发酵工程毕业论文完成版 - 图文

摘 要

高级醇是影响黄酒风味的重要物质之一，本文通过同源重组技术分别对支链氨基酸转氨酶的编码基因BAT1和BAT2，以及天冬氨酸β-半醛脱氢酶的编码基因HOM2进行敲除，研究这些基因敲除对黄酒酵母高级醇生成量的影响。主要研究内容及结果如下：

（1）构建重组质粒pUC-BABK，PCR扩增重组质粒pUC-BABK上的重组盒BA-KanMX-BB并将其转化黄酒酵母单倍体，筛选获得BAT1基因敲除突变株。将BAT1基因敲除突变株与出发菌株进行黄酒发酵实验，发酵结果显示BAT1基因敲除突变株的高级醇生成量和基本发酵性能均未发生明显变化。

（2）将重组盒BA-KanMX-BB转化BAT2基因敲除黄酒酵母单倍体突变株，筛选获得BAT1、BAT2基因双敲除突变株。将BAT1、BAT2双敲除突变株及其单敲除突变株与出发菌株同时进行黄酒发酵实验，发酵结果显示：BAT1、BAT2双敲除突变株的生长繁殖受到了抑制，CO2总失重平均降低了2.8 g，酒精度平均下降了1.5度。同时，BAT1、BAT2双敲除突变株产正丙醇、异丁醇和异戊醇含量较出发菌株分别平均降低了10.46%、52.16%、25.31%，较BAT1突变株分别平均降低了10.12%、51.82%、25.28%，较BAT2突变株分别平均降低了10.76%、24.71%、15.72%。

（3）构建重组质粒pUC-HABK，PCR扩增重组质粒pUC-HABK上的重组盒HA-KanMX-HB并将其转化黄酒酵母二倍体，筛选获得HOM2基因一个等位基因敲除的突变株RY1-1。将突变株RY1-1与出发菌株RY1同时进行黄酒发酵实验，发酵结果显示：RY1-1产异戊醇的含量明显下降，比RY1降低了30.39%，并且其基本发酵性能无明显变化。RY1-1去除KanMX抗性基因后，得到的突变株RY1-2的高级醇生成量和基本发酵性能与RY1-1相比基本保持不变。

（4）构建重组质粒pUC-HB1A1K，在突变株RY1-2的基础上再次敲除HOM2基因，获得HOM2基因两个等位基因敲除的突变株RY1-3。将突变株RY1-3与突变株RY1-2及出发菌株RY1同时进行黄酒发酵实验，发酵结果显示：与RY1和RY1-2相比，RY1-3的CO2总失重平均降低了5.0 g，酒精度平均下降了3.5度。同时，RY1-3产正丙醇、异丁醇和异戊醇含量较RY1分别降低63.27%、46.80%和66.98%，较RY1-2分别降低63.75%、42.09%和53.52%。

关键词：黄酒酵母；支链氨基酸转氨酶编码基因；天冬氨酸β-半醛脱氢酶编码基因；同源重组；黄酒发酵；高级醇

ABSTRACT

In this paper, higher alcohols as the main byproducts during yellow wine yeast fermentation were studied. BAT1 and BAT2 genes encoding branched-chain amino acid transaminase, HOM2 gene encoding aspartic β semi-aldehyde dehydrogenase were knocked out by the double homologous recombination, respectively. Then we studied the effect of gene deletion on the basic fermentation performances and higher alcohols production.

(1) Recombinant plasmid pUC-BABK were constructed. The disruption cassette BA-KanMX-BB from the recombinant plasmid pUC-BABK was transformed into yellow wine yeast haploid and the BAT1 deletion strains were successfully obtained. The yellow rice wine fermentation test of BAT1 deletion strains and parental strains showed that the higher alcohols production and the basic fermentation performances were not affected by the deletion of BAT1 gene.

(2) The disruption cassette BA-KanMX-BB was transformed into BAT2 deletion strains and the BAT1 and BAT2 double deletion strains were successfully obtained. The yellow rice wine fermentation test of BAT1 and BAT2 single and double deletions strains and parental strains showed that deletion of both BAT1 and BAT2 resulted in growth retardation and diminished higher alcohols production. The total loss of CO2 decreased by an average of 2.8 g, and ethanol production decreased by an average of 1.5 degrees. The content of propanol, isobutyl alcohol and isoamyl alcohol produced by BAT1 and BAT2 double deletion strains decreased by an average of 10.46%, 52.16% and 25.31% compared with parental strains, decreased 10.12%, 51.82 % and 25.28% compared with BAT1 deletion strains, decreased 10.76%, 24.71%, 15.72% compared with BAT2 deletion strains.

