Abstract
Covering: up to 2021Terpenoids are a diverse group of chemicals used in a wide range of industries. Microbial terpenoid production has the potential to displace traditional manufacturing of these compounds with renewable processes, but further titre improvements are needed to reach cost competitiveness. This review discusses strategies to increase terpenoid titres in Escherichia coli with a focus on alternative metabolic pathways. Alternative pathways can lead to improved titres by providing higher orthogonality to native metabolism that redirects carbon flux, by avoiding toxic intermediates, by bypassing highly-regulated or bottleneck steps, or by being shorter and thus more efficient and easier to manipulate. The canonical 2-C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways are engineered to increase titres, sometimes using homologs from different species to address bottlenecks. Further, alternative terpenoid pathways, including additional entry points into the MEP and MVA pathways, archaeal MVA pathways, and new artificial pathways provide new tools to increase titres. Prenyl diphosphate synthases elongate terpenoid chains, and alternative homologs create orthogonal pathways and increase product diversity. Alternative sources of terpenoid synthases and modifying enzymes can also be better suited for E. coli expression. Mining the growing number of bacterial genomes for new bacterial terpenoid synthases and modifying enzymes identifies enzymes that outperform eukaryotic ones and expand microbial terpenoid production diversity. Terpenoid removal from cells is also crucial in production, and so terpenoid recovery and approaches to handle end-product toxicity increase titres. Combined, these strategies are contributing to current efforts to increase microbial terpenoid production towards commercial feasibility.
Highlights
IntroductionArchaeal MVA pathways Archaeal MVA pathway I Archaeal MVA pathway II Archaeal MVA pathway III isopentenyl diphosphate (IPP)-bypass pathways Isoprenol/prenol to IPP/DMAPP pathways Terpenoids outside the ‘isoprene rule’ Alternative prenyl diphosphate synthases cis-Prenyl diphosphate synthases Non-head-to-tail prenyl diphosphate synthases, terpenoid cyclases and other prenyltransferases Bacterial terpenoid synthases and modifying enzymes Handling toxicity Mechanisms of toxicity Physical extraction Secretion Membrane engineering Excretion Adaptive laboratory evolution Bioderivatisation
Canonical pathways to isopentenyl diphosphate (IPP) and DMAPP The native methyl-D-erythritol 4-phosphate (MEP) pathway Regulation Alternative genes The MVA pathway Regulation Alternative genes Alternative precursor pathways Alternative entry points to the MEP and MVA pathways Pathways to D-xylulose 5-phosphate (DXP) Leucine shuntArchaeal MVA pathways Archaeal MVA pathway I Archaeal MVA pathway II Archaeal MVA pathway III IPP-bypass pathways Isoprenol/prenol to IPP/DMAPP pathways Terpenoids outside the ‘isoprene rule’ Alternative prenyl diphosphate synthases cis-Prenyl diphosphate synthases Non-head-to-tail prenyl diphosphate synthases, terpenoid cyclases and other prenyltransferases Bacterial terpenoid synthases and modifying enzymes Handling toxicity Mechanisms of toxicity Physical extraction Secretion Membrane engineering Excretion Adaptive laboratory evolution Bioderivatisation Review
DXP is converted to MEP by DXP reductoisomerase (DXR) using NADPH,[17] and MEP is activated with cytidine 50triphosphate (CTP) to produce 4-diphosphocytidyl-2-C-methylD-erythritol (CDP-ME) by CPD-ME synthase (CMS).[18]
Summary
Terpenoids form one of the largest and most diverse classes of chemicals comprising thousands of structures.[1]. Mauro Adriel Rinaldi received his BSc in Molecular Biology from the University of Buenos Aires and his PhD in Biochemistry and Cell Biology from Rice University studying plant peroxisomes and metabolism He is engineering microbes to make natural products from renewable carbon at the Manchester Institute of Biotechnology, Department of Chemistry, The University of Manchester. The use of alternative pathways can avoid toxic intermediate build-up, bypass highly-regulated or bottleneck steps, and present more energetically efficient or shorter routes to the desired product. Such pathways are likely to be easier targets for optimisation through pathway engineering.
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