ORCID Profile
0000-0002-0792-050X
Current Organisation
CSIRO
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Biochemistry and Cell Biology | Synthetic Biology | Microbial Genetics | Industrial Microbiology (incl. Biofeedstocks) | Systems Biology
Organic Industrial Chemicals (excl. Resins, Rubber and Plastics) | Expanding Knowledge in the Biological Sciences | Biofuel (Biomass) Energy | Human Pharmaceutical Treatments (e.g. Antibiotics) |
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.YMBEN.2018.02.005
Abstract: Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.YMBEN.2015.03.008
Abstract: Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).
Publisher: Springer Science and Business Media LLC
Date: 14-11-2009
DOI: 10.1007/S11120-009-9504-5
Abstract: In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of in idual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V(cmax) and J(max). Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.
Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2017
DOI: 10.1101/234252
Abstract: Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nano-scale beer production. S les included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.
Publisher: Elsevier BV
Date: 2023
DOI: 10.1016/J.TIBTECH.2022.07.002
Abstract: Biological homeostasis is a dynamic and elastic equilibrium of countless interlinked biochemical reactions. A key goal of life sciences is to understand these dynamics bioengineers seek to reconfigure such networks. Both goals require the ability to monitor the concentration of in idual intracellular metabolites with sufficient spatiotemporal resolution. To achieve this, a range of protein or protein/DNA signalling circuits with optical readouts have been constructed. Protein biosensors can provide quantitative information at subsecond temporal and suborganelle spatial resolution. However, their construction is fraught with difficulties related to integrating the affinity- and selectivity-endowing components with the signal reporters. We argue that development of efficient approaches for construction of chemically induced dimerisation systems and reporter domains with large dynamic ranges will solve these problems.
Publisher: Springer Science and Business Media LLC
Date: 04-2015
Publisher: Springer Science and Business Media LLC
Date: 06-2016
DOI: 10.1038/NBT.3593
Publisher: Wiley
Date: 22-05-2014
DOI: 10.1111/PCE.12350
Abstract: Isoprene strengthens thylakoid membranes and scavenges stress-induced oxidative species. The idea that isoprene production might also influence isoprenoid and phenylpropanoid pathways under stress conditions was tested. We used transgenic tobacco to compare physiological and biochemical traits of isoprene-emitting (IE) and non-emitting (NE) plants exposed to severe drought and subsequent re-watering. Photosynthesis was less affected by drought in IE than in NE plants, and higher rates were also observed in IE than in NE plants recovering from drought. Isoprene emission was stimulated by mild drought. Under severe drought, isoprene emission declined, and levels of non-volatile isoprenoids, specifically de-epoxidated xanthophylls and abscisic acid (ABA), were higher in IE than in NE plants. Soluble sugars and phenylpropanoids were also higher in IE plants. After re-watering, IE plants maintained higher levels of metabolites, but isoprene emission was again higher than in unstressed plants. We suggest that isoprene production in transgenic tobacco triggered different responses, depending upon drought severity. Under drought, the observed trade-off between isoprene and non-volatile isoprenoids suggests that in IE plants isoprene acts as a short-term protectant, whereas non-volatile isoprenoids protect against severe, long-term damage. After drought, it is suggested that the capacity to emit isoprene might up-regulate production of non-volatile isoprenoids and phenylpropanoids, which may further protect IE leaves.
Publisher: American Chemical Society (ACS)
Date: 07-02-2020
Publisher: Springer Science and Business Media LLC
Date: 10-12-2012
DOI: 10.1007/S10529-011-0821-3
Abstract: Escherichia coli is currently used by many research institutions and companies around the world as a platform organism for the development of bio-based production processes for bulk biochemicals. A given bulk biochemical bioprocess must be economically competitive with current production routes. Ideally the viability of each bioprocess should be evaluated prior to commencing research, both by metabolic network analysis (to determine the maximum theoretical yield of a given biocatalyst) and by techno-economic analysis (TEA to determine the conditions required to make the bioprocess cost-competitive). However, these steps are rarely performed. Here we examine theoretical yields and review available TEA for bulk biochemical production in E. coli. In addition, we examine fermentation feedstocks and review recent strain engineering approaches to achieve industrially-relevant production, using ex les for which TEA has been performed: ethanol, poly-3-hydroxybutyrate, and 1,3-propanediol.
Publisher: Springer Science and Business Media LLC
Date: 06-01-2011
Abstract: Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses. We have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties. The genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models.
