Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

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Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0314-z ◽

2013 ◽

Vol 170 (8) ◽

pp. 1807-1814 ◽

Cited By ~ 32

Author(s):

Jang Min Park ◽

Baek-Rock Oh ◽

Jeong-Woo Seo ◽

Won-Kyung Hong ◽

Anna Yu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Simultaneous Saccharification And Fermentation ◽

Efficient Production ◽

Fed Batch ◽

Palm Fruit ◽

Simultaneous Saccharification

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Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2310-2317.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2310-2317 ◽

Cited By ~ 39

Author(s):

Marc W. T. Werten ◽

Frits A. de Wolf

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Pichia Pastoris ◽

Single Gene ◽

Foreign Protein ◽

Heterologous Proteins ◽

Basic Residue ◽

Substrate Specificities ◽

Basic Amino Acids ◽

High Level

ABSTRACT Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae α-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the α-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the α-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

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Enhancing the translational capacity ofE. coliby resolving the codon bias

10.1101/318105 ◽

2018 ◽

Author(s):

Zoltan Lipinszki ◽

Viktor Vernyik ◽

Nora Farago ◽

Tobias Sari ◽

Laszlo G. Puskas ◽

...

Keyword(s):

Ribosomal Rna ◽

Codon Bias ◽

Dynamic Control ◽

Exponential Growth Phase ◽

Cell Physiology ◽

Trna Genes ◽

Efficient Production ◽

Heterologous Proteins ◽

Codon Composition ◽

Trna Species

AbstractEscherichia coliis a well-established, and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation.Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a ribosomal RNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in exponential growth phase.Results from expression assays of a panel of heterologous proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.ImportanceCodon composition not fitting the codon bias of the expression host frequently compromises the efficient production of foreign proteins inE. coli. Various attempts to remedy the problem (codon optimization by gene synthesis, expression of rare tRNAs from a plasmid) proved to be unsatisfying. Our new approach, adjusting the tRNA pool by co-expressing extra copies of rare tRNA genes with ribosomal RNA genes, does not affect normal cell physiology, and seems to be a superior solution in terms of simplicity, cost, and yield.

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Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae

Methods in Enzymology - Cell Cycle Control ◽

10.1016/s0076-6879(97)83025-x ◽

1997 ◽

pp. 313-322 ◽

Cited By ~ 21

Author(s):

Volker Rönicke ◽

Wolff Graulich ◽

Dominik Mumberg ◽

Rolf Müller ◽

Martin Funk

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Proteins

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Efficient production of levan using a recombinant yeast Saccharomyces cerevisiae hypersecreting a bacterial levansucrase

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-019-02206-1 ◽

2019 ◽

Vol 46 (11) ◽

pp. 1611-1620 ◽

Cited By ~ 2

Author(s):

Hyunjun Ko ◽

Jung-Hoon Bae ◽

Bong Hyun Sung ◽

Mi-Jin Kim ◽

Chul-Ho Kim ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Efficient Production ◽

Recombinant Yeast ◽

Yeast Saccharomyces Cerevisiae

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Molecular Basis for Transmission Barrier and Interference between Closely Related Prion Proteins in Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m110.183889 ◽

2011 ◽

Vol 286 (18) ◽

pp. 15773-15780 ◽

Cited By ~ 22

Author(s):

Evgenia G. Afanasieva ◽

Vitaly V. Kushnirov ◽

Mick F. Tuite ◽

Michael D. Ter-Avanesyan

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Basis ◽

Prion Proteins ◽

Heterologous Proteins ◽

Highly Sensitive ◽

Prion Transmission ◽

Patterns Of Interaction ◽

Interspecies Barrier

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI+] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI+] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI+] cells often resulted in frequent loss of the native [PSI+] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI+] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI+] variant. The observed loss of S. cerevisiae [PSI+] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.

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