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, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °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·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °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. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.

Techno-economical valorization of sugarcane bagasse for efficiently producing optically pure D-(-)-lactate approaching the theoretical maximum yield in low-cost salt medium by metabolically engineered Klebsiella oxytoca.

Sugarcane bagasse (SCB) was utilized for efficiently producing optically pure D-(-)-lactate by Klebsiella oxytoca KIS004-91T strain. Cellulase (15 U/g NaOH-treated SCB) sufficiently liberated high sugars with saccharifications of 79.8 % cellulose and 52.5 % hemicellulose. For separated hydrolysis and fermentation, D-(-)-lactate was produced at 53.5 ± 2.1 g/L (0.98 ± 0.01 g/g sugar utilized or 0.71 ± 0.01 g/g total sugars) while D-(-)-lactate at 47.2 ± 1.8 g/L (0.78 ± 0.03 g/g sugar used or 0.69 ± 0.01 g/g total sugars) was obtained under simultaneous saccharification and fermentation (SSF). D-(-)-lactate at 99.9 ± 0.9 g/L (0.97 ± 0.01 g/g sugar utilized or 0.78 ± 0.01 g/g total sugars) was improved via fed-batch SSF. Based on mass balance, raw SCB of 7 kg is required to produce 1 kg D-(-)-lactate. Unlike others, D-(-)-lactate production was performed in low-cost salt medium without requirements of rich nutrients. Costs regarding medium, purification, and waste disposal may be reduced. This unlocks economic capability of SCB bioconversion or agricultural and agro-industrial wastes into high valuable D-(-)-lactate.

The potential of the oleaginous yeast Rhodotorula paludigena CM33 to produce biolipids.

Sixty-seven yeast strains were isolated from castor beans then their endogenous lipids were stained by Nile Red (NR) fluorescence dye, and flow cytometry was used to obtain a strain with a high relative mean fluorescence intensity (MFI) value. The highest MFI value was obtained for strain CM33, which produced a maximum lipid content of 20.8 % dry cell weight (DCW). Based on the sequence of the ITS-5.8S-ITS rDNA and D1/D2 26S rDNA regions, CM33 showed 99 % identity with Rhodotorula paludigena. The potential of CM33 to assimilate various carbon sources was examined by growth on minimal media using glucose, glycerol, sucrose or xylose. CM33 was grown in glucose-based medium for 96 h and exhibited a maximum lipid content of 23.9 % DCW. Furthermore, when cells were cultured on molasses waste, their biomass, lipid content and lipid concentration reached 16.5 g/L, 37.1 % DCW and 6.1 g/L, respectively. These results demonstrated the potential of R. paludigena CM33 to contribute to a value-added carbon chain by converting renewable waste materials for biolipid production.

Genome Sequence of the Oleaginous Yeast Rhodotorula paludigena Strain CM33, a Potential Strain for Biofuel Production.

The genome sequence of Rhodotorula paludigena strain CM33, an oleaginous yeast isolated from castor bean (Ricinus sp.) in Thailand, is reported here. Genome sequencing and assembly yielded 20,657,327 bases with a 64.3% G+C content.