Publisher: American Chemical Society (ACS)
Date: 23-01-2018
DOI: 10.1021/ACSSYNBIO.7B00355
Abstract: The GAL promoters are applied in metabolic engineering and synthetic biology to control gene expression in the budding yeast Saccharomyces cerevisiae. In gal80Δ background strains, they show diauxie-inducible expression, a feature beneficial in metabolic pathway optimization. However, only a limited number of GAL promoters have been characterized and are available for engineering purposes. Multiple uses of the same promoters can result in genetic instability in engineered strains due to homologous recombination. Here, 11 GAL1/2 promoters from other Saccharomyces species were isolated and characterized in S. cerevisiae. They exhibited diauxie-inducible expression patterns with low strength in exponential growth phase and induction in the ethanol growth phase. These promoters represent an expansion to the collection of GAL promoters available for genetic engineering in S. cerevisiae, including an increased ersity of expression levels. This provides the capacity for increased numbers of genetic manipulations with a lower risk of genetic instability.
Publisher: Springer Science and Business Media LLC
Date: 17-04-2009
DOI: 10.1038/NCHEMBIO.158
Abstract: The sessile nature of plants has resulted in the evolution of an extraordinarily erse suite of protective mechanisms against biotic and abiotic stresses. Though volatile isoprenoids are known to be involved in many types of biotic interactions, they also play important but relatively unappreciated roles in abiotic stress responses. We review those roles, discuss the proposed mechanistic explanations and examine the evolutionary significance of volatile isoprenoid emission. We note that abiotic stress responses generically involve production of reactive oxygen species in plant cells, and volatile isoprenoids mitigate the effects of oxidative stress by mediating the oxidative status of the plant. On the basis of these observations, we propose a 'single biochemical mechanism for multiple physiological stressors' model, whereby the protective effect against abiotic stress is exerted through direct or indirect improvement in resistance to damage by reactive oxygen species.
Publisher: Wiley
Date: 11-2021
Publisher: Wiley
Date: 17-09-2013
DOI: 10.1111/NPH.12477
Abstract: Isoprene protects the photosynthetic apparatus of isoprene‐emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene‐emitting and non‐emitting tobacco ( Nicotiana tabacum ) plants, to examine: the response of isoprene emission to plant water deficit a possible relationship between concentrations of the drought‐induced phytohormone abscisic acid ( ABA ) and isoprene and whether isoprene affected foliar reactive oxygen species ( ROS ) and lipid peroxidation levels. Isoprene emission did not affect whole‐plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well‐watered controls, droughted non‐emitting plants significantly increased ROS content (31–46%) and lipid peroxidation (30–47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole‐plant water use efficiency ( WUE ). Droughted isoprene‐emitting plants showed no increase in ROS content or lipid peroxidation relative to well‐watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole‐plant WUE , non‐emitting plants had 8–24% more biomass under drought, implying that isoprene emission incurred a yield penalty.
Publisher: Oxford University Press (OUP)
Date: 02-2010
DOI: 10.1111/J.1365-2672.2009.04432.X
Abstract: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin(+) trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.
Publisher: American Chemical Society (ACS)
Date: 30-09-2021
Publisher: Springer Science and Business Media LLC
Date: 18-11-2019
DOI: 10.1038/S41477-019-0539-0
Abstract: Synthetic biology is here to stay and will transform agriculture if given the chance. The huge challenges facing food, fuel and chemical production make it vital to give synthetic biology that chance-notwithstanding the shifts in mindset, training and infrastructure investment this demands. Here, we assess opportunities for agricultural synthetic biology and ways to remove barriers to their realization.
Publisher: Springer Science and Business Media LLC
Date: 26-06-2015
Publisher: American Chemical Society (ACS)
Date: 28-07-2020
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.JBIOTEC.2011.07.003
Abstract: Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50% this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture.
