Recombinant bacterium for producing lactase and application thereof

Abstract

The invention relates to a recombinant bacterium for producing lactase and application of the recombinant bacterium, and belongs to the technical field of fermentation. A repression gene influencing lactase expression in a galactose metabolic pathway is firstly knocked out to increase the yield, then an iterative integration platform is constructed based on genome multi-copy sites and haploid insufficient sites, lactase expression is further enhanced, and finally, high yield of lactase is achieved through the obtained recombinant bacteria. In a fermentation mode of adopting initial glucose as a carbon source and subsequently supplementing glucose in a 5-liter bioreactor, the enzyme activity of the lactase produced by the recombinant strain disclosed by the invention reaches 556.5 U / mL. Under the condition that the whey by-product is used as a supplementary material for fermentation culture, the enzyme activity of the lactase reaches 473.9 U / mL, which is the highest lactase yield reported by using the whey by-product as a carbon source so far.

Description

A recombinant lactase-producing bacterium and its application Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to a recombinant lactase-producing bacterium and its applications. Background Technology

[0002] The industrial production of lactase has formed a complete industrial system covering microbial fermentation, genetic engineering, advanced immobilization, and diversified applications. Currently, industrial enzyme sources mainly rely on fungi, yeasts, and bacteria, obtained through liquid deep fermentation or genetic engineering methods (such as using Pichia pastoris heterologous expression to achieve a high yield of 6 g / L). At the application level, immobilization technologies such as encapsulation, nanoflower assembly, and 3D printed reactors are used to achieve stable recovery and continuous production of enzymes, which are widely used in the synthesis of low-lactose dairy products (hydrolysis rate >90%) and functional galactooligosaccharides (GOS).

[0003] Kluyveromyces lactis, a Krebutere-negative yeast, can achieve high-density growth with minimal fermentation byproducts and possesses moderate glycosylation modification capabilities. These characteristics make it an ideal platform for producing food-grade proteins, therapeutic proteins, and various natural products. Due to its high hydrolytic activity and safety of endogenous β-galactosidase, Kluyveromyces lactis has become a key microorganism for the production of lactose-free dairy products and shows broad potential in the production of natural products, biofuels, and industrial enzymes. Furthermore, this strain can efficiently utilize lactose as its sole carbon source, which is of significant value for the value-added utilization of whey waste. However, its biotechnological potential has not been fully explored, limited by factors such as the lack of genetic tools required for complex metabolic engineering, low recombinant protein expression efficiency, and unclear secretion mechanisms. Therefore, further exploration of the fermentation potential of Kluyveromyces lactis is needed to achieve efficient lactase production. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem of low lactase activity when using yeast fermentation to produce lactase in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a recombinant lactase-producing strain and its applications. This invention first increases lactase production by knocking out repressor genes affecting lactase expression in the galactose metabolic pathway. Then, based on multicopy sites and haploid deficiency sites in the genome, an iterative integration platform is constructed to further enhance lactase expression. The resulting recombinant strain achieves high lactase production and improved lactase activity. This invention not only expands the genetic manipulation toolbox of Kluyveromyces but also lays a solid technical foundation for developing it into a highly efficient microbial cell factory for protein production.

[0006] The first objective of this invention is to provide a recombinant strain that produces lactase, wherein the recombinant strain is based on Kluyveromyces lactis as the starting strain, the gene encoding the transcriptional regulator of galactose, GAL80, is knocked out, and the gene encoding β-galactosidase is integrated into the genomic rDNA site and the RPL25 site, respectively.

[0007] Furthermore, the NCBI number of the GAL80 is KLLA0_A08162g, and the NCBI number of the β-galactosidase is KLLA0_B14883g.

[0008] Furthermore, using the 18S rDNA or 26S rDNA of Kluyveromyces lactis as an integration site, the β-galactosidase encoding gene is integrated and expressed to enhance the enzyme activity of the β-galactosidase and / or the expression level of its encoding gene.

[0009] Furthermore, the RPL25 site of *Kluyveromyces lactis* was used as the integration site to integrate and express the β-galactosidase encoding gene, thereby enhancing the enzyme activity of the β-galactosidase and / or the expression level of its encoding gene.

