Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library

Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways.United States. Office of Naval Research. Young Investigator Program (Grant N000140510656)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center. Grant EEC-0540879)MIT Faculty Start-up FundCodon Devices, Inc

Pairwise selection assembly for sequence-independent construction of long-length DNA

The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than ∼10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases

Numerical integration techniques for curved-element discretizations of molecule-solvent interfaces

urface formulations of biophysical modeling problems offer attractive theoretical and computational properties. Numerical simulations based on these formulations usually begin with discretization of the surface under consideration; often, the surface is curved, possessing complicated structure and possibly singularities. Numerical simulations commonly are based on approximate, rather than exact, discretizations of these surfaces. To assess the strength of the dependence of simulation accuracy on the fidelity of surface representation, here methods were developed to model several important surface formulations using exact surface discretizations. Following and refining Zauhar’s work [J. Comput.-Aided Mol. Des. 9, 149 (1995) ], two classes of curved elements were defined that can exactly discretize the van der Waals, solvent-accessible, and solvent-excluded (molecular) surfaces. Numerical integration techniques are presented that can accurately evaluate nonsingular and singular integrals over these curved surfaces. After validating the exactness of the surface discretizations and demonstrating the correctness of the presented integration methods, a set of calculations are presented that compare the accuracy of approximate, planar-triangle-based discretizations and exact, curved-element-based simulations of surface-generalized-Born (sGB), surface-continuum van der Waals (scvdW), and boundary-element method (BEM) electrostatics problems. Results demonstrate that continuum electrostatic calculations with BEM using curved elements, piecewise-constant basis functions, and centroid collocation are nearly ten times more accurate than planar-triangle BEM for basis sets of comparable size. The sGB and scvdW calculations give exceptional accuracy even for the coarsest obtainable discretized surfaces. The extra accuracy is attributed to the exact representation of the solute-solvent interface; in contrast, commonly used planar-triangle discretizations can only offer improved approximations with increasing discretization and associated increases in computational resources. The results clearly demonstrate that the methods for approximate integration on an exact geometry are far more accurate than exact integration on an approximate geometry. A MATLAB implementation of the presented integration methods and sample data files containing curved-element discretizations of several small molecules are available online as supplemental material.National Institutes of Health (U.S.) (GM065418)National Institutes of Health (U.S.) (CA096504)Singapore-MIT AllianceNational Science Foundation (U.S.)Natural Sciences and Engineering Research Council of Canada (NSERC