Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells.

In this PrePrint study, a containment strategy is presented to ensure that genetically modified (GM) bacteria cannot survive outside the laboratory. The bacteria are equipped with a dependency on a certain synthetic compound. The GM bacteria can only grow if this particular substance is added to the culture medium. If the compound is not available, the bacteria die because they cannot produce it themselves, an effect that is also known as “synthetic auxotrophy”. This approach has often been discussed as a containment strategy. One problem with this strategy, however, is that the genetic information of bacteria changes very quickly and this positive selection pressure allows them to escape the genetic safety measures. Bacteria that can survive without the addition of the synthetic compound could therefore prevail.
The authors of the study have now found a way to prevent this effect: They modified genes in the bacterial genome that are necessary for their survival so that they need the synthetic substance biphenylalanine (BipA) to fulfill their functions. They cultivated these bacteria over a long period of time (100 days) in special incubators, which enable the bacteria to be grown continuously and automatically, a process also known as continuous evolution. The authors reduced the concentration of BipA and thus increased the selection pressure. Bacteria that are no longer dependent on the additive could therefore preferentially survive. However, it turned out that the synthetic auxothropy of the bacteria, and thus the dependence on BipA, remains stable for 100 days despite the selection pressure. There were no mutations in the synthetic auxotrophy markers. They found that mutations occurred in specific transporter genes that allow the bacteria to survive under a lower concentration of BipA. The mutations in these transporter genes likely cause BipA to stay longer in the bacteria and to move out of the bacteria less quickly.

Kunjapur AM, Napolitano MG, Hysolli E, Noguera K, Appleton EM, Schubert MG, Jones MA, Iyer S, Mandell DJ, Church GM (2020) Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells. bioRxiv:2020.2009.2027.315804. doi:10.1101/2020.09.27.315804