ORCID Profile
0000-0003-4130-6252
Current Organisation
Queensland University of Technology
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Microbial Genetics | Biochemistry and Cell Biology | Synthetic Biology
Organic Industrial Chemicals (excl. Resins, Rubber and Plastics) | Expanding Knowledge in the Biological Sciences | Human Pharmaceutical Treatments (e.g. Antibiotics) |
Publisher: Portland Press Ltd.
Date: 27-02-2018
DOI: 10.1042/EBC20170047
Abstract: Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource. Plant cells contain organelles called plastids that retain their own genome, harbour unique biosynthetic pathways and differentiate into distinct plastid types upon environmental and developmental cues. Chloroplasts, the plastid type hosting the photosynthetic processes in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis and storage of particular classes of compounds, might prove more suitable for engineering the production and storage of non-native metabolites without affecting plant fitness. This review provides the current state of knowledge on the molecular mechanisms involved in plastid differentiation and focuses on non-photosynthetic plastids as alternative biotechnological platforms for metabolic engineering.
Publisher: Portland Press Ltd.
Date: 20-03-2015
DOI: 10.1042/BJ20141493
Abstract: Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few ex les to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts.
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-321-3_16
Abstract: DNA family shuffling is an efficient method for creating libraries of novel enzymes, in which a high proportion of mutants exhibit correct folding and possess catalytic properties distinct from the starting material. The evolutionary arrangement of cytochromes P450 into subfamilies of enzymes with highly similar nucleotide sequences but distinct catalytic properties renders them excellent starting material for DNA family shuffling experiments. This chapter provides a general method for creating libraries of shuffled P450s from two or more related sequences and incorporates several recent improvements to previously published methods.
Publisher: American Chemical Society (ACS)
Date: 18-03-2019
DOI: 10.1021/ACSSYNBIO.8B00418
Abstract: Protein scaffolding is a useful strategy for controlling the spatial arrangement of cellular components via protein-protein interactions. Protein scaffolding has primarily been used to colocalize soluble proteins in the cytoplasm, but many proteins require membrane association for proper function. Scaffolding at select membrane domains would provide an additional level of control over the distribution of proteins within a cell and could aid in exploiting numerous metabolic pathways that contain membrane-associated enzymes. We developed and characterized a membrane-bound protein scaffolding module based on the thylakoid protein CURT1A. This scaffolding module forms homo-oligomers in the membrane, causing proteins fused to CURT1A to cluster together at membrane surfaces. It is functional in erse expression hosts and can scaffold proteins at thylakoid membranes in chloroplasts, endoplasmic reticulum in higher plants and Saccharomyces cerevisiae, and the inner membrane of Escherichia coli.
Publisher: Springer Berlin Heidelberg
Date: 15-11-2014
Publisher: Frontiers Media SA
Date: 09-01-2023
DOI: 10.3389/FPLS.2022.1049177
Abstract: Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical ersity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco ( Nicotiana benthamiana ) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1016/J.CELS.2017.04.008
Abstract: Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C. autoethanogenum shifts from acetate to ethanol production to maintain ATP homeostasis at higher biomass concentrations but reaches a limit at a molar acetate/ethanol ratio of ∼1. This regulatory mechanism eventually leads to depletion of the intracellular acetyl-CoA pool and collapse of metabolism. We accurately predict growth phenotypes using a genome-scale metabolic model. Modeling revealed that the methylene-THF reductase reaction was ferredoxin reducing. This work provides a reference dataset to advance the understanding and engineering of arguably the first carbon fixation pathway on Earth.
Publisher: Proceedings of the National Academy of Sciences
Date: 15-10-2021
Abstract: Chloroplast biogenesis is a fundamental process occurring during seedling ontogenesis and leading to plant autotrophy. Which membrane components sterically organize the light-triggered transition of etioplast prolamellar bodies (PLBs) into chloroplast thylakoids, and thus mediate cubic–lamellar transformation, is poorly understood. Here, we used combined two- and three-dimensional electron microscopy, spectroscopy, and biochemical methods to determine the role of CURT1 proteins in the formation of etioplast cubic membranes and their transformation to photosynthetically active chloroplast thylakoids. CURT1 proteins were previously recognized as significant contributors to thylakoid membrane folding. We found that CURT1 proteins are integral proteins of etioplast membranes and act as factors modulating PLBs and prothylakoid nanomorphology. They are also required for concerted thylakoid maturation under de-etiolation.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5GC02708J
Abstract: Insight into energy metabolism of gas-fermenting acetogens using a systems level approach for sustainable production of fuels and chemicals.
