Adaptive evolution for fast growth on glucose and the effects on the regulation of glucose transport system in Clostridium tyrobutyricum

Abstract

Laboratory adaptive evolution of microorganisms offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. By combining a fibrous-bed bioreactor, we successfully developed a simple and valuable adaptive evolution strategy in repeated-batch fermentation mode with high initial substrate concentration and evolved Clostridium tyrobutyricum mutant with significantly improved butyric acid volumetric productivity up to 2.25 g/(L h), which is the highest value in batch fermentation reported so far. Further experiments were conducted to pay attention to glucose transport system in consideration of the high glucose consumption rate resulted from evolution. Complete characterization and comparison of the glucose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) were carried out in the form of toluene-treated cells and cell-free extracts derived from both C. tyrobutyricum wide-type and mutant, while an alternative glucose transport route that requires glucokinase was confirmed by the phenomena of resistance to the glucose analogue 2-deoxyglucose and ATP-dependent glucose phosphorylation. Our results suggest that C. tyrobutyricum mutant is defective in PTS activity and compensates for this defect with enhanced glucokinase activity, resulting in the efficient uptake and consumption of glucose during the whole metabolism.

Identification and Characterization of a Novel Trehalose Synthase Gene Derived from Saline-Alkali Soil Metagenomes

“…To determine the effect of metal ions and different chemical reagents on TreS, its activity was also assayed in the presence of these ions or compounds at 1 mM, respectively. To determine the conversion efficiency of TreS, the 5-L reaction system consisted of a 5-L stirred-tank fermentor (B. Braun, B. Braun Biotech International, Melsungen, Germany) containing 2-L of a medium was employed [27]. …”

Genome Sequence of Clostridium tyrobutyricum ATCC 25755, a Butyric Acid-Overproducing Strain

“…Clostridium tyrobutyricum ATCC 25755 is the most promising acidogenic bacterium used for the production of butyric acid (2–6). A highest butyric acid concentration of 86.9 g/liter has been achieved by our team with an adapted strain of C. tyrobutyricum ATCC 25755 (7). Moreover, genetic manipulation with this type strain, such as the inactivation of ack and pta genes, which encode enzymes associated with the acetate formation pathway, has been proved to significantly improve butyric acid production (8, 9).…”

“…According to the genomic analysis, strain C. tyrobutyricum ATCC 25755 may have a powerful membrane transport capacity, including 20 protein-coding sequences (CDSs) involved in the phosphotransferase systems and the corresponding regulatory system analogs (e.g., Hpr), 106 CDSs involved in ATP-binding cassette (ABC)-type transporters, and 1 involved in P-type ATPases, as well as many uncharacterized transporters. They may play important roles in the production of butyric acid with high productivity (6, 7). Additionally, several putative enzymes were also annotated in the C. tyrobutyricum ATCC 25755 genome, such as glycosyltransferase, aspartate transaminase, sugar phosphate isomerase, amino acid permease, glucokinase, intracellular protease/amidase, amidohydrolase, helicase, and some regulatory proteins.…”

Long-term production of butyric acid through immobilization ofClostridium tyrobutyricumin a moving fibrous-bed bioreactor (MFBB)

“…The immobilized cells in this MFBB system exhibited good productivity under a high initial concentration of glucose, which was higher than that in a traditional FBB bioreactor . The production of butyric acid did not encounter substrate inhibition, and the increased glucose concentration up to 143 g L ‐1 resulted in an improvement in the final concentration of butyric acid (64.5 ± 1.2 g L ‐1 ) without markedly decreased productivity, and thus overcame the common problems existing in free‐cell fermentation . Moreover, two fermentative phases were recognized under high glucose concentration (batch 5 to batch 8).…”