Sustainable heterologous production of terpene hydrocarbons in cyanobacteria

Abstract

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial application. However, the slow catalytic activity of terpene synthases (k = 4 s or slower) makes them noncompetitive for the pool of available substrate, thereby limiting the rate and yield of product generation. Work in this paper applied transformation technologies in Synechocystis for the heterologous production of β-phellandrene (monoterpene) hydrocarbons. Conditions were defined whereby expression of the β-phellandrene synthase (PHLS), as a CpcB·PHLS fusion protein with the β-subunit of phycocyanin, accounted for up to 20 % of total cellular protein. Moreover, CpcB·PHLS was heterologously co-expressed with enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase, increasing carbon flux toward the terpenoid biosynthetic pathway and enhancing substrate availability. These improvements enabled yields of 10 mg of β-phellandrene per g of dry cell weight generated in the course of a 48-h incubation period, or the equivalent of 1 % β-phellandrene:biomass (w:w) carbon-partitioning ratio. The work helped to identify prerequisites for the efficient heterologous production of terpene hydrocarbons in cyanobacteria: (i) requirement for overexpression of the heterologous terpene synthase, so as to compensate for the slow catalytic turnover of the enzyme, and (ii) enhanced endogenous carbon partitioning toward the terpenoid biosynthetic pathway, e.g., upon heterologous co-expression of the MVA pathway, thereby supplementing the native metabolic flux toward the universal isopentenyl-diphosphate and dimethylallyl-diphosphate terpenoid precursors. The two prerequisites are shown to be critical determinants of yield in the photosynthetic CO to terpene hydrocarbons conversion process.

Harnessing transcription for bioproduction in cyanobacteria

“…In addition, short regulatory RNAs, like anti‐sense RNAs, might have potential to be developed to tightly controlling regulatory systems for induced protein production (Higo et al ). Furthermore, fast and ‘smooth’ translation has shown to enhance productivity (Formighieri and Melis , Formighieri and Melis ).…”

Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects

“…Standardised cloning methods, like shotgun cloning or cDNA cloning [110], have been employed to express exogenous genes or knockout native genes in Synechocystis 6803 [111,112,113,114,115,116]. For maximum expression levels of heterologous genes, codon optimisation has been utilised in several studies with favorable effects [16,116,117,118]. Additionally, the development of degradation tags used for the fine-tuning of protein levels and protein turnover times in this model organism have also been achieved [100].…”

“…The expression of ipi , crtR , and crtP and crtB led to a large increase in the corresponding transcript levels that led to the accumulation of zeaxanthin and myxoxanthophyll in the mutant strains [101]. A study involving increased production of carotenoids from Synechocystis 6803 reported a β-phellandrene yield reaching 1% of the dry cell weight [116]. This was accomplished by expressing β-phellandrene synthase as a fusion protein with the β-subunit of phycocyanin.…”

“…This was accomplished by expressing β-phellandrene synthase as a fusion protein with the β-subunit of phycocyanin. The expression of enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase (GPPS) increased substrate availability and overall terpenoid biosynthesis pathway utilisation [116]. Another study reported a pinene synthase mutant with an altered cofactor preference towards magnesium, which possess superior activity to the wild-type [119].…”

A Novel Application Potential of GH6 Cellobiohydrolase CtCel6 from Thermophilic Chaetomium thermophilum for Gene Cloning, Heterologous Expression and Biological Characterization

“…Pichia pastoris, as a convenient production system of yeast that could heterologously express proteins in high amounts, has been wildly used as a heterogeneous expression system Zhao et al, 2015). To satisfy the strong demand for the thermostable hydrolase production in enzyme preparation industry, heterologous expression using P. pastoris has become the primary way which significantly improves the protein expression level (Formighieri and Melis, 2016). However, as a significant part of glycoside hydrolases of thermophilic C. thermophilum, there are few reports about the thermostable GH6 cellobiohydrolases from C. thermophilum.…”