ORCID Profile
0000-0002-9141-3112
Current Organisation
Macquarie University
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Agricultural Molecular Engineering of Nucleic Acids and Proteins | Biochemistry and Cell Biology | Synthetic Biology
Expanding Knowledge in the Chemical Sciences | Environmentally Sustainable Animal Production not elsewhere classified | Expanding Knowledge in the Biological Sciences |
Publisher: Wiley
Date: 24-10-2020
DOI: 10.1002/BAB.1819
Abstract: The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector-based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae P
Publisher: Wiley
Date: 08-04-2013
DOI: 10.1002/JCTB.4068
Publisher: Cold Spring Harbor Laboratory
Date: 04-11-2020
DOI: 10.1101/2020.11.03.367615
Abstract: The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer cell development, the induction of the UPR has a significant impact on recombinant protein production yields in microbial cell factories, including the industrial workhorse Saccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimisation of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage, or lack updated systematic evaluation. To this end we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor, SM1, was only 98 bp in length, had minimal homology to other native yeast sequences and displayed superior sensor characteristics. Using this synthetic minimal UPR sensor, we demonstrate its ability to accurately discriminate between cells expressing different heterologous proteins and at varying production levels. Our sensor is thus a novel high-throughput tool for determining expression/engineering strategies for optimal heterologous protein production.
Publisher: Wiley
Date: 15-09-2017
Abstract: The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.
Publisher: Wiley
Date: 08-10-2017
DOI: 10.1002/BBB.1824
Publisher: Elsevier BV
Date: 02-2013
Publisher: Oxford University Press (OUP)
Date: 05-07-2019
Abstract: Decoding the genetic basis of lignocellulosic inhibitor tolerance in Saccharomyces cerevisiae is crucial for rational engineering of bioethanol strains with enhanced robustness. The genetic ersity of natural strains present an invaluable resource for the exploration of complex traits of industrial importance from a pan-genomic perspective to complement the limited range of specialised, tolerant industrial strains. Natural S. cerevisiae isolates have lately garnered interest as a promising toolbox for engineering novel, genetically encoded tolerance phenotypes into commercial strains. To this end, we investigated the genetic basis for lignocellulosic inhibitor tolerance of natural S. cerevisiae isolates. A total of 12 quantitative trait loci underpinning tolerance were identified by next-generation sequencing linked bulk-segregant analysis of superior interbred pools. Our findings corroborate the current perspective of lignocellulosic inhibitor tolerance as a multigenic, complex trait. Apart from a core set of genetic variants required for inhibitor tolerance, an additional genetic background-specific response was observed. Functional analyses of the identified genetic loci revealed the uncharacterised ORF, YGL176C and the bud-site selection XRN1/BUD13 as potentially beneficial alleles contributing to tolerance to a complex lignocellulosic inhibitor mixture. We present evidence for the consideration of both regulatory and coding sequence variants for strain improvement.
Publisher: Elsevier BV
Date: 2013
Publisher: Oxford University Press (OUP)
Date: 27-10-2018
Abstract: Strains of Saccharomyces cerevisiae with improved tolerance to plant hydrolysates are of utmost importance for the cost-competitive production of value-added chemicals and fuels. However, engineering strategies are constrained by a lack of understanding of the yeast response to complex inhibitor mixtures. Natural S. cerevisiae isolates display niche-specific phenotypic and metabolic ersity, encoded in their DNA, which has evolved to overcome external stresses, utilise available resources and ultimately thrive in their challenging environments. Industrial and laboratory strains, however, lack these adaptations due to domestication. Natural strains can serve as a valuable resource to mitigate engineering constraints by studying the molecular mechanisms involved in phenotypic variance and instruct future industrial strain improvement to lignocellulosic hydrolysates. We, therefore, investigated the proteomic changes between two natural S. cerevisiae isolates when exposed to a lignocellulosic inhibitor mixture. Comparative shotgun proteomics revealed that isolates respond by regulating a similar core set of proteins in response to inhibitor stress. Furthermore, superior tolerance was linked to NAD(P)/H and energy homeostasis, concurrent with inhibitor and reactive oxygen species detoxification processes. We present several candidate proteins within the redox homeostasis and energy management cellular processes as possible targets for future modification and study. Data are available via ProteomeXchange with identifier PXD010868.
Publisher: Wiley
Date: 13-01-2017
Publisher: Elsevier BV
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 03-11-2020
DOI: 10.1186/S13068-020-01823-8
Abstract: For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene ( CEL3A ) from Saccharomycopsis fibuligera and an endoglucanase gene ( CEL5A ) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of in idual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β- d -cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.
Publisher: American Chemical Society (ACS)
Date: 14-06-2021
DOI: 10.1021/ACSSYNBIO.0C00633
Abstract: The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer, the induction of the UPR has a significant impact on the recombinant protein production in eukaryotic cell factories, including the industrial workhorse
Publisher: MDPI AG
Date: 16-07-2018
DOI: 10.3390/FERMENTATION4030054
Abstract: Yeast—especially Saccharomyces cerevisiae—have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast—including S. cerevisiae—to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds.
Publisher: Research Square Platform LLC
Date: 15-04-2020
DOI: 10.21203/RS.3.RS-22334/V1
Abstract: Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene ( CEL3A ) from Saccharomycopsis fibuligera and an endoglucanase gene ( CEL5A ) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of in idual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that BPNPG5 (Megazyme®) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2 respectively (ratio of 1.6:1). Conclusions Here we describe a synthetic biology approach to rapidly optimize gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.
Start Date: 03-2023
End Date: 03-2026
Amount: $423,000.00
Funder: Australian Research Council
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