Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803

Abstract

Isoprenoids are diverse natural compounds, which have various applications as pharmaceuticals, fragrances, and solvents. The low yield of isoprenoids in plants makes them difficult for cost-effective production, and chemical synthesis of complex isoprenoids is impractical. Microbial production of isoprenoids has been considered as a promising approach to increase the yield. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 for sustainable production of a commercially valuable isoprenoid, limonene. Limonene synthases from the plants Mentha spicata and Citrus limon were expressed in cyanobacteria for limonene production. Production of limonene was two-fold higher with limonene synthase from M. spicata than that from C. limon. To enhance isoprenoid production, computational strain design was conducted by applying the OptForce strain design algorithm on Synechocystis 6803. Based on the metabolic interventions suggested by this algorithm, genes (ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) in the pentose phosphate pathway were overexpressed, and a geranyl diphosphate synthase from the plant Abies grandis was expressed to optimize the limonene biosynthetic pathway. The optimized strain produced 6.7 mg/L of limonene, a 2.3-fold improvement in productivity. Thus, this study presents a feasible strategy to engineer cyanobacteria for photosynthetic production of isoprenoids.

Photosynthetic generation of heterologous terpenoids in cyanobacteria

“…However, a yield of 0.3% was still a small fraction of the photosynthetic carbon flux through the cell, given that the terpenoid biosynthetic pathway itself fluxes about 5% of all photosynthetic carbon to meet the overall prenyl needs of the cell (Lindberg et al, ). Similarly, low yields were reported for the production of other heterologous terpenoids in cyanobacteria (Chaves et al, ; Davies, Work, Beliaev, & Posewitz, ; Englund, Andersen‐Ranberg, Miao, & Lindberg, ; Gao et al, ; Kiyota, Okuda, Ito, Yokota Hirai, & Ikeuchi, ; Leonard et al, ; Lin & Pakrasi, ; Lin, Saha, Zhang, & Pakrasi, ; Wang et al, ), underscoring the universal flux and yield restrictions of the process. Greater yields were recently reported for the heterologous synthesis of non‐prenyl compounds, including 2,3‐butanediol (Nozzi, Case, Carroll, & Atsumi, ), isobutanol (Miao, Xie, & Lindblad, ), and lactate (Du et al, ), underscoring the variable efficacy of different biosynthetic pathways and endogenous substrates to support carbon flux for the generation of heterologous product synthesis.…”

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy

“…However, it is anticipated that increased integration of computational design and automation with biology will rapidly shift this paradigm. Computational modeling can be used to predict non-intuitive approaches to optimize metabolic flux through heterologous pathways, as was demonstrated by the optimization of terpenoid production in cyanobacteria (Lin et al, 2017). Novel biological designs or complex combinatorial libraries can be rapidly assembled and evaluated in automated, highthroughput biofoundries, which are attracting investment from research institutions across the globe (Hillson et al, 2019).…”

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

“…Self‐replicating plasmids, which are more commonly used in model hosts such as E. coli and S. cerevisiae , are promising alternatives for high‐level expression of heterologous genes. For instance, Lin et al combined the utilization of both an endogenous plasmid and an exogenous plasmid to produce limonene, and Miao et al achieved higher yield and titer of isobutanol when a synthetic pathway was expressed on an RSF1010‐derived pEEK2 plasmid. In addition to high expression levels, self‐replicating plasmids are easy to manipulate, and hence can be used to rapidly generate different pathway designs for functional studies and optimization.…”

“…6803 ( Synechocystis 6803) . For instance, cyanobacteria have been engineered to produce fuels and fuel precursors, such as ethanol, isobutyraldehyde, butanol, alkenes, fatty acids and lipids, and the inventory of products has been expanded to value‐added chemicals for manufacturing, food, pharmaceuticals, and cosmetics, e.g., isoprene, squalene, l ‐lysine, limonene, and lactic acid . Besides the increasingly diverse products, mixotrophic growth of these photoautotrophic strains has been achieved by the installation of sugar‐utilizing pathways, thus establishing synthetic hybrid cyanobacterial platforms capable of continuous growth in diurnal conditions with significantly improved metabolic robustness .…”