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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

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