Hexanol biosynthesis from syngas by Clostridium carboxidivorans P7 – product toxicity, temperature dependence and in situ extraction

Abstract

Clostridium carboxidivorans converts syngas into industrial alcohols like hexanol, but titers may be limited by product toxicity. Investigation of IC 50 at 30 °C (17.5 mM) and 37 °C (11.8 mM) revealed increased hexanol tolerance at lower temperatures. To avoid product toxicity, oleyl alcohol was added as an extraction solvent, increasing hexanol production nearly 2.5-fold to 23.9 mM (2.4 g/L) at 30 °C. This titer exceeds the concentration that is acutely toxic in the absence of a solvent, confirming the hypothesis that current hexanol production is limited by product toxicity. The solvent however had no positive effect at 37 °C. Furthermore, C. carboxidivorans cell membranes adapted to the higher temperature by incorporating more saturated fatty acids, but surprisingly not to hexanol. Corn oil and sunflower seed oil were tested as alternative, inexpensive extraction solvents. Hexanol titers were similar with all solvents, but oleyl alcohol achieved the highest extraction efficiency.

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

“…After reduction of the acids butyrate and caproate to the respective aldehydes via the native AOR (aldehyde:ferredoxin oxidoreductase) the molecules can be further converted to the alcohols butanol and hexanol. These alternative pathways allow the conservation of energy in form of ATP production via substrate level phosphorylation and are shown in grey (adapted from [ 26 ]) B Schematic representation of pIM Hex#15. The integration cassette consisting of a butanol-hexanol biosynthesis cluster and the adjacent ermC sequence is flanked by the mycomar sites (ITR–inverted terminal repeats) which allow integration catalyzed by the xylose-inducible Himar1 transposase.…”

“…However, C. kluyveri is not able to grow on gaseous substrates. The highest titer of hexanol reported on a gaseous substrate thus far is 1.36 g L −1 without product extraction [ 25 ] and 2.4 g L −1 when the product is removed by in situ extraction [ 26 ]. In both cases, this was achieved using wild-type C. carboxidivorans .…”

Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria

“…In fermentations, we observed hexanol titers of ~200 mg□L -1 (2.0 mM) at a rate of ~125□mg□L -1 □d -1 in a continuous system. C. carboxidivorans a related acetogenic clostridia has recently been shown to natively produce butanol, butanoic acid, hexanol, hexanoic acid and the product spectrum in that organism is heavily growth condition dependent 29,30 . This suggests that yields and specificity of our generated C. autoethanogenum strains can be further improved through growth condition optimizations.…”

Extractive Fermentation Processes: Modes of Operation and Application

“…Furthermore, several advances in gasification-fermentation processes have demonstrated the use of ISPR liquid-liquid extraction techniques for the production of short to medium chain length acids and alcohols with increased yield and high carbon selectivity [3,85]. In this thermochemical-biological process, syngas can either be supplied to the microorganisms directly, or alternatively, biomass may be converted to syngas, and consequently fermented by the microorganisms to make the required products [86][87][88][89].…”

“…In situ product recovery is an attractive processing route, which has the potential to improve productivity and yield [3][4][5][6][7] through i) limiting product inhibition, ii) improving substrate conversion by recycling unconverted substrate, and iii) allowing continuous or longer-term fermentations. For example, the productivities of several processes have shown improvements over conventional fermentations when using integrated product extraction.…”