Abstract
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.
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Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
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Acknowledgements
The authors thank Mitr Phol Sugar Corporation, Ltd. (Thailand) for the provision of sugarcane molasses used in this study.
Funding
This work was financially supported by (i) Suranaree University of Technology (SUT), (ii) Thailand Science Research and Innovation (TSRI), and (iii) National Science Research and Innovation Fund (NSRF) (project code 90464) under the full-time researcher fellowship (Grant No.61/10/2562).
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KJ conceived, designed, and suggested methodology and research plan, supervised, and performed project administration and funding acquisition. CG investigated experiments, provided methodology, and wrote an original draft of the manuscript. PK helped investigating experiments. KJ also analyzed and validated data and edited, reviewed, and finalized the manuscript. All authors read and approved the manuscript.
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Gosalawit, C., Khunnonkwao, P. & Jantama, K. Genome engineering of Kluyveromyces marxianus for high D-( −)-lactic acid production under low pH conditions. Appl Microbiol Biotechnol 107, 5095–5105 (2023). https://doi.org/10.1007/s00253-023-12658-2
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DOI: https://doi.org/10.1007/s00253-023-12658-2