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Heterologous transporters from anaerobic fungi bolster fluoride tolerance in Saccharomyces cerevisiae.

ABSTRACT: Membrane-embedded transporters are crucial for the stability and performance of microbial production strains. Apart from engineering known transporters derived from model systems, it is equally important to identify transporters from nonconventional organisms that confer advantageous traits for biotechnological applications. Here, we transferred genes encoding fluoride exporter (FEX) proteins from three strains of early-branching anaerobic fungi (Neocallimastigomycota) to Saccharomyces cerevisiae. The heterologous transporters are localized to the plasma membrane and complement a fluoride-sensitive yeast strain that is lacking endogenous fluoride transporters up to 10.24?mM fluoride. Furthermore, we show that fusing an amino-terminal leader sequence to FEX proteins in yeast elevates protein yields, yet inadvertently causes a loss of transporter function. Adaptive laboratory evolution of FEX proteins restores fluoride tolerance of these strains, in one case exceeding the solute tolerance observed in wild type S. cerevisiae; however, the underlying molecular mechanisms and cause for the increased tolerance in the evolved strains remain elusive. Our results suggest that microbial cultures can achieve solvent tolerance through different adaptive trajectories, and the study is a promising step towards the identification, production, and biotechnological application of membrane proteins from nonconventional fungi.

SUBMITTER: Seppala S

PROVIDER: S-EPMC6475669 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Heterologous transporters from anaerobic fungi bolster fluoride tolerance in Saccharomyces cerevisiae.

Seppälä Susanna S Yoo Justin I JI Yur Daniel D O'Malley Michelle A MA

Metabolic engineering communications 20190411

Membrane-embedded transporters are crucial for the stability and performance of microbial production strains. Apart from engineering known transporters derived from model systems, it is equally important to identify transporters from nonconventional organisms that confer advantageous traits for biotechnological applications. Here, we transferred genes encoding fluoride exporter (FEX) proteins from three strains of early-branching anaerobic fungi (Neocallimastigomycota) to Saccharomyces ...[more]

PMID: 31016136

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Project description:When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.ImportanceEthanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Project description:BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.ConclusionsSaccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.

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Heterologous transporters from anaerobic fungi bolster fluoride tolerance in Saccharomyces cerevisiae.

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Heterologous transporters from anaerobic fungi bolster fluoride tolerance in <i>Saccharomyces cerevisiae</i>.

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