Conversion of Starch to Ethanol in a Recombinant Saccharomyces cerevisiae Strain Expressing Rice α-Amylase from a Novel Pichia pastoris Alcohol Oxidase Promoter

ABSTRACT In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s ) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast.

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Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution

Biotechnology and Bioengineering ◽

10.1002/bit.26585 ◽

2018 ◽

Vol 115 (7) ◽

pp. 1666-1674 ◽

Cited By ~ 18

Author(s):

Javier Viña-Gonzalez ◽

Katarina Elbl ◽

Xavier Ponte ◽

Francisco Valero ◽

Miguel Alcalde

Keyword(s):

Saccharomyces Cerevisiae ◽

Pichia Pastoris ◽

Directed Evolution ◽

Alcohol Oxidase ◽

Functional Expression

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Enhanced Production of α-Galactosyl Epitopes by Metabolically Engineered Pichia pastoris

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5238-5242.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5238-5242 ◽

Cited By ~ 15

Author(s):

Jun Shao ◽

Takahisa Hayashi ◽

Peng George Wang

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Pichia Pastoris ◽

Sucrose Synthase ◽

Large Scale ◽

Alcohol Oxidase ◽

Chromosomal Dna ◽

Transcription Terminator ◽

Enhanced Production

ABSTRACT A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine α-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P. pastoris strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole P. pastoris cells were used to synthesize α-galactosyl (α-Gal) trisaccharide (Galα1,3Galβ1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM α-Gal was accumulated in a 200-ml reaction. The Pichia system described here is simple and flexible. This work demonstrates that recombinant P. pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides.

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Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-005-0010-2 ◽

2005 ◽

Vol 32 (9) ◽

pp. 402-408 ◽

Cited By ~ 6

Author(s):

Yu-Jin Kim ◽

You-Kwan Oh ◽

Whankoo Kang ◽

Eun Yeol Lee ◽

Sunghoon Park

Keyword(s):

Saccharomyces Cerevisiae ◽

Pichia Pastoris ◽

Recombinant Saccharomyces Cerevisiae

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Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽

10.1093/genetics/151.4.1379 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1379-1391

Author(s):

Monique A Johnson ◽

Hans R Waterham ◽

Galyna P Ksheminska ◽

Liubov R Fayura ◽

Joan Lin Cereghino ◽

...

Keyword(s):

Pichia Pastoris ◽

Allyl Alcohol ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Direct Selection ◽

Toxic Exposure ◽

Peroxisome Biogenesis ◽

Yeast Pichia Pastoris ◽

Methylotrophic Yeast Pichia Pastoris ◽

Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

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