A scarcity of cofactors, necessary metabolites or substrates for in vivo enzymatic reactions are among the major barriers of product synthesis in metabolically engineered cells. This work compares our recently developed cofactor boosting strategy, which uses xylose reductase (XR) and lactose to increase the intracellular levels of reduced or oxidized nicotinamide adenine dinucleotide (phosphate) NAD(P)H, adenosine triphosphate (ATP) and acetyl coenzymeA (acetyl-CoA) with other previously reported methods. We demonstrated that the XR/lactose approach enhances levels of sugar alcohols and sugar phosphates which leads to elevated levels of crucial cofactors required by specific metabolic pathways. The patterns of cofactor enhancement are not uniform and depend upon the specific pathway components that are overexpressed. We term this model the “user-pool” model. Here, we investigated metabolite alteration in the fatty alcohol producing system in the presence of XR/lactose within an early time frame (5 mins after the bioconversion started). All metabolite data were analyzed using untargeted metabolomics. We found that the XR/lactose system could improve fatty alcohol production as early as 5 mins after the bioconversion started. The enhancement of key cofactors and intermediates such as hexitol, NAD(P)H, ATP, 3-phosphoglycerate, acetyl-CoA, 6-phosphogluconate (6-PG) and glutathione was consistent with those previously reported on a longer time scale (after 1 hr). However, measurements performed at the early time reported here showed detectable differences in metabolite enhancement patterns such as those of ATP, NADPH, acetyl-CoA and glutathione. These data can serve as a basis for future analysis of metabolic flux alteration by the XR/lactose system. Comparative analysis of the cofactor enhancement by XR and other methods suggests that the XR/lactose can serve as a simple tool to increase levels of various cofactors for microbial cell factories.