Development and application of serial cultivation techniques for strain development and improvement

Metabolic engineering, directed evolution, and evolutionary dynamics aim to study and modify the genetic composition of microorganisms. Metabolic engineering and directed evolution have influenced biotechnology by providing drastic manipulations of genetic codes. Evolutionary dynamics has remained a field for basic scientific research. In this work we develop a technique for blending all three fields for the development and improvement of biocatalysts.; The technique used in this work is based on the serial cultivation process. Serial cultivation is a semicontinuous bioreactor operation commonly encountered in traditional microbiological protocols in which a cultivation is prolonged by continuously starting a new culture using as an inoculum a fraction of a previous culture. Serial cultivation is readily automated and provides reduced waste generation rates and lower operation costs as compared to continuous culture. As it obviates washouts, serial cultivation is particularly suited for long term cultivation under strong selection pressures. We have established a strong selection pressure that couples growth with the expression of a target pathway, and we have used the selection pressure to evolve novel physiological properties for ethanol production.; The selection pressure is based on the NAD/NADH balance requirement. We developed a triple mutant strain of E. coli that is unable to oxidize NADH under anaerobic conditions. The pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) was introduced into the mutant strains to complement anaerobic growth. We evolved the mutant strain for over 1000 generations using the serial cultivation method. The evolved strain displayed fermentative properties that were an overall improvement over the parent strain. Among the resulting phenotypic properties obtained were increased enzymatic activities of the enzymes encoded by the pet operon, higher glucose consumption, higher ethanol titers, faster growth, and higher cell densities.; With a separate serial cultivation, we evolved a strain that did not require supplementation with yeast extract. The parent strain could not grow anaerobically without yeast extract supplementation. A serial cultivation line in which the concentration of yeast extract in the medium was gradually reduced yielded a strain that did not require yeast extract. The strain maintained the high ethanol titers obtained from the first line of cultivation. Histograms of the serial cultivations demonstrated we were able to obtain sequential improvements in the strain by first evolving the increased ethanol titers, and subsequently introducing the ability to grow without yeast extract.; Serial cultivation is a powerful tool for the development and improvement of biotechnology platforms. Full automation of the serial cultivation process allows the development of a massively parallel technique to study and apply evolutionary processes.