Thesis

Production of value-added isoprenoids by expressing Pinus sabiniana methylbutenol (MBO) synthase gene in Escherichia coli and Nostoc punctiforme

Non-renewable fossil fuels are responsible to meet more than 80% of global energy demand and its increasing price has ignited the interest in renewable biofuel production. 2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcoholic compound produced by several pine species. MBO has the potential to be used as biofuel. Higher energy content and less solubility in water make MBO superior than bioethanol in terms of both energy output and cost effectiveness. In pine chloroplast, methyl-erythritol-4-phosphate (MEP) isoprenoid pathway produce dimethylallyl pyrophosphate (DMAPP) which is utilized by MBO synthase for production of MBO. MBO production from its natural host is challenging due to its volatile nature. However, the volatile nature of MBO makes its recovery easier when produced through bacterial cultures in closed system bioreactor. The aim of this study is to produce MBO from Escherichia coli and also from the photosynthetic microorganism Nostoc punctiforme. MBO production in E. coli was attained by metabolic engineering with mevalonate (MVA) dependent pathway to increase the intracellular supply of DMAPP substrate and co-transformation with codon optimized Pinus sabiniana MBO synthase gene. Production was characterized and quantified using gas chromatography (GC) analysis. Further, MBO production was optimized using different culture media and conditions. MBO toxicity to the host cells was also studied to estimate the maximum amount of MBO that can be produced from E. coli culture. N. punctiforme was used as another host to produce photosynthetically derived MBO by expressing the MBO synthase gene from a plasmid under control of an indigenous petE promoter. Reverse transcriptase (RT)-PCR and SDS PAGE were performed to analyze the MBO synthase gene expression at mRNA and protein level. Although, the transcription and translation of MBO synthase were confirmed, detectable level of MBO production was not detected through gas chromatograph-mass spectrometry (GC-MS). Instead, enhanced production of phytols in the transgenic strain was observed and confirmed through a GC-MS analysis of total extracted lipids. To explain the enhanced production of phytols in transgenic strains, two plausible hypothesis were proposed; first, presence of an indigenous broad range substrate specific prenyltrasferases and second, appropriation of a MBO synthase metabolic intermediate by a native geranyl diphosphate synthase. To study the location of phytol production and accumulation in N. punctiforme, cell fractionation was performed using French pressure cell press and ultracentrifugation. Phytols were found to be present in cytoplasmic fraction. This study demonstrates feasibility of MBO production through bioengineering of E. coli however further work is required to improve its production to an economically efficient level. At the same time, this work also highlights the challenges of bioengineering a non-native cyanobacterial host, N. punctiforme, for production of useful compounds.

Relationships

Items