Engineering Clostridium acetobutylicum to permit simultaneous utilization of glucose and xylose: elimination of carbon catabolite repression

Abstract

The industrial gram-positive anaerobe Clostridium acetobutylicum is a valued ABE (acetone, butanol, ethanol) 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 bi-phasic 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 glucose and xylose by C. acetobutylicum. First, we utilized an in vivo gene reporter assay to demonstrate that an identified 14-nucleotide catabolic 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.