Cyanamide-inducible expression of homing nuclease I−SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae

In synthetic biology,microbial chassis including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale.Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements,such as the galactose-inducible(GAL)promoter in S.cerevisiae.Here,we prototyped the cyanamide-induced I−SceI expression,which triggered double-strand DNA breaks(DSBs)for selectable marker removal.We further combined cyanamide-induced I−SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution,which simplified the molecular cloning procedure for promoter characterisation.We then characterised three tetracycline-inducible promoters showing differential strength,a non-leakyβ-estradiol-inducible promoter,cyanamide-inducible DDI2 promoter,bidirectional MAL32/MAL31 promoters,and five pairs of bidirectional GAL1/GAL10 promoters.Overall,alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.

Profiling proteomic responses to hexokinase-II depletion in terpene-producing Saccharomyces cerevisiae

Hexokinase II(Hxk2)is a master protein in glucose-mediated transcriptional repression signaling pathway.De-grading Hxk2 through an auxin-inducible protein degradation previously doubled sesquiterpene(nerolidol)pro-duction at gram-per-liter levels in Saccharomyces cerevisiae.Global transcriptomics/proteomics profiles in Hxk2-deficient background are important to understanding genetic and molecular mechanisms for improved nerolidol production and guiding further strain optimization.Here,proteomic responses to Hxk2 depletion are investi-gated in the yeast strains harboring a GAL promoters-controlled nerolidol synthetic pathway,at the exponential and ethanol growth phases and in GAL80-wildtype and gal80Δbackgrounds.Carbon metabolic pathways and amino acid metabolic pathways show diversified responses to Hxk2 depletion and growth on ethanol,including upregulation of alternative carbon catabolism and respiration as well as downregulation of amino acid synthesis.De-repression of GAL genes may contribute to improved nerolidol production in Hxk2-depleted strains.Seven-teen transcription factors associated with upregulated genes are enriched.Validating Ash1-mediated repression on the RIM4 promoter shows the variation on the regulatory effects of different Ash1-binding sites and the syner-gistic effect of Ash1 and Hxk2-mediated repression.Further validation of individual promoters shows that HXT1 promoter activities are glucose-dependent in hxk2Δbackground,but much weaker than those in HXK2-wildtype background.In summary,inactivating HXK2 may relieve glucose repression on respiration and GAL promoters for improved bioproduction under aerobic conditions in S.cerevisiae.The proteomics profiles provide a better genetics overview for a better metabolic engineering design in Hxk2-deficient backgrounds.