Publisher: American Chemical Society (ACS)
Date: 26-07-2022
DOI: 10.1021/ACSSYNBIO.2C00117
Abstract: Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons and for the creation of biocatalytic nanoreactors for
Publisher: Elsevier BV
Date: 11-2020
Publisher: Oxford University Press (OUP)
Date: 08-2005
DOI: 10.1093/PCP/PCI129
Abstract: Agrobacterium-based transformation was used to introduce a promoter-less glucuronidase uidA gene (beta-glucuronidase GUS) into Lotus japonicus. Transgenic plants were screened for GUS activation at different stages after inoculation with its symbiont, Mesorhizobium loti. Functional GUS fusion frequencies ranged from about 2 to 5% of the total number of transgenic lines. These lines provide excellent histological markers for tissue ontogeny analysis. Some of the activations generated GUS expression patterns that correspond to well-known tissue types, such as lateral root and nodule primordia, root tips and developing nodules (line CHEETAH). Others generated GUS activation associated with predictable but previously unknown (i) tissue types, such as the vascular bundle of the nodule (line VASCO) or (ii) expression domains, such as pericycle, nodule primordia, nodule and flower connective/vascular tissue (line FATA MORGANA) or inner root cortex cells in the vicinity of a curled root hair, nodule primordia and nodule cortex (line TIMPA). Putative members of two gene superfamilies, EH (Esp homolog) and AAA ATPase (ATPase associated with various cellular activities), were located next to the CHEETAH and VASCO insertions, respectively, and a nodulin gene, LjENOD40-2, was located next to the FATA MORGANA insertion. We utilized promoter GUS fusions to investigate the genetic regulation of LjENOD40-2 and FATA MORGANA GUS. The LjENOD40-2 promoter defined a novel expression domain and the FATA MORGANA nodule expression was reiterated by the 2 kb sequence upstream of the T-DNA insertion.
Publisher: Oxford University Press (OUP)
Date: 15-10-2014
Publisher: Springer Science and Business Media LLC
Date: 07-2003
DOI: 10.1007/S00299-003-0627-4
Abstract: The use of barley grains as bioreactors for high-level production of cellulase (1,4-beta-glucanase) was investigated. A hybrid cellulase gene, cel-hyb1, driven by the rice GluB-1 promoter was expressed specifically in developing endosperm. Codon usage optimisation of cel-hyb1 increased its expression in barley grains 527-fold and led to cellulase production of up to 1.5% of total grain protein. CEL-HYB1 enzyme in barley grains was highly stable during post-harvest storage. Selectable marker gene ( hph) was subsequently eliminated from transgenic lines through segregation of hph from synthetic cel-hyb1 ( syn.cel-hyb1) in T1 progeny, using a binary plasmid containing hph and syn.cel-hyb1 in separate T-DNAs. These data suggest that barley grains can potentially be used for the commercial production of cellulase.
Publisher: Springer Science and Business Media LLC
Date: 2013
Publisher: American Society for Microbiology
Date: 15-01-2013
DOI: 10.1128/AEM.02544-12
Abstract: Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in Escherichia coli , however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism ( csc ) genes in E. coli W were examined by knockout and overexpression experiments. At low sucrose concentrations, the csc genes are repressed and cells cannot grow. Removal of either the repressor protein ( cscR ) or the fructokinase ( cscK ) gene facilitated derepression. Furthermore, combinatorial knockout of cscR and cscK conferred an improved growth rate on low sucrose. The invertase ( cscA ) and sucrose transporter ( cscB ) genes are essential for sucrose catabolism in E. coli W, demonstrating that no other genes can provide sucrose transport or inversion activities. However, cscK is not essential for sucrose utilization. Fructose is excreted into the medium by the cscK -knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of cscA , cscAK , or cscAB could complement the WΔ cscRKAB knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing cscAB , and full growth rate complementation in WΔ cscRKAB also required cscAB . Our understanding of sucrose utilization can be used to improve E. coli W and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/S00299-003-0667-9
Abstract: Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene ( sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUS Plus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.
Publisher: American Chemical Society (ACS)
Date: 27-04-2020
Publisher: Springer Science and Business Media LLC
Date: 21-02-2017
Publisher: Elsevier BV
Date: 11-2010
Publisher: American Chemical Society (ACS)
Date: 30-03-2023
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.JBIOTEC.2017.07.014
Abstract: The rapid expansion in the number of sequenced genomes and metagenomes provides an exceptional resource for mining of the enzymes with biotechnologically relevant properties. However, the majority of protein production and analysis methods are not sufficiently cost-efficient and scalable to experimentally verify the results of computational genomic mining. Here, we present a pipeline based on Leishmania tarentolae cell-free system that was used to characterize 30 putative thermostable endo-1,4-β-glucanases and xylanases identified in public genomic databases. In order to analyse the recombinant proteins without purification, novel high-throughput assays for glucanase and xylanase activities were developed. The assays rely on solubilisation of labelled particulate substrates performed in multiwell plates. Using this approach both acidophilic and thermophilic enzymes were identified. The developed approach enables rapid discovery of new biotechnologically useful enzymes.
