Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli.

The Calvin Cycle is the primary conduit for the fixation of carbon dioxide into the biosphere; ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting fixation step. Our goal is to direct the evolution of RuBisCO variants with improved kinetic and biophysical properties. The Calvin Cycle was partially reconstructed in Escherichia coli; the engineered strain requires the Synechococcus PCC6301 RuBisCO for growth in minimal media supplemented with a pentose. We randomly mutated the gene encoding the large subunit of RuBisCO (rbcL), co-expressed the resulting library with the small subunit (rbcS) and the Synechococcus PCC7492 phosphoribulokinase (prkA), and selected hypermorphic variants. The RuBisCO variants that evolved during three rounds of random mutagenesis and selection were over-expressed, and exhibited 5-fold improvement in specific activity relative to the wild-type enzyme. These results demonstrate a new strategy for the artificial selection of RuBisCO and other non-native metabolic enzymes.

Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors.

The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.