Genetic analysis of bacterial formaldehyde-fixing enzyme system and tis application of useful compound production

1)细菌甲醛固定酶系统的遗传分析：从兼性甲基化菌Mycobacterium gastri MB 19和专性甲基化菌Methylomonas aminofaciens 77a中克隆到单磷酸核酮糖途径中3-己糖-6-磷酸合成酶（EPS）和6-磷酸-3-己糖异构酶（PHI）的甲醛固定酶系统基因。这两个菌株在基因组织和基因表达调控机制上存在差异。2)短芽孢杆菌（Bacillus brevis）中HPS和PHI的纯化及其基因分析：从短芽孢杆菌中纯化HPS和PHI，并克隆出含有hpi和PHI的DNA片段。从片段的遗传分析来看，基因簇是hps/phi的操纵子，只有甲醇或甲醛存在时才会表达。3)枯草芽孢杆菌中甲醛固定（hps/phi）操纵子的表达调控：纯化并鉴定了位于hps/phi操纵子上游的基因产物yckH。发现了几个与yckH特异性结合的核苷酸序列。由此得出结论，YckH对hps/phi操纵子的转录至关重要。4)超嗜热古细菌堀koshii焦球菌HPS和PHI的表达和纯化：从堀koshii焦球菌的DNA片段（PH1938）中构建相应的HPS和PHI的DNA片段。证实这两个DNA片段和PH1938足以表达甲醛固定的活性酶。这是第一个证据表明，甲醛固定基因是活跃的古菌。

1) Genetic analysis of bacterial formaldehyde-fixing enzyme system : The genes for formaldehydefixing enzyme system in the ribulose monophosphate pathway, involving 3-hexulose-6-phosphate synthase (EPS) and 6-phospho-3-hexuloisomerase (PHI), were cloned from the facultative methylotroph, Mycobacterium gastri MB 19, and the obligate methylotroph, Methylomonas aminofaciens 77a. The two strain were different in the gene organization and regulation mechanism for the gene expression. The homologous proteins to HPS and PHI are found in a variety of strains of Archaea and Bacteria.2) Purification of HPS and PHI from a methylotrophic and thermophilic bacterium, Bacillus brevis, and their gene analyses : HPS and PHI were purified from B. brevis and characterized A DNA fragment containing hpi and phi was cloned. From the genetic analysis for the fragment, gene cluster is an operon for hps/phi, whose expression occurred only the presence of methanol or formaldehyde.3) Regulation in the expression of formaldehyde-fixing (hps/phi) operon in Bacillus subtilist : The gene product of yckH, which locates upstream of hps/phi operon, was purified and characterized. Several nucleotide sequence on which yckH bound specifically were found. From these and another results, it is concluded that YckH is essential for the transcription of hps/phi operon.4) Expression and purification of HPS and PHI from a hyper thermophilic Archaean, Pyrococcus horikoshii : DNA fragments corresponding hps and phi were constructed from a DNA fragment (PH1938) of P. horikoshii. It has been confirmed that the two DNA fragments and PH1938 are enough to expression of the active enzymes for formaldehyde fixation. This is the first evidence that genes for formaldehyde fixation are active in an Archaeon.