Chin Jonathan W, Khankal Reza, Monroe Caroline A, Maranas Costas D, Cirino Patrick C
Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biotechnol Bioeng. 2009 Jan 1;102(1):209-20. doi: 10.1002/bit.22060.
Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.
先前有报道称，表达来自博伊丁假丝酵母的NADPH依赖性木糖还原酶（CbXR）的大肠杆菌菌株PC09（ΔxylB，不依赖cAMP的CRP（crp*）突变体）在代谢葡萄糖的同时能利用木糖生产木糖醇[西里诺等人（2006年），《生物技术与生物工程》95(6): 1167 - 1176]。本研究旨在了解NADPH供应在木糖醇产量中的作用以及关键的中心碳代谢酶对木糖醇生产的贡献。对CbXR或木糖转运蛋白表达增加的研究表明，酶活性和木糖转运并非PC09中木糖醇生产的限制因素。基于约束的化学计量代谢网络模型被用于理解中心碳代谢反应和木糖转运动力学对理论最大摩尔木糖醇产量（每消耗的葡萄糖产生的木糖醇）的作用，并且通过各种PC09衍生菌株的静息细胞生物转化来测量木糖醇产量（Y(RPG)）。对于木糖 - 质子同向转运的情况，从模型中省略Zwf（葡萄糖 - 6 - 磷酸脱氢酶）或PntAB（膜结合转氢酶）反应或三羧酸循环活性，理论最大产量分别从9.2降至8.8、3.6和8.0摩尔木糖醇/（摩尔葡萄糖）⁻¹。在实验中，从菌株PC09中删除pgi（编码磷酸葡萄糖异构酶）可使产量从3.4提高到4.0摩尔木糖醇/（摩尔葡萄糖）⁻¹，而删除大肠杆菌转氢酶sthA和pntA中的一个或两个对测量产量没有显著影响。删除zwf或sucC（三羧酸循环）分别显著降低产量，从3.4降至2.0和2.3摩尔木糖醇/（摩尔葡萄糖）⁻¹。表达具有宽松辅因子特异性的木糖还原酶可使产量提高到4.0。理论最大值与实验测定值之间的巨大差异表明，生物催化受到竞争还原当量和消耗能量的途径的影响。在大肠杆菌生物催化过程中转氢酶的代谢作用在很大程度上仍未明确。我们的结果证明了在中心代谢中由利用NADP⁺的酶直接供应NADPH对于驱动异源NADPH依赖性反应的重要性，并表明可用于生物转化的还原辅因子库不能通过转氢酶轻易互换。