[0010] Furthermore, the β-galactosidase is expressed by promoter LAC4, the gene sequence of which is shown in SEQ ID NO.1.

[0011] Furthermore, SEQ ID NO.1:

[0012]

[0013] Furthermore, the rDNA site is an 18S rDNA or 26S rDNA site.

[0014] Furthermore, the Kluyveromyces lactis includes Kluyveromyces lactis JNXR-2101.

[0015] Furthermore, the Kluyveromyces lactis JNXR-2101 has the accession number CCTCC NO: M 20211628.

[0016] A second objective of this invention is to provide a microbial agent containing the aforementioned recombinant bacteria.

[0017] A third objective of this invention is to provide the application of the above-mentioned recombinant bacteria or the above-mentioned microbial agents in the production of lactase.

[0018] A fourth objective of this invention is to provide a method for producing lactase, wherein the method involves inoculating the recombinant bacteria or the microbial agent into a fermentation medium for fermentation.

[0019] Furthermore, during the fermentation process, whey byproducts are added for fed-batch fermentation, wherein the whey byproducts include galactose, lactose, protein, and glucose.

[0020] Furthermore, the fermentation medium includes mannitol.

[0021] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0022] This invention enhances lactase production by knocking out repressor genes affecting lactase expression in the galactose metabolism pathway. Then, an iterative integration platform is constructed based on multicopy sites and haploid deficiency sites in the genome to further strengthen lactase expression. The resulting recombinant strain achieved high lactase production and activity. In a 5-liter bioreactor using initial glucose as the carbon source and subsequent glucose supplementation, the lactase activity reached 556.5 U / mL. Under conditions of fed-batch fermentation using whey byproducts, the recombinant strain achieved a lactase activity of 473.9 U / mL, which is the highest lactase yield reported to date using whey byproducts as the carbon source. Attached Figure Description

[0023] 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.

[0024] Figure 1 shows the enzyme activity of GAL80 knockout strains under different carbon source conditions;

[0025] Figure 2 is a schematic diagram of the rDNA site integration mechanism;

[0026] Figure 3 is a schematic diagram of the PRL25 site integration mechanism;

[0027] Figure 4 is a statistical diagram of intracellular lactase activity in recombinant bacteria obtained by integrating β-galactosidase at different integration sites.

[0028] Figure 5 shows the expression and transcription levels of lactase protein in strain E6.

[0029] Figure 6 shows the experimental results of optimizing fermentation conditions;

[0030] Figure 7 shows the genetic stability of strain E6 and the enzyme activity during fermentation in a 5L tank. Detailed Implementation

[0031] 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.

[0032] The materials and methods involved in the embodiments of the present invention are as follows:

[0033] (1) Culture medium:

[0034] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10.

[0035] YPD medium (g / L): yeast extract 10, tryptone 20, glucose 20.

[0036] YPD / screening medium: YPD medium containing 0.2 g / L hygromycin B is used to screen strains carrying the Klebsiella pneumoniae hphR (HYG) resistance marker.

[0037] YPL medium (g / L): yeast extract 10, tryptone 20, lactose 20 g / L.

[0038] YNB / screening medium: 0.5 g / L leucine is added for screening LEU2 auxotrophic strains.

[0039] YNB / Fluorescence Screening Medium: Supplement with MUGAL to a final concentration of 0.06 g / L in YNB medium.

[0040] Fed-batch culture (g / L): glucose 30, yeast extract 16.7, tryptone 33.3, corn steep liquor 40, manganese sulfate 0.3 and magnesium sulfate 0.5.

[0041] (2) Fluorescence screening of multi-copy strains based on MUGal screening

[0042] MUGal screening was performed according to established methods. After integration, recombinant strains were randomly selected and inoculated into 24-well deep-well plates (each well containing 2 mL of YPD medium) and cultured at 30°C and 220 rpm for 24 hours with shaking. The seed culture was then transferred at a 1% inoculum to new deep-well plates containing 4 mL of fresh YPD medium and fermented for another 60 hours. 20 μL of the culture was added to 96-well plates (each well containing 180 μL of YNB medium supplemented with 0.06 g / L MUGal) and incubated at 30°C and 380 rpm for 2 hours with shaking. Fluorescence intensity and OD were measured using a microplate reader (BioTek, Winnowski, Vermont, USA). 600 Value. A culture medium sample without yeast inoculation was used as a negative control.

