Towards a platform organism for terpenoid production – in silico analysis of metabolic networks of potential hosts and in vivo validation

Terpenoids are mostly plant derived compounds with industrial and medicinal applications. These compounds can be provided via biotechnological production as environmental friendly alternative to chemical synthesis or plant extraction. However, productivities from microbial hosts require improvement in order to be economically competitive. Therefore, microbial hosts are compared and new metabolic engineering targets aimed at increasing terpenoid production are investigated by stoichiometric metabolic network analysis and selected strategies are validated in vivo. The in silico analysis of the metabolic networks of Escherichia coli and Saccharomyces cerevisiae (yeast) led to several promising metabolic engineering targets with potential to increase the theoretical maximum terpenoid yield. Production of the sesquiterpenoid patchoulol in yeast was chosen for the in vivo validation study. A two-phase cultivation method for terpenoid capture with dodecane overlay was established and the produced spectrum of sesquiterpenoids was determined. Precursor metabolite flux towards the desired product was increased via overexpression of a truncated HMG-CoA reductase and fusion of FPP synthase with patchoulol synthase. Two in silico identified strategies were implemented: (i) disruption of α-ketoglutarate dehydrogenase gene redirected the metabolic flux as predicted, however, the intermediate acetate was produced in high amounts instead of the desired product; (ii) expression of ATP-citrate lyase from Arabidopsis did not increase terpenoid production due to insufficient in vivo activity. The findings of this thesis contribute to an increased knowledge about enhancing terpenoid production in both E. coli and S. cerevisiae, as well as metabolic behavior of yeast. The in silico stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. This study highlights that kinetics, regulation and cultivation conditions may interfere with predictions, resulting in poor in vivo performance. These findings promote developments of metabolic modelling to increase the predictive power and accelerate microbial strain improvement

Fermentation and purification strategies for the production of betulinic acid and its lupane-type precursors in Saccharomyces cerevisiae

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182mg/L, and total triterpenoid concentrations of 854mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering

Genome-scale modeling of yeast: chronology, applications and critical perspectives

Over the last 15 years, several genome-scale metabolic models (GSMMs) were developed for different yeast species, aiding both the elucidation of new biological processes and the shift toward a bio-based economy, through the design of in silico inspired cell factories. Here, an historical perspective of the GSMMs built over time for several yeast species is presented and the main inheritance patterns among the metabolic reconstructions are highlighted. We additionally provide a critical perspective on the overall genome-scale modeling procedure, underlining incomplete model validation and evaluation approaches and the quest for the integration of regulatory and kinetic information into yeast GSMMs. A summary of experimentally validated model-based metabolic engineering applications of yeast species is further emphasized, while the main challenges and future perspectives for the field are finally addressedThis work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. grant (PD/BD/52336/2013), of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01–0145FEDER-006684) and also in the context of the EU-funded initiative ERA-NET for Industrial Biotechnology (ERA-IB-2/0003/2013), in addition to the BioTecNorte operation (NORTE-01–0145FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Advances in Pathway Engineering for Natural Product Biosynthesis

Biocatalysts provide an efficient, inexpensive and environmentally friendly alternative to traditional organic synthesis, especially for compounds with complex stereochemistries. The past decade has seen a significant rise in the use of biocatalysts for the synthesis of compounds in an industrial setting; however, the incorporation of single enzymatically catalysed steps into organic synthesis schemes can be problematic. The emerging field of synthetic biology has sparked interest in the development of whole-cell factories that can convert simple, common metabolites into complex, high-value molecules with a range of applications such as pharmaceuticals and biofuels. This Review summarises conventional methods and recent advances in metabolic engineering of pathways in microorganisms for the synthesis of natural products

In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo.Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production.Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to meet the required activities for an increased product formation in vivo

Sesquiterpenoid spectrum of patchoulol synthase produced by yeast.

<p>A representative GC/FID analysis of the patchoulol synthase products in the organic phase of a yeast two-phase cultivation heterologously expressing patchoulol synthase is shown. 22 Peaks with nominal masses of 222 (sesquiterpenoids with hydroxy-group) and 204 (sesquiterpenoids without hydroxy-group) were detected. The peak marked with an asterisk (*) was identified as patchoulol.</p

Effect of ATP-citrate lyase (<i>ACL</i>) overexpression on terpenoid production.

<p>Terpenoid formation, <i>i</i>.<i>e</i>. (a) ergosterol content, (b) squalene content, (c) sesquiterpenoid titer and (d) sesquiterpenoid yield on biomass of yeast strains carrying pSP-FPt and pSP-FPt-ACL during glucose-phase (glc-phase) and ethanol-phase at four and six days (4 d, 6 d). Shown are mean values and standard deviations of three experiments.</p

Western blot analysis of yeast proteins.

<p>A representative western blot of protein from yeast strain carrying pSP-FPt-ACL (ACL) and pSP-FPt as control (C) is shown. The protein ladder is shown on the left (M). The predicted position of AclA-1-T2A is indicated with an arrow.</p