Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Appl Microbiol Biotechnol. 2020 Aug;104(16):7067-7078. doi: 10.1007/s00253-020-10709-6. Epub 2020 Jun 29.
The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.
硫酸化多糖的合成涉及到更简单多糖底物的硫酸化，这是通过硫酸转移酶利用辅助因子 3'-磷酸腺苷-5'-磷酸硫酸（PAPS）来完成的。有三种酶对于 PAPS 的体外合成是必不可少的，即焦磷酸酶（PPA）、腺苷 5'-磷酸硫酸激酶（APSK）和三磷酸腺苷硫酸化酶（ATPS）。使用新型合成生物学工具 ePathBrick 来评估酶表达比例的优化和对 PAPS 合成的影响，ePathBrick 可以将多个基因组装在单个载体中。在不同位置引入多个启动子和终止密码子可以使细菌系统精细调节插入基因的表达水平。表达 PPA（U39393.1）、ATPS（CP021243.1）和 PPA（CP047127.1）的重组载体用于发酵，产生了 400-1380mg/L 的体积产率，其中可溶部分积累了 34-66%。未进一步纯化的可溶性部分中的酶用于 PAPS 合成。使用 PAPS 进行肝素硫酸多糖的化学酶合成，并与 PAPS-ASTIV 再生系统偶联。ASTIV 催化 PAPS 的再生。使用表达来自挪威鼠的酶 ASTIV（ASTIV）的重组载体进行发酵，产生了 1153mg/L 酶的体积产率，其中可溶部分积累了 48%。总之，我们成功地利用代谢工程方法优化了整体 PAPS 合成的生产力。此外，我们还证明了 ePathBrick 系统可用于研究和改善酶合成条件。同时，我们还成功地展示了一种自动诱导微生物发酵方法，用于生产哺乳动物酶（ASTIV）。关键点：
ePathBrick 用于优化酶的表达水平。
已经建立了用于生产重组酶的方案。
采用高细胞密度分批补料发酵，获得高产量的可溶性酶。
成功将稳健的发酵方案转移到合同制造和研究设施。