[0043] (3) Lactase activity assay

[0044] Enzyme activity units are defined as the amount of enzyme required per minute to hydrolyze o-nitrophenyl-β-galactoside (ONPG) to produce 1 μmol of o-nitrophenol under standard conditions (30°C, pH 6.5). For the assay, 300 μL of enzyme solution was mixed with 1.5 mL of ONPG substrate solution (2.5 mg / mL), incubated at 30°C for 10 minutes, and then the reaction was terminated by adding 600 μL of sodium carbonate solution (50 mg / mL). The absorbance of the released o-nitrophenol at 420 nm was measured using a spectrophotometer to quantify lactase activity.

[0045] (3) Construction of plasmids

[0046] Detailed information on the plasmids and strains used in this study is listed in Table 1. Genome editing was performed using the CRISPR-Cas9 system. Targeted gene knockout in Kluyveromyces was performed using the PUDP025 plasmid carrying CRISPR / Cas9, and a specific sgRNA was designed for the GAL80 gene. Gibson assembly was performed using the ClonExpress Ultra One Step Cloning Kit, purchased from Vazyme (Nanjing, Jiangsu, China). Three donor fragments were constructed using the T-vector as a backbone: a fragment containing a seamless gene knockout homologous arm, a gene expression cassette fragment targeting the PRL25 site for integration, and a recombinant fragment for site-specific rDNA insertion.

[0047] (4) Construction of strains

[0048] Kluyveromyces jNXR-2101 was used as the initial host for gene modification. Based on this strain, the derivative strain obtained by knocking out GAL80 as a single gene was named G1, while the derivative strain obtained by knocking out both GAL80 and LEU2 genes was named G2. Multiple strain series were obtained through multi-copy integration screening of the lactase gene: starting from G1, C1-C10 and D0-D18 transformants were obtained through integration at the 26S rDNA and 18S rDNA sites, respectively; starting from G2, R1-R7 transformants (expression driven by the constitutive HSP promoter) and P8-P14 transformants (expression driven by the inducible LAC4 promoter) were obtained through integration at the PRL25 site; further, using strain D9 as the host, multi-copy integration at the PRL25 site constructed the F1-F7 series controlled by the HSP promoter and the E6-E13 series controlled by the LAC4 promoter, respectively. The strain information involved in the following examples is shown in Table 1, where D0-D18 represent different transformants that integrate at the 18S site.

[0049] Table 1. Information on the strains involved in the following examples.

[0050]

[0051] Example 1: CRISPR-based knockout of GAL80 repressor protein

[0052] Kluyveromyces is considered a highly promising non-traditional yeast due to its natural lactose metabolism. Previous studies have shown that lactose is not only a carbon source for cell growth but also a key inducer for regulating lactase expression. In this study, a parent strain of Kluyveromyces JNXR-2101 (CCTCC NO: M20211628) was isolated in a previous study, with an intracellular lactase activity of 13.8 U / mL. Based on this parent strain, this study used the previously established CRISPR / Cas9 editing tool to knock out the GAL80 gene involved in lactase expression and its regulatory pathway. The results are shown in Figure 1. In YPD medium, relieving GAL80-mediated inhibition increased lactase activity from 15.0 U / mL to 31.8 U / mL; similarly, the GAL80-deficient strain cultured in YPL medium also showed a considerable increase in enzyme activity (32.7 U / mL).

