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.

Benchmarking two Saccharomyces cerevisiae laboratory strains for growth and transcriptional response to methanol

One-carbon compounds,such as methanol,are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals,fuels,foods,and pharmaceuticals.Efficient biological production often requires extensive genetic manipulation of a microbial host strain,making well-characterised and geneticallytractable model organisms like the yeast Saccharomyces cerevisiae attractive targets for the engineering of methylotrophic metabolism.S.cerevisiae strains S288C and CEN.PK are the two best-characterised and most widely used hosts for yeast synthetic biology and metabolic engineering,yet they have unpredictable metabolic phenotypes related to their many genomic differences.We therefore sought to benchmark these two strains as potential hosts for engineered methylotrophic metabolism by comparing their growth and transcriptomic responses to methanol.CEN.PK had improved growth in the presence of methanol relative to the S288C derivative BY4741.The CEN.PK transcriptome also had a specific and relevant response to methanol that was either absent or less pronounced in the BY4741 strain.This response included up-regulation of genes associated with mitochondrial and peroxisomal metabolism,alcohol and formate dehydrogenation,glutathione metabolism,and the global transcriptional regulator of metabolism MIG3.Over-expression of MIG3 enabled improved growth in the presence of methanol,suggesting that MIG3 is a mediator of the superior CEN.PK strain growth.CEN.PK was therefore identified as a superior strain for the future development of synthetic methylotrophy in S.cerevisiae.

Harnessing genetic variation at regulatory regions to fine-tune traits for climate-resilient crops

Climate change is making it more challenging to meet the food demands of a growing global population.Increased food produc-tion relies on continual crop improvements to generate higher and more stable yields,especially with increasingly unpredictable environments and less arable land.The improvement of traits that promote climate resilience and resource utilization,for example,greater photosynthetic capacity,increased nitrogen use efficiency,and optimized root and shoot architecture,repre-sents a promising avenue for engineering crops to yield more with less(Evans and Lawson,2020).A key challenge for crop engineering is optimizing performance in specific environments.