Scheffersomyces stipitis发酵单糖能力及相关基因初步研究

发布时间：2018-08-29 19:21

【摘要】：本论文以树干毕赤酵母(Scheffersomyces stipitis)CBS 6054为研究对象,用4种不同单糖进行发酵实验,测定发酵过程中菌体生长曲线、糖消耗情况、乙醇生成情况;并对树干毕赤酵母的Ss GSF2基因和NG68基因进行初步研究。研究结果表明:1.树干毕赤酵母在木糖和葡萄糖培养基中的生长曲线类似,菌体浓度较高,其次是乳糖培养基、阿拉伯培养基。树干毕赤酵母利用葡萄糖和木糖的能力相当,发酵48 h糖浓度降至较低水平半乳糖培养基和阿拉伯糖培养基发酵72 h时糖的利用率分别为90.9%、21.6%。葡萄糖的乙醇产率为0.4774 g/g(乙醇/消耗的糖),达到理论值的93.6%。木糖的乙醇产率为0.3968 g/g,达到理论值的86.3%。半乳糖培养基发酵到72 h的乙醇产率可达0.3395 g/g,阿拉伯糖几乎未产生乙醇,反而产生少量甲醇。2.相对于葡萄糖培养基,在木糖培养基中Ss GSF2基因在24 h出现表达高峰,其他时间点与葡萄糖培养基相似。构建Ss GSF2表达载体、Ss GSF2和GFP融合表达载体,然后转化酿酒酵母S132菌株。进行m RNA表达检测,结果显示原始菌株S132中无Ss GSF2基因表达,工程菌株中SsGSF2基因表达良好。3.原始菌株S132菌液浓度从高到低依次是半乳糖、葡萄糖、木糖、阿拉伯糖,葡萄糖培养基中菌体生长最快,阿拉伯培养基菌体生长最慢。Ss GSF2基因转化子在葡萄糖、木糖、半乳糖培养基中生长曲线基本一致,在阿拉伯糖培养基中生长较慢,菌浓度最低。在己糖培养基中,S132菌株菌浓度均高于Ss GSF2基因转化子;在戊糖培养基中,Ss GSF2基因转化子生长都快于原始菌株S132。Ss GSF2基因转化子对于己糖的利用率均低于原始菌,对于戊糖的利用率均高于原始菌。4.通过显微镜观察和培养试验发现,两种菌株的菌体形态有差异,工程菌株在发酵时分散状态较好,不易沉降和凝聚,这些特点有利于氧气和糖分的充分利用,用于工业生产时可降低发酵所需能耗。5.生物信息学分析结果显示NG68基因属于核基因,ORF长度为4371 bp,编码1456个氨基酸。该基因位于酵母基因组3号染色体,GC含量为44%,含有144 bp的内含子,序列中有多处重复序列。NG68蛋白含有信号肽以及5个Candida ALS结构域,蛋白序列的末端有跨膜螺旋结构,除末端外其他序列位于膜外。6.NG68基因在己糖培养基中表达量类似;在戊糖培养基中发酵前期均出现表达高峰,而且在木糖和阿拉伯糖培养基中表达情况存在差异,说明该基因与戊糖代谢有关,且对不同戊糖响应程度不同。
[Abstract]:In this paper, we take Pichia pastoris (Scheffersomyces stipitis) CBS 6054 as the research object, use four kinds of monosaccharides to carry on the fermentation experiment, measure the growth curve of the cell, sugar consumption, ethanol production in the fermentation process; The Ss GSF2 and NG68 genes of Pichia pastoris were studied. The results of the study show that 1: 1. The growth curve of Pichia pastoris in xylose and glucose medium was similar, and the cell concentration was higher, followed by lactose medium and Arabic medium. The ability of Pichia pastoris to make use of glucose and xylose was similar. The sugar utilization ratio of Pichia pastoris was 90.9% and 21.6g respectively when the glucose concentration decreased to a lower level after 48 h fermentation and that of arabinose medium decreased to a lower level after 72 h fermentation. The ethanol yield of glucose is 0.4774 g / g (ethanol / consumed sugar), which is 93.6% of the theoretical value. The ethanol yield of xylose is 0.3968 g / g, which is 86.3% of the theoretical value. The ethanol yield reached 0.3395 g / g after fermentation on galactose medium for 72 h, and the arabinose produced little methanol instead of ethanol. Compared with glucose medium, Ss GSF2 gene expression peaked in xylose medium at 24 h, and was similar to glucose medium at other time points. Ss GSF2 expression vector, Ss GSF2 and GFP, were constructed and transformed into Saccharomyces cerevisiae S132 strain. The expression of m RNA was detected. The results showed that there was no Ss GSF2 gene expression in the original strain S132, and the SsGSF2 gene expression was good in the engineering strain. 3. The concentration of the original strain S132 was in the order of galactose, glucose, xylose, arabinose, the fastest growth of bacteria in glucose medium, and the slowest growth of SS-Ss GSF2 gene transformant in Arabic medium was in glucose and xylose. The growth curve of galactose medium was basically the same, and the growth rate was slower and the concentration of bacteria was the lowest in the arabinose medium. In hexose medium, the concentration of S132 strain was higher than that of Ss GSF2 gene transformant, and in pentose medium, the transformants of Ss GSF2 gene grew faster than the original strain S132.Ss GSF2 gene transformants, and the utilization rate of hexose was lower than that of the original strain S132.Ss GSF2 gene transformants. The utilization rate of pentose was higher than that of original bacteria. Through microscope observation and culture experiment, it was found that the two strains had different bacterial morphology, and the engineering strain was in a good dispersion state during fermentation, which was not easy to settle and agglomerate. These characteristics were beneficial to the full utilization of oxygen and sugar. When used in industrial production, it can reduce the energy consumption of fermentation. Bioinformatics analysis showed that NG68 gene belonged to nuclear gene and encoded 1456 amino acids with a length of 4371 bp,. The GC content of the gene located on chromosome 3 of yeast genome is 44 and contains an intron of 144 bp. There are several repeat sequences. NG68 protein contains signal peptide and five Candida ALS domains, and the end of the protein sequence has transmembrane helical structure. The expression levels of NG68 gene were similar in hexose medium, peak expression occurred in pentose medium at the early stage of fermentation, and there were differences between xylose and arabinose medium. The results showed that the gene was related to pentose metabolism and had different response to different pentose.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ920.1

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