Session 1

Fischer et al. (2011), Metabolic Engineering of Monoterpene Synthesis in Yeast, Biotechnology and Bioengineering.

Key findings:
Wild S. cerevisiae strains are – with a few exceptions for winemaking strains up to 5 µg/L – not able to produce monoterpenes. Unlike plants microorganisms usually do not carry a specific geranyl diphosphate (GPP) synthetase.

In yeast one enzyme catalyzes the GPP and farnesyl diphosphate (FPP), the farnesyl diphosphate synthase. Thus the catalytic steps cannot be easily separated. FPP is the precursor for several essential yeast metabolites such as ergosterol, ubiquinone, dolichol and others, whereas GPP has no specific role other than to be a precursor for FPP. The GPP remains bound to the catalytic site and is transformed to FPP. The Gene for FPPS is essential for yeast and disruption is lethal. This leaves little room for technological improvement.
A mutant was isolated with a 14-fold lower affinity of FFPS. This was due to a single nucleotide change in the erg20 gene resulting in a K[197] -> E substitution. This site is usually highly conserved amongst FPPS of different origins. A multialignment made with 200 available sequences showed no divergence in this site. This mutant was studied and showed several unusual properties such as excreting the terpenols geraniol and linalool. Also a correlation between the FFPS activity and the produced amount of ergosterol and cell growth could be shown.
Through this observation knowledge was gained that the tight binding of geraniol could be lowered led to the isolation of mutant that secreted geraniol. Former experiments could increase the production of geraniol to 1 mg/L by the heterologous expression of a geraniol synthase(GES) from basilic. Now a set of mutations at the K197 position was generated to assess the potentially differing GPPS and FPPS activities, and to assess whether their monoterpenol production would differ upon expressing of a heterologous monoterpene synthase. This was combined with the evaluation of the effect of the production on cell viability and gross metabolism.

Physiological behavior
Some of the mutations could not be generated. The mutation resulted in one strain with no significant change in doubling time and 16 strains with an increase in doubling time compared to K197K. No strain had faster doubling time. An increasing of the chain length of the AA resulted in increased doubling times. However some strains also showed little effect although the AA had different physiochemical properties. No simple correlation could be found between doubling time and a single property
GES expression increased the doubling time significant for six strains. The change was not significant for 11 strains and one strain showed a significant faster doubling time after GES expression.
The cells exhibited a great variety of sterol content. The mutation resulted in 12 strains with significant higher sterol contend and five strains with no significant change. No strain had significant lower sterol content. Basic AA gave the lowest sterol content, high contents were observed with aliphatic, aromatic, sulfur and acidic AA. Mixed response was observed with aliphatic hydroxyl amino acids.
GES expression had mostly no significant affect on sterol synthesis.
Strains with very high doubling times (> 9,25h) also had significant higher sterol content.

Monoterpene Content
The Yeast cells exhibited a great variance of monoterpenol production, with geraniol and linalool as main products in similar quantities. The mutation resulted in 10 strains with significant higher terpenol production than the original K197K and 7 strains with no significant change.
GES expression could enhance the monoterpenol production 10-20 fold and resulted in a more contrasted profile variation between yeast with high and low terpenol content. A linear relationship was observed between basic production and GES expression for high and intermediate producers.
The expression of GES had no effect on the production of linalool but a great impact on citronellol content.
Terpenol production could be observed for aliphatic, sulphydryl, aliphatic hydroxyl and acidic AA.
The results also indicated that geraniol synthesis through GES is linked to GPP availability.

Outcome of the experiments
FPPS is essential for S. cerevisiae so lethality of K197 mutations could be assumed, however only two mutated strains showed no sufficient FFP synthesis.
The general fitness of the mutated strains was assessed, however the highest fitness could be observed with the native FPPS enzyme. Almost any modification had a strong effect on the doubling time of the strains.
The FFPS influences the amount of synthesized ergosterol by making the needed precursor available. But previous studies showed only a slight change in ergosterol when the FFPS activity changed. This on the other side means that big changes in the ergosterol level indicate big changes in the FFPS activity. In this study variations in the ergosterol level up to 50-60% could be detected. Also a correlation of sterol content and growth rate for small growth rates could be found. The dependence is however unclear.
The Modeling revealed the toleration of changes of physical behavior between different AA at the mutated site. The results however don’t explain why different AA are not tolerated or similar AA behave so differently.
Leaky mutant strains with heterologous GES expression were able to produce terpenols. The study revealed the most suited mutations for this task. Monoterpene production however remains dependant from non GES expressing GPP production.
GES expression enhances the ratio of geraniol to linalool. The formation of linalool and citronellol could be linked to endogenous yeast enzymes or chemical instability.
Geraniol is catalyzed from GPP by GES but is also derived by hydrolysis of GPP. Linalool derives mainly from GPP but not from geraniol.

Relevance of the article:
The study showed the possibility to enhance the monoterpene production in S. cerevisiae through a combined approach of affecting existing metabolic pathways of the yeast by mutation and introducing new pathways by heterologous protein expression. The production of monoterpenes could be enhanced by 10 fold.
The study also showed that an increase of the doubling time by 20% is enough for the selection of one specific form of an enzyme, as 200 examined forms of different FPPS all display the same AA at the observed site.
With some mutated AA an increased doubling time could be observed alongside with little monoterpene production with additional GES expression. This could suggest a different role of GPP in the yeast metabolism.

Rating (‘Exceptional – a must read paper’, ‘Very good – worth spending the time’, ‘Good – only if you have time to spare’).
Very good – worth spending the time

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