Publisher: Wiley
Date: 17-11-2016
Publisher: Cold Spring Harbor Laboratory
Date: 11-02-2022
DOI: 10.1101/2022.02.10.479872
Abstract: Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons, and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside virus-like particles can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here we present a set of acceptor vectors that provide a quick and efficient way to build, test and optimise cargo loading inside P22 virus-like particles. We prototyped the system using yellow fluorescent protein then applied it to mevalonate kinases, a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different mevalonate kinases required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimise cargo enzyme behaviour in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behaviour.
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2021
DOI: 10.1101/2021.01.30.428974
Abstract: Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme athway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast ( Saccharomyces cerevisiae ) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing ‘free’ MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1038/S42003-022-03070-Z
Abstract: Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast ( Saccharomyces cerevisiae ) suffers from several drawbacks specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae . Trans - and cis - modules, including eukaryotic trans -activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L −1 in flask cultivation. These negative ositive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.
Publisher: Elsevier BV
Date: 12-2018
Publisher: Springer Science and Business Media LLC
Date: 21-01-2022
Publisher: Informa UK Limited
Date: 12-2008
Publisher: Wiley
Date: 26-09-2023
Publisher: Springer Science and Business Media LLC
Date: 26-11-2019
DOI: 10.1038/S41564-018-0305-5
Abstract: Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms
Publisher: Springer Science and Business Media LLC
Date: 14-05-2010
DOI: 10.1007/S11103-010-9642-3
Abstract: Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.
Publisher: Oxford University Press (OUP)
Date: 12-2004
Abstract: Previous studies have shown that the promoter from the barley (Hordeum vulgare) phosphate transporter gene, HvPht1 , activates high levels of expression in rice (Oryza sativa) roots and that the expression level was induced by up to 4-fold in response to phosphorus (P) deprivation. To identify promoter regions controlling gene regulation specificities, successive promoter truncations were made and attached to reporter genes. Promoters of between 856 and 1,400 nucleotides activated gene expression in a number of cell types but with maximal expression in trichoblast (root hair) cells. For shorter promoters the trichoblast specificity was lost, but in other tissues the distribution pattern was unchanged. The low P induction response was unaffected by promoter length. Domain exchange experiments subsequently identified that the region between −856 and −547 nucleotides (relative to the translational start) is required for epidermal cell expression. A second region located between 0 and −195 nucleotides controls root-tip expression. The HvPht1 promoter contains one PHO-like motif and three motifs similar to the dicot P1BS element. Analysis of promoters from which the PHO-like element was eliminated (by truncation) showed no change in the gene induction response to P deficiency. In contrast, mutation of the P1BS elements eliminated any induction of gene expression in response to low P. An internal HvPht1 promoter fragment, incorporating a single P1BS element, had an increased response to P deprivation in comparison with the unmodified promoter (containing three elements). Together these findings further our understanding of the regulation of the HvPht1 gene and provide direct evidence for a functional role of the P1BS element in the expression of P-regulated genes.
Publisher: Elsevier BV
Date: 10-2017
DOI: 10.1016/J.CBPA.2017.05.017
Abstract: Isoprenoids (terpenes/terpenoids) have many useful industrial applications, but are often not produced at industrially viable level in their natural sources. Synthetic biology approaches have been used extensively to reconstruct metabolic pathways in tractable microbial hosts such as yeast and re-engineer pathways and networks to increase yields. Here we review recent advances in this field, focusing on central carbon metabolism engineering to increase precursor supply, re-directing carbon flux for production of C10, C15, or C20 isoprenoids, and chemical decoration of high value diterpenoids (C20). We also overview other novel synthetic biology strategies that have potential utility in yeast isoprenoid pathway engineering. Finally, we address the question of what is required in the future to move the field forwards.
