL centrifuge tubes. Each centrifuge tube contains 100 μL of 1% chitosan (adjusted to pH=5.5 with 50 mM NaAc) and 100 μL of fermentation supernatant diluted with 50 mM NaAc (pH=5.5); 2) Immediately after aliquoting, place the tubes in a 55℃ water bath for 15 min; 3) Immediately add 200 μL of DNS reagent to the centrifuge tubes, incubate in a boiling water bath for 10 min, and then cool; 4) Transfer 100 μL of the reaction system from the centrifuge tubes to a flat-bottomed 96-well plate and detect the absorbance at 540 nm using a microplate reader; 5) Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of chitosan or release 1 μmol of reducing sugar per minute under the conditions of 55℃ and pH=5.5. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of chitosan or release 1 μmol of reducing sugar per minute in the reaction system.

[0057] The results of chitosanase activity assays in the supernatants of recombinant strains FIM-1ura3Δ / PJY15 and FIM-1ura3ΔMNN11Δ / PJY15 showed that, compared with strain FIM-1, the secretion and expression levels of chitosanase PJY15 increased by 452%. Figure 4 The SDS-PAGE electrophoresis results of the supernatant samples also confirmed that the expression level of chitosanase PJY15 in the FIM-1ura3ΔMNN11Δ strain was significantly higher than that in the FIM-1ura3Δ strain.

[0058] Example 5: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses saccharifying enzyme AaGlA

[0059] The AaGlA gene (SEQ ID No. 9) was inserted into the Sma I and Spe I sites of the *Kluyveromyces martensii* expression vector pUKDN132 to construct the recombinant expression vector pUKDN132 / AaGlA. This vector was then transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains to construct the AaGlA-expressing recombinant strains FIM-1ura3Δ / AaGlA and FIM-1ura3ΔMNN11Δ / AaGlA. The AaGlA-expressing recombinant strains were inoculated into 50 mL of YP medium and cultured at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the AaGlA activity was detected. The detection method is as follows: 1) Detect enzyme activity using 2mL centrifuge tubes. Each centrifuge tube contains 337.5μL of sodium acetate buffer (20mM, pH=5.5), 375μL of 1% soluble starch (w / v, freshly prepared), and 37.5μL of fermentation supernatant; 2) Immediately after aliquoting, place the tubes in a 60℃ water bath for 5min; 3) Immediately add 750μL of DNS reagent to each 2mL centrifuge tube, incubate in a boiling water bath for 5min, and then cool; 4) Transfer 100μL of the reaction system from the centrifuge tubes to a flat-bottomed 96-well plate and detect the absorbance at 540nm using a microplate reader; 5) Enzyme activity is defined as the amount of enzyme required to convert 1μmol of substrate soluble starch or release 1μmol of reducing sugar per minute under the conditions of 60℃ and pH=5.5. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1μmol of substrate soluble starch or release 1μmol of reducing sugar per minute in the reaction system.

[0060] The results showed that, compared with the recombinant strain FIM-1ura3Δ / AaGlA, the yield of saccharifying enzyme AaGlA in the supernatant of the FIM-1ura3ΔMNN11Δ / AaGlA strain was increased by 76.3%. Figure 5 ).

[0061] Example 6: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses xylanase Xyn-CDBFV

[0062] The xylanase Xyn-CDBFV gene (SEQ ID No. 11) was inserted into the Sma I and Spe I sites of the expression vector pUKDN132 to construct the recombinant expression vector pUKDN132 / Xyn-CDBFV. This was then transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains, respectively, to obtain the recombinant xylanase Xyn-CDBFV expressing strains FIM-1ura3Δ / CDBFV and FIM-1ura3ΔMNN11Δ / CDBFV. The recombinant expression strains were inoculated into 50 mL of YP medium and cultured at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the xylanase activity was detected. The detection method is as follows: 1) Use 1.5 mL EP tubes to detect enzyme activity. Each tube contains 135 μL of 2% arabinoxylan (adjusted to pH=5.5 with 50 mM NaAc) and 15 μL of fermentation supernatant diluted with 50 mM NaAc (pH=5.5); 2) Immediately after aliquoting, place the tubes in a 65℃ water bath for 10 min; 3) Immediately add 150 μL of DNS reagent to the EP tubes, mix by pipetting, boil in a water bath for 6 min, cool, and transfer 100 μL to a 96-well plate; 4) Use a microplate reader to detect the absorbance at 570 nm wavelength; 5) Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of substrate arabinoxylan or release 1 μmol of reducing sugar per minute under the conditions of 65℃ and pH=5.5. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of substrate arabinoxylan or release 1 μmol of reducing sugar per minute in the reaction system.

