Valentin H E, Mitsky T A, Mahadeo D A, Tran M, Gruys K J
Monsanto Company, St. Louis, Missouri 63167, USA.
Appl Environ Microbiol. 2000 Dec;66(12):5253-8. doi: 10.1128/AEM.66.12.5253-5258.2000.
The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381-1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K(m) of 50 microM and a specific activity of 120 micromol. min(-1). mg(-1) were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590-6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291-299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.
鼠伤寒沙门氏菌中用于丙酸降解的遗传操纵子包含一个名为prpE的开放阅读框，其编码一种丙酰辅酶A（丙酰-CoA）合成酶（A. R. Horswill和J. C. Escalante-Semerena，《微生物学》145:1381 - 1388，1999）。在本文中，我们报告了通过PCR克隆prpE、其在大肠杆菌中的过表达以及该酶的底物特异性。当以丙酸盐作为PrpE的底物时，在饱和底物浓度下发现其K(m)为50微摩尔，比活性为120微摩尔·分钟⁻¹·毫克⁻¹。PrpE还能将乙酸盐、3 - 羟基丙酸盐（3HP）和丁酸盐激活为它们相应的辅酶A酯，但效率远低于丙酸盐。当prpE与来自真养产碱菌的聚羟基脂肪酸酯（PHA）生物合成基因在重组大肠杆菌中共表达时，当在生长培养基中提供3HP时，会积累含有3HP单元的PHA共聚物。为了比较酰基辅酶A合成酶与酰基辅酶A转移酶在PHA生产中的效用，构建了产生PHA的重组菌株，使其将PHA生物合成基因与prpE、acoE（来自真养产碱菌的乙酰辅酶A合成酶基因[H. Priefert和A. Steinbüchel，《细菌学杂志》174:6590 - 6599，1992]）或orfZ（来自丙酸梭菌的乙酰辅酶A:4 - 羟基丁酸 - CoA转移酶基因[H. E. Valentin，S. Reiser和K. J. Gruys，《生物技术与生物工程》67:291 - 299，2000]）共表达。在这三种酶中，PrpE和OrfZ使3HP掺入PHA的水平相似，而AcoE在这方面的效果明显较差。
