Protease-Deficient<i>Saccharomyces</i><i>cerevisiae</i>Strains for the Synthesis of Human-Compatible Glycoproteins

Tomimoto, Kazuya; Fujita, Yuka; Iwaki, Toru; Chiba, Yasunori; Jigami, Yoshifumi; Nakayama, Kenichi; Nakajima, Yoshihiro; Abe, Hiroko

doi:10.1271/bbb.130588

Cited by 29 publications

(17 citation statements)

References 31 publications

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“…Vacuolar proteases often degrade heterologous proteins expressed in S. cerevisiae. The proteinase gene PEP4 is among the major functional proteases, and disruption of PEP4 is able to increase heterologous protein production [ 27 ]. Considering that disruption of PEP4 may reduce strain biomass and robustness, we replaced the promoter of PEP4 using the inducible GAL1 promoter in the strain JZH-InuMKC, resulting in the strain JZH-InuMKCP.…”

Section: Resultsmentioning

confidence: 99%

Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing

Wang

2016

Biotechnol Biofuels

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BackgroundThe yeast Saccharomyces cerevisiae is an important eukaryotic workhorse in traditional and modern biotechnology. At present, only a few S. cerevisiae strains have been extensively used as engineering hosts. Recently, an astonishing genotypic and phenotypic diversity of S. cerevisiae was disclosed in natural populations. We suppose that some natural strains can be recruited as superior host candidates in bioengineering. This study engineered a natural S. cerevisiae strain with advantages in inulin utilization to produce ethanol from inulin resources by consolidated bioprocess. Rational engineering strategies were employed, including secretive co-expression of heterologous exo- and endo-inulinases, repression of a protease, and switch between haploid and diploid strains.ResultsResults from co-expressing endo- and exo-inulinase genes showed that the extracellular inulinase activity increased 20 to 30-fold in engineered S. cerevisiae strains. Repression of the protease PEP4 influenced cell physiology in late stationary phase. Comparison between haploid and diploid engineered strains indicated that diploid strains were superior to haploid strains in ethanol production albeit not in production and secretion of inulinases. Ethanol fermentation from both inulin and Jerusalem artichoke tuber powder was dramatically improved in most engineered strains. Ethanol yield achieved in the ultimate diploid strain JZD-InuMKCP was close to the theoretical maximum. Productivity achieved in the strain JZD-InuMKCP reached to 2.44 and 3.13 g/L/h in fermentation from 200 g/L inulin and 250 g/L raw Jerusalem artichoke tuber powder, respectively. To our knowledge, these are the highest productivities reported up to now in ethanol fermentation from inulin resources.ConclusionsAlthough model S. cerevisiae strains are preferentially used as hosts in bioengineering, some natural strains do have specific excellent properties. This study successfully engineered a natural S. cerevisiae strain for efficient ethanol production from inulin resources by consolidated bioprocess, which indicated the feasibility of natural strains used as bioengineering hosts. This study also presented different properties in enzyme secretion and ethanol fermentation between haploid and diploid engineering strains. These findings provided guidelines for host selection in bioengineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0511-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing

Wang

2016

Biotechnol Biofuels

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“…These results, together with a recent report on reorganization of carbon metabolism for optimized lipid yield (31), indicate the engineering potential of redirecting cellular resources toward protein synthesis and secretion. Our screens also emphasize the importance of protein modification and turnover processes in strain improvement for optimized protein production, which have been demonstrated by disruption of glycosylation (32,33), protein sorting (34), and degradation (16,35). A beneficial effect from down-regulation of chaperone SSA1, unlike that from overexpression of SSA4 in Pichia pastoris (36), indirectly stressed SSA1's functional specificity in the recognition of misfolded protein for ER-associated degradation (ERAD) (37) and destabilization of correctly folded multimeric protein (38).…”

Section: Discussionmentioning

confidence: 85%

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

Wang

Björk

Huang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

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“…developed in other yeast species, including H. polymorpha and P. pastoris, demonstrating that aspartyl proteases localized at the cell surface are principally responsible for aberrant proteolytic cleavage of secretory recombinant proteins (Sohn et al, 2010;Wu et al, 2013). In addition, reducing major intracellular protease activities by the disruption of vacuolar protease genes, such as PEP4 and PRB1, also enhanced the secretory production of human interferon-b by c. 10-fold in S. cerevisiae (Tomimoto et al, 2013).…”

Section: Host Strain Engineering For Secretory Production Of Recombinmentioning

confidence: 98%

Yeast synthetic biology for the production of recombinant therapeutic proteins

2014

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The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.

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Protease-DeficientSaccharomycescerevisiaeStrains for the Synthesis of Human-Compatible Glycoproteins

Cited by 29 publications

References 31 publications

Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing

Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

Yeast synthetic biology for the production of recombinant therapeutic proteins

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