Blocking sporulation by inhibiting SpoIIE
We have shown that the control of solventogenesis and sporulation can be genetically uncoupled in C. acetobutylicum . In strain 824(pASspo), the absence of SpoIIE causes sporulation to be blocked at stage II. The cell remains in a vegetative state, and this allows solvent production to proceed for longer and for solvents to accumulate more rapidly and to a higher concentration. The characteristic drop in OD600 observed in wild type and control strains of C. acetobutylicum after 48-72 hours as the cells transition from the solventogenic phase to sporulation is notably absent in the fermentations of 824(pASspo). Mutant S (wild type background, spoIIE disrupted), Mutant BS (Mutant B background, spoIIE disrupted), Mutant HS (Mutant H background, spoIIE disrupted) and Mutant bukS (buk- background, spoIIE disrupted) were generated to create stable solvent producing bacteria with complete inactivation of the SpoIIE protein. Similarity between the SpoIIE protein of C. acetobutylicum, B. subtilis , and other Clostridial species indicates that the techniques used in C. acetobutylicum can be applied to other solvent producing Clostridia.
1. A recombinant solvent producing Clostridium , said Clostridium being a Clostridium acetobutylicum and having a SpoIIE gene disruption sufficient to inhibit sporulation and wherein said SpoIIE disruption increases the production of ethanol, acetone, and/or butanol.
2. The solvent producing Clostridium strain of claim 1 , wherein said disruption is constructed from the SpoIIE gene of SEQ ID NO: 1.
3. The solvent producing Clostridium strain of claim 1 , wherein said disruption is selected from the group consisting of a mutation, deletion, and interruption of the SpoIIE gene.
4. The solvent producing Clostridium strain of claim 1 , wherein said disruption is mutation of a conserved SpoIIE residue.
5. The solvent producing Clostridium strain of claim 1 , wherein said disruption is a mutation of a conserved residue selected from the group consisting of asp-610, asp-628, asp-746, gly-747 and asp-795, wherein the amino acids are numbered with reference to SEQ ID NO: 26 as numbered in FIG. 7 .
6. A method for producing an organic solvent comprising:
a) culturing the solvent producing strain of Clostridium of any of claims 1 to 5 under conditions suitable for solventogenesis in order to generate a culture media; and
b) purifying an organic solvent from said culture media.
7. The method of claim 6 , wherein said organic solvent is ethanol, acetone, butanol or combinations thereof.