Increased activity of a model heterologous protein in Saccharomyces cerevisiae strains with reduced vacuolar proteinases

1993 ◽  
Vol 39 (2) ◽  
Author(s):  
JonathanM. Wingfield ◽  
J.Richard Dickinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Protein ◽  
Increased Activity

scholarly journals Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

2009 ◽  
Vol 75 (8) ◽  
pp. 2320-2325 ◽  
Author(s):  
Derek A. Abbott ◽  
Erwin Suir ◽  
Giang-Huong Duong ◽  
Erik de Hulster ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Reference Strain ◽  
Specific Growth ◽  
Pyruvate Decarboxylase ◽  
Additional Stress ◽  
Batch Cultures ◽  
Native Promoter ◽  
Specific Growth Rates ◽  
Increased Activity

ABSTRACT Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Δ reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h−1 in anaerobic batch cultures containing lactic acid but only 0.16 h−1 in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.

scholarly journals Compartmentalized Reconstitution of Post-squalene Pathway for 7-Dehydrocholesterol Overproduction in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Jing Guo ◽  
Ming-Dong Yao ◽  
Wen-Hai Xiao ◽  
Ying Wang ◽  
Guang-Rong Zhao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vitamin D ◽  
Endoplasmic Reticulum ◽  
Shake Flask ◽  
Metabolic Flux ◽  
Heterologous Protein ◽  
Subcellular Location ◽  
Product Yield ◽  
Lipid Bodies ◽  
Level Production

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D3. Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE_Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE_Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway.

scholarly journals Effective Production of Heterologous Protein Using Saccharomyces cerevisiae PGK Promoter.

1991 ◽  
Vol 17 (3) ◽  
pp. 680-686 ◽  
Author(s):  
Kong-Hua Lin ◽  
Shinji Iijima ◽  
Shih-Yow Huang ◽  
Fumio Hishinuma ◽  
Takeshi Kobayashi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Protein

Increased Activity of Wall Hydrolytic Enzymes May Explain the Phenotype of Saccharomyces cerevisiae Fragile Mutants

1998 ◽  
Vol 37 (6) ◽  
pp. 365-367 ◽  
Author(s):  
José Ruiz-Herrera ◽  
Claudia León-Ramírez ◽  
P. Elizabeth Alvarez ◽  
Pencho Venkov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrolytic Enzymes ◽  
Increased Activity
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