Li Tinggang, Yan Yu, He Jianzhong
Department of Civil and Environmental Engineering, National University of Singapore, Block E2-02-13, 1 Engineering Drive 3, Singapore, 117576 Singapore.
Biotechnol Biofuels. 2015 Oct 12;8:166. doi: 10.1186/s13068-015-0351-7. eCollection 2015.
The main challenge of cassava-based biobutanol production is to enhance the simultaneous saccharification and fermentation with high hyperamylolytic activity and butanol yield. Manipulation of cofactor [e.g., Ca(2+) and NAD/(P)H] levels as a potential tool to modulate carbon flux plays a key role in the cassava hydrolysis capacity and butanol productivity. Here, we aimed to develop a technology for enhancing butanol production with simultaneous hydrolysis of cassava (a typical model as a non-cereal starchy material) using a cofactor-dependent modulation method to maximize the production efficacy of biobutanol by Clostridium sp. stain BOH3.
Supplementing CaCO3 to the medium containing cassava significantly promotes activities of α-amylase responsible for cassava hydrolysis and butanol production due to the role of Ca(2+) cofactor-dependent pathway in conversion of cassava starch to reducing sugar and its buffering capacity. Also, after applying redox modulation with l-tryptophan (a precursor as de novo synthesis of NADH and NADPH), the levels of cofactor NADH and NADPH increased significantly by 67 % in the native cofactor-dependent system of the wild-type Clostridium sp. stain BOH3. Increasing availability of NADH and NADPH improved activities of NADH- and NADPH-dependent butanol dehydrogenases, and thus could selectively open the valve of carbon flux toward the more reduced product, butanol, against the more oxidized acid or acetone products. By combining CaCO3 and l-tryptophan, 17.8 g/L butanol with a yield of 30 % and a productivity of 0.25 g/L h was obtained with a hydrolytic capacity of 88 % towards cassava in a defined medium. The metabolic patterns were shifted towards more reduced metabolites as reflected by higher butanol-acetone ratio (76 %) and butanol-bioacid ratio (500 %).
The strategy of altering enzyme cofactor supply may provide an alternative tool to enhance the stimulation of saccharification and fermentation in a cofactor-dependent production system. While genetic engineering focuses on strain improvement to enhance butanol production, cofactor technology can fully exploit the productivity of a strain and maximize the production efficiency.
木薯基生物丁醇生产的主要挑战在于提高具有高淀粉水解活性和丁醇产量的同步糖化和发酵过程。作为调节碳通量的潜在工具，操纵辅因子（如Ca(2+)和NAD/(P)H）水平在木薯水解能力和丁醇生产率方面起着关键作用。在此，我们旨在开发一种技术，通过使用依赖辅因子的调节方法，在同步水解木薯（一种典型的非谷物淀粉原料模型）的同时提高丁醇产量，以最大化梭菌属菌株BOH3生产生物丁醇的效率。
向含有木薯的培养基中添加CaCO3可显著促进负责木薯水解和丁醇生产的α-淀粉酶活性，这是由于Ca(2+)依赖辅因子的途径在木薯淀粉转化为还原糖及其缓冲能力方面的作用。此外，在用L-色氨酸（一种作为NADH和NADPH从头合成前体）进行氧化还原调节后，野生型梭菌属菌株BOH3的天然依赖辅因子系统中辅因子NADH和NADPH的水平显著提高了67%。NADH和NADPH可用性的增加提高了依赖NADH和NADPH的丁醇脱氢酶的活性，因此可以选择性地打开碳通量的阀门，使其朝着更还原的产物丁醇流动，而不是朝着更氧化的酸或丙酮产物流动。通过结合CaCO3和L-色氨酸，在限定培养基中获得了17.8 g/L的丁醇，产率为30%，生产率为0.25 g/L·h，对木薯的水解能力为88%。代谢模式向更多还原代谢物转变，这体现在更高的丁醇-丙酮比（76%）和丁醇-生物酸比（500%）上。
改变酶辅因子供应的策略可能为在依赖辅因子的生产系统中增强糖化和发酵刺激提供一种替代工具。虽然基因工程专注于菌株改良以提高丁醇产量，但辅因子技术可以充分利用菌株的生产力并最大化生产效率。
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