In Vitro Bioconversion of Pyruvate to n-Butanol with Minimized Cofactor Utilization

Abstract

Due to enhanced energy content and reduced hygroscopicity compared with ethanol, n-butanol is flagged as the next generation biofuel and platform chemical. In addition to conventional cellular systems, n-butanol bioproduction by enzyme cascades is gaining momentum due to simplified process control. In contrast to other bio-based alcohols like ethanol and isobutanol, cell-free n-butanol biosynthesis from the central metabolic intermediate pyruvate involves cofactors [NAD(P)H, CoA] and acetyl-CoA-dependent intermediates, which complicates redox and energy balancing of the reaction system. We have devised a biochemical process for cell-free n-butanol production that only involves three enzyme activities, thereby eliminating the need for acetyl-CoA. Instead, the process utilizes only NADH as the sole redox mediator. Central to this new process is the amino acid catalyzed enamine–aldol condensation, which transforms acetaldehyde directly into crotonaldehyde. Subsequently, crotonaldehyde is reduced to n-butanol applying a 2-enoate reductase and an alcohol dehydrogenase, respectively. In essence, we achieved conversion of the platform intermediate pyruvate to n-butanol utilizing a biocatalytic cascade comprising only three enzyme activities and NADH as reducing equivalent. With reference to previously reported cell-free n-butanol reaction cascades, we have eliminated five enzyme activities and the requirement of CoA as cofactor. Our proof-of-concept demonstrates that n-butanol was synthesized at neutral pH and 50°C. This integrated reaction concept allowed GC detection of all reaction intermediates and n-butanol production of 148 mg L−1 (2 mM), which compares well with other cell-free n-butanol production processes.

To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

“…Besides acting as an important enzyme for ethanol production, PDC has also been applied in the in vitro production of nbutanol (Scheme 1). 10,11 Butanol together with other longerchain alcohols, such as 1-propanol, isobutanol and Scheme 1 Simplified pathway to produce ethanol and 1-butanol from lignocellulosic biomass via pyruvate decarboxylase (PDC). There are two major pathways for producing 1-butanol via pyruvate: CoAdependent or proline-dependent condensation.…”

“…17,18 This effect, however, is less pronounced for the enzymes catalysing butanol production in vitro. 10,19 Many enzymes can already tolerate higher organic solvent concentrations than the corresponding microorganisms. 20 Furthermore, engineering enzyme stability towards organic solvents is less challenging than evolving microorganisms to withstand the same concentration of organic solvents.…”

Recent Progress in the Microbial Production of Pyruvic Acid

“…Having both keto and carboxylic groups in its structure, pyruvate is a potential precursor for many chemicals, pharmaceuticals, food additives, and polymers. For example, pyruvate has been used in the biochemical synthesis of L-DOPA [1], N-acetyl-D-neuraminic acid [2], (R)-phenylacetylcarbinol [3], butanol using a three-enzyme cascade [4], and has been proposed as a starting material for the enzymatic synthesis of propionate [5]. A recent assessment of Escherichia coli as a cell factory concluded that pyruvate was one of the most useful metabolic precursors to a wide range of non-native commercial products [6].…”

Applied in situ product recovery in ABE fermentation

“…To overcome these problems, various strategies have been investigated, including: strain modifications to improve butanol tolerance, in vitro production using immobilized enzymes, and fermentation process developments to remove the butanol from the fermentation broth as it is produced have been developed . Via such product removal techniques, the butanol concentration in the fermentation broth will be maintained below inhibitory levels, leading to increased productivity and overall titers.…”

“…5 This is compounded by the complex separation of butanol from water due to the azeotrope forming at 55.5 wt% butanol at 101.3 kPa. 6 To overcome these problems, various strategies have been investigated, including: strain modifications to improve butanol tolerance, 2,4 in vitro production using immobilized enzymes, 7 and fermentation process developments to remove the butanol from the fermentation broth as it is produced have been developed. 2 Via such product removal techniques, This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.…”