Genetics of Prokaryotes, Faculty of Biology & Center for Biotechnology, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Department of Biotechnology, NTNU, Norwegian University of Science and Technology, Trondheim, Norway.
BMC Microbiol. 2020 Mar 24;20(1):63. doi: 10.1186/s12866-020-01750-6.
The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus.
The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed K of 0.74 mM and V of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts.
While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.
革兰氏阳性兼性甲基营养菌巴氏芽孢杆菌利用核酮糖-1,5-二磷酸（RuBP）循环的 sedoheptulose-1,7-双磷酸酶（SBPase）变体来生长甲醇作为 C 碳源。先前对生理上不同的巴氏芽孢杆菌野生型菌株 MGA3 和 PB1 的全基因组测序揭示了所有假定的 RuMP 循环基因，随后对 MGA3 的几种 RuMP 循环酶进行了生物化学表征。在这项研究中，重点是研究转醛酶（Ta）及其在巴氏芽孢杆菌 RuMP 循环中的可能作用。
巴氏芽孢杆菌 MGA3 和 PB1 的 Ta 基因在大肠杆菌中重组表达，并对基因产物进行了纯化和表征。发现 PB1 Ta 蛋白以同源二聚体的形式发挥活性，分子量为 54kDa，以果糖-6-磷酸为底物时 K 值为 0.74mM，V 值为 16.3U/mg。相比之下，编码缺少 80 个 N 端氨基酸的截断 Ta 蛋白的 MGA3 Ta 基因没有 Ta 活性。构建了表达各种全长 MGA3 Ta 蛋白的 7 种不同突变基因，所有基因产物均显示 Ta 活性。此外，MGA3 细胞在粗提物中显示出与 PB1 细胞相似的 Ta 活性。
尽管已经证实巴氏芽孢杆菌可以使用 RuMP 循环的 SBPase 变体，但本研究表明巴氏芽孢杆菌具有 Ta 活性，并且可能还可以利用 RuMP 的 Ta 变体。
