Toward improved terpenoids biosynthesis: strategies to enhance the capabilities of cell factories

Abstract

Terpenoids form the most diversified class of natural products, which have gained application in the pharmaceutical, food, transportation, and fine and bulk chemical industries. Extraction from naturally occurring sources does not meet industrial demands, whereas chemical synthesis is often associated with poor enantio-selectivity, harsh working conditions, and environmental pollutions. Microbial cell factories come as a suitable replacement. However, designing efficient microbial platforms for isoprenoid synthesis is often a challenging task. This has to do with the cytotoxic effects of pathway intermediates and some end products, instability of expressed pathways, as well as high enzyme promiscuity. Also, the low enzymatic activity of some terpene synthases and prenyltransferases, and the lack of an efficient throughput system to screen improved high-performing strains are bottlenecks in strain development. Metabolic engineering and synthetic biology seek to overcome these issues through the provision of effective synthetic tools. This review sought to provide an in-depth description of novel strategies for improving cell factory performance. We focused on improving transcriptional and translational efficiencies through static and dynamic regulatory elements, enzyme engineering and high-throughput screening strategies, cellular function enhancement through chromosomal integration, metabolite tolerance, and modularization of pathways.

Keywords: Cellular tolerance; Chromosomal integration; Dynamic regulation; Modular engineering; Promoter engineering; Protein engineering; RBS engineering; Terpenoids.

Fig. 1

The MVA pathway and MEP pathway for isoprenoids biosynthesis. The isoprenoid biosynthetic pathway can be grouped into the central carbon pathway, upstream isoprenoid pathway, and downstream isoprenoid pathway. PEP, phosphoenolpyruvate; HMG-CoA, S-3-hydroxy-3-methylglutaryl-CoA; DXP, 1-deoxy-d-xylulose 5-phosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; CDP-ME, 4-(cytidine 5ʹ-diphospho)-2-C-methyl-d-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5ʹ-diphospho)-2-C-methyl-d-erythritol; MEcPP, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl pyrophosphate; NPP, neryl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; CCP, central carbon pathway; C5, hemiterpenoids; C10, monoterpenoids; C15, sesquiterpenoids; C20, diterpenoids; C30, triterpenoids; C40, tetraterpenoids

Fig. 2

Proposed mechanism of terpenoid carbonation. Terpenoids undergo a wide range of cyclization and rearrangements to ensure final product synthesis

Fig. 3

A Illustration of dynamic control of LuxI/LuxR Quorum Sensing (QS) system. (i) At low cell density, the transcriptional regulator, LuxR, binds the P_lux promoter to repress the transcription of the target gene. (ii) At high cell density, LuxI protein synthesizes acyl-homoserine lactone (AHL) which binds the transcriptional regulator resulting in dissociation from the promoter. The target gene is subsequently expressed. B Graphical representation of putative enzymes joined with a linker. Sequential pathway enzymes can be modified through enzyme fusion to improve enzymatic reaction. The fusion of enzymes ensures substrates are channelled from one active site to the other

Fig. 4

Schematic diagram for producing enzyme, promoter, and RBS library construction and screening. Directed evolution, site-directed mutagenesis, DNA shuffling can be employed to enhance enzyme and regulatory elements efficiency. Engineered promoters and RBS can be used to fine-tune biosynthetic pathways

Fig. 5

Schematic illustration of adaptive laboratory evolution (ALE), atmospheric and room-temperature plasma (ARTP), and modular co-culture. A Microorganisms are exposed to a desired selective mechanism and or environment for an iterative period enabling natural selection to optimize variants with enhanced fitness. Genome sequencing and transcriptome can be used to analyze mutant variants. B Modular co-culture engineering. Segregating pathway into modules ensures a holistic assessment of each part for efficient optimization and improvement

Fig. 6

Mechanism of action of riboswitches. A (i, ii) Binding of ligand to a riboswitch triggers the formation of a hairpin loop that terminates transcription. A (iii, iv) Binding of ligand to a riboswitch generates the formation of a helix that sequesters the RBS to inhibit the translational process. B (i, ii, iii) Application of riboswitches as biosensors. Riboswitches can be linked to colorimetric reporters (for example GFP) to screen for high-producing strains depending on the concentration of the compound of interest. There is a high expression of the reporter gene when the concentration of a ligand is high (B ii) and vice versa