Grimaldi Joseph, Collins Cynthia H, Belfort Georges
Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180-3590.
Biotechnol Prog. 2016 Jan-Feb;32(1):66-73. doi: 10.1002/btpr.2197. Epub 2015 Dec 8.
Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system.
由于反馈抑制、细胞不稳定性以及缺乏经济的产物回收工艺，细胞培养生产燃料和化学中间体受到低产物浓度（≤0.2%（v/v））的严重限制。我们基于无细胞固定化酶系统开发了一种替代的简化生产方案。两种固定化酶（酮酸脱羧酶（KdcA）和乙醇脱氢酶（ADH））以及一种溶液中的酶（用于NADH循环的甲酸脱氢酶（FDH））产生的异丁醇滴度比以mol/mol为基础报道的酿酒酵母的最高滴度高8至20倍。通过将酶（ADH）和酶融合体（fKdcA）共价固定在甲基丙烯酸酯树脂上，实现了这些高转化率和低蛋白质渗漏。在没有原位去除异丁醇的情况下，新的酶系统在浓度为0.135时（每消耗一摩尔酮异戊酸产生的异丁醇摩尔数）实现了酮异戊酸向异丁醇55%的转化率。进一步提高滴度将需要使用原位回收系统连续去除异丁醇。
