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Metabolic engineering of Taxol biosynthesis in yeast Saccharomyces cerevisiae using a yeast artificial chromosome (YAC)

David, Anand (2013) MOBN15 20112
Degree Projects in Molecular Biology
Abstract
Enhancing stability of YAC in S. cerevisiae

The aim of the study was to develop S. cerevisiae strain that can maintain yeast artificial chromosome (YAC) for prolonged period. The enzymes for taxadiene biosynthesis were cloned on YAC and introduced into engineered strain. Ultimately heterologous pathway was superimposed on native ergosterol pathway.

The strain development was achieved by targeting ergosterol, which maintains cell viability. The squalene synthase (erg9) is crucial for ergosterol synthesis in S. cerevisiae. The expression of erg9 was controlled by galactose-inducible promoter. Thus erg9 was available only when cells were grown on growth medium with galactose (YPG). These cells were not viable on medium with glucose... (More)
Enhancing stability of YAC in S. cerevisiae

The aim of the study was to develop S. cerevisiae strain that can maintain yeast artificial chromosome (YAC) for prolonged period. The enzymes for taxadiene biosynthesis were cloned on YAC and introduced into engineered strain. Ultimately heterologous pathway was superimposed on native ergosterol pathway.

The strain development was achieved by targeting ergosterol, which maintains cell viability. The squalene synthase (erg9) is crucial for ergosterol synthesis in S. cerevisiae. The expression of erg9 was controlled by galactose-inducible promoter. Thus erg9 was available only when cells were grown on growth medium with galactose (YPG). These cells were not viable on medium with glucose (YPD) due to deregulated ergosterol pathway. The (YAC) cloned with C. albicans erg9 (C.a.-erg9) was used to complement function of native erg9. Consequently the cells were forced to stably maintain YAC for survival on YPD.

Basic building blocks for diterpenoid (Taxol) biosynthesis are provided by enzymes in upstream of ergosterol pathway in S. cerevisiae. These YAC construct also contained genes encoding enzymes for taxadiene biosynthesis from different species. As a result heterologous taxadiene pathway was superimposed on endogenous ergosterol pathway. The transcription, translation of each target gene and product formation was monitored by different techniques to study the engineered strain.

Improving taxol production in transgenic S. cerevisiae
The results proved that linear YAC with functional telomeres were more stable than circular version. Linearization did not interfere with transcription of transgene on YAC. Instead transcription was observed to be solely dependent on promoter-terminator combination. The basal level of C.a.-erg9 expression was found to complement function of native erg9. The results also showed the presence of an interesting mechanism in S. cerevisiae to down regulate transcription of endogenous erg9 in presence of C.a.-erg9.

The transgenes for taxadiene biosynthesis were expressed more prominently in YPD medium, indicating stable maintenance of YAC. The study also revealed expression of p450 reductase which was important for converting taxadiene to taxol. The codon optimization of 2 genes encoding enzymes for essential building blocks results in poor translation. Consequently taxadiene was not detected in engineered strain.

The stable maintenance of YAC has been successfully achieved in this study. The method for improving taxol producing strain is also suggested. The use of such engineered strain for fermentation would reduce manufacturing cost of taxol, which is a highly demanded anti-cancer drug. An array of valuable terpenoids can also be produced by including specific enzymes on YAC.


Advisor: Stefan Jennewein, (Fraunhofer IME)
Master´s Degree Project: 45 credits in Cell and Molecular Biology, 2013
Department of Biology., Lund University (Less)
Please use this url to cite or link to this publication:
author
David, Anand
supervisor
organization
course
MOBN15 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3812527
date added to LUP
2013-06-13 10:54:29
date last changed
2013-06-13 10:54:29
@misc{3812527,
  abstract     = {Enhancing stability of YAC in S. cerevisiae 

The aim of the study was to develop S. cerevisiae strain that can maintain yeast artificial chromosome (YAC) for prolonged period. The enzymes for taxadiene biosynthesis were cloned on YAC and introduced into engineered strain. Ultimately heterologous pathway was superimposed on native ergosterol pathway. 

The strain development was achieved by targeting ergosterol, which maintains cell viability. The squalene synthase (erg9) is crucial for ergosterol synthesis in S. cerevisiae. The expression of erg9 was controlled by galactose-inducible promoter. Thus erg9 was available only when cells were grown on growth medium with galactose (YPG). These cells were not viable on medium with glucose (YPD) due to deregulated ergosterol pathway. The (YAC) cloned with C. albicans erg9 (C.a.-erg9) was used to complement function of native erg9. Consequently the cells were forced to stably maintain YAC for survival on YPD. 

Basic building blocks for diterpenoid (Taxol) biosynthesis are provided by enzymes in upstream of ergosterol pathway in S. cerevisiae. These YAC construct also contained genes encoding enzymes for taxadiene biosynthesis from different species. As a result heterologous taxadiene pathway was superimposed on endogenous ergosterol pathway. The transcription, translation of each target gene and product formation was monitored by different techniques to study the engineered strain.
 
Improving taxol production in transgenic S. cerevisiae 
The results proved that linear YAC with functional telomeres were more stable than circular version. Linearization did not interfere with transcription of transgene on YAC. Instead transcription was observed to be solely dependent on promoter-terminator combination. The basal level of C.a.-erg9 expression was found to complement function of native erg9. The results also showed the presence of an interesting mechanism in S. cerevisiae to down regulate transcription of endogenous erg9 in presence of C.a.-erg9.

The transgenes for taxadiene biosynthesis were expressed more prominently in YPD medium, indicating stable maintenance of YAC. The study also revealed expression of p450 reductase which was important for converting taxadiene to taxol. The codon optimization of 2 genes encoding enzymes for essential building blocks results in poor translation. Consequently taxadiene was not detected in engineered strain. 

The stable maintenance of YAC has been successfully achieved in this study. The method for improving taxol producing strain is also suggested. The use of such engineered strain for fermentation would reduce manufacturing cost of taxol, which is a highly demanded anti-cancer drug. An array of valuable terpenoids can also be produced by including specific enzymes on YAC. 


Advisor: Stefan Jennewein, (Fraunhofer IME)
Master´s Degree Project: 45 credits in Cell and Molecular Biology, 2013
Department of Biology., Lund University},
  author       = {David, Anand},
  language     = {eng},
  note         = {Student Paper},
  title        = {Metabolic engineering of Taxol biosynthesis in yeast Saccharomyces cerevisiae using a yeast artificial chromosome (YAC)},
  year         = {2013},
}