Development of bio-production process from biogas

(1)高产酒精Moorella sp.的选育HUC22-1：提高Moorella sp.乙醇产量。对HUC22-1的遗传转化体系的建立进行了尝试。结果表明，利用同源重组和电穿孔转化系统，获得了一株缺失脱氢酶脱氢酶基因(PYRD)和转磷酶基因(PYREE)的尿嘧啶营养缺陷菌。目前，我们正在进行以尿嘧啶营养缺陷菌、PYRD和PYRE为宿主和选择标记的遗传转化体系的开发。(2)Moarella sp.代谢特性分析。不同底物中的HUC22-1：Moorella sp.HUC22-1发酵乙醛生成乙酸乙酯，粗略按2乙氧基ATP醋酸酯2 CO_2进行发酵。根据分批培养的数据，计算得到异化底物→的产率为1.11m o l/m o l，高于单一的Wood-Ljugdahl途径。乙醛培养细胞粗提物催化二磷酸腺苷-…丙酮酸和乙酰辅酶A的缩合为丙酮酸和CO_2，丙酮酸转化为乙酰辅酶A和CO_2。在该途径的关键酶反应中也检测到了ATP的生成。此外，该菌还消耗了MAIL-CoA途径的中间体L-苹果酸，并产生了乙酸酯。这些发现表明，Moorella sp.HUC22-1可以通过malyl-CoA途径在乙醛分解代谢过程中底物水平的磷酸化生成ATP。但乙醛酸、乙醇酸、草酸和苹果酸等不能在所有供试底物中产生乙醇。(3)分批培养中，我们用CO代替H_2/CO_2作为发酵底物生产乙醇。虽然在第一批培养中细胞不能生长到20%CO，但在CO气体中反复驯化后可以生长出100%CO。在100%CO气体底物条件下，细胞生长可提高8倍，提高乙酸乙酯/乙醇比可使产物转化为乙醇。(4)静息细胞体系乙酸乙酯转化为乙醇：在静息细胞体系中进行乙酸乙酯转化为乙醇。结果表明，在pH值为7的甲基紫精存在下，机械振荡可使乙醇产量提高到147g/kg干细胞。较少

(1) Breeding of high ethanol producing Moorella sp. HUC22-1:To improve the ethanol productivity of Moorella sp. HUC22-1, development of its genetic transformation system was tried. As the results, an uracil auxotroph that lacks dehydrogenase dehydrogenam gene(pyrD) and orotate phosphonlosyltransferaro gene(pyreE) was obtained by the transformation system using homologous recombination and electroporation. Now, we are carrying on a development of genetic transformation system by using the uracil auxotroph, pyrD and pyrE as a host and selection markers.(2) Analysis of metabolic characterization of Moarella sp. HUC22-1 in various substrates:Moorella sp. HUC22-1 ferments glyoxylate to acetate roughly according to 2 glyoxylate→acetate 2 CO_2. Based on the data obtained in a batch culture, the ATP yield per substrate dissimilated was calculated to be 1.11 mol/mol, which was higher than that obtained from only Wood-Ljungdahl pathway. Crude extracts of glyoxylate-grown cells catalyzed the ADP- … More and NADP-dependent condensation of glyoxylate and acetyl-CoA to pyruvate and CO_2 as well as the conversion of pyruvate to acetyl-CoA and CO_2. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, L-malate, an intermediate in the malyl-CoA pathway, was consumed and acetate was produced in this bacterium. These findings suggest that Moorella sp. HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway. But, ethanol was not produced in all substrates tested including glyoxylate, glycolate, oxalate and malate.(3) Ethanol production in gaseous substrate in batch culture:In this year, we used CO as substrate instead of H_2/CO_2. Although the cell could not grow up to 20% CO in the first batch culture, it can grow 100% CO after repeating the acclimatization in the CO gas. At the 100% CO gas substrate, cell growth could increase 8 times and the product could be sifted to ethanol production by increasing the acetate/ethanol ratio.(4) Conversion of acetate to ethanol in resting cell system:Conversion of acetate to ethanol was carried out in the resting cell system. As the results, ethanol production was increased to 147 g/kg dry cell under mechanical shaking in the presence of methyl-viologen at pH7. Less