RESEARCH INTERESTS:

The focus of our research is to expand the structural and functional diversity of proteins and natural product molecules by enzyme directed evolution. We evolve and engineer protein posttranslational modification enzymes to install new chemical functionalities on proteins. We also aim to reprogram the catalytic activity of natural product biosynthetic enzymes to produce new chemical structures with desired biological activities. Protein posttranslational modification (PTM) plays a key role in regulating all the cellular processes. In parallel, many natural product molecules demonstrate their biological activities by targeting key proteins in the cell. Generating enzymes to control protein functions by specific modification or to synthesize natural product molecules of diverse structures and bioactivities would enable us to manipulate and correct various cellular processes at the molecular level. To achieve this goal, we have been working in three areas since August 2006:

(1)   We have developed an M13 phage display method for the directed evolution of PTM enzymes based on catalytic turnover. We successfully used this method to acquire mutants of Sfp phosphopantetheinyl transferase that catalyzes protein modification with coenzyme A (CoA) analogues. We are currently using this method to evolve kinase, glycosyltransferase, acetyltransferase, methyltransferase, ubiquitin ligase and other PTM enzymes to install new modifications on their target proteins in order to control their biological activities.

(2) We have developed an M13 phage display method to evolve the substrate specificity of adenylation (A) domains in nonribosomal peptide synthase (NRPS) and acyltransferase (AT) domains in polyketide synthase (PKS). We are currently using this method to change the substrate specificity of A and AT domains involved in the biosynthesis of antibiotics rifamycin, novobiocin, and gramicidin etc.

(3) We have developed a T7 phage display method to directly clone fragments of NRPS and PKS gene clusters from the soil metagenome in order to identify natural product biosynthetic genes from uncultivable bacteria. Using this method, we have discovered numerous novel NRPS and PKS gene fragments from the soil metagenome and we are in the process to clone the corresponding full length NRPS and PKS gene clusters.

Selected References

1.  Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem Biol 2, 337-46 (2007).

2.  High-throughput profiling of posttranslational modification enzymes by phage display. Biotechniques 43, 31, 33, 35 passim (2007).

3.  Enzyme catalyzed site-specific protein labeling and cell imaging with quantum dots. Chem Commun (Camb), 5927-9 (2008).

4.  Alkyne-functionalized chemical probes for assaying the substrate specificities of the adenylation domains in nonribosomal peptide synthetases. Chembiochem 9, 2804-10 (2008).

5.  Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display. Bioorg Med Chem Lett 18, 5664-7 (2008).

6.  Chapter 10 using phosphopantetheinyl transferases for enzyme posttranslational activation, site specific protein labeling and identification of natural product biosynthetic gene clusters from bacterial genomes. Methods Enzymol 458, 255-75 (2009).

7.  Site specific protein labeling by enzymatic posttranslational modification. Org Biomol Chem 7, 3361-71 (2009).

8.  Catalytic turnover-based phage selection for engineering the substrate specificity of Sfp phosphopantetheinyl transferase. J Mol Biol 387, 883-98 (2009).

9.  Identifying natural product biosynthetic genes from a soil metagenome by using T7 phage selection. Chembiochem 10, 2599-606 (2009).

10. Phosphopantetheinyl transferase catalyzed site-specific protein labeling with ADP conjugated chemical probes. J Am Chem Soc 131, 7548-9 (2009).