|dc.description.abstract||Metabolic engineering is receiving widespread attention in the chemical industry because of the increasing ability to manipulate the metabolic pathways of microorganisms with the goal of raising the efficiency of substrate utilisation, improve the growth of microorganisms, and, most significantly, produce higher yields of desirable products.
Sustainable production of valuable chemicals has garnered interest in the use of low-value carbon sources as feedstock in biological processes. However, the ability of a microorganism to survive in a low-carbon source media and convert the low-value feedstock to high-value products is hampered by the strict regulation of carbon flux within the microbial genome. The work presented in this thesis concentrates on the application of cutting-edge metabolic engineering methods to improve the production of a high-value product (lovastatin) from a low-value feedstock (glycerol) using Aspergillus terreus fungi.
Recent developments to improve lovastatin production from A. terreus have concentrated mainly on fermentation and the optimisation of its environment. Strain modification has been limited to just random mutation techniques and lovastatin gene cluster perturbations by increasing the copy number for the enhanced production of lovastatin. The research in this thesis, as far as the author is aware, presents, an alternative approach to bolster the metabolic pathway of A. terreus towards lovastatin biosynthesis.
The ultimate aim here was to increase the product carbon flow towards lovastatin production by carefully controlling the metabolism of the key intermediate metabolites, acetyl-CoA and malonyl-CoA. Advances in the understanding of the lovastatin production pathway were hindered by a lack of information on the carbon metabolic network of A. terreus. Therefore, metabolic pathways from other similar microorganism were reviewed from previous literature and compiled to facilitate the identification of the elementary limiting reaction steps and competing pathway(s) in lovastatin production.
This thesis outlines a successful sequence of steps to construct genetically modified A. terreus strains (ATCC 20542) that have superior capabilities in lovastatin production. Modification of targeted genes was achieved by inserting a single continuous (SC) DNA construct through homologous recombination. The overlapping extension polymerase chain reaction (OE-PCR) technique was optimised and applied specifically for A. terreus to assemble heterologous DNA fragments to obtain SC DNA. The SC DNA construct was inserted through the advance of the biolistic process. Optimisation of the biolistic parameters and A. terreus spore conditions was also carried out to ensure high transformation frequency of SC DNA construct homologous recombination.
Three separate strains were constructed in sequential order; the accox, Δpks, and accox+Δpks strains. The wild-type (WT) and modified strains were tested for improved lovastatin production using glycerol (G) and the combination of glycerol and lactose (GL) as feedstock. The genetically modified accox strain was developed by overexpressing the acetyl-CoA carboxylase (ACCase), an enzyme that controls the conversion of acetyl-CoA to malonyl-CoA. The strong constitutive promoter, PadhA, from Aspergillus nidulans was inserted into the A. terreus genome to increase enzymatic expression of ACCase. It was observed that the accox strain was able to increase the accumulation of acetyl-CoA and malonyl-CoA and consequently enhance the production of lovastatin or (+)-geodin concentration (depending on the type of carbon sources used). In this way, the concentration of the primary precursors, acetyl-CoA and malonyl-CoA, were confirmed to be influential in the production of lovastatin.
The genetically modified Δpks strain was developed to eliminate (+)-geodin biosynthesis, an unwanted metabolite that competes with the production of lovastatin. It is known from other microorganisms that the biosynthesis of (+)-geodin is initiated by enzymatic expression of emodin anthrone polyketide synthase (PKS), which utilises similar precursors to lovastatin. In this study, emodin anthrone PKS expression was genetically disrupted by homologous recombination of the SC DNA construct, partially deleting the emodin anthrone PKS gene sequences. The Δpks strain was found to accumulate significantly higher concentrations of acetyl-CoA and malonyl-CoA. A maximum increment of 80% and 29% in lovastatin production cultivated with the GL substrate was achieved with the Δpks strain relative to the production of lovastatin from the WT and accox strains, respectively.
Lastly, a double mutant (accox+Δpks) strain was developed by combining the overexpression of ACCase and disruption of the emodin anthrone PKS in a single strain A. terreus. In this accox+Δpks strain, the accumulation of acetyl-CoA and malonyl-CoA greatly exceeded the accox, Δpks, and WT strains by a significant margin. The rise in these primary precursors resulted in a boost to lovastatin production. For instance, elevations by 270% (119 mg/L) and 143% (152 mg/L) with the G and GL as substrate were observed, respectively, compared to the WT strain. Additionally, the biosynthesis of (+)-geodin was completely inhibited, indicating that the re-routing of carbon flow from (+)-geodin to the lovastatin biosynthetic pathway was successful.
A series of suggestions are proposed for further research that includes: i) additional genetic modifications that may contribute to higher accumulation of acetyl-CoA and malonyl-CoA. A number of genes have been identified based on the investigation of previous publications, such as pantothenate kinase (pank), acetyl-CoA synthase (acs), and ethanol oxidoreductase (adhE). Additionally, other competing pathways have also been recognised, especially those that utilise similar precursors as the lovastatin pathway. For example, regulating fatty acid biosynthesis and tricarboxylic acid (TCA) can potentially increase the accumulation of precursors; and ii) the enhancement of the accox+Δpks strain through optimising substrate formulation and culture conditions to further amplify lovastatin biosynthesis. This would also include optimisation of fermentation in bioreactors that would further be implemented in industrial scale.
In summary, the results of the work presented here have demonstrated the re-routing of carbon flow to attain higher accumulation of precursors that can subsequently be directed towards improved production of lovastatin. In light of these metabolic alterations and associated responses, this work emphasises the importance of understanding the A. terreus metabolism network to discover and implement modifications that enhance the production of a valuable metabolite - lovastatin. The knowledge developed in this thesis paves the way for better control of A. terreus metabolism that might lead to a broad potential to overproduce native or non-native high-value products from cheap and renewable carbon sources, including crude glycerol from the waste of palm oil industry.||en_AU|
|dc.publisher||University of Sydney||en_AU|
|dc.publisher||Faculty of Engineering and Information Technologies||en_AU|
|dc.publisher||School of Chemical and Biomolecular Engineering||en_AU|
|dc.rights||The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.||en_AU|
|dc.subject||acetyl-CoA and genetic engineering||en_AU|
|dc.title||Metabolically-Engineered Aspergillus Terreus To Improve Production Of Lovastatin||en_AU|
|dc.type.pubtype||Doctor of Philosophy Ph.D.||en_AU|
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