Korotkova N, Lidstrom M E
Department of Chemical Engineering, University of Washington, Seattle, Washington 98195-1750, USA.
J Bacteriol. 2001 Feb;183(3):1038-46. doi: 10.1128/JB.183.3.1038-1046.2001.
Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.
从基因组序列数据中鉴定出了几个包含参与聚-β-羟基丁酸酯（PHB）生物合成与降解以及脂肪酸降解相关基因的DNA区域，并已在丝氨酸循环兼性甲基营养菌甲基营养型芽孢杆菌AM1中进行了表征。参与PHB生物合成的基因包括编码β-酮硫解酶（phaA）、NADPH连接的乙酰乙酰辅酶A（乙酰辅酶A）还原酶（phaB）和PHB合酶（phaC）的基因。phaA和phaB在染色体上紧密相连，还有第三个与PHB颗粒相关蛋白的调节因子具有同源性的基因，称为orf3。phaC与phaA和phaB不连锁。参与PHB降解的基因包括两个不连锁的基因，预测它们编码细胞内PHB解聚酶（depA和depB）。这些基因在DNA和氨基酸水平上彼此具有高度同源性。此外，还鉴定出了一个编码β-羟基丁酸脱氢酶（hbd）的基因。将插入突变引入depA、depB、phaA、phaB、phaC和hbd，以及一个预测编码参与脂肪酸降解的巴豆酸酶（croA）的基因中，以研究它们在PHB循环中的作用。depA、depB、hbd和croA的突变体均产生正常水平的PHB，观察到的唯一生长表型是hbd突变体无法在β-羟基丁酸上生长。然而，phaA、phaB和phaC突变体在PHB合成方面均表现出缺陷。令人惊讶的是，这些突变体在以C(1)和C(2)化合物为碳源的培养基上生长也存在缺陷，对于phaB突变体，通过添加乙醛酸可挽救这些缺陷。这些结果表明，β-羟基丁酰辅酶A是甲基营养菌和链霉菌属中未知途径（该途径将乙酰辅酶A转化为乙醛酸）的一个中间产物。
