Li Zonglin, Sun Chuanqi, Lou Longwei, Li Zhimin
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai, China.
Biotechnol Bioeng. 2022 Jun;119(6):1405-1415. doi: 10.1002/bit.28061. Epub 2022 Feb 23.
The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway-related substances. Here, we constructed a four-enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2-fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2-fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two-stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.
嘧啶代谢途径在微生物中受到严格调控，这使得通过代谢工程生产与该途径相关物质的成功率有限。在此，我们构建了一个用于体外生产三磷酸尿苷（UTP）的四酶偶联系统。所使用的酶包括核苷激酶、尿苷酸激酶、核苷二磷酸激酶和用于能量再生的多磷酸激酶。所有这些酶均来源于嗜极微生物。为了提高产物的总产量和单位时间产量，除多磷酸激酶外的三种酶分别通过多种蛋白质工程策略进行了改造。分别获得了比活性从2.7提高到36.5 U/mg的核苷激酶变体、热稳定性提高5.2倍（比活性为37.1 U/mg）的尿苷酸激酶变体以及比活性提高2倍至超过900 U/mg的核苷二磷酸激酶变体。对偶联系统的反应条件进行了进一步优化，并采用两阶段方法避免酶促pH适应性不匹配的问题。在最佳条件下，该系统可在3.0小时内生产超过65 mM的UTP（31.5 g/L）。底物转化率超过98%，最大UTP生产率达到40 mM/h。