[0053] Example 2: rDNA site integration based on homologous recombination

[0054] Based on the GAL80 knockout strain, this example further investigated the integration of lactase into the genome. To enhance lactase expression levels, a multi-copy genome integration strategy for the endogenous lactase expression cassette based on rDNA sites was first employed. The Kluyveromyces genome contains approximately 60 rDNA repeat units, each consisting of a transcribed region and an untranscribed spacer (NTS). By constructing a vector using rDNA as the homologous recombination site, stable multi-copy integration of the lactase expression cassette into the genome can be achieved under selection pressure. In the experiment, 18S and 26S rDNA sequences carrying the hygromycin resistance gene were used to drive the integration of the lactase expression cassette. For rapid preliminary screening, the previously established MUGAL method was used, which detects lactase activity based on the linear relationship between its catalytic product fluorescence intensity. All colonies grown on the selection plate were fermented in deep-well plates for 24 hours, then cultured in MUGAL and their fluorescence intensity was measured. The fluorescence intensity of each strain showed a gradient distribution, with the fluorescence intensity of the integrated strain being approximately 1-4 times that of the wild type. The strain with the highest fluorescence intensity was subjected to shake-flask level secondary screening, ultimately yielding the C5 strain (26S rDNA::p) which underwent multiple copy integration at the 26S rDNA site. LAC4 -LAC4) enzyme activity increased by 23.5% (to 40.4 U / mL); D9 strain, which performed multicopy integration at the 18S rDNA site (18S rDNA::p LAC4 -LAC4 enzyme activity increased by 38.8% (to 45.4 U / mL) (Figures 4a and b).

[0055] Example 3: Integration of RPL25 site based on in vivo autonomous amplification

[0056] Although lactase integration at rDNA sites enhanced the enzyme activity of engineered strains, the enhancement effect remained relatively limited. Based on the artificial integration module of the HapAmp system established in Saccharomyces cerevisiae, we developed a genome integration platform suitable for Kluyveromyces kuhlii and validated its functional effectiveness. First, based on the haploid-deficient RPL25 locus, we constructed an integration cassette through seamless cloning and designed two constructs for multi-copy lactase integration (3'pRPL25-LEU2-RPL25t-ARS305-p). LAC4 -LAC4-pBTS1-5'pRPL25 and 3'pRPL25-LEU2-RPL25t-ARS305-p HSP2The expression levels driven by different promoters were compared and evaluated using the LEU2 auxotrophic host strain and GFP as a reporter gene. Iterative genome integration of the integration cassette under weak promoter drive was achieved through a single transformation. After subculturing, the growth of the transformants recovered to a level comparable to that of the wild-type strain. A series of lactase-integrating strains were obtained through a similar process of single transformation combined with multiple subculturing. Using the previously established MUGal fluorescence screening method, transformants with significantly enhanced fluorescence were screened based on differences in fluorescence signal. Subsequently, these dominant transformants with the highest fluorescence intensity were subjected to shake-flask rescreening (Figure 4c). The final transformed strain R7 (RPL25::pHSP-LAC4) had an enzyme activity of 67 U / mL, while the transformed strain P8 (RPL25::pLAC4-LAC4) had an enzyme activity of 71.4 U / mL.

[0057] Finally, based on the multi-copy integration strain D9 constructed using the rDNA site, we further performed a second round of multi-copy integration at the RPL25 site, ultimately obtaining the engineered strain E6 (Figures 4d and 5a). This dual-integrated strain achieved a total enzyme activity of 86.4 U / mL, with intracellular enzyme activity of 59.5 U / mL and extracellular enzyme activity of 26.9 U / mL (Figures 5b and c). RT-PCR analysis showed that the transcriptional level of strain E6 was approximately 5 times that of the wild type (Figure 5d).

[0058] Table 2 Primers used in strain construction

[0059]

[0060] Example 4: Optimization of Fermentation Conditions

[0061] This embodiment improves strain growth and protein yield by optimizing carbon sources and supplementing antioxidants.

[0062] First, the dual carbon source ratio was optimized. The optimization included three groups of experiments: lactose (Lac) 15 g / L + glucose (Glu) 15 g / L, lactose (Lac) 30 g / L, and glucose (Glu) 30 g / L. Strain E6 exhibited a higher growth rate in the glucose-containing medium and achieved a maximum enzyme activity of 283.5 U / mL (Figures 6a and c). As the most readily available carbon source, glucose provides a survival advantage in the early stages of growth, laying a solid physiological foundation for subsequent protein synthesis.

