Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum

Abstract

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in kcat/KM was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

De Novo Biosynthesis of Lutein in Yarrowia lipolytica

“…9,10 Among them, the lutein synthesis pathway in yeast consists of three metabolic modules: lycopene synthesis from acetyl-CoA via the MVA pathway, α-carotene synthesis through β-cyclization and ε-cyclization of lycopene, and lutein synthesis by β-hydroxylation and ε-hydroxylation of α-carotene (Figure 1). 10,11 Lycopene biosynthesis has been exhaustively investigated and remarkable efficient synthesis of lycopene has been achieved in various microbial hosts. 12,13 However, α-carotene biosynthesis, the second metabolic module, is one of the primary bottlenecks in lutein biosynthesis owing to the lack of an enzyme capable of effective catalysis of asymmetric cyclization of lycopene to α-carotene.…”

“…The lutein biosynthesis pathway mainly includes MEP pathways in prokaryotes and chloroplasts and MVA pathways in most of the eukaryotes. , Among them, the lutein synthesis pathway in yeast consists of three metabolic modules: lycopene synthesis from acetyl-CoA via the MVA pathway, α-carotene synthesis through β-cyclization and ε-cyclization of lycopene, and lutein synthesis by β-hydroxylation and ε-hydroxylation of α-carotene (Figure ). , Lycopene biosynthesis has been exhaustively investigated and remarkable efficient synthesis of lycopene has been achieved in various microbial hosts. , However, α-carotene biosynthesis, the second metabolic module, is one of the primary bottlenecks in lutein biosynthesis owing to the lack of an enzyme capable of effective catalysis of asymmetric cyclization of lycopene to α-carotene. , In the third metabolic module, α-carotene is enzymatically converted by two P450 monooxygenases via β-hydroxylation and ε-hydroxylation, along with cytochrome P450 reductase (CPR), to produce lutein . Currently, lutein biosynthesis has been realized in Chlorella sorokiniana, Escherichia coli, and Saccharomyces cerevisiae. , C.…”