Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae

Abstract

BackgroundSesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for α-santalene production.ResultsA multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall α-santalene yield of a 0.0052 Cmmol (Cmmol glucose)-1 corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final α-santalene productivity of 0.036 Cmmol (g biomass)-1 h-1.ConclusionsThe results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene α-santalene. This represents a starting point toward the construction of a yeast “sesquiterpene factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…4). In contrast to previous transcriptionally-mediated approaches to regulate squalene synthase (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014), the destabilization strategy does not require either addition of repressors which may increase the cost of the bioprocess (in the case of repressible promoters), or appropriately repressing fermentation conditions (in the case of glucose-responsive promoter). Destabilisation of squalene synthase does not inhibit the maximum specific growth rate.…”

“…For metabolic engineering applications, transcription/RNA-level engineering has been used to regulate the flux-competing nodes, e.g., selecting a weaker promoter (Scalcinati et al, 2012a) or applying RNA interference (Williams et al, 2015a). To direct more FPP toward sesquiterpene production, transcription of the native squalene synthase gene (ERG9) is commonly decreased by replacing the native promoter with a weaker promoter or a promoter that can be specifically down-regulated (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014). Here we investigated a novel alternative strategy to regulate the level of Erg9p: a protein degradation mechanism to decrease protein half-life.…”

“…Consequently, microbial fermentation from sugars using microbial cell factories is being pursued as a competitive approach for their bulk production (Ajikumar et al, 2010;Scalcinati et al, 2012b;Shiba et al, 2007;Vickers et al, 2015a).…”

“…Conditional down-regulation of ERG9 at the transcriptional level has therefore been used to constrain squalene/sterol synthesis and increase heterogeneous sesquiterpene production. This has been achieved by replacing the native ERG9 promoter on the genome with various repressible promoters: MET3 (repressed upon addition of methionine to the medium), CRT3 (repressed on highconcentration copper) or HXT1 (induced by high glucose and reduced under low glucose; transcription decreases after the exponential phase of batch cultivation) (Asadollahi et al, 2008;Paddon et al, 2013;Scalcinati et al, 2012a;Scalcinati et al, 2012b;Xie et al, 2014). However, these strategies require strict fermentation conditions and/or addition of repressors to achieve regulation, conditions that can increase the complexity and/or cost in industrial processes.…”

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

“…Hence, several antioxidants have been used to reduce oxidative stress or damage in human body, animal models and cell cultures (Turkez 2011;Dirican et al 2012;Jain et al 2012;Sumathi et al 2012;Farah et al 2013;Sozio et al 2013;Cacciatore et al 2012). Sesquiterpenes, one of the most common terpenes, are a class of natural products with a diverse range of attractive industrial properties (Scalcinati et al 2012). They contain three isoprene units, which is fifteen carbons and twenty-four hydrogens per molecule (C 15 H 24 ).…”