Study on the genetics of acetic acid bacteria for the efficient vinegar production

高效制醋醋酸菌的遗传学研究

• 批准号: 09556016
• 负责人: HORINOUCHI Sueharu
• 金额: $ 7.87万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09556016/
• 关键词: acetic acid fermentation; acetic acid resistance; 挿入配列; acetic acid fermentation; acetic acid resistance; 挿入配列

1. Two acetic acid-sensitive mutants (AS101 and AS102) of Acetobacter acetisbsp. AcetiAS10 were isolated by mutagenesis with N-metyl-N-nitro-N-nitrosoguanidine. By shotgun cloning of Sau3Al-digested chromosome DNA from the parental-type A.aceti AS10 into mutant AS101 and AS102 with a Acetobacter-E.colishattle vector, pMV24, we isolated DNA fragments that complemented these mutations. One of the recombinant plamids contained a 6.6kb fragment showing the presence of five ORFs which is highly homologous with SecD, SecF, MscL, OmpR and EnvZ, respectively. secF and ompR among these genes were sufficient to complement mutation of AS102. Other recombinant plasmid contained a 2.2kb fragnent showing the presence of a ORF which is homologous with OmpR.ompR gene were complemented mutation of AS101, but not complemented these of AS102.2. Exposure of A.aceti 10-8S2 to 3% ethanol or 1% acetate induced the expression of seven proteins and five proteins, respectively. Two of five proeins induced by 1% acetate were revealed to be aconitase and isocitrate dehydrogenase by analysis of N-terminal amino acid sequence of each proteins. We cloned aconitase gene by southern hybridization, and is going to do overexpression of it in A.aceti. In addition, we are going to clone isocitrate dehydrogenase gene. Other three proteins were shown to have no homology with other proteins by analysis of N-terminal amino acid sequence.

1。乙酰杆菌的两个乙酸敏感突变体（AS101和AS102）。用N-甲基-N-硝基-N-硝基杜烷氨酸通过诱变分离乙酰基10。通过将来自父母型A.Aceti AS10的Sau3Al消化的染色体DNA克隆到突变体AS101和AS102中，用乙酰杆菌-E.Colishattle Vector，PMV24，我们分离了这些突变的DNA片段，这些DNA碎片互补了这些突变。一个重组的石质质量之一包含一个6.6kb的片段，显示了五个ORF的存在，分别与SECD，SECF，MSCL，OMPR和ENVZ高度同源。这些基因中的SECF和OMPR足以补充AS102的突变。其他重组质粒包含一个2.2kb脆弱的片段，显示了与OmpR.OMPR基因同源的ORF的存在是AS101的突变，但没有补充AS102.2。 A.Aceti 10-8S2暴露于3％乙醇或1％乙酸盐会诱导七种蛋白质和五种蛋白质的表达。通过分析每种蛋白质的N末端氨基酸序列，乙酸1％诱导的五个蛋白质中的五个蛋白酶中有2个是丙酸酶和异氯酸酯脱氢酶。我们通过南部杂交克隆了刺刺酶基因，并将在a.aceti中对其进行过表达。另外，我们将要克隆异位酸脱氢酶基因。通过分析N末端氨基酸序列，其他三种蛋白质与其他蛋白质没有同源性。