Publisher: Wiley
Date: 07-04-2009
DOI: 10.1111/J.1365-3040.2009.01946.X
Abstract: Isoprene emission represents a significant loss of carbon to those plant species that synthesize this highly volatile and reactive compound. As a tool for studying the role of isoprene in plant physiology and biochemistry, we developed transgenic tobacco plants capable of emitting isoprene in a similar manner to and at rates comparable to a naturally emitting species. Thermotolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene the effect was very mild and could only be identified over repetitive stress events. However, isoprene-emitting plants were highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls. In ozone-treated plants, accumulation of toxic reactive oxygen species (ROS) was inhibited, and antioxidant levels were higher. Isoprene-emitting plants showed remarkably decreased foliar damage and higher rates of photosynthesis compared to non-emitting plants immediately following oxidative stress events. An inhibition of hydrogen peroxide accumulation in isoprene-emitting plants may stall the programmed cell death response which would otherwise lead to foliar necrosis. These results demonstrate that endogenously produced isoprene provides protection from oxidative damage.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2014
DOI: 10.1007/S00253-014-5956-4
Abstract: Sugarcane is the most efficient large-scale crop capable of supplying sufficient carbon substrate, in the form of sucrose, needed during fermentative feedstock production. However, sucrose metabolism in Escherichia coli is not well understood because the two most common strains, E. coli K-12 and B, do not grow on sucrose. Here, using a sucrose utilizing strain, E. coli W, we undertake an in-depth comparison of sucrose and glucose metabolism including growth kinetics, metabolite profiling, microarray-based transcriptome analysis, labelling-based proteomic analysis and (13)C-fluxomics. While E. coli W grew comparably well on sucrose and glucose integration of the omics, datasets showed that during growth on each carbon source, metabolism was distinct. The metabolism was generally derepressed on sucrose, and significant flux rearrangements were observed in central carbon metabolism. These included a reduction in the flux of the oxidative pentose phosphate pathway branch, an increase in the tricarboxylic acid cycle flux and a reduction in the glyoxylate shunt flux due to the dephosphorylation of isocitrate dehydrogenase. But unlike growth on other sugars that induce cAMP-dependent Crp regulation, the phosphoenol-pyruvate-glyoxylate cycle was not active on sucrose. Lower acetate accumulation was also observed in sucrose compared to glucose cultures. This was linked to induction of the acetate catabolic genes actP and acs and independent of the glyoxylic shunt. Overall, the cells stayed highly oxidative. In summary, sucrose metabolism was fast, efficient and led to low acetate accumulation making it an ideal carbon source for industrial fermentation with E. coli W.
Publisher: Springer Science and Business Media LLC
Date: 09-08-2007
DOI: 10.1007/S10529-007-9467-6
Abstract: A binary vector containing two reporter gene cassettes has been developed. This vector is ideal for optimising new plant transformation systems. Following optimisation, one of the reporter genes can be replaced with a gene of interest the second can be used as a marker to confirm transgenic lines, and to estimate locus number and determine zygosity. This allows simple, efficient and economical screening for homozygous single-insert lines and azygous controls.
Publisher: Wiley
Date: 07-02-2018
DOI: 10.1002/BIT.26530
Abstract: Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2021
DOI: 10.1038/S41467-021-21313-1
Abstract: In metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L −1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism.
Publisher: Public Library of Science (PLoS)
Date: 28-02-2014
Publisher: Springer Science and Business Media LLC
Date: 24-05-2022
DOI: 10.1038/S41467-022-30529-8
Abstract: Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene lification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L −1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Publisher: Elsevier BV
Date: 11-2010
Publisher: Springer Science and Business Media LLC
Date: 26-11-2015
Publisher: Springer Science and Business Media LLC
Date: 23-08-2019
DOI: 10.1038/S41467-019-11290-X
Abstract: Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae , where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.
Publisher: Oxford University Press (OUP)
Date: 23-07-2013
DOI: 10.1093/JXB/ERT202
Publisher: Elsevier BV
Date: 05-2023
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/BT13209
Abstract: The Kimberley region of Western Australia is recognised for its high bio ersity and many endemic species, including the charismatic boab tree, Adansonia gregorii F.Muell. (Malvaceae: Bombacoideae). In order to assess the effects of biogeographic barriers on A. gregorii, we examined the genetic ersity and population structure of the tree species across its range in the Kimberley and adjacent areas to the east. Genetic variation at six microsatellite loci in 220 in iduals from the entire species range was examined. Five weakly ergent populations, separated by west–east and coast–inland ides, were distinguished using spatial principal components analysis. However, the predominant pattern was low geographic structure and high gene flow. Coalescent analysis detected a population bottleneck and significant gene flow across these inferred biogeographic ides. Climate cycles and coastline changes following the last glacial maximum are implicated in decreases in ancient A. gregorii population size. Of all the potential gene flow vectors, various macropod species and humans are the most likely.
