A common challenge in the assembly and optimization of plant natural product biosynthetic pathways in recombinant hosts is the identification of gene orthologues that will result in best production titers. Here, we describe the modular assembly of a naringenin biosynthetic pathway in Saccharomyces cerevisiae that was facilitated by optimized naringenin-inducible prokaryotic transcription activators used as biosensors. The biosensors were designed and developed in S. cerevisiae by a multiparametric engineering strategy, which further was applied for the in vivo, high-throughput screening of the established yeast library. The workflow for assembling naringenin biosynthetic pathways involved Golden gate-directed combinatorial assembly of genes and promoters, resulting in a strain library ideally covering 972 combinations in S. cerevisiae. For improving the performance of our screening biosensor, a series of fundamental components was optimized, affecting the efficiency as nuclear localization signal (NLS), the detector module and the effector module. One biosensor (pTDH3 NLS_FdeR-N_tPGKl-pGPM1-fdeO_mcherry_tTDH1-MV2) showed better performance, defined as better dynamic range and sensitivity than others established in this study as well as other previously reported naringenin biosensors. Using this biosensor, we were able to identify a recombinant S. cerevisiae strain as the most efficient candidate for the production of naringenin from the established naringenin biosynthetic library. This approach can be exploited for the optimization of other metabolites derived from the flavonoid biosynthetic pathways and more importantly employed in the characterization of putative flavonoid biosynthetic genes.
[1]MOE Key Lab Standardizat Chinese Med, Shanghai 201203, Peoples R China.
[2]Shanghai Univ Tradit Chinese Med, SATCM Key Lab New Resources & Qual Evaluat Chines, Inst Chinese Mat Med, Shanghai 201203, Peoples R China.
[3]Rensselaer Polytech Inst, Dept Chem & Biol Engn, Ctr Biotechnol & Interdisciplinary Studies, Troy, NY 12180 USA.
[4]Rensselaer Polytech Inst, Dept Biol Sci, Troy, NY 12180 USA.
[5]Pohang Univ Sci & Technol, Dept Chem Engn, Pohang 37673, Gyeongbuk, South Korea.
(8.0+极速模式)