scholarly journals Combination ofERG9Repression and Enzyme Fusion Technology for Improved Production of Amorphadiene inSaccharomyces cerevisiae

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Rama Raju Baadhe ◽  
Naveen Kumar Mekala ◽  
Sreenivasa Rao Parcha ◽  
Yalavarthy Prameela Devi
Keyword(s):  
Gene Fusion ◽  
Mevalonate Pathway ◽  
Artemisia Annua ◽  
Combinatorial Biosynthesis ◽  
Plant Origin ◽  
Fusion Technology ◽  
Precursor Molecule ◽  
Yeast Strains ◽  
Intermediate Metabolite ◽  
Enzyme Fusion

The yeast strain (Saccharomyces cerevisiae) MTCC 3157 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene. Our main objective was to overproduce amorpha 4-11-diene, which is a key precursor molecule of artemisinin (antimalarial drug) produced naturally in plantArtemisia annuathrough mevalonate pathway. Farnesyl diphosphate (FPP) is a common intermediate metabolite of a variety of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols, dolichol and ubiquinone, and so forth. In our studies, FPP converted to amorphadiene (AD) by expressing heterologous amorphadiene synthase (ADS) in yeast. First,ERG9(squalane synthase) promoter of yeast was replaced with repressible methionine (MET3) promoter by using bipartite gene fusion method. Further to overcome the loss of the intermediate FPP through competitive pathways in yeast, fusion protein technology was adopted and farnesyldiphosphate synthase (FPPS) of yeast has been coupled with amorphadiene synthase (ADS) of plant origin (Artemisia annuaL.) where amorphadiene production was improved by 2-fold (11.2 mg/L) and 4-fold (25.02 mg/L) in yeast strains YCF-002 and YCF-005 compared with control strain YCF-AD (5.5 mg/L), respectively.

Synergistic re-channeling of mevalonate pathway for enhanced artemisinin production in transgenic Artemisia annua

Plant Science ◽  
2009 ◽  
Vol 177 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Li-Ling Feng ◽  
Rui-Yi Yang ◽  
Xue-Qin Yang ◽  
Xiao-Mei Zeng ◽  
Wen-Jie Lu ◽  
...  
Keyword(s):  
Mevalonate Pathway ◽  
Artemisia Annua

scholarly journals A genomic approach to gene fusion technology

2001 ◽  
Vol 98 (5) ◽  
pp. 2555-2560 ◽  
Author(s):  
T. K. Van Dyk ◽  
Y. Wei ◽  
M. K. Hanafey ◽  
M. Dolan ◽  
M. J. G. Reeve ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Fusion Technology ◽  
Genomic Approach

scholarly journals Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream Biosynthetic Pathways

2020 ◽  
Author(s):  
Shanhui Xu ◽  
Curtis Chen ◽  
Yanran Li
Keyword(s):  
Plasma Membranes ◽  
Mevalonate Pathway ◽  
Plant Secondary Metabolites ◽  
Structural Diversity ◽  
Ergosterol Biosynthesis ◽  
Engineering Strain ◽  
Growth Deficiency ◽  
Yeast Strains ◽  
Plant Enzymes ◽  
Low Efficiency

AbstractAs essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ~40mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains with decent growth and titer of campesterol (~ 7mg/L), β-sitosterol (~2mg/L), 22-hydroxycampesterol (~1mg/L), and 22-hydroxycampest-4-en-3-one (~4mg/L). This work resolves the technical bottlenecks in phytosterol-derived pathway reconstitution in the backer’s yeast and opens up opportunities for efficient bioproduction and pathway elucidation of this group of phytochemicals.

Insertional gene fusion technology

FEBS Letters ◽  
1999 ◽  
Vol 457 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Nobuhide Doi ◽  
Hiroshi Yanagawa
Keyword(s):  
Gene Fusion ◽  
Fusion Technology

scholarly journals Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

1998 ◽  
Vol 180 (7) ◽  
pp. 1913-1919 ◽  
Author(s):  
Frédérique Ness ◽  
Tilman Achstetter ◽  
Catherine Duport ◽  
Francis Karst ◽  
Roberto Spagnoli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mevalonate Pathway ◽  
Sterol Content ◽  
Wild Type ◽  
Sterol Uptake ◽  
Yeast Strains ◽  
Partial Activity ◽  
The Relationship ◽  
Cytochrome P 450

ABSTRACT The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation inerg auxotrophs.

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