To increase the butanol titers and selectivity in <em>Clostridium acetobutylicum</em> we replaced the promoter of the alcohol/aldehyde dehydrogenase (<em>aad</em>) gene with the phosphotranbutyrylase (<em>ptb</em>) promoter and combined this with CoAT downregulation to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers and overall solvent titers of 30 g/L. To increase then the carbon flux towards butanol, we examined thiolase (<em>thl</em>) overexpression. The combined <em>thl</em> overexpression with <em>aad</em> overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. <em>thl</em> overexpression in strains with CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to achieve improved butanol titers and selectivity. The <em>aad</em> with the <em>ptb</em> promoter was also used in the M5 strain and restored butanol production to wildtype levels. Thiolase overexpression was combined with <em>aad</em> overexpression aiming to enhance butanol formation. While acetate was reduced, butanol titers were not improved. We generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5, reducing the respective acid formation. We could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 <em>aad</em> overexpression strain. The difficulty in generating high butanol producers without acetone and acid production is likely hindered by the inability to control the electron flow, which may be affected by unknown pSOL1 genes. Using antisense RNA, we investigated the downregulation of the histidine kinase coded by CAC0654 on solventogenesis and sporulation. Our findings suggest the two-component system coded by CAC0654 and CAC0653 (a kinase and its cognate response regulator) is a putative negative regulator of sporulation. pH-controlled fermentations of the CAC0654 asRNA strain construct targeting show reduced glucose uptake and an 80% reduction in butanol production compared to the plasmid control strain. Transcriptional profiling of 824(pHK654) using microarrays shows profound upregulation of the sporulation cascade downstream of Spo0A, the master regulator of sporulation, through the expression of <em>sigK</em>. This two-component system may act affect the phosphorylation state of Spo0A repressing <em>sigF</em>, <em>spoIIE</em>, and <em>sigE</em>.

Last modified

• 10/01/2018

Creator

• William Sillers

DOI

• https://doi.org/10.21985/N2615M

Subject

• Chemical and Biological Engineering

Keyword

• metabolic engineering
• Clostridium acetobutylicum

Date created

• 2008-12-04

Resource type

• Dissertation

Rights statement

• In Copyright