Characterization of chromosomal integration sites for heterologous gene expression inSaccharomyces cerevisiae

Yeast ◽  
2009 ◽  
Vol 26 (10) ◽  
pp. 545-551 ◽  
Author(s):  
Dongmei Bai Flagfeldt ◽  
Verena Siewers ◽  
Le Huang ◽  
Jens Nielsen
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Chromosomal Integration ◽  
Integration Sites

scholarly journals Characterization of the Human Artemis Promoter by Heterologous Gene Expression In Vitro and In Vivo

2011 ◽  
Vol 30 (10) ◽  
pp. 751-761 ◽  
Author(s):  
Megan M. Multhaup ◽  
Sweta Gurram ◽  
Kelly M. Podetz-Pedersen ◽  
Andrea D. Karlen ◽  
Debra L. Swanson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum

Extremophiles ◽  
2021 ◽  
Author(s):  
Ilma Fauziah Ma’ruf ◽  
Yuka Sasaki ◽  
Anastasia Kerbs ◽  
Jochen Nießer ◽  
Yu Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Thermoplasma Acidophilum

scholarly journals Targeted and Repetitive Chromosomal Integration Enables High-Level Heterologous Gene Expression in Lactobacillus casei

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Yongping Xin ◽  
Yingli Mu ◽  
Jian Kong ◽  
Tingting Guo
Keyword(s):  
Gene Expression ◽  
Genetic Instability ◽  
Lactobacillus Casei ◽  
Heterologous Gene Expression ◽  
Stable Expression ◽  
Bioactive Molecules ◽  
Heterologous Gene ◽  
Chromosomal Integration ◽  
Content Type ◽  
High Level

ABSTRACT Lactobacillus casei is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in Lb. casei by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with the Cre/loxP site-specific recombination system, with an efficiency of ∼3.7 × 103 CFU/µg DNA. A gfp gene was successfully integrated into six selected chromosomal sites, and the relative fluorescence intensities (RFUs) of the resulting integrants varied up to ∼3.7-fold depending on the integrated site, among which the LCABL_07270 site gfp integration showed the highest RFU. However, integrants with gfp gene(s) integrated into the LCABL_07270 site showed various RFUs, ranging from 993 ± 89 to 7,289 ± 564 and corresponding to 1 to 13.68 ± 1.08 copies of gfp gene integration. Moreover, the integrant with 13.68 ± 1.08 copies of the gfp gene had a more stable RFU after 63 generations compared to that of a plasmid-engineered strain. To investigate the feasibility of this system for bioactive molecules with high expression levels, the fimbrial adhesin gene, faeG, from Escherichia coli was tested and successfully integrated into the LCABL_07270 site with 5.51 ± 0.25 copies, and the integrated faeG achieved stable expression. All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location in Lb. casei. IMPORTANCE Lactic acid bacteria (LAB), including Lactobacillus casei, have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in Lb. casei without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.

Cloning and characterization of two 3-phosphoglycerate kinase genes of Rhizopus niveus and heterologous gene expression using their promoters

1994 ◽  
Vol 25 (6) ◽  
pp. 524-530 ◽  
Author(s):  
Naoki Takaya ◽  
Koji Yanai ◽  
Hiroyuki Horiuchi ◽  
Akinori Ohta ◽  
Masamichi Takagi
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Phosphoglycerate Kinase ◽  
Heterologous Gene ◽  
Rhizopus Niveus

scholarly journals Among Multiple Phosphomannomutase Gene Orthologues, Only One Gene Encodes a Protein with Phosphoglucomutase and Phosphomannomutase Activities in Thermococcus kodakaraensis

2004 ◽  
Vol 186 (18) ◽  
pp. 6070-6076 ◽  
Author(s):  
Naeem Rashid ◽  
Tamotsu Kanai ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Gene Expression ◽  
Recombinant Protein ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Expression And Purification ◽  
Purification And Characterization ◽  
Orthologous Genes ◽  
Hyperthermophilic Archaeon ◽  
In Cells

ABSTRACT Four orthologous genes (TK1108, TK1404, TK1777, and TK2185) that can be annotated as phosphomannomutase (PMM) genes (COG1109) have been identified in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. We previously found that TK1777 actually encodes a phosphopentomutase. In order to determine which of the remaining three orthologues encodes a phosphoglucomutase (PGM), we examined the PGM activity in T. kodakaraensis cells and identified the gene responsible for this activity. Heterologous gene expression and purification and characterization of the recombinant protein indicated that TK1108 encoded a protein with high levels of PGM activity (690 U mg−1), along with high levels of PMM activity (401 U mg−1). Similar analyses of the remaining two orthologues revealed that their protein products exhibited neither PGM nor PMM activity. PGM activity and transcription of TK1108 in T. kodakaraensis were found to be higher in cells grown on starch than in cells grown on pyruvate. Our results clearly indicate that, among the four PMM gene orthologues in T. kodakaraensis, only one gene, TK1108, actually encodes a protein with PGM and PMM activities.

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