l-Lactic Acid Production via Sustainable Neutralizer-Free Route by Engineering Acid-Tolerant Yeast Pichia kudriavzevii

l-Lactic acid (l-LA) is a platform chemical obtained via microbial fermentation at a near-neutral pH value. Large amounts of neutralizers are required during this process, which increases the production costs in downstream processing as well as environmental burden. To address this challenge, an acid-tolerant yeast Pichia kudriavzevii E1 was isolated and metabolically engineered to produce l-LA without neutralizers. The genome of strain E1 was sequenced and a CRISPR-Cas9 system was developed in this newly isolated strain. Subsequently, the gene encoding pyruvate decarboxylase (pdc) was knocked out to subdue ethanol formation. Furthermore, the l-lactate dehydrogenase gene from Weizmannia coagulans 2-6 and the codon-optimized L-ldhA gene from Bos taurus were introduced into P. kudriavzevii E1 chromosome to redirect the ethanol fermentation pathway to l-LA production. Deletion of the dld(chr3) gene further increased the optical purity of l-LA. After optimizing fermentation conditions, the maximum titer of l-LA in the 5 L fermenter reached 74.57 g/L without any neutralizers, with an optical purity of 100% and a maximum yield of 0.93 g/g glucose. This is the first report of optically pure l-LA production without neutralizers and the engineered acid-tolerant yeast paves the way for the sustainable production of l-LA via a green route.