Microbial Synthetic Biology, Systems Metabolic Engineering and Enzyme Engineering for Advanced Microbial Biodiesel Production with Saccharomyces cerevisiae

Sammanfattning: The continuous requirement of transportation biofuels has brought the necessity to establish alternatives permitting low-cost production of biodiesel while being environmentally friendly. Biodiesel production was achieved utilizing Saccharomyces cerevisiae by employing respective enzymes that catalyze the synthesis of fatty acid ethyl esters (FAEEs) based on fatty acyl-CoA molecules and ethanol. Five acyltransferases/wax ester synthases were tested and heterologously introduced in yeast by expressing their codon-optimized gene for expression in a yeast host under the strong promoter TEF1p using plasmid pSP-GM2. In conclusion MhWS2 from oil bacteria Marinobacter hydrocarbonoclasticus was the highliest active with 8.1 pmol/(mg protein•min). Through Metabolically Engineering, metabolism was widely modified for increasing biodiesel production by eliminating fatty acid-consumption competitive pathways, therefore augmenting the fatty acid pool. This was achieved by deleting genes ARE1, ARE2, LRO1, DGA1 and POX1, which conferred a 5-fold increase of FAEEs formation (17.2 mg/L). Right after, MhWS2 was overproduced in yeast by chromosomal integration of its codon-optimized ws2. Then gene copy number was enhanced by integrating it in δ-regions, conferring 7.5-fold higher biodiesel production in a gradually evolved strain tolerant to 20 mg/mL antibiotic G418. Furthermore, Protein Engineering of two natural catalysts (MhWS2 and α/β-hydrolase Eeb1p homolog to yeast Saccharomyces cerevisiae) was addressed. In these subprojects, directed evolution of these two enzymes was achieved for favoring the synthesis of biodiesel by augmenting their efficiency and altering selectivity towards biocatalyzing FAEEs of desired chain length (C16 and C18, either saturated or monounsaturated). Starting with random mutagenesis of the respective codifying genes (ws2 and EEB1) allowed libraries of random point-mutations. Then library screening was conducted for reducing the CFU (colony formation unit) number; since lipotoxicity was employed as screening method due the condition of the yeast mutants, modifying to a weaker promoter was needed: KEX2p was then further applied. Ultimate selection of the best evolved variants of these enzymes was performed: variants MhWS2-v11 (65.3%) increment when compared to natural MhWS2) and Eeb1p-v04 (45.7% increment). MhWS2-v11 possesses five residue substitutions, while Eeb1p-v04 has 19 residue substitutions. In this case of scientific and technological studies, an advanced biofuel of an upcoming generation has been produced.