[0063] The results showed that the production of xylanase Xyn-CDBFV in the supernatant of the FIM-1ura3ΔMNN11Δ / Xyn-CDBFV strain was 28.6% higher than that of the control strain FIM-1ura3Δ / CDBFV. Figure 6 ).

[0064] Example 7: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses mannanase Man330

[0065] The mannanase gene Man330 (SEQ ID No. 13) was inserted into the Sma I and Spe I sites of the expression vector pUKDN132 to construct the recombinant expression vector pUKDN132 / Man330. This vector was then transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains, respectively, to obtain the recombinant Man330 expressing strains FIM-1ura3Δ / Man330 and FIM-1ura3ΔMNN11Δ / Man330. The recombinant expression strains were inoculated into 50 mL of YP medium and cultured at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the mannanase activity was detected. The detection method is as follows: 1) Use 1.5 mL EP tubes to detect enzyme activity. Each tube contains 135 μL of 0.3% locust bean gum (adjusted to pH=5.5 with 50 mM NaAc) and 15 μL of fermentation supernatant diluted with 50 mM NaAc (pH=5.5); 2) Immediately after aliquoting, place the tubes in a 65℃ water bath for 10 min; 3) Immediately add 150 μL of DNS reagent to the EP tubes, mix by pipetting, boil in a water bath for 6 min, cool, and transfer 100 μL to a 96-well plate; 4) Use a microplate reader to detect the absorbance at 570 nm wavelength; 5) Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of the substrate locust bean gum or release 1 μmol of reducing sugar per minute under the conditions of 65℃ and pH=5.5. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of the substrate locust bean gum or release 1 μmol of reducing sugar per minute in the reaction system.

[0066] The results showed that the mannanase Man330 production in the supernatant of the culture medium of strain FIM-1ura3ΔMNN11Δ / Man330 was 137% higher than that of the control strain FIM-1ura3Δ / Man330. Figure 7 ).

[0067] Example 8: Kluyveromyces martensii FIM-1ura3ΔMNN11Δ strain overexpresses cellulase RuCelA

[0068] The cellulase RuCelA ​​gene (SEQ ID No. 15) was inserted into the Sma I and SpeI sites of the expression vector pUKDN132 to construct the recombinant expression vector pUKDN132 / RuCelA. This was then transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains, respectively, to obtain recombinant cellulase RuCelA-expressing strains FIM-1ura3Δ / RuCelA ​​and FIM-1ura3ΔMNN11Δ / RuCelA. The recombinant expression strains were inoculated into 50 mL of YP medium and cultured at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the cellulase activity was detected. The detection method is as follows: 1) Use 1.5 mL EP tubes to detect enzyme activity. Each tube contains 135 μL of 0.3% locust bean gum (adjusted to pH=5.5 with 50 mM NaAc) and 15 μL of fermentation supernatant diluted with 50 mM NaAc (pH=5.5); 2) Immediately after aliquoting, place the tubes in a 65℃ water bath for 10 min; 3) Immediately add 150 μL of DNS reagent to the EP tubes, mix by pipetting, boil in a water bath for 6 min, cool, and transfer 100 μL to a 96-well plate; 4) Use a microplate reader to detect the absorbance at 570 nm wavelength; 5) Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of the substrate locust bean gum or release 1 μmol of reducing sugar per minute under the conditions of 65℃ and pH=5.5. One enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of the substrate locust bean gum or release 1 μmol of reducing sugar per minute in the reaction system.

[0069] The results showed that the cellulase RuCelA ​​production in the supernatant of the culture medium of strain FIM-1ura3ΔMNN11Δ / RuCelA ​​was 37% higher than that of the control strain FIM-1ura3Δ / RuCelA. Figure 8 ).

