GDP-MANNOSE DEHYDROGENASE IS THE KEY REGULATORY ENZYME IN ALGINATE BIOSYNTHESIS IN PSEUDOMONAS-AERUGINOSA - EVIDENCE FROM METABOLITE STUDIES
GDP-MANNOSE DEHYDROGENASE IS THE KEY REGULATORY ENZYME IN ALGINATE BIOSYNTHESIS IN PSEUDOMONAS-AERUGINOSA - EVIDENCE FROM METABOLITE STUDIES
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DOI：
10.1099/13500872-140-7-1745
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发表时间：
1994-07-01
期刊：
影响因子：
2.8
通讯作者：
GACESA, P
中科院分区：
文献类型：
--
作者：
TATNELL, PJ;RUSSELL, NJ;GACESA, P
铜绿假单胞菌酶cdp -甘露糖脱氢酶（GMD）是由algD基因编码的，以往的遗传学研究表明它是藻酸多糖生物合成的关键调控和承诺步骤。本研究将algD基因克隆到广宿主表达载体pMMB66EH和GMD中，在P. aeruginosa粘液样菌株和遗传相关的非粘液样菌株中过表达。P. J. Tatnell， N. J. Russell和P. Gacesa (1993), J Gen Microbiol 139, 119-127的代谢方法被用于研究GMD过表达对细胞内关键代谢物gdp -甘露糖和gdp -甘露酸盐浓度的影响，这两种代谢物与GMD活性和总海藻酸盐产量有关。与野生型菌株相比，在粘液样菌株和非粘液样菌株中过表达algD导致GMD活性升高；gdp -甘露糖浓度随之降低，而gdp -甘露糖酸浓度则大大增加。然而，值得注意的是，海藻酸盐生物合成仅在粘液样菌株中检测到，GMD过表达仅导致胞外多糖产量的边际增加。过表达GMD的粘液样菌株的gdp -甘露糖酸浓度始终显著高于gdp -甘露糖浓度，表明GMD不再是这些转基因菌株生物合成海藻酸盐的主要动力学控制点。这些菌株海藻酸盐产量的微小但显著的增加以及gdp -甘露酸盐浓度的增加被解释为海藻酸盐途径的后期酶已经成为主要的动力学控制点，现在决定了海藻酸盐的生产程度。本研究提供了直接的代谢证据，证明GMD是铜绿假单胞菌藻酸盐生物合成的关键调控酶。
The Pseudomonas aeruginosa enzyme CDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell and P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.