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RNA-seq of strains grown under the control of native galactose regulon or synthetic xylose regulon vs constitutive expression of galactose and xylose metabolic genes

ABSTRACT: In this study, we first assess the role of the GAL regulon in enabling efficient galactose utilization for cell growth by decoupling its regulatory responses from sugar catabolism. We provide evidence that regulon-controlled galactose assimilation is more efficient than constitutive expression of the catabolic genes in supporting fast growth rates to higher cell densities. Next, we assessed whether a regulon could enable more complete and efficient utilization of a nutrient that is non-native to this yeast – xylose. We first adapt the GAL regulon to respond to xylose through directed evolution of Gal3p, enabling coupling of nutrient stimulus with sensing, computation, and regulatory actuation. Next, by using a rational, model-guided approach, we test two different positive feedback signal transduction loop designs for the regulon and demonstrate their individual merits and weaknesses. We also show that implementation of a GAL-type xylose-responsive regulon can regulate multiple genes across the yeast genome and enable more homogeneous population-wide gene expression. By integrating a minimal set of heterologous catabolic genes into the synthetic regulon we demonstrate high cellular growth rates and high final cell densities on xylose as well as better growth in non-inducing carbon sources. Finally, we compare the genome-wide expression profiles of strains grown with regulon assistance and conventionally engineered strains to identify mechanistic reasons that account for the different phenotypes observed. We posit that this study strongly supports the need to re-evaluate how nutrient assimilation systems are currently implemented and introduces a new and unexplored paradigm of adapting a native regulon for efficient non-native sugar assimilation.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE110818 | GEO | 2018/05/30

REPOSITORIES: GEO

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Project description:BACKGROUND: Converting carbon dioxide (CO2) into value-added chemicals using engineered cyanobacteria is a promising strategy to tackle the global warming and energy shortage issues. However, most cyanobacteria are autotrophic and use CO2 as a sole carbon source, which makes it hard to compete with heterotrophic hosts in either growth or productivity. One strategy to overcome this bottleneck is to introduce sugar utilization pathways to enable photomixotrophic growth with CO2 and sugar (e.g., glucose and xylose). Advances in engineering mixotrophic cyanobacteria have been obtained, while a systematic interrogation of these engineered strains is missing. This work aimed to fill the gap at omics level.RESULTS: We first constructed two engineered Synechococcus elongatus YQ2-gal and YQ3-xyl capable of utilizing glucose and xylose, respectively. To investigate the metabolic mechanism, transcriptomic and metabolomic analysis were then performed in the engineered photomixotrophic strains YQ2-gal and YQ3-xyl. Transcriptome and metabolome of wild-type S. elongatus were set as baselines. Increased abundance of metabolites in glycolysis or pentose phosphate pathway indicated that efficient sugar utilization significantly enhanced carbon flux in S. elongatus as expected. However, carbon flux was redirected in strain YQ2-gal as more flowed into fatty acids biosynthesis but less into amino acids. In strain YQ3-xyl, more carbon flux was directed into synthesis of sucrose, glucosamine and acetaldehyde, while less into fatty acids and amino acids. Moreover, photosynthesis and bicarbonate transport could be affected by upregulated genes, while nitrogen transport and assimilation were regulated by less transcript abundance of related genes in strain YQ3-xyl with utilization of xylose.CONCLUSIONS: Our work identified metabolic mechanism in engineered S. elongatus during photomixotrophic growth, where regulations of fatty acids metabolism, photosynthesis, bicarbonate transport, nitrogen assimilation and transport are dependent on different sugar utilization. Since photomixotrophic cyanobacteria is regarded as a promising cell factory for bioproduction, this comprehensive understanding of metabolic mechanism of engineered S. elongatus during photomixotrophic growth would shed light on the engineering of more efficient and controllable bioproduction systems based on this potential chassis.

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RNA-seq of strains grown under the control of native galactose regulon or synthetic xylose regulon vs constitutive expression of galactose and xylose metabolic genes

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