Genome engineering of Kluyveromyces marxianus for high D-(-)-lactic acid production under low pH conditions

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D- lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree- negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KM Delta pdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 degrees C provided the highest D-lactate titer of 42.97 +/- 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 +/- 0.01 g/g, 0.90 +/- 0.01 g/(L center dot h), and 1.06 +/- 0.00 g/(L center dot h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 +/- 0.68 g/L, 1.38 +/- 0.05 g/(L center dot h), and 1.22 +/- 0.00 g/(L center dot h), respectively, were higher at 42 degrees C compared to 30 degrees C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 +/- 0.81 g/L and 0.91 +/- 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale.

Automated summary

In this study, a genetically engineered yeast strain was developed to efficiently produce D-lactate, a high purity and high yield of D-lactate was achieved under suitable fermentation conditions, which has the potential to reduce production costs and increase D-lactate production.