丁二酸产生菌的代谢及选育研究
首发时间:2008-08-18
摘要:在矩阵分析代谢通量基础上,围绕柔性节点下的副产物乙酸及乙醇的降低分别实施软X诱变及定点突变选育,并对比分析了突变株与出发株相关酶活及基因序列变化。针对出发株的流量分析显示产物丁二酸的代谢通量为1.78 mmol•g-1•h-1,主要副产物乙酸与乙醇的代谢通量分别为0.60 mmol•g-1•h-1和1.04 mmol•g-1•h-1,并发现乙醇代谢加剧了丁二酸合成中的H电子供体的不足;筛选出的氟乙酸抗性突变株S.JST1的乙酸代谢通量降低了96%,为0.024 mmol•g-1•h-1,酶活检测表明磷酸乙酰转移酶(Pta)的酶比活力从602降低到74,进一步的序列对比分析发现pta突变基因中产生了一个突变位点;adh定点复合突变株S.JST2的乙醇代谢通量降低了98%,为0.020 mmol•g-1•h-1,酶活检测表明Adh的酶比活力从585降低到62,最终突变株S.JST2丁二酸累积产量达65.7 g/L。
关键词: 产丁二酸放线杆菌 代谢通量 选育 磷酸乙酰转移酶 乙醇脱氢酶
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Metabolism and Breeding of a Succinic-acid-producing Strain
Abstract:The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the fulx of succinic acid was 1.78 mmol•g-1•h-1 and that the flux of acetic acid and ethanol were 0.60 mmol•g-1•h-1 and 1.04 mmol•g-1•h-1, respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 mmol•g-1•h-1, decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 mmol•g-1•h-1, decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 g•L-1.
Keywords: Actinobacillus succinogenes, metabolic flux, breeding, phosphotransacetylase, alcohol dehydrogenase
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