Iwasaki Yuki, Kita Akihisa, Yoshida Koichiro, Tajima Takahisa, Yano Shinichi, Shou Tomohiro, Saito Masahiro, Kato Junichi, Murakami Katsuji, Nakashimada Yutaka
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
CREST, JST, Kawaguchi, Saitama, Japan.
Appl Environ Microbiol. 2017 Mar 31;83(8). doi: 10.1128/AEM.00247-17. Print 2017 Apr 15.
For the efficient production of target metabolites from carbohydrates, syngas, or H-CO by genetically engineered , the control of acetate production (a main metabolite of ) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in , this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 () and Moth_1181 (), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from ATCC 33223 (). The acetate production from fructose as the sole carbon source by the deletion mutant was not deficient, whereas the disruption of significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate. This is the first report, to our knowledge, on the experimental identification of PTA genes in and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.
为了通过基因工程从碳水化合物、合成气或H-CO高效生产目标代谢产物，需要控制乙酸盐的产生（乙酸盐是一种主要代谢产物）。尽管丙二醇利用蛋白（PduL）被预测为参与乙酸盐产生的磷酸转乙酰酶（PTA），但这尚未得到证实。我们在此描述的研究结果直接表明，由Moth_0864（）和Moth_1181（）编码的两种假定的PduL蛋白作为PTA参与乙酸盐的形成。为了破坏这些基因，我们用来自嗜热栖热菌ATCC 33223（）的乳酸脱氢酶基因替换了每个基因。嗜热栖热菌缺失突变体以果糖作为唯一碳源时的乙酸盐产生并不缺乏，而Moth_1181的破坏显著降低了乙酸盐产量，降至野生型菌株的约三分之一。双缺失（两个基因均缺失）突变体不产生乙酸盐，而是仅产生乳酸作为果糖的终产物。这些结果表明，两个基因均通过乙酰辅酶A（acetyl-CoA）与乙酸盐形成相关，并且它们的破坏能够使同型乙酸途径通过丙酮酸转变为基因合成的同型乳酸途径。据我们所知，这是关于嗜热栖热菌中PTA基因的实验鉴定以及通过在糖平台上破坏这些基因使天然同型乙酸途径转变为基因合成的同型乳酸途径的首次报道。
