Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes

Abstract

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

“…4B, viii). It is unclear why it was possible to express ERG20 as a fusion with CFP and not as a fusion to the PcPs-YFP as has been described in yeast (Albertsen et al, 2011), however, this was not investigated further in this work. The relative productivity of patchoulol normalized to enzyme expression was indeed higher for the strains expressing the ispA-PcPs fusion.…”

“…It has been previously demonstrated that fusion of the yeast ERG20 FPPs to a PcPs resulted in higher titers of patchoulol production in the yeast host, with the assumption that close proximity of FPP substrate generation to the PcPs active site increases substrate channelling to the synthase (Albertsen et al, 2011). Since high titers of the PcPs synthase were desired for robust productivity, and the CrFPPs exhibited specialized localization in the cell, only ispA and ERG20 were chosen for fusion trials with the PcPs.…”

“…It has been previously shown that some terpene synthases are capable of fusion to fluorescent reporters without effecting product generation (Albertsen et al, 2011). To increase the efficiency of identification of transformants with robust patchoulol synthase expression, the codon optimized PcPs gene was expressed in frame with YFP (mVenus) so that the fluorescent reporter was in fusion to the C-terminus of the patchoulol synthase.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…Interaction domain Reference chimeric adapters protein [143] fusion enzymes protein [144] GBD-PDZ-SH3 scaffold protein [10] MAPK scaffold protein [145] two-component systemsp rotein [146] Thec lassical approach wase stablished due to historical developments as the first microorganisms were used as wild-type strains andt heir natural ability for production of certain chemicals was exploited. Fermentative production of ethanol by the yeast S. cerevisiae represents the most prominent example.…”

Development of bio-based fine chemical production through synthetic bioengineering

“…Valencene, cubebol, patchoulol, and α-santalene are produced by expressing heterologous sesquiterpene synthase genes in S. cerevisiae [42][43][44][45][46][47]. Coexpression of the genes encoding valencene synthase gene and a P450 mono-oxygenase in S. cerevisiae generates nootkatone that is industrially produced as a flavoring agent and fragrance [48].…”