Elimination of carbon catabolite repression in Clostridium acetobutylicum—a journey toward simultaneous use of xylose and glucose
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The industrial Gram-positive anaerobe Clostridium acetobutylicum is a valued acetone, butanol, and ethanol (ABE) solvent producer that is able to utilize a vast array of carbon sources in fermentation. When glucose is present in the growth medium, however, C. acetobutylicum, like many Gram-positive organisms, exhibits biphasic growth characteristics in which glucose is used preferentially over secondary carbon sources, a phenomenon known as carbon catabolite repression (CCR). The secondary carbon source is only utilized when the supply of glucose is exhausted, resulting in inefficient use of complex carbon sources. As biofuel production is sought from cheap feedstock, attention has turned to lignocellulosic biomass. Growth of C. acetobutylicum on lignocellulose, however, can be limited by CCR. Here, we present a method to relieve the inhibitory effect of CCR and allow simultaneous utilization of the lignocellulosic sugars of glucose and xylose by C. acetobutylicum. First, we utilized an in vivo gene reporter assay to demonstrate that an identified 14-nucleotide catabolite responsive element (CRE) sequence was sufficient to introduce CCR-mediated transcriptional inhibition, while subsequent mutation of the CRE sequence relieved the inhibitory effect. Next, we demonstrated that C. acetobutylicum harboring a CRE-less plasmid-borne xylose and pentose phosphate pathway operon afforded a 7.5-fold increase in xylose utilization in the presence of glucose as compared to a wild-type CRE plasmid-borne operon, effectively overcoming native CCR effects. The methodology presented here should translate to other members of Clostridium that exhibit CCR to enable simultaneous utilization of a vast array of carbon sources.
KeywordsBiofuel Butanol Clostridium acetobutylicum Carbon catabolite repression Catabolic responsive element Lignocellulose Xylose
This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (NSERC; grant number STPGP 430106–12) and the Canada Research Chairs (CRC; grant number 950–211471) programs.
Conflict of interest
DAC is a founder and employee of Neemo Inc., at which MB has also been employed. Neemo Inc. has a financial interest in the production of biofuels using clostridial microorganisms. The remaining authors declare no competing interests.
MB helped conceive the study, participated in its design and coordination, carried out the experiments, and drafted the manuscript. MMY participated in the study design and coordination. DAC and CPC helped conceive the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.
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