Publisher: Springer International Publishing
Date: 2015
DOI: 10.1007/10_2014_303
Abstract: Isoprene is a volatile C5 hydrocarbon. It is produced by a wide variety of organisms and has been shown to play a role in protection of plants under abiotic stress conditions. It also has many different uses as an industrial chemical: most notably as a precursor for synthetic rubbers, but also for production of elastomers, copolymers, adhesives, and specialised chemicals. Modifying and/or engineering isoprene production in plants has the potential to contribute to engineered stress resistance. Moreover, as petrochemical sources of isoprene increase in price and become more scarce, bioproduction routes through microbial processes are becoming more attractive. Here we examine biotechnological aspects of isoprene production and review the current state of the art for both microbial-based industrial bioprocesses and plant engineering.
Publisher: American Chemical Society (ACS)
Date: 12-10-2021
Publisher: Springer Science and Business Media LLC
Date: 09-05-2019
Publisher: Wiley
Date: 08-08-2006
Publisher: Wiley
Date: 03-09-2008
DOI: 10.1111/J.1365-3040.2008.01849.X
Abstract: Isoprene (C(5)H(8), 2-methyl 1,3-butadiene) is synthesized and emitted by many, but not all, plants. Unlike other related volatile organic compounds (monoterpenes and sesquiterpenes), isoprene has not been shown to mediate plant-herbivore interactions. Here, for the first time, we show, in feeding choice tests using isoprene-emitting transgenic tobacco plants (Nicotiana tabacum cv. Samsun) and non-emitting azygous control plants, that isoprene deters Manduca sexta caterpillars from feeding. This avoidance behaviour was confirmed using an artificial (isoprene-emitting and non-emitting control) diet. Both in vivo and in vitro experiments showed that isoprene can activate feeding avoidance behaviour in this system with a dose-response effect on caterpillar behaviour and an isoprene emission threshold level of <6 nmol m(-2) s(-1).
Publisher: Oxford University Press (OUP)
Date: 07-2019
DOI: 10.1104/PP.19.00345
Publisher: Springer Science and Business Media LLC
Date: 17-08-2006
DOI: 10.1007/S11103-006-9014-1
Abstract: To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5' deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCATCATGT, was required for ES specificity and substantially contributed to expression strength of the AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.
Publisher: MDPI AG
Date: 23-10-2018
DOI: 10.3390/GENES9110520
Abstract: Terpenoids are a group of natural products that have a variety of roles, both essential and non-essential, in metabolism and in biotic and abiotic interactions, as well as commercial applications such as pharmaceuticals, food additives, and chemical feedstocks. Economic viability for commercial applications is commonly not achievable by using natural source organisms or chemical synthesis. Engineered bio-production in suitable heterologous hosts is often required to achieve commercial viability. However, our poor understanding of regulatory mechanisms and other biochemical processes makes obtaining efficient conversion yields from feedstocks challenging. Moreover, production from carbon dioxide via photosynthesis would significantly increase the environmental and potentially the economic credentials of these processes by disintermediating biomass feedstocks. In this paper, we briefly review terpenoid metabolism, outline some recent advances in terpenoid metabolic engineering, and discuss why photosynthetic unicellular organisms—such as algae and cyanobacteria—might be preferred production platforms for the expression of some of the more challenging terpenoid pathways
Publisher: American Chemical Society (ACS)
Date: 03-02-2204
DOI: 10.1021/JACS.8B12298
Abstract: Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.
Publisher: Elsevier BV
Date: 12-2016
Publisher: eLife Sciences Publications, Ltd
Date: 12-03-2020
DOI: 10.7554/ELIFE.48685
Abstract: Volatile isoprenoids produced by plants are emitted in vast quantities into the atmosphere, with substantial effects on global carbon cycling. Yet, the molecular mechanisms regulating the balance between volatile and non-volatile isoprenoid production remain unknown. Isoprenoids are synthesised via sequential condensation of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), with volatile isoprenoids containing fewer isopentenyl subunits. The DMAPP:IPP ratio could affect the balance between volatile and non-volatile isoprenoids, but the plastidic DMAPP:IPP ratio is generally believed to be similar across different species. Here we demonstrate that the ratio of DMAPP:IPP produced by hydroxymethylbutenyl diphosphate reductase (HDR/IspH), the final step of the plastidic isoprenoid production pathway, is not fixed. Instead, this ratio varies greatly across HDRs from phylogenetically distinct plants, correlating with isoprenoid production patterns. Our findings suggest that adaptation of HDR plays a previously unrecognised role in determining in vivo carbon availability for isoprenoid emissions, directly shaping global biosphere-atmosphere interactions.
