【作者】 郭钦；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品安全， 2009， 博士

【摘要】 基因工程酵母菌在食品工业中的应用逐渐普遍,但重组菌抗生素抗性标记的存在对食品安全具有潜在隐患。此外,较低的生产成本、简单方便的下游操作技术和高水平的表达量等是工业大规模生产重组蛋白必须具备的特点。针对这些问题,本课题根据半乳糖-葡萄糖对GAL基因家族诱导-抑制效应以及GAL4p对GAL基因的调控机制,采用同源重组技术分步敲除了工业多倍体酿酒酵母MS-1染色体上的GAL1基因,并将GAL4基因整合到GALl基因位点,构建了不同GAL基因型的重组酵母SG1、DG115.DPG65和GQ21。同时,选用α-半乳糖苷酶作为食品级选择标记、β-1,3-1,4-葡聚糖酶作为报告基因构建了在GALl启动子和MFαl信号肽控制指导下的食品级分泌表达载体YGMPA-PM:并以α-凝集素C-端320个氨基酸为锚定序列,构建了能够展示表达p-1,3-1,4-葡聚糖酶的食品级展示表达载体YGMPNA-PM.将构建的载体和不同GAL基因型的重组酵母相结合,得到了适合工业化生产的食品级高效分泌表达系统和食品级高效展示表达系统。主要研究结果如下：扩增了GAL4结构基因全长序列,并构建了包含GAL4基因+KanMX表达盒的模板质粒PMD18-TGK.通过同源重组以GAL4+KanMX表达盒替换了工业三倍体酵母MS-1的三个等位的GALl基因中的一个,得到具有双GAL4的重组酵母。以KanMX为敲除框架,敲除了MS-l染色体上的一个GALl基因。利用LFH原理,设计了另外两套基因敲除框架KanMX+GALls和GALlt+KanMX,依次敲除了双GAL4重组菌株染色体上剩余的两个GALl等位基因。考虑到重组菌株应用的安全性,利用Cre/loxP系统切除了构建重组酿酒酵母菌株时使用的KanMX抗性表达盒,并通过传代使Zeocin抗性质粒pSH47/ZEO丢失,得到不同GAL基因型的重组酵母SGl、DG115.DPG65和GQ21。其中,重组菌株GQ21染色体上三个GALl基因全部缺失,且其中一个GALl基因位点被整合了一个GAL4基因拷贝。对不同GAL基因型的重组酵母SG1、DG115、DPG65和GQ21在含有不同碳源的培养基中的生长、发酵性能以及抗性和遗传稳定性进行了测试。结果表明,重组酵母的GAL基因型在传代过程中保持稳定,没有发生回复突变且抗性基因已经被彻底删除。GALl基因的缺失对重组菌株对葡萄糖的利用有轻微地影响。不同的培养方式影响重组酵母对半乳糖的利用。在YPG培养基中,GALl基因敲除对酵母利用半乳糖的影响并不明显,MS-1.SGl.DG115在20 h内将培养基中的半乳糖全部消耗完毕,DPG65则在26 h内将半乳糖利用完毕。在YPGL培养基中,MS-1、SG1.DG115和DPG65利用完半乳糖的时间分别为20 h、44 h、44 h和68 h。在两种培养模式中,GQ21培养基中的半乳糖浓度一直维持不变,表明GALl基因确实已经被完全敲除。GALl基因的敲除和GAL4基因拷贝的增加没有改变重组菌株的热致死温度。重组菌株和对照菌株的热致死温度仍然是54℃。构建了PGKl启动子控制下的α-半乳糖营酶表达单元,并将此表达单元克隆到多拷贝质粒YEPlacl 81上构建了食品级克隆载体YPM.YPM转化酵母后得到的重组子可以在以蜜二糖为唯碳源的培养基中生长,并能在涂有X-α-gal的培养上显蓝色,表明α-半乳糖苷酶已经被有效表达。重组酵母/YPM在YPD培养基和MSD培养基中表达的a-半乳糖苷酶酶活最高为104 U/m1和41.72 U/ml；对照菌株安琪酵母表达的α-半乳糖苷酶存在蜜二糖／葡萄糖诱导-抑制效应,在YPD培养基和MSD培养基中的最高酶活为29.79 U/ml和266.38U/mI。在MSD培养基中,安琪酵母12h进入对数生长期,32 h进入稳定期；重组酵母YPM在68 h后才开始快速生长,164 h后进入稳定期。两者在YPD培养基中的生长性能一致,8 h进入对数生长期,32 h达到稳定期。扩增了GALl启动子、MFal信号肽以及ADHl终止子基因序列,以p-1,3-1,4-葡聚糖酶为报告基因在克隆载体YPM的基础上构建了食品级分泌表达载体YGMPA-PM.以α-半乳糖苷酶为筛选标记,将载体YGMPA-PM转入所构建的重组酵母菌株SG1、DG115、DPG65和GQ21以及MS-1,在蜜二糖平板(MSD)挑选出了转化子。测定不同GAL基因型酵母中p-1,3-1,4-葡聚糖酶的表达水平。结果表明,SG1、DG115、DPG65和GQ21的β-1,3-1,4-葡聚糖酶最高酶活分别为1523.48 U/ml、2480.43 U/ml、3161.53 U/ml和3991.00 U/ml,为MS-1 (846.37U/ml)的1.8倍、2.93倍、3.73和4.72倍,说明酵母染色体上GALI基因的敲除和GAL4基因拷贝的增加确实有利于提高外源蛋白表达的水平。重组分泌表达的β-1,3-1,4-葡聚糖酶的最适温度为40℃,最适pH为6.0。50℃保温2 h后p-1,3-1,4-葡聚糖酶残留酶活为51.90%；60℃保温2 h后的活力下降为初始酶活的20.14%。在pH4-6的范围内于4℃放置24 h,酶活仍能保持80%以上。扩增了编码α-凝集素C-末端320个氨基酸的基因序列并在载体YGMPA-PM的基础上构建了食品级展示表达载体YGMPNA-PM.将其与不同GAL基因型的重组酵母相结合构建了食品级展示表达系统。此展示表达系统将p-1,3-1,4-葡聚糖酶成功展示表达在酿酒酵母细胞表面。通过平板鉴定和酶活测定确证了β-1,3-1,4-葡聚糖酶的正确定位。SG1、DG115、DPG65和GQ21展示表达的β-1,3-1,4-葡聚糖酶最高酶活分别为84.98 U/ml、118 U/ml、161.55 U/ml和201.87 U/ml,分别为MS-1 (45.10 U/ml)的1.88倍、2.62倍、3.56倍和4.48倍。展示表达的β-1,3-1,4-葡聚糖酶的酶学性质发生改变。最适温度变为60℃,热稳定性也被增强。50℃保温3 h对酶活几乎没有影响；60℃保温1 h残留酶活为初始酶活的129.2%,60℃保温3 h后的酶活为初始酶活的64.6%。70℃保温1 h,酶活增加到初始酶活的109.2%,3 h后残留酶活仅为初始酶活的35.8%。展示表达的β-1,3-1,4-葡聚糖酶最适pH为6.0,在pH4-7的范围内稳定性较好。更多还原

【Abstract】 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.更多还原