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：从兼性甲硫杆菌MB 19和专性甲氧单胞菌77a中克隆了一磷酸核酮糖途径中甲醛固定酶系统的基因，包括3-己酮糖-6-磷酸合成酶(EPS)和6-磷酸-3-己糖异构酶(PHI)。两个菌株在基因组织和基因表达调控机制上存在差异。HPS和PHI的同源蛋白存在于多种古生菌和芽孢杆菌中。2)从短芽孢杆菌中分离纯化HPS和PHI，并进行基因分析：从短芽孢杆菌中分离纯化HPS和PHI，并对其进行鉴定。对该片段的遗传分析表明，基因簇是HPS/Phi操纵子的操纵子，仅在甲醇或甲醛存在时表达。3)芽孢杆菌固定甲醛(HPS/Phi)操纵子表达的调控：纯化并鉴定了位于HPS/Phi操纵子上游的yck H基因产物。发现了几个与yck H特异结合的核苷酸序列。4)HPS和PHI的表达和纯化：Horikokoshii的一个DNA片段(PH1938)构建了对应于HPS和Phi的DNA片段。已证实这两个DNA片段和PH1938足以表达具有固定甲醛活性的酶。这是古猿体内甲醛固定基因活跃的第一个证据。

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.