Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids

Abstract

Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, combinatorial biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.

Keywords: Combinatorial biosynthesis; Hemiterpenes; Isoprenoids; Synthetic biology; Terpenes.

Fig. 1.

Biosynthesis of diverse isoprenoid natural products.

Fig. 2.

Combinatorial synthetic biology and chemo-enzymatic approaches to isoprenoids and their non-natural designer analogues.

Fig. 3.

Enzymatic biosynthesis pathways to isoprenoid precursors, DMAPP and IPP. a Native DXP pathway. b Native MVA pathway. c de novo pathways. Enzymatic steps highlighted in blue have been identified for critical metabolic engineering described in the text. DXS, DXP synthase; DXR, DXP reductoisomerase; CMS, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, HMB pyrophosphate synthase; HDR, HMB pyrophosphate reductase. AACT – acetoacetyl-CoA thiolase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate-5-phosphate decarboxylase; MPK, mevalonate-3-phosphate-5-kinase; MPD, mevalonate phosphate decarboxylase; IPK, isopentenyl phosphate kinase; BPMK, bisphosphate mevalonate; CK, choline kinase.

Fig. 4.

Biologically and chemoenzymatically derived non-natural prenyl diphosphates. Structural differences from the natural diphosphates are highlighted in red. See text for references.

Fig. 5.

Mechanism of C10 isoprenoid pyrophosphate formation. a GPP and NPP are formed by the condensation of one unit each of DMAPP and IPP, with the formation of an E- or Z- olefin, respectively. b LPP and CPP are formed by the head-to-middle condensation of two units of DMAPP. LPP undergoes a simple elimination, while CPP undergoes an intramolecular cyclopropanation to form the final product.

Fig. 6.

Non-native promiscuity and regioselectivity of FgaPT2 with unnatural alkyl diphosphates on indole acceptor.

Fig. 7.

Promiscuity and regioselectivity of PTases. a Alkylation of amino acids. b Alkylation of DKPs. c Alkylation of flavonoids. d Alkylation of phenylpropanoids. PTases shown are wild-type unless labeled with ‘Δ’.

Fig. 8.

Rational engineering of EIZS to produce different product profiles. Shown are the major products that result from some of the individual point mutations evaluated with the percent fraction of the indicated product of the total products shown [113, 115]. *denotes temperature dependence of the product distribution.

Fig. 9.

Proposed mechanism of sesquiterpene formation by γ-humulene synthase [117].

Fig. 10.

New-to-nature cyclized isoprenoids derived from precursor-directed biosynthesis.

Fig. 11.

High throughput screening platforms for isoprenoids. a Colorimetric product biosynthesis to enhance the production of precursor levels or colorimetric product titers. b Antibiotic (denoted with grey circles) selection markers for optimizing native or bioorthogonal pathways to enable growth. c Transcriptional regulation by a repressor protein prevents the expression of a downstream reporter gene such as green fluorescent protein (GFP). In the presence of the target small molecule, the protein binds the ligand, causing a conformational change in protein structure to yield a quantitative signal. These biosensing platforms have numerous applications related to isoprenoid synthetic biology.