Characterization of a Clostridium beijerinckii spo0A mutant and its application for butyl butyrate production

Abstract

Spo0A is a master regulator that governs the metabolic shift of solventogenic Clostridium species such as Clostridium beijerinckii. Its disruption can thus potentially cause a significant alteration of cellular physiology as well as metabolic patterns. To investigate the specific effect of spo0A disruption in C. beijerinckii, a spo0A mutant of C. beijerinckii was characterized in this study. In a batch fermentation with pH control at 6.5, the spo0A mutant accumulated butyrate and butanol up to 8.96 g/L and 3.32 g/L, respectively from 60 g/L glucose. Noticing the unique phenotype of the spo0A mutant accumulating both butyrate and butanol at significant concentrations, we decided to use the spo0A mutant for the production of butyl butyrate that can be formed by the condensation of butyrate and butanol during the ABE fermentation in the presence of the enzyme lipase. Butyl butyrate is a value-added chemical that has numerous uses in the food and fragrance industry. Moreover, butyl butyrate as a biofuel is compatible with Jet A-1 aviation kerosene and used for biodiesel enrichment. In an initial trial of small-scale extractive batch fermentation using hexadecane as the extractant with supplementation of lipase CalB, the spo0A mutant was subjected to acid crash due to the butyrate accumulation, and thus produced only 98 mg/L butyl butyrate. To alleviate the butyrate toxicity, the biphasic medium was supplemented with 10 g/L CaCO and 5 g/L butanol. The butyl butyrate production was then increased up to 2.73 g/L in the hexadecane layer. When continuous agitation was performed to enhance the esterification and extraction of butyl butyrate, 3.32 g/L butyl butyrate was obtained in the hexadecane layer. In this study, we successfully demonstrated the use of the C. beijerinckii spo0A mutant for the butyl butyrate production through the simultaneous ABE fermentation, condensation, and extraction. Biotechnol. Bioeng. 2017;114: 106-112. © 2016 Wiley Periodicals, Inc.

Genomic, Transcriptional, and Phenotypic Analysis of the Glucose Derepressed Clostridium beijerinckii Mutant Exhibiting Acid Crash Phenotype

“…A similar result was observed in the previous study that adc and ptb gene expression controlled by Spo0A binding in C. beijerinckii was disrupted in the absence of the 0A box motifs . Also, the phenotype of BA105 is similar to that of either the spo0A disruption mutant of C. beijerinckii or disruption of the abrB homologue expression in C. acetobutylicum . Even if we confirmed the overexpression of the AbrB in BA105 by RNA sequencing, further investigations still require testing whether the up‐regulation of the AbrB in BA105 is caused by the point mutation that we identified by genome sequencing.…”

“…The accumulated acetate and butyrate were not reassimilated during the late stage of the fermentation, indicating a failure of solventogenesis. Under the same conditions, the wild‐type strain demonstrated normal cell growth and acid reassimilation, and was able to accumulate 10.9 g L −1 butanol that is similar to the previously reported levels . To check whether it is pleiotropic phenotype which may be resulted from improper handling or variations, we repeated batch fermentations of BA105 mutant with special attention to the variable factors such as anaerobiosis, medium components, and the age of inoculum.…”

“…The acid crash phenomenon occasionally occurs in batch fermentations by solventogenic clostridia without pH control for physiological or regulatory reasons causing a fast growth rate with a high metabolic rate . The delay in the switch from acidogenesis to solventogenesis is also involved in the acid crash phenotype even under the typical fermentation conditions . Genome sequence and transcriptional analysis in this study suggested BA105 showed the acid crash phenotype due to the fast growth rate and delay in the metabolic switch.…”

“…The precultures of the C. beijerinckii NCIMB 8052 wild‐type strain and the C. beijerinckii BA105 mutant were prepared using the TGY medium (30 g L −1 tryptone, 20 g L −1 glucose, 10 g L −1 yeast extract, and 1 g L −1 l ‐cysteine) by the previously described method . The growing cultures were inoculated into the P2 or P2YE medium containing sugars in BioFlo® 115 benchtop bioreactors (New Brunswick Scientific Co., Enfield, CT) at a 5% (v/v) inoculum level . For the P2YE medium, 1 g L −1 yeast exact was additionally added to the P2 medium.…”

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…Esters can be produced extracellularly during the conversion process, for example by lipase CALB 146,147 . The necessary alcohol can be added or produced by ω-oxidation of n-alkanes.…”