Selective n-Butanol Production by Clostridium sp. MTButOH1365 During Continuous Synthesis Gas Fermentation Due to Expression of Synthetic Thiolase, 3-Hydroxy Butyryl-CoA Dehydrogenase, Crotonase, Butyryl-CoA Dehydrogenase, Butyraldehyde Dehydrogenase, and NAD-Dependent Butanol Dehydrogenase

Abstract

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60% CO and 40% H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms

“…Recently, Tyrurin and coworkers reported in a series of publications on completely abolishing acetate production in an undisclosed Clostridium strain, in favor of acetone [75], ethanol [76,77], butanol [78], mevalonate [78], methanol, or formate [79] production during autotrophic growth on syngas. However, eliminating acetate synthesis by deletion of the phosphotransacetylase and/or acetate kinase (ack) genes presumably prevents the concurrent synthesis of ATP (via SLP) by Ack.…”

New challenges for syngas fermentation: towards production of biopolymers

“…Predicted metabolic engineering changes can lead to delete undesired genes and/or to overexpress genes that are poorly expressed or even absent in the host strain. A few studies on overexpressing important genes to enhance strain productivity from syngas were published recently and thus underline this point . For example, Berzin et al .…”

“…A few studies on overexpressing important genes to enhance strain productivity from syngas were published recently and thus underline this point . For example, Berzin et al . reported on a Clostridium species ( Clostridium sp.…”

“…300 mmol L −1 ) from syngas. This was the first report on elimination of acetate and ethanol production genes and expression of a synthetic gene cluster encoding n‐butanol biosynthesis pathway in an acetogenic biocatalyst without the need for antibiotic selection to maintain the introduced genes …”

“…A few studies on overexpressing important genes to enhance strain productivity from syngas were published recently and thus underline this point. 96 For example, Berzin et al 96 reported on a Clostridium species (Clostridium sp. MTButOH1365) that was metabolically engineered so that it lost production of ethanol and acetate while initiating the production of n-butanol (approx.…”