Publisher: American Chemical Society (ACS)
Date: 13-12-2016
DOI: 10.1021/ACS.CHEMRESTOX.6B00396
Abstract: The 30 years since the inception of Chemical Research in Toxicology, game-changing advances in chemical and molecular biology, the fundamental disciplines underpinning molecular toxicology, have been made. While these have led to important advances in the study of mechanisms by which chemicals damage cells and systems, there has been less focus on applying these advances to prediction, detection, and mitigation of toxicity. Over the last ∼15 years, synthetic biology, the repurposing of biological "parts" in systems engineered for useful ends, has been explored in other areas of the biomedical and life sciences, for such applications as detecting metabolites, drug discovery and delivery, investigating disease mechanisms, improving medical treatment, and producing useful chemicals. These ex les provide models for the application of synthetic biology to toxicology, which, for the most part, has not yet benefited from such approaches. In this perspective, we review the synthetic biology approaches that have been applied to date and speculate on possible short to medium term and "blue sky" aspirations for synthetic biology, particularly in clinical and environmental toxicology. Finally, we point out key hurdles that must be overcome for the full potential of synthetic biology to be realized.
Publisher: American Chemical Society (ACS)
Date: 29-08-2012
DOI: 10.1021/TX300281G
Publisher: Frontiers Media SA
Date: 15-04-2021
DOI: 10.3389/FMICB.2021.649273
Abstract: Cytochrome P450 enzymes, or P450s, are haem monooxygenases renowned for their ability to insert one atom from molecular oxygen into an exceptionally broad range of substrates while reducing the other atom to water. However, some substrates including many organohalide and nitro compounds present little or no opportunity for oxidation. Under hypoxic conditions P450s can perform reductive reactions, contributing electrons to drive reductive elimination reactions. P450s can catalyse dehalogenation and denitration of a range of environmentally persistent pollutants including halogenated hydrocarbons and nitroamine explosives. P450-mediated reductive dehalogenations were first discovered in the context of human pharmacology but have since been observed in a variety of organisms. Additionally, P450-mediated reductive denitration of synthetic explosives has been discovered in bacteria that inhabit contaminated soils. This review will examine the distribution of P450-mediated reductive dehalogenations and denitrations in nature and discuss synthetic biology approaches to developing P450-based reagents for bioremediation.
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: Elsevier BV
Date: 11-2011
DOI: 10.1016/J.YMBEN.2011.09.001
Abstract: Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11 ± 4 (mean ± SD) recombinations and 1 ± 1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6L batch culture of one such mutant enabled the facile isolation of 0.3mg of the minor metabolite 1β-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.
Publisher: Cold Spring Harbor Laboratory
Date: 26-11-2019
DOI: 10.1101/852020
Abstract: Stable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step. Adding a combination of terbinafine (1 µM) and timentin (200 mg/L) to solid media delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from ten different wild-type and mutant Arabidopsis thaliana accessions. The method was also compatible with Nicotiana tabacum germination. Seedlings sown in non-sterile conditions could be maintained on antimicrobial media for up to a week without observable contamination. The antimicrobial cocktail was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as four days after stratification and transferred to soil before the onset of visible microbial contamination. The antimicrobial cocktail presented here delays microbial growth for long enough to permit germination of non-sterile Arabidopsis thaliana seedlings on solid media and it is compatible with rapid screening methods. We were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-1053-3_12
Abstract: DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates. Subsequent studies demonstrated that directed evolution could be further accelerated by shuffling similar native protein-coding sequences from the same gene family, rather than mutated variants of a single gene. Generally at least 65-75 % global identity between parental sequences is required in DNA family shuffling, with recombination mostly occurring at sites with at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I digestion and avoids the risk that parental sequences may be over-digested into unusable very small fragments. Restriction enzyme-mediated fragmentation also reduces the occurrence of undigested parental sequences that would otherwise reduce the number of unique variants in the resulting library. In the current chapter, we provide a brief overview of the alternative methods currently available for DNA shuffling as well as a protocol presented here that improves on several previous implementations of restriction enzyme-mediated DNA family shuffling, in particular with regard to purification of DNA fragments for reassembly.
Publisher: Wiley
Date: 2012
DOI: 10.1002/BMB.20576
Publisher: Wiley
Date: 14-03-2020
DOI: 10.1111/PPL.13079
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1016/J.YMBEN.2017.04.007
Abstract: Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO
Publisher: Elsevier BV
Date: 04-2020
DOI: 10.1016/J.TIBTECH.2019.10.009
Abstract: Protein scaffolding is a natural phenomenon whereby proteins colocalize into macromolecular complexes via specific protein-protein interactions. In the case of metabolic enzymes, protein scaffolding drives metabolic flux through specific pathways by colocalizing enzyme active sites. Synthetic protein scaffolding is increasingly used as a mechanism to improve product specificity and yields in metabolic engineering projects. To date, synthetic scaffolding has focused primarily on soluble enzyme systems, but many metabolic pathways for high-value secondary metabolites depend on membrane-bound enzymes. The compositional ersity of biological membranes and general challenges associated with modifying membrane proteins complicate scaffolding with membrane-requiring enzymes. Several recent studies have introduced new approaches to protein scaffolding at membrane surfaces, with notable success in improving product yields from specific metabolic pathways.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.YMBEN.2019.01.003
Abstract: Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-2011
Publisher: Springer Science and Business Media LLC
Date: 22-10-2018
Start Date: 11-2020
End Date: 11-2027
Amount: $35,000,000.00
Funder: Australian Research Council
View Funded Activity