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• Engineering a natural diploid S. cerevisiae for increased FAEE production. • Rational allocation of metabolic fluxes towards expanding FAEE precursor pools. • Process optimization in shake flask increased the titer to ∼40-folds. • Scale-up production in fed-batch bioreactor yielded a final 5.0 g/L FAEE titer. Developing microbial chassis for synthesizing value-added compounds is the most eco-friendly approach to minimize the impacts of overusing fossil fuels and other harsh chemicals. Recent research has focussed on harnessing microbial fatty acid metabolism to generate oleochemicals for use in industries as diverse as fuel production, cosmetics, lubricants and more. Considering the economic viability of generating "green biocatalysts", we reconfigured the fatty acid biosynthesis pathway in an environmentally robust strain of Saccharomyces cerevisiae MTCC4796 to produce fatty acid ethyl ester (FAEE) with high yield and titer. We applied the push–pull-block strategy wherein a heterologous wax ester synthase (WS2) was initially integrated to establish a FAEE biosynthetic pathway (pull) with additional rewiring including overexpression of PDH-bypass pathway genes for increasing the supply of precursor fatty acyl-CoA (push) and single allele disruption of non-essential genes from competing pathways (block) to maximize the metabolic flux towards FAEE overproduction. Our final engineered strain NGYT carrying 11 genetic modifications produced 26 mg/L and 1 g/L of FAEE before and after glucose optimization respectively. This titer was further scaled-up to reach 5 g/L in fed-batch bioreactors, which, to our knowledge, is the highest reported FAEE titer achieved from Saccharomyces cerevisiae with glucose as carbon source. Additionally, the FAEE's produced by NGYT strain showed a higher percentage of monounsaturated fatty acids, which are ideally suitable for biodiesel applications. Given the global emphasis on the development of cleaner fuels, the findings from this study expedite our progress towards industrial scale-FAEE production as a substitute to conventional diesel. [ABSTRACT FROM AUTHOR]