Abstract
Isoprenoids comprise one of the most chemically diverse family of natural products with high commercial interest. The structural diversity of isoprenoids is mainly due to the modular activity of three distinct classes of enzymes, including prenyl diphosphate synthases, terpene synthases, and cytochrome P450s. The heterologous expression of these enzymes in microbial systems is suggested to be a promising sustainable way for the production of isoprenoids. Several limitations are associated with native enzymes, such as low stability, activity, and expression profiles. To address these challenges, protein engineering has been applied to improve the catalytic activity, selectivity, and substrate turnover of enzymes. In addition, the natural promiscuity and modular fashion of isoprenoid enzymes render them excellent targets for combinatorial studies and the production of new-to-nature metabolites. In this review, we discuss key individual and multienzyme level strategies for the successful implementation of enzyme engineering towards efficient microbial production of high-value isoprenoids. Challenges and future directions of protein engineering as a complementary strategy to metabolic engineering are likewise outlined.
Highlights
Isoprenoids comprise one of the most diverse and important classes of natural products with a broad spectrum of biological activities
The basic isoprene units isopentenyl diphosphate (IPP) and DMAPP are condensed in an incremental manner by prenyl diphosphate synthases leading to the formation of various size linear prenyl chains, such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), the isoprenoid precursors of monoterpenes, sesquiterpenes, and diterpenes, respectively (Fig. 1)
Co-expression of the Erg20p variant with 8-hydroxy copalyl diphosphate synthase from Cistus creticus resulted in a more than 70-fold improvement in sclareol production (15.4 mg/L) in shake-flask culture (Ignea et al, 2015b). These results show that canonical prenyltransferases can be engineered to become specific for unusual substrates providing a series of new-to-nature isoprenoids
Summary
Isoprenoids ( known as terpenoids) comprise one of the most diverse and important classes of natural products with a broad spectrum of biological activities. Isoprenoids are derived from two distinct metabolic pathways, the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which is present in most bacteria and plastids of plant cells, and the mevalonate (MVA) pathway, which functions in eukaryotes, archaea, and certain bacteria (“upstream” pathways of isoprenoids) Both routes lead to the formation of the five-carbon constitutional isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. The basic terpenoid skeleton of terpenes is further diversified in a regio- and stereoselective manner through a series of postmodifications These modifications commonly start with oxidation catalyzed by heme-containing enzymes, namely cytochrome P450 monooxygenases (CYP450s, CYPs), which introduce one atom of molecular oxygen into nonactivated C–H bonds (Urlacher and Girhard, 2019). We discuss pitfalls associated with these engineering strategies and provide solutions on how these limitations could be overcome in this fascinating and highly evolving field
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