Abstract
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
Highlights
Porphobilinogen (PBG) is a pyrrole-containing intermediate in the metabolic pathways for biosynthesis of essential porphyrin/tetrapyrrole compounds known as “pigments of life”, including heme, cobalamin, chlorophyll, siroheme, heme d1, etc., in almost all types of biological cells (Frankenberg et al 2003)
Carbon flux direction from the tricarboxylic acid (TCA) pathways to the Shemin/C4 pathway The Shemin/C4 pathway was implemented in E. coli via heterologous expression of hemA from R. sphaeroides in BW∆ldhA (Miscevic et al 2021)
In this study, we demonstrated that implementation of the non-native C4 pathway in E. coli was effective to supply carbon flux from the natural TCA pathways for PBG biosynthesis via succinyl-coenzyme A (CoA)
Summary
Porphobilinogen (PBG) is a pyrrole-containing intermediate in the metabolic pathways for biosynthesis of essential porphyrin/tetrapyrrole compounds known as “pigments of life”, including heme, cobalamin, chlorophyll, siroheme, heme d1, etc., in almost all types of biological cells (Frankenberg et al 2003). The precursor of PBG, i.e., 5-aminolevulinic acid (5-ALA), is synthesized via either of the two unrelated metabolic routes, i.e., the Beale/ C5 pathway and the Shemin/C4 pathway (Zhang et al 2015). Found in most bacteria (including Escherichia coli) and all archaea and plants, the C5 pathway starts with the C5-skeleton of glutamate for conducting two enzymic reactions, i.e., initial reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA) via NADPH-dependent glutamyl-tRNA reductase (GluTR) and subsequent transamination of GSA via glutamate-1-semialdehyde2,1-aminomutase (GSAM), to form 5-ALA (Jahn et al.1992). PBG is synthesized via a common reaction for molecular condensation of two 5-ALA molecules catalyzed by ALA dehydratase (ALAD or HemB, encoded by hemB) (Layer et al 2010)
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