Isobutanol Production by Autotrophic Acetogenic Bacteria

Abstract

Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

“…Weitz et al . [ 55 ] were able to increase the isobutanol yield from 30 mg L −1 to 74 mg L −1 by feeding with the precursor isovalerate. Woolston et al .…”

Toward bioproduction of oxo chemicals from C1 feedstocks using isobutyraldehyde as an example

“…The probability of the hybrid microbial electrosynthesis method has been verified in R. eutropha using an electro-bioreactor, yielding 0.85 g/L isobutanol [ 33 ]. Other than aerobic R. eutropha , the acetogenic C. ljungdahlii that grows well on syngas (CO + H 2 + CO 2 mixture) has also been engineered to produce isobutanol under autotropic conditions [ 57 ]. Regarding the relationship between isobutanol and isobutyraldehyde, these microbial hosts show promise in isobutyraldehyde production regardless of the energy demands for hydrogen cost [ 58 ].…”

Bioisobutanol as a Promising Feedstock for Production of “Green” Hydrocarbons and Petrochemicals (A Review)

“…Along with carbohydrates, methanol [38] and gas mixtures containing СО, Н 2 , and СО 2 [39,40] can also be used for bioisobutanol production using microorganisms. Ma et al [38] used Methylorubrum extorquens for isobutanol production from methanol.…”

“…Additional blocking of the valine synthesis allowed the productive capacity for isobutanol to be increased by a factor of 6.5 to 0.13 g L -1 . For producing isobutanol from gas mixtures containing СО, Н 2 , and СО 2 , Weitz et al [40] used acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. The isobutanol yield was low, and additional introduction of ketoisovalerate was required for increasing it.…”