Biology subjects

Walls, L. E.

Publications and source records attributed to Walls, L. E..

3 recordsLinked to original sources

bioRxiv2023

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol (R) (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 {+/-} 33 mg/L. The best T5-yl Acetate yield obtained was up to 95 {+/-} 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resins treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.

bioengineering↗

bioRxiv2022

Toward sustainable jet fuels: bioconversion of cellulose into isoprenoid biojet candidates using rumen bacteria and non-conventional yeast

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 {+/-} 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 {+/-} 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/500214v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1662a84org.highwire.dtl.DTLVardef@717f66org.highwire.dtl.DTLVardef@1633b36org.highwire.dtl.DTLVardef@1c46c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

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↗