Publisher: Springer Science and Business Media LLC
Date: 23-08-2013
Abstract: Monoterpenes are a class of natural C 10 compounds with a range of potential applications including use as fuel additives, fragrances, and chemical feedstocks. Biosynthesis of monoterpenes in heterologous systems is yet to reach commercially-viable levels, and therefore is the subject of strain engineering and fermentation optimization studies. Detection of monoterpenes typically relies on gas chromatography/mass spectrometry this represents a significant analytical bottleneck which limits the potential to analyse combinatorial sets of conditions. To address this, we developed a high-throughput method for pre-screening monoterpene biosynthesis. An optimised DPPH assay was developed for detecting monoterpenes from two-phase microbial cultures using dodecane as the extraction solvent. The assay was useful for reproducible qualitative ranking of monoterpene concentrations, and detected standard preparations of myrcene and γ-terpinene dissolved in dodecane at concentrations as low as 10 and 15 μM, respectively, and limonene as low as 200 μM. The assay could not be used quantitatively due to technical difficulties in capturing the initial reaction rate in a multi-well plate and the presence of minor DPPH-reactive contaminants. Initially, limonene biosynthesis in Saccharomyces cerevisiae was tested using two different limonene synthase enzymes and three medium compositions. The assay indicated that limonene biosynthesis was enhanced in a supplemented YP medium and that the Citrus limon limonene synthase (CLLS) was more effective than the Mentha spicata limonene synthase (MSLS). GC-MS analysis revealed that the DPPH assay had correctly identified the best limonene synthase (CLLS) and culture medium (supplemented YP medium). Because only traces of limonene were detected in SD medium, we subsequently identified medium components that improved limonene production and developed a defined medium based on these findings. The best limonene titres obtained were 1.48 ± 0.22 mg limonene per L in supplemented YP medium and 0.9 ± 0.15 mg limonene per L in a pH-adjusted supplemented SD medium. The DPPH assay is useful for detecting biosynthesis of limonene. Although the assay cannot be used quantitatively, it proved successful in ranking limonene production conditions qualitatively and thus is suitable as a first-tier screen. The DPPH assay will likely be applicable in detecting biosynthesis of several other monoterpenes and for screening libraries of monoterpene-producing strains.
Publisher: Cold Spring Harbor Laboratory
Date: 25-11-2022
DOI: 10.1101/2022.11.24.517869
Abstract: Enzyme spatial organisation and compartmentalisation are naturally evolved mechanisms for facilitating multi-step biocatalysis. We explored the synthetic in vivo co-encapsulation of two different cargo proteins in yeast using a self-assembling virus-like particle. Co-encapsulation was verified using single particle techniques for both end-to-end fusion of the cargo proteins with the encapsulation anchor at one end, and coexpression of each cargo protein with their in idual anchors. The co-encapsulation of a bifunctional geranyl diphosphate/farnesyl diphosphate synthase and a bifunctional linalool/nerolidol synthase delivered nerolidol titres up to 30 times that of an unorganised ‘free’ enzyme control, a remarkable improvement from a single engineering step. Interestingly, striking differences in the ratio of products (linalool and nerolidol) were observed with each spatial organisation approach. This work presents the largest reported titre fold increases from in vivo enzyme compartmentalisation and suggests that enzyme spatial organisation could be used to modulate the product profile of promiscuous enzymes.
Publisher: Elsevier BV
Date: 12-2016
Publisher: Cold Spring Harbor Laboratory
Date: 09-11-2022
DOI: 10.1101/2022.11.08.515726
Abstract: The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable -fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), which corresponds to increases of a similar magnitude in terpene synthase expression. Therefore, increased in vivo enzyme levels – resulting from improved expression and/or stability – is likely to be a major driver of catalytic enhancement from enzyme fusion.
Publisher: eLife Sciences Publications, Ltd
Date: 16-08-2019
Publisher: American Chemical Society (ACS)
Date: 07-01-2013
DOI: 10.1021/SB300110B
Abstract: Population-density-dependent control of gene expression, or quorum sensing, is widespread in nature and is used to coordinate complex population-wide phenotypes through space and time. We have engineered quorum sensing in S. cerevisiae by rewiring the native pheromone communication system that is normally used by haploid cells to detect potential mating partners. In our system, populations consisting of only mating type "a" cells produce and respond to extracellular α-type pheromone by arresting growth and expressing GFP in a population-density-dependent manner. Positive feedback quorum sensing dynamics were tuned by varying α-pheromone production levels using different versions of the pheromone-responsive FUS1 promoter as well as different versions of pheromone genes (mfα1 or mfα2). In a second system, pheromone communication was rendered conditional upon the presence of aromatic amino acids in the growth medium by controlling α-pheromone expression with the aromatic amino acid responsive ARO9 promoter. In these circuits, pheromone communication and response could be fine-tuned according to aromatic amino acid type and concentration. The genetic control programs developed here are responsive to dynamic spatiotemporal and chemical cellular environments, resulting in up-regulation of gene expression. These programs could be used to control biochemical pathways for the production of fuels and chemicals that are toxic or place a heavy metabolic burden on cell growth.