(3) Recombinant plasmid pUC-HABK were constructed. The disruption cassette HA-KanMX-HB from the recombinant plasmid pUC-HABK was transformed into yellow wine yeast diploid and obtained one allele HOM2 gene deletion strain RY1-1. The yellow rice wine fermentation test of mutant strain RY1-1 and parental strain RY1 showed that the content of isoamyl alcohol produced by RY1-1 decreased 30.39% compared with RY1, and the basic fermentation performance of RY1-1 did not change. The KanMX of RY1-1 was deleted via Cre/Loxp system, then the mutant strain RY1-2 was obtained. The basic fermentation performances and the higher alcohols production of RY1-2 remained unchanged compared with RY1-1.

(4) Recombinant plasmid pUC-HA1B1K were constructed. The disruption cassette HA1-KanMX-HB1 from the recombinant plasmid pUC-HA1B1K was transformed into mutant strain RY1-2 and obtained two allele HOM2 gene deletion strain RY1-3. The yellow rice wine fermentation test of mutant strain RY1-3, RY1-2 and parental strain RY1 showed

that the once again deletion of HOM2 resulted in severe growth retardation and diminished higher alcohols production greatly. The total loss of CO2 produced by RY1-3 decreased by an average of 5.0 g, and ethanol production decreased by an average of 3.5 degrees. The content of propanol, isobutyl alcohol and isoamyl alcohol produced by RY1-3 decreased 63.27%, 46.80% and 66.98% compared with RY1, decreased 63.75%, 42.09% and 53.52% compared with RY1-2.

Key words: Yellow wine yeast, branched-chain amino acid transaminase encoding genes (BAT1 and BAT2), aspartic β semi-aldehyde dehydrogenase encoding gene (HOM2), homologous recombination, yellow rice wine fermentation, higher alcohols

目 录

1 前 言 ...............................................................................................................................1 1.1 黄酒概述.......................................................................................................................1 1.2 黄酒风味物质概述.......................................................................................................1 1.2.1 高级醇对黄酒风味的影响 ....................................................................................2 1.2.2 酯类对黄酒风味的影响 ........................................................................................3 1.2.3 其他类物质对黄酒风味的影响 ............................................................................3 1.3 黄酒发酵过程中高级醇的形成与控制.......................................................................3 1.3.1 高级醇的形成机理 ................................................................................................3 1.3.2 影响黄酒中高级醇含量的主要因素 ....................................................................6 1.3.3 降低黄酒中高级醇含量的主要措施 ....................................................................7 1.4 低产高级醇酵母菌株的研究进展...............................................................................8 1.4.1 诱变育种 ................................................................................................................8 1.4.2 细胞融合育种 ........................................................................................................9 1.4.3 基因工程育种 ........................................................................................................9 1.5 本课题的立题依据及研究内容.................................................................................10 1.5.1本课题的立体依据 ...............................................................................................10 1.5.2 本课题的主要研究内容 ...................................................................................... 11 2 材料与方法 .....................................................................................................................13 2.1 材料与仪器.................................................................................................................13 2.1.1 主要试剂 ..............................................................................................................13 2.1.2 主要实验仪器 ......................................................................................................14 2.1.3 菌种与质粒 ..........................................................................................................15 2.1.4 主要培养基 ..........................................................................................................15 2.1.5 主要溶液 ..............................................................................................................16 2.2 分析方法.....................................................................................................................17 2.2.1 CO2失重的测定....................................................................................................17 2.2.2 酒精度的测定 ......................................................................................................17 2.2.3 还原糖的测定 ......................................................................................................17 2.2.4 挥发性风味物质含量的测定 ..............................................................................18 2.2.5 生长曲线的测定 ..................................................................................................18 2.3 实验方法.....................................................................................................................19 2.3.1 引物设计 ..............................................................................................................19 2.3.2 各目的基因片段的获得 ......................................................................................21 2.3.3 重组质粒的构建 ..................................................................................................23

I

百度搜索“77cn”或“免费范文网”即可找到本站免费阅读全部范文。收藏本站方便下次阅读，免费范文网，提供经典小说教育文库发酵工程毕业论文完成版 - 图文在线全文阅读。