Valentin H E, Reiser S, Gruys K J
Monsanto Co., 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, USA.
Biotechnol Bioeng. 2000 Feb 5;67(3):291-9.
To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.
为了在大肠杆菌中从谷氨酸生成聚（3-羟基丁酸酯-co-4-羟基丁酸酯）提供4-羟基丁酰辅酶A，对来自克氏梭菌的乙酰辅酶A：4-羟基丁酸辅酶A转移酶、来自真养产碱菌的4-羟基丁酸脱氢酶、来自大肠杆菌的γ-氨基丁酸：2-酮戊二酸转氨酶以及来自拟南芥或大肠杆菌的谷氨酸脱羧酶进行了克隆，并通过在大肠杆菌中单个基因的表达来测试其功能，以验证酶活性，然后在乳糖启动子的控制下将它们独特地组装成操纵子。将这些操纵子独立转化到携带用于聚羟基脂肪酸酯（PHA）生产的失控复制载体pJM9238的大肠杆菌CT101中。质粒pJM9238包含在tac启动子控制下的真养产碱菌的PHA生物合成操纵子。通过对氯仿萃取和乙醇沉淀的聚酯进行核磁共振（NMR）光谱分析来监测聚羟基脂肪酸酯的形成。通过使用在4HB-CoA生物合成途径中提供不同前体的各种碳源进行补料实验，证明了共聚物生产生物合成途径的功能。
