Cloning of l-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485
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The gene encoding l-lactate dehydrogenase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an l-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.
We thank Dr. J. Wiegel for providing us with the wild-type strain Thermoanaerobacterium saccharolyticum JW/SL-YS485 and its genomic DNA library. The support of the National Institute of Standards and Technology (grant no. 60NANB1D0064) and the Link Foundation is gratefully acknowledged. The experiments reported in this study comply with the current laws of the country in which they were performed.
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