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.YMBEN.2016.12.003
Abstract: Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100mgL
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.BIOTECHADV.2011.08.019
Abstract: Sucrose has economic and environmental advantages over glucose as a feedstock for bioprocesses. E. coli is widely used in industry, but the majority of current industrial E. coli strains cannot utilize sucrose. Previous attempts to transfer sucrose catabolic capabilities into non-sucrose-utilizing strains have met with limited success due to low growth rates on sucrose and phenotypic instability of the engineered strains. To address these problems, we developed a transferrable sucrose utilization cassette which confers efficient sucrose catabolism when integrated onto the E. coli chromosome. The cassette was based on the csc genes from E. coli W, a strain which grows very quickly on sucrose. Both plasmid-borne expression and chromosomal integration of a repressor-less sucrose utilizing cassette were investigated in E. coli strains K-12, B and C. In contrast to previous studies, strains harboring chromosomal cassettes could grow at the same rate as they do on glucose. Interestingly, we also discovered that spontaneous chromosomal integration of the csc genes was required to allow efficient growth from plasmid-transformed strains. The ability to engineer industrial strains for efficient sucrose utilization will allow substitution of sucrose for glucose in industrial fermentations. This will encourage the use of sucrose as a carbon source and assist in transition of our petrochemical-based economy to a bio-based economy.
Publisher: Springer Science and Business Media LLC
Date: 15-11-2010
DOI: 10.1038/NCHEMBIO.484
Abstract: Hyper-performing whole-cell catalysts are required for the renewable and sustainable production of petrochemical replacements. Chassis cells—self-replicating minimal machines that can be tailored for the production of specific chemicals—will provide the starting point for designing these hyper-performing 'turbo cells'.
Publisher: Wiley
Date: 25-06-2022
DOI: 10.1111/NPH.18285
Abstract: The strigolactone (SL) class of phytohormones shows broad chemical ersity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the ersification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several ersifying oxidation reactions on the structure. Evidence was obtained for functional ergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone ersification in early land plants, which may have extended to the ersification of strigol-type SLs.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-2011
Publisher: Springer Science and Business Media LLC
Date: 14-08-2010
DOI: 10.1007/S00253-010-2825-7
Abstract: Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical ex le of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered L-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L L-threonine by fed-batch culture, resulting in an overall yield of 0.284 g L-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.
Publisher: Oxford University Press (OUP)
Date: 16-12-2021
Abstract: A biofoundry provides automation and analytics infrastructure to support the engineering of biological systems. It allows scientists to perform synthetic biology and aligned experimentation on a high-throughput scale, massively increasing the solution space that can be examined for any given problem or question. However, establishing a biofoundry is a challenging undertaking, with numerous technical and operational considerations that must be addressed. Using collated learnings, here we outline several considerations that should be addressed prior to and during establishment. These include drivers for establishment, institutional models, funding and revenue models, personnel, hardware and software, data management, interoperability, client engagement and biosecurity issues. The high cost of establishment and operation means that developing a long-term business model for biofoundry sustainability in the context of funding frameworks, actual and potential client base, and costing structure is critical. Moreover, since biofoundries are leading a conceptual shift in experimental design for bioengineering, sustained outreach and engagement with the research community are needed to grow the client base. Recognition of the significant, long-term financial investment required and an understanding of the complexities of operationalization is critical for a sustainable biofoundry venture. To ensure state-of-the-art technology is integrated into planning, extensive engagement with existing facilities and community groups, such as the Global Biofoundries Alliance, is recommended.
Publisher: Wiley
Date: 07-04-2011
DOI: 10.1111/J.1365-3040.2011.02303.X
Abstract: Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.
Location: Australia
Start Date: 2014
End Date: 12-2017
Amount: $376,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 11-2020
End Date: 11-2027
Amount: $35,000,000.00
Funder: Australian Research Council
View Funded Activity