| The application of the genetically-modified yeasts in food industry becomes more and more popular. However, the presences of antibiotic genes for the selection of yeast transformation make it undesirabl traits for the recombinant yeast. Besides, it is necessary for industrialisation to be provided with lower production costs, simple and convenient downstream processions and higher expression levels of foreign proteins.Based on the above problems, according to the induction-repression effects of galactose-glucose on the GAL family and the regulation mechanisms of GAL4 gene, the GAL1 genes in the genome of industrial yeast MS-1 were disrupted by homologous recombination and a duplicate GAL4 gene was integrated into one of the GAL gene loci in this paper, yielding different GAL yeasts SG1, DG115, DPG65 and GQ21. Meanwhile, the food-grade secretion vector YGMPA-PM under the control of GAL1 promoter and MFal signal peptide was constructed using a-galactosidase as selection marker andβ-1,3-1,4-glucanase as reporter gene. The food-grade display vector YGMPNA-PM was also constructed using a-agglutinin as anchor protein. The combination of constructed vectors and recombinant yeasts formed the high-efficient food-grade secretion/display systems, which could meet the industrial criteria. The main results are listed as following.The full-length sequence of GAL4 structure gene was amplified and the template plasmid PMD18-TGK containing GAL4 gene and KanMX expression cassette was constructed. One of the GAL1 genes in MS-1 genome was replaced by GAL4+KanMX, producing yeast DG115 with double GAL4 in genome. According to LFH theory, the other two gene-disruption cassettes, KanMX+GALls and GALlt+KanMX, were designed to knockout the rest of two GAL1 genes in yeast DG115. Considering the safety of the recombinant yeast in the application of industry, the KanMX expression cassette in the recombinant yeast was removed by Cre/loxP system using plasmid pSH47/ZEO. The resulting yeasts with different GAL genotypes were denominated as SGI (yeast with one disrupted-GALI gene), DG115 (yeast with one GAL1 gene replaced by GAL4), DPG65 (yeast with one GAL1 gene replaced by GAL4 and the other one disrupted) and GQ21 (double GAL4 yeast without GAL1 gene), respectively.The physiological characteristics and the genetic stability of GAL genotype of the recombinant yeasts in medium containing various carbon sources were evaluated. The results showed that the GAL genotypes were stable during cultivation and no recovery mutation occured. The ability of yeast to ferment glucose was slightly affected by the deficiency of GAL1 genes and the cultivation modes had also impacts on galactose utilization of the recombinant yeasts. In YPG medium, galactose was exhausted within 20 h by MS-1, SGI and DG115 and within 26 h by DPG65, while the depletion time of galactose of MS-1, SGI, DG115 and DPG65 in YPGL medium was 20 h,44 h,44 h and 68 h, respectively. In both cultivation modes, the galactose concentration of GQ21 in medium was constant, indicating that GAL1 genes were completely knockouted. However, the deletion of GAL1 genes and the insertion of GAL4 gene had no effects on the heat-resistance temperature of yeast.We constructedα-galactosidase expression cassette under the control of PGK1 promoter and then cloned it into multicopy plasmid YEPlac181 to create food-grade cloning vector YPM. The transformants harbouring YPM could be grown in the media containg melibiose as a sole carbon source, and the colonies could turn into blue in X-a-gal-containing plate. The highest activity of a-galactosidase of the recombinant/YPM in YPD medium and MSD medium was 104 U/ml and 41.72 U/ml, respectively. For Anqi yeast, donor strain of a-galactosidase gene, the highest activity of a-galactosidase in YPD medium and MSD medium was 29.79 U/ml and 266.38 U/ml, which maybe involved the induction-repression effect of galactose-glucose on the native a-galactosidase. In MSD medium, the growth of Anqi yeast and the recombinant yeast/YPM were influenced by the expression of a-galactosidase, which resulted in the growth lag of yeast/YPM. But in YPD medium, the growth for Anqi yeast was similar to yeast/YPM due to the independence of expression of a-galactosidase.The sequences of GALI promoter, MFal signal peptide and ADHl terminator were amplified using MS-1 genomic DNA as template. The gene encodingβ-1,3-1,4-glucanase from Bacillus subtilis mutant ZJF-1A5 was cloned. Subsequently, the food-grade serection vector YGMPA-PM was constructed using YPM as backbone vector and a-galactosidase as selection marker. YGMPA-PM was then introduced into yeast SGI, DG115, DPG65, GQ21 and MS-1 and the transformants were screened in MSD plate. Theβ-1,3-1,4-glucanase activities secreted by different GAL yeasts were measured. The highest activity ofβ-1,3-1,4-glucanase in SGI, DG115, DPG65 and GQ21 was 1523.48 U/ml,2480.43 U/ml,3161.53 U/ml and 3991.00 U/ml, respectively, which was 1.8-fold,2.93-fold,3.7-fold and 4.72-fold of MS-1(846.37 U/ml). The enhancedβ-1,3-1,4-glucanase activity in the recombinant yeasts indicated the beneficial effect of GALI genes disruption and the increased GAL4 gene copy on the expression of heterologous proteins. The optimal temperature and pH of secretedβ-1,3-1,4-glucanase was 40℃and 6.0. After incubation for 2 h at 50℃and 60℃, the residual avtivity was 51.90%and 20.14%of the initial activity and was hardly detected at 70℃after the 2 h incubation. When the secreted enzyme was incubated for 24 h in pH ranging from 4-6 at 4℃, the residual activity was still above 80%.The cell surface-display of enzymes is one of the most attractive applications in yeasts. To construct food-grade display vector YGMPNA-PM, a-agglutinin gene containing the 3'half of the region encoding 320 amino acids and a 238-bp flanking region was amplified, ligated withβ-1,3-1,4-glucanase gene and cloned into YGMPA-PM. The recombinant GAL yeasts were transformed by YGMPNA-PM to form food-grade display systems.β-1,3-1,4-glucanase was successfully immobilized on the cell wall of yeast/YGMPNA-PM by a-agglutinin anchor system and could hydrolyzeβ-glucan efficiently. The highest activity ofβ-1,3-1,4-glucanase in cells of SGI, DG 115, DPG65 and GQ21 was 84.98 U/ml,118 U/ml,161.55 U/ml and 201.87 U/ml, respectively, which was 1.88-fold,2.62-fold, 3.56-fold and 4.48-fold of MS-1 (45.10U/ml). The properties of displayed enzyme changed. The optimal temperature and pH of displayed enzyme was 60℃and 6.0. The thermal stability ofβ-1,3-1, 4-glucanase displayed in the recombinant yeast cells was enhanced compared to the free enzyme. The residual activity ofβ-1,3-1,4-glucanase in yeast/YGMPNA-PM cells increased with incubation time and reached 129.2% at 60℃and 109.2% at 70℃at 1 h, and then gradually decreased. After incubation for 3 h at 60℃and 70℃, the enzyme activity still remained at 64.6% and 35.8%, respectively, which indicated an improved thermostability.
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