Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052

Abstract

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stressdirected evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon-and energy-limiting substrates, sucrose and the products of its hydrolysis D-glucose and D-fructose and only D-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l "1 of butanol and 26.3 g l "1 of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g "1 , respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in D-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the D-fructose phosphotransferase transport system and delayed sporulation.

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

“…The results suggest that BA105 may be an offspring of the C. beijerinckii SA‐1 strain, not the wild‐type 8052 strain, which is inconsistent with the previous report . The mutations shared by both C. beijerinckii BA105 and SA‐1 strains may be involved in the increased butanol production . However, the shared mutations may not be responsible for the acid crash phenotype of BA105 as the SA‐1 strain did not show the acid crash phenotype under the typical fermentation conditions unlike what was observed in BA105 .…”

“…The SA‐1 strain is a butanol‐tolerant mutant developed by a serial enrichment with increased butanol in the medium. The genome sequencing of SA‐1 mutant was previously performed to identify the genomic variations responsible for the increased butanol tolerance phenotype . Interestingly, BA105 mutant contained all the nine SNPs and one insertion between Cbei_1788 and Cbei_1789 that was the additional copy of the sequence ISCb1 from the IS4 family found in SA‐1 mutant (Table ) .…”

“…The genome sequencing of SA‐1 mutant was previously performed to identify the genomic variations responsible for the increased butanol tolerance phenotype . Interestingly, BA105 mutant contained all the nine SNPs and one insertion between Cbei_1788 and Cbei_1789 that was the additional copy of the sequence ISCb1 from the IS4 family found in SA‐1 mutant (Table ) . One additional insertion containing the copy of ISCb1 between Cbei_4611 and Cbei_4612 was found in BA105 genome.…”

“…The duplicated read and quality (Phred) filtering were applied to the Illumina paired‐end sequences. For comparative analysis with the wild‐type strain, corrections in reference genome of C. beijerinckii NCIMB 8052 (GenBank accession number NC_009617) were applied based on the previous study that identified four misannotations in the reference genome . The alignments of the filtered sequence were performed with SNAP aligner program (University of California at Berkeley, AMP Lab).…”

The Phosphotransferase System in Solventogenic Clostridia

“…The range of PTSs is an essential component of the metabolic flexibility of the clostridia, although, particularly in C. beijerinckii , it can be anticipated that there is likely to be some degree of overlap in substrate specificity between multiple systems belonging to the same PTS family. The continually increasing availability of genome sequences of different solventogenic strains, including representatives of the species C. saccharobutylicum and C. saccharoperbutylacetonicum [Bao et al, 2011;Del Cerro et al, 2013;Poehlein et al, 2013Poehlein et al, , 2014Sandoval-Espinola et al, 2013] will further increase understanding of the diversity of PTSs within the solventogenic group.…”