The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
College of Life Sciences, Northwest University, Xi'an, 710069, Shanxi, China.
Appl Microbiol Biotechnol. 2022 Sep;106(17):5479-5493. doi: 10.1007/s00253-022-12110-x. Epub 2022 Aug 6.
Spermidine is an important polyamine that can be used for the synthesis of various bioactive compounds in the food and pharmaceutical fields. In this study, a novel efficient whole-cell biocatalytic method with an NADPH self-sufficient cycle for spermidine biosynthesis was designed and constructed by co-expressing homoserine dehydrogenase (HSD), carboxyspermidine dehydrogenase (CASDH), and carboxyspermidine decarboxylase (CASDC). First, the enzyme-substrate coupled cofactor regeneration system from co-expression of NADP-dependent ScHSD and NADPH-dependent AfCASDH exactly provides an efficient method for cofactor cycling. Second, we identified and characterized a putative CASDC with high decarboxylase activity from Butyrivibrio crossotus DSM 2876; it showed an optimum temperature of 35 °C and an optimum pH of 7.0, which make it better suited for the designed synthetic route. Subsequently, the protein expression level of each enzyme was optimized through the variation of the gene copy number, and a whole-cell catalyst with high catalytic efficiency was constructed successfully. Finally, a yield of 28.6 mM of spermidine was produced in a 1-L scale of E. coli whole-cell catalytic system with a 95.3% molar conversion rate after optimization of temperature, the ratio of catalyst-to-substrate, and the amount of NADP, and a productivity of 0.17 g·L·h was achieved. In summary, this novel pathway of constructing a whole-cell catalytic system from L-homoserine and putrescine could provide a green alternative method for the efficient synthesis of spermidine. KEY POINTS: • A novel pathway for spermidine biosynthesis was developed in Escherichia coli. • The enzyme-substrate coupled system provides an NADPH self-sufficient cycle. • Spermidine with 28.6 mM was obtained using an optimized whole-cell system.
精胺是一种重要的多胺，可以用于食品和制药领域中各种生物活性化合物的合成。在这项研究中，通过共表达高丝氨酸脱氢酶（HSD）、羧化精胺脱氢酶（CASDH）和羧化精胺脱羧酶（CASDC），设计并构建了一种新型高效的全细胞生物催化方法，用于精胺生物合成，并具有 NADPH 自给循环。首先，NADP 依赖性 ScHSD 和 NADPH 依赖性 AfCASDH 共表达的酶-底物偶联辅酶再生系统为辅酶循环提供了一种高效的方法。其次，我们从丁酸梭菌 DSM 2876 中鉴定并表征了一种具有高脱羧酶活性的推定 CASDC；它的最适温度为 35°C，最适 pH 为 7.0，使其更适合设计的合成路线。随后，通过改变基因拷贝数优化了每种酶的蛋白表达水平，并成功构建了具有高催化效率的全细胞催化剂。最后，通过优化温度、催化剂与底物的比例以及 NADP 的用量，在 1 L 规模的大肠杆菌全细胞催化体系中获得了 28.6 mM 的精胺产量，摩尔转化率为 95.3%，产物得率为 0.17 g·L·h。总之，从 L-高丝氨酸和腐胺构建全细胞催化体系的这条新途径为精胺的高效合成提供了一种绿色替代方法。 关键点： • 在大肠杆菌中开发了一种新的精胺生物合成途径。 • 酶-底物偶联系统提供了 NADPH 自给循环。 • 使用优化的全细胞系统获得了 28.6 mM 的精胺。
