Ty1‐fused protein‐body formation for spatial organization of metabolic pathways in Saccharomyces cerevisiae

Abstract

Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in three and fourfold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast.

Refining Metabolic Mass Transfer for Efficient Biosynthesis of Plant Natural Products in Yeast

“…To solve these problems, some artificial scaffolds (including nucleic acid scaffolds and protein scaffolds) have been developed to form substrate channeling for the cascade enzymatic reactions, which can enhance the membrane free mass transfer process and improve the mass transfer efficiency. Han employed the retrotransposon element Ty1 as a scaffold to spatially organize enzymes involved in biosynthesis of farnesene and farnesol, forming the substrate channel and resulting in threefold and fourfold increased titers, respectively (Han et al, 2018). The key enzymes in resveratrol synthesis pathway including 4CL1 and STS were recruited to the protein scaffold to relocated in S. cerevisiae, and the yield of resveratrol was increased by five times (Wang and Yu, 2012).…”

In vitro and in vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

“…The plasmids constructed by in vitro recombination might be lost during the fermentation process with YPD medium,which might lead to the reduction of astaxanthin accumulation. There were multiple Ty1 retrotransposon sites in yeast genome [30], which could be used for the integration of multiple copies of heterologous modules by in vivo recombination methods. To integrate the crtZ and crtW into the genome, all the crtZ and crtW were flanked by about 500 base pair homologous sequences selected from the TyA (Additional file 1: Figure S4).…”

In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

“…The plasmids constructed by in vitro recombination may be lost during the fermentation process with YPD medium which may lead to the reduction of astaxanthin accumulation. There are multiple Ty1 retrotransposition sites in yeast genome [30], which can be used for the integration of multiple copies of heterologous modules by in vivo recombination methods. To integrate the crtZ and crtW into the genome, all the crtZ and crtW were flanked by about 500 base pair homologous sequences from the TyA (additional Fig.4).…”