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bioRxiv · 10.1101/2024.04.18.589993

Evolution engineering of methylotrophic E. coli enables faster growth than native methylotrophs

Nieh, L.-Y.·Chen, F. Y.- H.·Jung, H.-W.·Su, K.-Y.·Tsuei, C.-Y.·Lin, C. T.·Lee, Y.-Q.·Liao, J. C.
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Abstract

As methanol can be derived from either CO2 or methane, methanol economy may play a role in combating climate change. In this scenario, rapid utilization of methanol by an industrial microorganism is the first and crucial step for efficient utilization of the C1 feedstock chemical. Here, we report the development of a methylotrophic E. coli strain (SM6) with a doubling time of 3.5 hours, outpacing that of common native methylotrophs. We accomplish this using evolution engineering with dynamic copy number variation (CNV). We developed a bacterial artificial chromosome (BAC) with dynamic CNV to facilitate overcoming the formaldehyde-induced DNA-protein cross-linking (DPC) problem in the evolution process. The growth rate of the organism in methanol minimal medium improved significantly after it acquired a loss-of-function mutation in mutS. We tracked the genome variations of 72 cultures along the evolution process by next-generation sequencing, and identified the metabolic features of the fast-growing strain. This study illustrates the potential of dynamic CNV as an evolution tool and synthetic methylotrophs as a platform for sustainable biotechnological applications.

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Nieh, L.-Y., Chen, F. Y.- H., Jung, H.-W., Su, K.-Y., Tsuei, C.-Y., Lin, C. T., Lee, Y.-Q., Liao, J. C.. 2024-04-18. Evolution engineering of methylotrophic E. coli enables faster growth than native methylotrophs. https://doi.org/10.1101/2024.04.18.589993

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