Changes in expression of adhEII, spo0A and hydrogenase gene may contribute to enhanced solvent formation in EA 2018
Comparative genomic analysis identified a set of solvent-relevant genes with variations within their coding sequences (Table 2). And most of which were SNV variations, such as genes encoding phosphotransacetylase and acetyl-CoA acetyltransferase. In addition, comparative genomics analysis also identified some variations up-stream of solvent-relevant genes (Table 3), which could potentially affect expression level of these genes. For example, a SNV site was found 84 bases upstream of the start codon of the AdhEII encoded gene CEA_P0034 (CA_P0035 in ATCC 824), under the s1 transcription start point [25]. Consistent with the variations at genomic level, we also found that transcription level of solvents formation genes, such as adhEII were highly expressed in EA 2018 relative to ATCC 824(Table 5 and Figure 3).
Figure 3  Expression profiles of variation genes involved in sporulation related two-component system and solvent formation pathway of C. acetobutylicum EA 2018 versus ATCC 824.   Spo0A is a central regulator of sporulation and solvent formation in C. acetobutylicum. Previous studies showed that C. acetobutylicum spo0A inactivation mutant stops producing spores and solvent, while over-expression of spo0A gene can enhance solvent production since 0A binding boxes have been identified in the promoter regions of solvent formation genes [26]. Therefore, it is speculative that the higher expression of spo0A could be one of the reasons for the higher butanol formation in EA 2018. Comparative transcriptomic analysis confirmed our speculation that higher transcriptional level of spo0A was found in EA 2018 (Table 4 and Figure 3). There were three ways for Spo0A to be phosphorylated (i.e. by a sensory kinase; through a novel phosphorylation system; by butyryl-P or acetyl-P) [27]. Using trans-membrane domain along with transcriptional analysis, four orphan kinases (CAC0437, CAC0323, CAC0903, and CAC2730) were identified as plausible kinases that might phosphorylate Spo0A in B. subtilis [27]. In our study, we also found that the transcriptional level of an orphan kinase CEA_G0344 (corresponding to CAC0323 in ATCC 824) was higher in EA 2018 (Figure 3), and the result is consistent well with the spo0A gene expression data.
During solvent fermentation process in C. acetobutylicum, a considerable amount of NADH was consumed by hydrogenase via reduced Fd (FeH2) to form hydrogen [28]. Previous reports showed that butanol production by C. acetobutylicum can be elevated by inhibiting hydrogen formation through adding viologen dyes or increasing hydrogen partial pressure [28], and knockdown of hupCBA cluster which encoded hydrogen uptake genes in C. saccharoperbutylacetonicum strain N1-4 decreased butanol formation (to 75.6% compared to the control strain) successfully [29]. Biochemical analysis showed that hydrogen formation in EA 2018 was nearly 29% lower than in ATCC 824 (Table 6). Interestingly, comparative genomic analysis also revealed SNVs in NiFe-hydrogenase coded gene CEA_P0140 (CA_P0141 in ATCC 824) and in the promoter of Fe-only hydrogenase coded gene CEA_G0028 (CAC 0028 in ATCC 824) (Table 2, 3). The variation site of CEA_G 0028 was located on the 12th base upstream of the start codon of hydA and altered the ribosome binding site (RBS) of this important gene (GGGAGG in ATCC 824 versus AGGAGG in EA 2018). In addition, the higher expression level of hydrogen uptake genes mbhs and mbhl were also revealed in EA 2018 (Figure 3). The result showed that hydrogen uptake could be an important factor for butanol formation, and increased expression level of hydrogen uptake gene mbhs and mbhl was closely correlated to the lower hydrogen formation in EA 2018, which can eventually help balance the NAD(P)H needed for higher production of butanol.
Table 6  Hydrogen production of C. acetobutylicum EA 2018 and C. acetobutylicum ATCC 824 in 6% glucose contained P2 medium   Using non-replicating plasmid pO1X, putative solvent formation repressor solR gene was inactivated in ATTC 824, and its fermentation experiment revealed that more solvent were produced in the solR inactivation mutant [5]. Although there are different speculations on the function of SolR [25], it has been confirmed that low expression of solR will enhance solvent formation [26]. Transcriptomic analysis revealed a lower expression level of solR, especially in the solventogenic phase in EA 2018, which might be related to hyper-butanol formation (Figure 3).
It has been suggested that the onset of solvent production is closely related to the accumulation of acid end products [30], and the addition of acetate and butyrate might result in a rapid induction of solventogenesis [31]. For example, it was reported that the concentration of undissociated butyric acid might play an important role in the induction of solventogenesis [32]. Transcriptomic analysis showed that expression of ack, pta, buk and ptb were all lower in EA 2018 than in ATCC 824 (Figure 3), consistent with the biochemical analysis (Figure 1). In addition, the results also suggested that the transition to the solventogenesis took place at a lower acetate and butyrate acid concentration in EA 2018 compared to ATCC 824.