scholarly journals Cloning of the gene for a carbohydrate oxidase from Lactuca sativa in the yeasts Saccharomyces cerevisiae and Pichia pastoris

2015 ◽  
Vol 69 (6) ◽  
pp. 689-701
Author(s):  
Vojin Tadic ◽  
Ana Balaz ◽  
Marija Petric ◽  
Snezana Milosevic ◽  
Nevena Zelenovic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Lactuca Sativa ◽  
Gene Sequence ◽  
Extracellular Expression ◽  
Empty Vector ◽  
Two Samples ◽  
Foreign Genes ◽  
Carbohydrate Oxidase

We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5? strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme further studies on purification and refolding of expressed protein are necessary.

scholarly journals ScatLay: utilizing transcriptome-wide noise for identifying and visualizing differentially expressed genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Thuy Tien Bui ◽  
Daniel Lee ◽  
Kumar Selvarajoo
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Mus Musculus ◽  
Embryonic Stem ◽  
Cell Types ◽  
Objective Method ◽  
Time Points ◽  
Two Samples

Abstract Differential expressed (DE) genes analysis is valuable for understanding comparative transcriptomics between cells, conditions or time evolution. However, the predominant way of identifying DE genes is to use arbitrary threshold fold or expression changes as cutoff. Here, we developed a more objective method, Scatter Overlay or ScatLay, to extract and graphically visualize DE genes across any two samples by utilizing their pair-wise scatter or transcriptome-wide noise, while factoring replicate variabilities. We tested ScatLay for 3 cell types: between time points for Escherichia coli aerobiosis and Saccharomyces cerevisiae hypoxia, and between untreated and Etomoxir treated Mus Musculus embryonic stem cell. As a result, we obtain 1194, 2061 and 2932 DE genes, respectively. Next, we compared these data with two widely used current approaches (DESeq2 and NOISeq) with typical twofold expression changes threshold, and show that ScatLay reveals significantly larger number of DE genes. Hence, our method provides a wider coverage of DE genes, and will likely pave way for finding more novel regulatory genes in future works.

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

2003 ◽  
Vol 69 (9) ◽  
pp. 5238-5242 ◽  
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.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

2019 ◽  
Author(s):  
Thomas Siemon ◽  
Zhangqian Wang ◽  
Guangkai Bian ◽  
Tobias Seitz ◽  
Ziling Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Chemical Synthesis ◽  
Building Block ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Economical Method ◽  
Englerin A ◽  
Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

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