Lactic acid production at low pH is therefore especially studied in yeasts and fungi, which generally have higher tolerances for lactic acid at low pH values than bacteria. Lactic acid inhibition, however, makes it difficult to reach high titers and productivities. Production of lactic acid at pH values below its p Ka value of 3.85, allows direct extraction of lactic acid from the culture broth, reducing overall production costs (Juturu and Wu, 2015). Ca(lactate) 2 is the initial product and it is converted into lactic acid in the downstream process by treatment with H 2SO 4 (Juturu and Wu, 2015). To mitigate this inhibition, the lactic acid production process can be carried out at neutral pH by titrating with base, usually Ca(OH) 2. Lactic acid has an inhibitory effect on microbial growth, especially at low pH values. as building block of poly lactic acid (PLA). Lactic acid is also used in technical applications, e.g. Lactic acid is a microbial product traditionally used to conserve food and feed products. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. Its genome and transcriptomes were sequenced and annotated. A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8.
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