[0063] Secondly, this embodiment optimizes the lactose concentration. The lactose concentration optimization includes three sets of experiments with lactose (Lac) concentrations of 30 g / L, 75 g / L, and 150 g / L. The experimental results are shown in Figures 6b and 6c. When using a lactose concentration of 75 g / L as the carbon source, the enzyme activity of strain E6 reaches 223.4 U / mL.

[0064] Finally, this example also investigated the effect of mannitol on lactase. When 1 g / L mannitol was added, the total enzyme activity of strain E6 decreased by 15.3% compared with the unadded group (Figure 6d). Although the intracellular enzyme activity only increased by 7%, it is noteworthy that the extracellular enzyme activity was significantly enhanced by 51.1% compared with the control group. This change in the ratio of intracellular to extracellular enzyme activity suggests that the addition of antioxidants may improve the efficiency of redox-dependent protein processing, promote the correct folding and modification of large lactase proteins containing disulfide bonds, and thus promote the extracellular secretion of mature lactase.

[0065] In summary, the optimized culture medium composition includes: 30 g / L glucose, 50 g / L compound nitrogen source, 40 g / L corn steep liquor, and 1 g / L mannitol.

[0066] The glucose-containing supplemental culture medium includes: glucose 30 g / L, compound nitrogen source 50 g / L, corn steep liquor 40 g / L, and mannitol 1 g / L.

[0067] The composition of fed culture medium containing whey byproducts includes: 1% galactose, 80% lactose, 6% protein, and 10% glucose.

[0068] Example 5: Fed-batch fermentation in 5L tanks with different carbon sources

[0069] Example 3 successfully constructed a high-yield lactase strain E6, and its excellent genetic stability was confirmed after 10 consecutive generations of long-term stability evaluation (Figure 7A). Therefore, we used strain E6 to optimize the subsequent fermentation process to achieve higher lactase yield. In a 5-liter bioreactor using glucose as the initial carbon source and subsequent glucose supplementation, the enzyme activity of strain E6 reached 556.5 U / mL (Figure 7B), which is the highest lactase yield reported to date in Kluyveromyces. When strain E6 was cultured in a 5-liter bioreactor with whey by-products (1% galactose, 80% lactose, 6% protein, and 10% glucose) as feed, the final enzyme activity reached 473.9 U / mL (Figure 7B), which is the highest lactase yield reported to date using whey by-products as the carbon source.

[0070] 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 recombinant bacteria that produces lactase, characterized in that, The recombinant strain was Kluyveromyces lactis as the starting strain. The gene encoding the transcription factor that regulates galactose, GAL80, was knocked out, and the gene encoding β-galactosidase was integrated into the Kluyveromyces lactis genome rDNA site and RPL25 site, respectively.

2. The recombinant bacteria according to claim 1, characterized in that, The NCBI number of the GAL80 is KLLA0_A08162g, and the NCBI number of the β-galactosidase is KLLA0_B14883g.

3. The recombinant bacteria according to claim 1, characterized in that, The β-galactosidase is expressed by promoter LAC4, the gene sequence of which is shown in SEQ ID NO.

1.

4. The recombinant bacteria according to claim 1, characterized in that, The rDNA site is either an 18S rDNA site or a 26S rDNA site.

5. The recombinant bacteria according to claim 1, characterized in that, The Kluyveromyces lactis includes Kluyveromyces JNXR-2101.

6. A microbial agent containing the recombinant bacteria as described in any one of claims 1-5.

7. The use of the recombinant bacteria according to any one of claims 1-5 or the microbial agent according to claim 6 in the production of lactase.

8. A method for producing lactase, characterized in that, The method involves inoculating the recombinant bacteria according to any one of claims 1-5 or the microbial agent according to claim 6 into a fermentation medium for fermentation culture.

9. The method according to claim 8, characterized in that, During the fermentation process, whey byproducts are added for fed-batch fermentation, wherein the whey byproducts include galactose, lactose, protein, and glucose.

10. The method according to claim 8, characterized in that, The fermentation medium also includes mannitol.

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