Biology subjects

Nowrouzi, B.

Publications and source records attributed to Nowrouzi, B..

3 recordsLinked to original sources

bioRxiv2023

Rewiring Saccharomyces cerevisiae metabolism for optimised Taxol(R) precursors production

Saccharomyces cerevisiae has been recognised as a convenient host for the production of early precursors to the Taxol(R) anticancer drug. Recent studies have highlighted the harmful impact of oxidative stress as a result of the activity of Taxol(R) first cytochrome P450-reductase enzymes (Taxus spp. CYP725A4-POR). Here, we evolved a new oxidative stress-tolerant yeast strain on galactose, which led to a three-fold higher titre of the CYP725A4 enzyme substrate, taxadiene. We comprehensively analysed the performance of the evolved and parent strain in galactose-limited chemostat cultures before and during oxidative stress induction. Integrating the transcriptomics and metabolite profiling data in an enzyme-constrained genome scale model enabled a more accurate prediction of changes that occurred to biological pathways as a response to/consequence of evolution and oxidative stress. The analyses showed a better performance of the evolved strain with improved respiration and reduced overflow metabolites production. The strain was robust to re-introduction of the oxidative stress, potentially due to the cross-protection mechanism, which contributed to likely better heme, flavin and NADPH availability for an optimal expression of CYP725A4 and POR in yeast. The increased level of taxadiene production has potentially occurred due to the antioxidant properties of taxadiene or as a mechanism to overcome the toxicity of geranylgeranyl diphosphate, the precursor to taxadiene synthase. HighlightsO_LIThe antioxidant properties of taxadiene promotes its production in Saccharomyces cerevisiae C_LIO_LIS. cerevisiae ALE on H2O2 and galactose regulates Flavin, iron and NADPH metabolism as well as carbon and protein recycling pathways through cross-protection and anticipation mechanisms C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/543533v1_fig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1758c64org.highwire.dtl.DTLVardef@543681org.highwire.dtl.DTLVardef@c67339org.highwire.dtl.DTLVardef@b6c56e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract of the study. Figure was created with BioRender.com. C_FIG

synthetic biology↗

bioRxiv2021

In situ solid-liquid extraction enhances recovery of taxadiene from engineered S. cerevisiae cell factories

A novel in situ solid phase adsorption strategy was investigated for enhanced recovery of taxadiene, a precursor to the blockbuster anticancer drug, paclitaxel, from engineered Saccharomyces cerevisiae. A synthetic adsorbent resin (HP-20) was employed to capture taxadiene across a range of cultivation scales. Cultivations from 12 % (w/v) resin concentration resulted in bead fragmentation which were found to be detrimental to cellular growth. After cultivation, the use of acetone for desorption captured intracellular and secreted taxadiene, achieving an integration of the bioprocess. Implementation of the proposed method at microscale (2 mL) and benchtop bioreactor scale (250 mL) resulted in 1.9-fold and 1.4-fold increments in taxadiene titer, respectively, compared to the extraction method using a dodecane overlay. Taxadiene was found to be distributed between resin beads and biomass in a ratio of 50 %. Finally, a maximum taxadiene titer of 76 {+/-} 19 mg/L was achieved in the benchtop bioreactor cultivations.

bioengineering↗

bioRxiv2020

Enhanced production of taxadiene in Saccharomyces cerevisiae

Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask scale. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Apertor Labs, Ansa Biotechnologies, and Berkeley Brewing Sciences.AbbreviationsBTS1Geranylgeranyl diphosphate synthasecrtEGeranylgeranyl diphosphate synthaseDMAPPDimethylallyl pyrophosphateERG8Phosphomevalonate kinaseERG9Farnesyl-diphosphate farnesyl transferase (squalene synthase)ERG103-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG12Mevalonate kinaseERG133-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG19Mevalonate pyrophosphate decarboxylaseERG20Farnesyl pyrophosphate synthetaseFPPFarnesyl diphosphateGGOH(E,E,E)-geranylgeraniolGGPP(E,E,E)-Geranylgeranyl diphosphateGPPGeranyl diphosphateHMG13-hydroxy-3-methylglutaryl-coenzyme A reductase 1HMG23-hydroxy-3-methylglutaryl-coenzyme A reductase 2IDIIsopentenyl-diphosphate delta-isomeraseIPPIsopentenyl pyrophosphateMBPMaltose binding proteinmvaEAcetyl-CoA acetyltransferasemvaSHydroxymethylglutaryl-CoA synthaseMVA pathwayMevalonate pathwayTASYTaxadiene synthase.View Full Text

synthetic biology↗