amyP, a reporter gene to study strain degeneration inClostridium acetobutylicumATCC 824

Abstract

Clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus alpha-amylases. Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures. These results are in agreement with the extracellular alpha-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon.

Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum

“…Considering that no potential CRE site can be found upstream of the amyA2 gene, and the fact that gene amyA2 showed no obvious change in transcriptional level when cells were cultivated with and without starch [37] and amyA2 -encoded α-amylase could be readily purified from the cells grown on d -glucose [52], this gene is unlikely subject to CcpA-dependent glucose repression in C. acetobutylicum . It should be noted here that amyA1 and amyA2 likely act as the major genes responsible for starch degradation in C. acetobutylicum , because the loss of the megaplasmid pSOL1 (harboring gene amyA1 and amyA2 ) caused serious defect in starch degradation [53]. …”

Development of Real-Time PCR Primer and Probe Sets for Detecting Degenerated and Non-degenerated Forms of the Butanol-Producing Bacterium Clostridium acetobutylicum ATCC 824

“…The DGS set was designed from the sequence of the amyP gene. The amyP gene, which encodes α-amylase, is located in pSOL1 and is transcribed during solventogenesis [4]. Table 1 shows the characteristics of the C. aceto set and the DGS set.…”

“…Cornillot et al reported that the degeneration of C. acetobutylicum ATCC 824, the most well-known butanol-producing bacteria, is related to the loss of the pSOL1 plasmid [8]. Because the pSOL1 plasmid has all of the genes (including ctfA, ctfB, adc, and aad) that are related to solvent formation, its loss leads to the degeneration of C. acetobutylicum ATCC 824 and the loss of its solvent production capabilities [4,5,8]. Therefore, the detection and quantification of the pSOL1 plasmid should be one of the approaches for monitoring degenerated C. acetobutylicum.…”

“…In this sense, Sabathe et al investigated amyP, a gene in the pSOL1 plasmid that encodes an extracellular α-amylase that is transcribed during solventogenesis. They showed that amyP could be a good marker for detecting the degeneration of butanol-producing bacteria [4]. Schuster et al developed a method for monitoring the degeneration of solventogenic Clostridium strains using Fourier transform infrared spectroscopy of bacterial cells [2].…”

“…However, solventogenic clostridia lose their solvent formation capabilities during repeated sub-culturing of batch cultures and during continuous fermentation. This phenomenon is known as "degeneration", and it is one of the most important factors for optimizing butanol production [1,4]. Therefore, a method for detecting and quantifying degenerated cells will enhance and stabilize butanol production during continuous fermentation.…”