Department of Chemical Engineering, Massachusetts Institute ofTechnology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
J Bacteriol. 2010 Oct;192(20):5319-28. doi: 10.1128/JB.00207-10. Epub 2010 Aug 20.
The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.
产碱杆菌 Ralstonia eutropha H16 通过β-酮硫解酶（PhaA）、乙酰乙酰辅酶 A 还原酶（PhaB）和聚羟基烷酸合酶（PhaC）催化的反应，从乙酰辅酶 A（acetyl-CoA）合成聚羟基丁酸酯（PHB）。在产碱杆菌 R. eutropha 中发现了编码这些酶促步骤的三个基因的操纵子，并对其进行了深入研究。对产碱杆菌 R. eutropha 基因组的测序和分析揭示了 PHB 生物合成基因的每个基因的假定同工基因，其中许多基因从未被表征过。除了先前鉴定的 phaB1 基因外，基因组还包含同工基因 phaB2 和 phaB3 以及 15 个其他潜在的乙酰乙酰辅酶 A 还原酶。我们通过单独和组合从基因组中删除这三个 phaB 同工基因来研究它们的作用。结果发现，phaB1 和 phaB3 的基因产物都有助于果糖最小培养基中的 PHB 生物合成，但在植物油最小培养基和丰富培养基中，phaB3 似乎没有表达。这就提出了关于 phaB3 表达调控的有趣问题。在测试条件下，删除基因 phaB2 不会导致可观察的表型，尽管该基因确实编码一种活性还原酶。将单个还原酶基因添加到 ΔphaB1 ΔphaB2 ΔphaB3 菌株的基因组中恢复了 PHB 的产生，在我们的互补实验过程中，我们偶然创造了一个 PHB 高产突变体。突变菌株的 PhaB 和 PhaA 活性的测量表明，在氮限制条件下以果糖为碳源生长时，硫解酶反应是 PHB 生物合成的限速步骤。
