Understanding the evolution of a new metabolic capability in full mechanistic detail is challenging, as causative mutations may be masked by non-essential "hitchhiking" mutations accumulated during the evolutionary trajectory. We have previously used adaptive laboratory evolution of a rationally engineered ancestor to generate an Escherichia coli strain able to utilize CO2 fixation for sugar synthesis. Here, we reveal the genetic basis underlying this metabolic transition. Five mutations are sufficient to enable robust growth when a non-native Calvin-Benson-Bassham cycle provides all the sugar-derived metabolic building blocks. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO2 fixation cycle toward biomass (prs, serA, and pgi), or in key regulators of carbon metabolism (crp and ppsR). Using suppressor analysis, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the enzymatic constraints that are essential to the metabolic stability of autocatalytic cycles and are relevant to future efforts in constructing non-native carbon fixation pathways.
Bibliographical noteFunding Information:
We thank Elad Noor, Arren Bar Even, Avi Flamholz, Gal Ofir, Gil Amitai, Rotem Sorek, Avigdor Eldar, Ahuva Cooperstein, Shaked Cahanovitc, Elad Itzhaki, Amit Bachar, Sagit Yahav, Lior Zelcbuch, Adar Lopez, Ghil Jona, and the bacteriology team, Shlomit Gilad, Sima Benjamin, and the INCPM genomics team, Yishai Levin, Dan Yakir, Roy Kishony, Idan Yelin, Dan Tawfik, and Tomer Shlomi. This work was funded by the European Research Council (projects SYMPAC 260392 and NOVCARBFIX 646827), Israel Science Foundation 740/16, Dana and Yossie Hollander, the Helmsley Charitable Foundation, the Larson Charitable Foundation, the Estate of David Arthur Barton, the Anthony Stalbow Charitable Trust, and Stella Gelerman, Canada. R.M. is the Charles and Louise Gartner professional chair. D.G.W. is supported by the United States–Israel Education Foundation.
© 2017 The Author(s).