Lawford H G, Rousseau J D
Department of Biochemistry, University of Toronto, Ontario, Canada.
Appl Biochem Biotechnol. 1997 Spring;63-65:221-41. doi: 10.1007/BF02920427.
The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-O-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) by Escherichia coli B (ATCC 11303) and two different ethanologenic recombinants--a strain in which pet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-type E. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.
从生物质和废弃物大规模生产燃料乙醇的经济学要求有效利用木质纤维素原料杂聚半纤维素组分水解产生的所有糖类。葡萄糖醛酸和4-O-甲基葡萄糖醛酸是禾本科植物和阔叶树木聚糖中的主要侧链，这些植物已被视为生产纤维素乙醇的潜在原料。这些酸的含量与阿拉伯糖相似，阿拉伯糖现在被视为生物质衍生乙醇生产中的另一种潜在底物。本研究比较了大肠杆菌B（ATCC 11303）以及两种不同的产乙醇重组菌——一种通过多拷贝质粒（pLOI297）表达pet的菌株和一种染色体整合构建体KO11菌株，在以D-葡萄糖（Glc）和D-葡萄糖醛酸（GlcUA）（作为唯一碳源和能源）发酵时的终产物分布。使用含有2%（w/v）Glc或GlcUA的改良LB培养基进行pH稳态分批发酵，pH控制设定点为6.3或7.0。未转化的宿主培养物仅从葡萄糖产生乳酸，但GlcUA发酵产生乙醇、乙酸和乳酸的混合物，其中乙酸是主要终产物。两种重组菌GlcUA发酵的乙醇产率相似，但乙酸是一种重要的副产物。将pH从6.3提高到7.0会提高葡萄糖醛酸发酵速率，但也会使乙醇质量产率从0.22降至0.19 g/g，这主要是由于乙酸产量增加。在所有发酵中，碳质量平衡都能很好地闭合，但携带质粒pLOI297的重组菌是个例外，它从GlcUA产生了一种未鉴定的产物。这种代谢工程构建体对GlcUA的代谢仍未明确。这些结果为野生型和工程菌株中的代谢通量以及丙酮酸分解代谢的调控提供了见解。未转化的野生型大肠杆菌B和重组菌株KO11对葡萄糖醛酸代谢的终产物分布表明，丙酮酸-甲酸裂解酶并非唯一负责从丙酮酸产生乙酰辅酶A的酶，而去阻遏的丙酮酸脱氢酶可能在GlcUA的代谢中起重要作用。
