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

Vogeli, B.; Schulz, L.; Garg, S.; Tarasava, K.; Clomburg, J. M.; Lee, S. H.; Gonnot, A.; Moully, E. H.; Kimmel, B. R.; Tran, L.; Zeleznik, H.; Brown, S. D.; Simpson, S. D.; Mrksich, M.; Karim, A. S.; Gonzalez, R.; Koepke, M.; Jewett, M.

doi:10.1101/2022.03.28.486064

bioRxiv · 10.1101/2022.03.28.486064

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

Vogeli, B.·Schulz, L.·Garg, S.·Tarasava, K.·Clomburg, J. M.·Lee, S. H.·Gonnot, A.·Moully, E. H.·Kimmel, B. R.·Tran, L.·Zeleznik, H.·Brown, S. D.·Simpson, S. D.·Mrksich, M.·Karim, A. S.·Gonzalez, R.·Koepke, M.·Jewett, M.

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Abstract

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse {beta}-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimized and implemented r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screened 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generated designer strains for the selective production of butanoic acid (4.9 {+/-}0.1 gL-1), hexanoic acid (3.06 {+/-} 0.03 gL-1) and 1-hexanol (1.0 {+/-} 0.1 gL-1) at the best performance reported to date in this bacterium. We also generated Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5-L continuous fermentation. Our strategy enables optimization of rBOX derived products for biomanufacturing and industrial biotechnology.

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Vogeli, B., Schulz, L., Garg, S., Tarasava, K., Clomburg, J. M., Lee, S. H., Gonnot, A., Moully, E. H., Kimmel, B. R., Tran, L., Zeleznik, H., Brown, S. D., Simpson, S. D., Mrksich, M., Karim, A. S., Gonzalez, R., Koepke, M., Jewett, M.. 2022-03-29. Cell-free prototyping enables implementation of optimized reverse beta-oxidation pathways in heterotrophic and autotrophic bacteria. https://doi.org/10.1101/2022.03.28.486064

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