[0070] Example 9: Kluyveromyces martensii strain FIM-1ura3ΔMNN11Δ highly expresses β-amylase Bce

[0071] The β-amylase Bce gene (SEQ ID No. 17) was inserted into the Sma I and Spe I sites of the expression vector pUKDN132 to construct the recombinant expression vector pUKDN132 / Bce. This vector was then transformed into the FIM-1ura3Δ and FIM-1ura3ΔMNN11Δ strains, respectively, to obtain the recombinant β-amylase Bce expression strains FIM-1ura3Δ / Bce and FIM-1ura3ΔMNN11Δ / Bce. The recombinant expression strains were inoculated into 50 mL of YP medium and cultured at 30°C and 220 rpm for 72 h. The supernatant was then centrifuged, and the cellulase activity was detected. The method for detecting β-amylase is as follows: 1) Detect enzyme activity using 2 mL centrifuge tubes. Each centrifuge tube contains 337.5 μL of phosphate buffer (20 mM, pH = 5.50) and 375 μL of 1% soluble starch. After incubating in a 60°C water bath for 10 min, add 37.5 μL of fermentation supernatant. 2) Immediately place the tube in a 60°C water bath for 10 min. 3) Immediately add 750 μL of DNS reagent to the 2 mL centrifuge tube, incubate in a boiling water bath for 5 min, and then cool. 4) Transfer 100 μL of the reaction mixture from the centrifuge tube to a flat-bottomed 96-well plate and detect the absorbance at 540 nm using a microplate reader. 5) The amount of enzyme required to produce 1 μmol of reducing sugar at 60°C and pH = 5.5 is defined as one enzyme activity unit (U).

[0072] The results showed that the production of β-amylase Bce in the supernatant of the culture medium of strain FIM-1ura3ΔMNN11Δ / Bce was 45% higher than that of the control strain FIM-1ura3Δ / Bce. Figure 9 ).

[0073] The above embodiments are examples provided to clearly illustrate the present invention and are not intended to limit the implementation. It should be understood that those skilled in the art can make other variations or modifications based on the concept of the present invention without creative effort. Therefore, all obvious technical solutions derived therefrom should be within the scope of protection defined by the claims.

Claims

1. A Kluyveromyces martensii expression host strain that efficiently secretes and expresses biological enzymes, characterized in that, It is achieved by knocking out key genes in the uracil synthesis pathway. URA3 Kluyveromyces masculinus FIM-1 ura3 Δ, as the starting strain, is FIM-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 10621. It is a gene-deleted mutant strain obtained by seamlessly knocking out a gene in the protein synthesis, secretion, and transport pathway of *Kluyveromyces martensii* genome using CRISPR / Cas9 gene editing technology. The knocked-out gene is... MNN11 The strain was designated FIM-1. ura3 Δmnn 11 Δ.

2. The strain according to claim 1, characterized in that, The bioenzyme is selected from one or more of α-galactosidase, ferulic acid esterase, chitosanase, saccharifying enzyme, xylanase, mannanase, cellulase, and β-amylase.

3. The method for constructing the strain according to claim 1, characterized in that, The specific steps are as follows: (1) Key candidate genes regulating the secretory expression of Kluyveromyces marxi were screened through bioinformatics analysis; (2) Using Kluyveromyces martensii FIM-1 ura3 Using Δ as the starting strain, genes involved in the protein synthesis, secretion, and transport pathways in the genome of *Kluyveromyces martensii* were knocked out without scarring using CRISPR / Cas9 gene editing technology, resulting in a gene deletion mutant strain.

4. The method for constructing the strain according to claim 3, characterized in that, Specifically: The 20bp target sequence preceding the NGG in the CDS of the gene to be edited was inserted into the CRISPR-Cas9 plasmid pARS1-CRISPR-2. Using the Kluyveromyces martensii genome as a template, 500bp fragments upstream and downstream of the ORF of the gene to be knocked out were amplified by PCR. Then, the upstream and downstream fragments were fused together using overlap extension PCR to form a 1kb knockout DNA fragment. The pARS1-CRISPR-2 containing the target sequence and the knockout DNA fragment were co-transformed into Kluyveromyces martensii FIM-1 cells using lithium acetate chemical transformation. URA3 The Δ strain was then screened for positive clones on SC-Ura plates and verified by PCR to obtain mutant strains with the target gene knocked out.

5. Use of the strain according to claim 1 or 2 in the preparation of a bioenzyme, wherein the bioenzyme is one or more selected from α-galactosidase, ferulic acid esterase, chitosanase, saccharifying enzyme, xylanase, mannanase, cellulase and β-amylase.

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