Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae

Abstract

Spatial clustering of enzymes has proven an elegant approach to optimize metabolite transfer between enzymes in synthetic metabolic pathways. Among the multiple methods used to promote colocalisation, enzyme fusion is probably the simplest. Inspired by natural systems, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of Xanthophyllomyces dendrorhous carotenoid enzymes produced in Saccharomyces cerevisiae. Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties considerably impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains, all concurring to either diminish or boost specific enzymatic steps of the metabolic pathway. Remarkably, the yield of β-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes.

Improved Astaxanthin Production with Corynebacterium glutamicum by Application of a Membrane Fusion Protein

“…Application of fusion proteins for carotenoid production has recently gained more interest. It has been shown that application of a tridomain protein comprising CrtB, CrtI and CrtY doubled β-carotene production by S. cerevisiae [26]. Both, the domain order and the linker properties, were claimed to influence the stability and/or expression of such enzymes [26].…”

“…It has been shown that application of a tridomain protein comprising CrtB, CrtI and CrtY doubled β-carotene production by S. cerevisiae [26]. Both, the domain order and the linker properties, were claimed to influence the stability and/or expression of such enzymes [26]. Interestingly, the natural astaxanthin producer Xanthophyllomyces dendrorhous possesses bifunctional enzymes in carotenogenesis.…”

Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene

“…For instance, the CCD1 could be fused to the lycopene cyclase domain of the CrtYB, avoiding the access to other intermediates. A recent report of heterologous production of β-carotene in S. cerevisiae has shown promise for the optimization of carotenoids in yeast (Rabeharindranto et al, 2019).…”

Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments

“…The exact same coding sequences were used for cloning into pYeDP60 50 vector for S. cerevisiae expression, in association with a FLAG-tag instead of the previous hexahistidine-tag. We used YHR01 and YHR02 from Rabeharindranto et al 28 , using the protocol for yeast transforma- www.nature.com/scientificreports/ tion adapted from Gietz et al 51 . YHR01 and YHR02 correspond to S. cerevisiae BY4741 engineered for GGPP production (over-expression of GGPP synthase, CrtE, from P. rhodozyma and truncated form of HMG-CoA reductase, HMG1) 28 .…”

“…We used YHR01 and YHR02 from Rabeharindranto et al 28 , using the protocol for yeast transforma- www.nature.com/scientificreports/ tion adapted from Gietz et al 51 . YHR01 and YHR02 correspond to S. cerevisiae BY4741 engineered for GGPP production (over-expression of GGPP synthase, CrtE, from P. rhodozyma and truncated form of HMG-CoA reductase, HMG1) 28 . Phytoene production in YHR02 is mediated by the expression of bifunctional phytoene synthase/lycopene cyclase, CrtYB and in YHR01 thanks to the expression of the truncated form of CrtYB having only the phytoene synthase activity.…”