Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Chemical Engineering, Federal University of São Carlos, São Carlos, São Paulo, 13565-905, Brazil.
Department of Chemical Engineering, Federal University of São Carlos, São Carlos, São Paulo, 13565-905, Brazil.
Metab Eng. 2021 Mar;64:15-25. doi: 10.1016/j.ymben.2021.01.002. Epub 2021 Jan 14.
Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.
绿脓菌素是铜绿假单胞菌产生的一种次生代谢产物，属于吩嗪类化合物，是具有多种生物学功能的芳香含氮化合物。除了具有抗真菌和抗菌活性外，绿脓菌素还是一种显著的氧化还原活性分子，具有广泛的应用潜力，从制药工业到微生物燃料电池的发展都有涉及。然而，绿脓菌素的生产一直局限于铜绿假单胞菌菌株，限制了其实际应用。在本研究中，首次对异源宿主中该化合物的高水平生产进行了绿脓菌素生物合成途径的工程改造。携带该途径的 9 个基因并分为两个质粒的大肠杆菌细胞能够产生和分泌比一些铜绿假单胞菌菌株更高水平的绿脓菌素。评估了培养和诱导参数的影响，优化条件使绿脓菌素积累增加了 3.5 倍。通过测试一组具有不同拷贝数的质粒来实现途径平衡，以优化绿脓菌素生物合成基因的表达水平，从而在工程菌株之间产生 4 倍的产物滴度差异。通过共表达血晶素血红蛋白 Vhb 进一步改进，这缓解了氧气限制，最终绿脓菌素的产量达到 18.8mg/L。这些结果表明，使用大肠杆菌生产吩嗪类化合物具有前景，并且这里开发的工程菌株有可能用于电发酵系统，其中绿脓菌素作为电子穿梭体发挥作用。
