Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO3-associated recovery of solvent production.

Degenerate Clostridium beijerinckii strain (DG-8052) can be partially recovered by supplementing CaCO3 to fermentation media. Genome resequencing of DG-8052 showed no general regulator mutated. This study focused on transcriptional analysis of DG-8052 and its response to CaCO3 treatment via microarray. The expressions of 5168 genes capturing 98.6% of C. beijerinckii NCIMB 8052 genome were examed. The results revealed that with addition of CaCO3 565 and 916 genes were significantly up-regulated, and 704 and 1044 genes significantly down-regulated at acidogenic and solventogenic phase of DG-8052, respectively. These genes are primarily responsible for glycolysis to solvent/acid production (poR, pfo), solventogensis (buk, ctf, aldh, adh, bcd) and sporulation (spo0A, sigE, sigma-70, bofA), cell motility and division (ftsA, ftsK, ftsY, ftsH, ftsE, mreB, mreC, mreD, rodA), and molecular chaperones (grpE, dnaK, dnaJ, hsp20, hsp90), etc. The functions of some altered genes in DG-8052, totalling 5.7% at acidogenisis and 8.0% at sovlentogenisis, remain unknown. The response of the degenerate strain to CaCO3 was suggested significantly pleiotropic. This study reveals the multitude of regulatory function that CaCO3 has in clostridia and provides detailed insights into degeneration mechanisms at gene regulation level. It also enables us to develop effective strategies to prevent strain degeneration in future.

Proteomic analysis to elucidate degeneration of Clostridium beijerinckii NCIMB 8052 and role of Ca(2+) in strain recovery from degeneration.

Degeneration of solventogenic Clostridium strains is one of the major barriers in bio-butanol production. A degenerated Clostridium beijerinckii NCIMB 8052 strain (DG-8052) was obtained without any genetic manipulation. Supplementation of CaCO3 to fermentation medium could partially recover metabolism of DG-8052 by more than 50 % increase of cell growth and solvent production. This study investigated the protein expression profile of DG-8052 and its response to CaCO3 treatment. Compared with WT-8052, the lower expressed proteins were responsible for disruption of RNA secondary structures and DNA repair, sporulation, signal transduction, transcription regulation, and membrane transport in DG-8052. Interestingly, accompanied with the decreased glucose utilization and lower solvent production, there was a decreased level of sigma-54 modulation protein which may indicate that the level of sigma-54 activity may be associated with the observed strain degeneration. For the addition of CaCO3, proteomic and biochemical study results revealed that besides buffer capacity, Ca(2+) could stabilize heat shock proteins, increase DNA synthesis and replication, and enhance expression of solventogenic enzymes in DG-8052, which has a similar contribution in WT-8052.