Microbial metabolism can be harnessed to convert sugars and other carbonaceous feedstocks into a variety of chemicals, fuels, and drugs. Recently, the development of CRISPR based genome editing technology, makes it possible to modify the yeast genome in a modular, parallel, and high-throughput manner. CRISPR-mediated genome editing in yeasts involves creation of double-strand DNA break (DSB) in the target loci followed by homology-directed repair (HDR). Depending on the purpose of genome editing the donor for HDR, thus mediating genetic manipulations such as gene disruption, deletion, insertion, replacement and assembly. In gene editing and transcriptional regulation, the Cas9 is used to either to create targeted DSB or upregulate/downregulate genes based on the sequence encoded by guide RNA. For gene editing, techniques such as multiplexing or HI-CRISPR have been developed that allow fast genome manipulations in short time. In addition to genome editing, transcriptional regulation is also a powerful approach and often adopted for metabolic engineering for production chemicals and biofuels. However, microbial metabolism is non-trivial to redirect into abundant production of the desired products. Recent developments in metabolomics have enabled us to evaluate the metabolic state of the producing microorganism based on Metabolic Flux Analysis (MFA) , which are able to discern the flux through the different metabolic pathways in its organism. We use the knowledge to shed light into microbial metabolism and improve metabolic engineering efforts for fatty acid-derived biofuel production. Genome wide co-factor balance helped us to identify potential targets for overexpression, down-regulation, deletion or temporal/spatial adjustment. Rational engineering of yeast metabolism was performed based on flux distributions.