Metabolic Engineering of an Acid-Tolerant Yeast Pichia Kudriavzevii NG7 for Gypsum-Free Production of D-Lactic Acid

Lactic acid is a main building block of polylactic acid (PLA), a biodegradable bioplastic. Conventional lactic acid fermentation uses calcium hydroxide for the neutralization of acidic pH and eventually produces equal masses of insoluble by-product (gypsum), which decrease quality and value of the product in the product recovery process. To develop a robust yeast platform for gypsum-free lactic acid production, an acid-tolerant yeast strain was isolated from grape skins, and named as Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain was able to grow at pH 2.0 and 42°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the fermentation pathway from pyruvate to ethanol was redirected to lactic acid. Two copies of pyruvate decarboxylase gene in a diploid P. kudriavzevii NG7 were replaced with the D-lactate dehydrogenase gene derived from Lactobacillus plantarum. Then the engineered strain was sequentially exposed to the high concentration of lactic acid for adapted evolution. Final adapted strain produced 40 g/L of lactic acid without neutralization in flask cultivation. In a fed-batch fermentation, it produced 121 g/L of lactic acid with productivity over 4 g/L/h at a final pH of 3.5.