Exploiting tandem repetitive promoters for high-level production of 3-hydroxypropionic acid

High-level biosynthesis of desired metabolites is challenging due to complexity of metabolic networks. Here, we report that platform chemical 3-hydroxypropionic acid (3-HP) can be overproduced through promoter engineering and growth-sustaining cultivation, two parallel strategies relying on RNA polymerases (RNAPs). First, we screened a promoter library and revealed that IPTG-inducible tac promoter was most effective for overexpression of PuuC, an endogenous aldehyde dehydrogenase catalyzing 3-HP biosynthesis in Klebsiella pneumoniae. Next, tandem repetitive tac promoters were harnessed to accommodate adequate RNAPs. When three tandem repetitive tac promoters were recruited to overexpress PuuC, up to 102.61 g/L 3-HP was produced. This is the highest 3-HP titer reported so far. In addition, lactic acid completely vanished during the late stage of fermentation. The backflow of lactic acid to pyruvic acid saves the trouble of downstream separation of lactic acid from 3-HP, which has long been a mission impossible because they are small-molecule isomers. Furthermore, timely removal of acid stress and replenishment of nitrogen source are crucial for 3-HP biosynthesis. A mathematical model shows that RNAPs modulate the tradeoff between bacterial growth and 3-HP formation. Overall, promoter engineering and growth-promoting cultivation jointly leverage RNAPs to maximize 3-HP. This study provides a paradigm for maximizing growth-coupled metabolites.