Dataset Information

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate.

ABSTRACT:

Background

Baker's yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity.

Results

Here, we employ CRISPR interference in S. cerevisiae to identify genes modulating fermentative growth in plant hydrolysate and in presence of lignocellulosic toxins. We find that at least one-third of hydrolysate-associated gene functions are explained by effects of known toxic compounds, such as the decreased growth of YAP1 or HAA1, or increased growth of DOT6 knock-down strains in hydrolysate.

Conclusion

Our study confirms previously known genetic elements and uncovers new targets towards designing more robust yeast strains for the utilization of lignocellulose hydrolysate as sustainable feedstock, and, more broadly, paves the way for applying CRISPRi screens to improve industrial fermentation processes.

SUBMITTER: Gutmann F

PROVIDER: S-EPMC7874482 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate.

Gutmann Friederike F Jann Cosimo C Pereira Filipa F Johansson Andreas A Steinmetz Lars M LM Patil Kiran R KR

Biotechnology for biofuels 20210210 1

<h4>Background</h4>Baker's yeast is a widely used eukaryotic cell factory, producing a diverse range of compounds including biofuels and fine chemicals. The use of lignocellulose as feedstock offers the opportunity to run these processes in an environmentally sustainable way. However, the required hydrolysis pretreatment of lignocellulosic material releases toxic compounds that hamper yeast growth and consequently productivity.<h4>Results</h4>Here, we employ CRISPR interference in S. cerevisiae ...[more]

PMID: 33568224

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Project description:Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

Project description:BackgroundMicrobial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH).ResultsTwenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains.ConclusionsThere is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose.

Dataset Information

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate.

Background

Results

Conclusion

Publications

CRISPRi screens reveal genes modulating yeast growth in lignocellulose hydrolysate.

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