College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, 700 Changcheng Road, Qingdao, 266109, China.
Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao, 266101, China.
J Ind Microbiol Biotechnol. 2018 Feb;45(2):111-121. doi: 10.1007/s10295-018-2002-z. Epub 2018 Jan 10.
As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.
作为一种重要的辅酶，NADH 是大多数氧化还原反应和生物燃料电池所必需的。然而，由于 NADH 再生系统的生产效率低、成本高，以及 NADH 稳定性低，外源 NADH 的供应受到了挑战。在这里，我们构建了一种新型的细胞表面多酶共展示系统，以可控的比例和空间方式作为外源 NADH 再生系统，用于可持续地从低成本生物质中生产 NADH。融合了 dockerin 的葡萄糖淀粉酶（GA）和葡萄糖脱氢酶（GDH）在工程细菌表面表达和组装，这些酶展示了具有不同粘着蛋白组合的蛋白质支架。当 GA 和 GDH 的比例为 3:1 时，以淀粉为底物的全细胞生物催化剂的 NADH 生产速率达到最高水平，是游离酶混合物的三倍，这表明由于酶的比例和接近效应，酶的高度有序的空间组织会促进反应。为了确认所建立的 NADH 再生系统的性能，该系统高效合成了 L-乳酸（L-LA），其 L-LA 的产量（16 g/L）是游离酶混合物的两倍。多酶共展示系统在循环利用中表现出良好的稳定性。总之，新型可持续的 NADH 系统将为从低成本生物质中再生辅助因子提供一种具有成本效益的策略。
