Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

Bioresources and Bioprocessing

Marc Aucoin¹  Mark Bruder¹  Davinder Lall¹  C. Perry Chou¹  Adam Westbrook¹  Murray Moo-Young¹  Dragan Miscevic¹ 

[1] Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, N2L 3G1, Waterloo, ON, Canada;

关键词: Escherichia coli;    Glycerol;    Glyoxylate shunt;    Porphobilinogen (PBG);    Strain engineering;    Succinyl-CoA;    Tricarboxylic acid (TCA) cycle;   

DOI  :  10.1186/s40643-021-00482-3

来源: Springer

【 摘 要 】

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L−1 glycerol, we achieved high PBG titers up to 209 mg L−1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization.Graphical Abstract

【 授权许可】

CC BY