Abstract
Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. Operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L−1. Likewise, yields of the sesquiterpene alcohols (−)-patchoulol and (−)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L−1 and 96.3 ± 2.2 mg * L−1, respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDC system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. Together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria.
Highlights
Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals
For ectopic expression of the terpene synthases, codon optimised coding sequences (CDS) of bisabolene synthase from Abies grandis (AgBIS, GenBank accession no.: MG052654), patchoulol synthase from Pogostemon cablin (PcPs, GenBank accession no.: KX097887) and (−)-α-bisabolol synthase from Matricaria recutita (MrBBS, GenBank accession no.: KJ020282) were each linked to the promoter:RBS module PpetE:BCD2, which mediates strong Cu2+-inducible expression (Fig. 1b)
The expression module was further linked to a codon optimised version of the fluorescence reporter mVenus[40], which was deployed as isogenic negative control for sesquiterpene accumulation and quantitative expression proxy during all experiments
Summary
Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. Further strategies sought to challenge the problem of inherently weak terpene synthase activities by increasing cellular enzyme titers, concurrently pushing carbon flux towards the heterologous metabolic sink To this end, major improvements were reported by systematic, genetic optimisation of terpene synthase expression cassettes, and/or by designing highly expressed fusion proteins[13,14,15,20,21]. Major improvements were reported by systematic, genetic optimisation of terpene synthase expression cassettes, and/or by designing highly expressed fusion proteins[13,14,15,20,21] Aside from these specific metabolic and physiological bottlenecks, general drawbacks of photoautotrophic production strategies arise from inherent technical limitations of the established cultivation systems, regarding the supply of essential nutrients and light[22,23]. This system facilitates rapid, sustainable biomass accumulation and was demonstrated to further raise the potential for the recovery of natural products from cyanobacteria[28,29,30]
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