Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct ad-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes,HXT7,HXK2,PGI1,PFK1,PFK2,FBA1,TPI1,TDH3,PGK1,GPM1,ENO2, andPYK2, andd-lactate dehydrogenase (d-LDH) fromLeuconostoc mesenteroideswere randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L ofd-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L ofd-LA from 100 g/L glucose with 20 g/L CaCO(3)in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA.