Engineered dynamic distribution of malonyl-CoA flux for improving polyketide biosynthesis in Komagataella phaffii

• Published in: Journal of biotechnology 2020-08, Vol.320, p.80-85
• Main Authors: Wen, Jiao, Tian, Lin, Liu, Qi, Zhang, Yuanxing, Cai, Menghao
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Dynamic control; FapR/fapO; Komagataella phaffii; Malonyl Coenzyme A - metabolism; Malonyl-CoA; Metabolic Engineering - methods; Polyketides - metabolism; Promoter Regions, Genetic - genetics; Saccharomycetales - genetics; Saccharomycetales - metabolism; Synthetic Biology; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2020.06.012

[Display omitted] •A synthetic malonyl-CoA independent module allowed continuous polyketide synthesis.•A synthetic malonyl-CoA dependent module dynamically regulated fatty acid synthesis.•An integrated biosystem redirected malonyl-CoA flux to improve polyketide synthesis. Malonyl-CoA is a basic but limited precursor for the biosynthesis of various bioactive compounds and life-supporting fatty acids in cells. This study develops a biosynthetic system to dynamically redirect malonyl-CoA flux and improve production of malonyl-CoA derived polyketide (6-MSA) in Komagataella phaffii. A synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to work with a hybrid promoter (-7)fapO-cPAOX1 and activate gene expression. Expression mode by the Prm1-FapR/(-7)fapO-cPAOX1 device was not affected by intracellular malonyl-CoA levels. Further, 9 promoter variants of PGAP with insertion of fapO at various sites were tested with the Prm1-FapR. It generated a biosensor of Prm1-FapR/PGAP-(+2)fapO with regulation behavior of malonyl-CoA-low-level repression/high-level derepression. Both devices were subsequently integrated into a single cell, for which fatty acid synthesis module was driven by Prm1-FapR/PGAP-(+2)fapO but 6-MSA synthesis module was expressed by Prm1-FapR/(-7)fapO-cPAOX1. The integrated system allowed continuous polyketide synthesis but malonyl-CoA-high-level “on”/low-level “off” fatty acid synthesis. This design finally increased 6-MSA production capacity by 260 %, proving the positive effects of dynamic malonyl-CoA distribution to the target compounds. It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts.