Adenosine triphosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in control of metabolic flux. Considering the importance of ATP in regulation of the glycolytic pathway, the use of ATP-oriented manipulation is a rational and efficient route to regulate metabolic flux. In this paper, a series of efficient ATP-oriented regulation methods, such as changing ambient temperature and altering reduced nicotinamide adenine dinucleotide (NADH), was developed. To satisfy the different demand for ATP at different phases in directed biosynthesis of uridine-phosphoryl compounds, a multiphase ATP supply regulation strategy was also used to enhance to yield of target metabolites.