Scheffersomyces stipitis發(fā)酵單糖能力及相關(guān)基因初步研究
發(fā)布時(shí)間：2018-08-29 19:21
【摘要】：本論文以樹干畢赤酵母(Scheffersomyces stipitis)CBS 6054為研究對(duì)象,用4種不同單糖進(jìn)行發(fā)酵實(shí)驗(yàn),測定發(fā)酵過程中菌體生長曲線、糖消耗情況、乙醇生成情況;并對(duì)樹干畢赤酵母的Ss GSF2基因和NG68基因進(jìn)行初步研究。研究結(jié)果表明:1.樹干畢赤酵母在木糖和葡萄糖培養(yǎng)基中的生長曲線類似,菌體濃度較高,其次是乳糖培養(yǎng)基、阿拉伯培養(yǎng)基。樹干畢赤酵母利用葡萄糖和木糖的能力相當(dāng),發(fā)酵48 h糖濃度降至較低水平半乳糖培養(yǎng)基和阿拉伯糖培養(yǎng)基發(fā)酵72 h時(shí)糖的利用率分別為90.9%、21.6%。葡萄糖的乙醇產(chǎn)率為0.4774 g/g(乙醇/消耗的糖),達(dá)到理論值的93.6%。木糖的乙醇產(chǎn)率為0.3968 g/g,達(dá)到理論值的86.3%。半乳糖培養(yǎng)基發(fā)酵到72 h的乙醇產(chǎn)率可達(dá)0.3395 g/g,阿拉伯糖幾乎未產(chǎn)生乙醇,反而產(chǎn)生少量甲醇。2.相對(duì)于葡萄糖培養(yǎng)基,在木糖培養(yǎng)基中Ss GSF2基因在24 h出現(xiàn)表達(dá)高峰,其他時(shí)間點(diǎn)與葡萄糖培養(yǎng)基相似。構(gòu)建Ss GSF2表達(dá)載體、Ss GSF2和GFP融合表達(dá)載體,然后轉(zhuǎn)化釀酒酵母S132菌株。進(jìn)行m RNA表達(dá)檢測,結(jié)果顯示原始菌株S132中無Ss GSF2基因表達(dá),工程菌株中SsGSF2基因表達(dá)良好。3.原始菌株S132菌液濃度從高到低依次是半乳糖、葡萄糖、木糖、阿拉伯糖,葡萄糖培養(yǎng)基中菌體生長最快,阿拉伯培養(yǎng)基菌體生長最慢。Ss GSF2基因轉(zhuǎn)化子在葡萄糖、木糖、半乳糖培養(yǎng)基中生長曲線基本一致,在阿拉伯糖培養(yǎng)基中生長較慢,菌濃度最低。在己糖培養(yǎng)基中,S132菌株菌濃度均高于Ss GSF2基因轉(zhuǎn)化子;在戊糖培養(yǎng)基中,Ss GSF2基因轉(zhuǎn)化子生長都快于原始菌株S132。Ss GSF2基因轉(zhuǎn)化子對(duì)于己糖的利用率均低于原始菌,對(duì)于戊糖的利用率均高于原始菌。4.通過顯微鏡觀察和培養(yǎng)試驗(yàn)發(fā)現(xiàn),兩種菌株的菌體形態(tài)有差異,工程菌株在發(fā)酵時(shí)分散狀態(tài)較好,不易沉降和凝聚,這些特點(diǎn)有利于氧氣和糖分的充分利用,用于工業(yè)生產(chǎn)時(shí)可降低發(fā)酵所需能耗。5.生物信息學(xué)分析結(jié)果顯示NG68基因?qū)儆诤嘶?ORF長度為4371 bp,編碼1456個(gè)氨基酸。該基因位于酵母基因組3號(hào)染色體,GC含量為44%,含有144 bp的內(nèi)含子,序列中有多處重復(fù)序列。NG68蛋白含有信號(hào)肽以及5個(gè)Candida ALS結(jié)構(gòu)域,蛋白序列的末端有跨膜螺旋結(jié)構(gòu),除末端外其他序列位于膜外。6.NG68基因在己糖培養(yǎng)基中表達(dá)量類似;在戊糖培養(yǎng)基中發(fā)酵前期均出現(xiàn)表達(dá)高峰,而且在木糖和阿拉伯糖培養(yǎng)基中表達(dá)情況存在差異,說明該基因與戊糖代謝有關(guān),且對(duì)不同戊糖響應(yīng)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：TQ920.1
本文編號(hào)：2212168
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：TQ920.1
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