PhD Student, University of Manchester
New strains of E. coli will be created to have the capability of utilizing feedstocks derived from agricultural wastes to synthesize gaseous biofuels. The catalytic properties of the key enzymes involved in the butane and isobutane biosynthesis pathways will also be investigated, exploiting magnetic nanoparticles as a means of concentrating, delivering and enhancing the properties of immobilised biocatalysts for their production. In sum, the pathway engineering, synthetic biology, and bioreactor engineering will be carried out with the intention of opening up new routes to butane and isobutane production from widely available and inexpensive feedstocks.
Abstract: Propane, a major component of liquid petroleum gas (LPG) derived from fossil fuels, has widespread applications in vehicles, cooking, and ambient heating. Given the concerns about fossil fuel depletion and carbon emission, exploiting alternative and renewable source of propane have become attractive. In this study, we report the construction of a novel propane biosynthetic pathway in Escherichia coli.We constructed an aldehyde reductases (ALR)-deprived E. coli strain BW25113(DE3) Δ13 via genetic engineering, which produced sufficient isobutyraldehyde precursors and finally achieved de novo synthesis of propane (91 μg/L) by assembling the engineered valine pathway and cyanobacterial aldehyde-deformylating oxygenase (ADO). Additionally, after extensive screening of ADO mutants generated by engineering the active center to accommodate branched-chain isobutyraldehyde, we identified two ADO mutants (I127G, I127G/A48G) which exhibited higher catalytic activity for isobutyraldehyde and improved propane productivity by three times (267 μg/L).The propane biosynthetic pathway constructed here through the engineered valine pathway can produce abundant isobutyraldehyde for ADO and overcome the low availability of precursors in propane production. Furthermore, the rational design aiming at the ADO active center illustrates the plasticity and catalytic potential of ADO. These results together highlight the potential for developing a microbial biomanufacturing platform for propane.
Pub.: 01 Apr '16, Pinned: 28 Jul '17
Abstract: Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.
Pub.: 30 Sep '16, Pinned: 28 Jul '17
Abstract: 5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of L-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of L-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than L-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient L-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose.Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36 promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid.Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.
Pub.: 09 Oct '16, Pinned: 28 Jul '17
Abstract: It is increasing clear that biofuels can be a viable source of renewable energy in contrast to the finite nature, geopolitical instability, and deleterious global effects of fossil fuel energy. Collectively, biofuels include any energy-enriched chemicals generated directly through the biological processes or derived from the chemical conversion from biomass of prior living organisms. Predominantly, biofuels are produced from photosynthetic organisms such as photosynthetic bacteria, micro- and macro-algae and vascular land plants. The primary products of biofuel may be in a gas, liquid, or solid form. These products can be further converted by biochemical, physical, and thermochemical methods. Biofuels can be classified into two categories: primary and secondary biofuels. The primary biofuels are directly produced from burning woody or cellulosic plant material and dry animal waste. The secondary biofuels can be classified into three generations that are each indirectly generated from plant and animal material. The first generation of biofuels is ethanol derived from food crops rich in starch or biodiesel taken from waste animal fats such as cooking grease. The second generation is bioethanol derived from non-food cellulosic biomass and biodiesel taken from oil-rich plant seed such as soybean or jatropha. The third generation is the biofuels generated from cyanobacterial, microalgae and other microbes, which is the most promising approach to meet the global energy demands. In this review, we present the recent progresses including challenges and opportunities in microbial biofuels production as well as the potential applications of microalgae as a platform of biomass production. Future research endeavors in biofuel production should be placed on the search of novel biofuel production species, optimization and improvement of culture conditions, genetic engineering of biofuel-producing species, complete understanding of the biofuel production mechanisms, and effective techniques for mass cultivation of microorganisms.
Pub.: 22 Dec '16, Pinned: 28 Jul '17