Cho Changhee, Choe Donghui, Jang Yu-Sin, Kim Kyung-Jin, Kim Won Jun, Cho Byung-Kwan, Papoutsakis E Terry, Bennett George N, Seung Do Young, Lee Sang Yup
Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Department of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Biotechnol J. 2017 Feb;12(2). doi: 10.1002/biot.201600457. Epub 2017 Jan 16.
Previously the development of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re-sequenced by a high-throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA ) is showed a 32% higher activity than that of the wild-type thiolase (thlA ). In batch fermentation, butanol production is increased by 26% and 23% when the thlA gene is overexpressed in the wild-type C. acetobutylicum ATCC 824 and in its derivative, the thlA-knockdown TKW-A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5 residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.
此前有报道称，通过对亲本菌株PJC4BK进行随机诱变，开发出了一种高产丙酮 - 丁醇 - 乙醇（ABE）的丙酮丁醇梭菌BKM19菌株，该亲本菌株PJC4BK是丙酮丁醇梭菌ATCC 824菌株的buk突变体，BKM19菌株能够多产生30.5%的总溶剂。在此，利用高通量测序技术对BKM19和PJC4BK菌株进行重新测序，以了解导致溶剂产量提高的突变。与丙酮丁醇梭菌PJC4BK相比，在丙酮丁醇梭菌BKM19基因组中鉴定出13个单核苷酸变异（SNV）、1个缺失和1个回复突变SNV。除了在大质粒中发现的1个SNV外，所有突变均存在于BKM19的染色体中。其中，对编码硫解酶的thlA基因中的一个突变进行了酶活性和丁醇生产方面的进一步研究。突变型硫解酶（thlA ）的活性比野生型硫解酶（thlA ）高32%。在分批发酵中，当thlA基因在野生型丙酮丁醇梭菌ATCC 824及其衍生物thlA基因敲低的TKW - A菌株中过表达时，丁醇产量分别提高了26%和23%。基于结构分析，硫解酶中的突变对调节决定区（RDR）没有直接影响。然而，第5位的突变似乎影响了RDR的稳定性，因此，增加了酶活性并提高了BKM19菌株的溶剂产量。
