Glycerol as substrate and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase enable higher production of 3-hydroxypropionic acid through the β-alanine pathway in E. coli.

Microbial engineering is a promising way to produce3-HP using biorenewable substrates such as glycerol. However, theglycerol pathway to obtain 3-HPrequires vitamin B-12, which hinders its economic viability. The present work showed that 3-HP can be efficiently produced from glycerol through the β-alanine pathway. To develop a cell factory for this purpose, glycerol was evaluated as a substrate and showed more than two-fold improved 3-HP production compared to glucose. Next, the reducing power was modulated by overexpression of an NADP+ -dependent glyceraldehyde-3-phosphate dehydrogenase coupled with CRISPR-based repression of the endogenous gapA gene, resulting in a 91 % increase in 3-HP titer. Finally, the toxicity of 3-HP accumulation was addressed by overexpressing a putative exporter (YohJK). Fed-batch cultivation of the final strain yielded 72.2 g/L of 3-HP and a productivity of 1.64 g/L/h, which are the best results for the β-alanine pathway and are similar to those found for other pathways.