Hypocrea jecorina Cellobiohydrolase I Stabilizing Mutations Identified Using Noncontiguous Recombination

Abstract

Noncontiguous recombination (NCR) is a method to identify pieces of structure that can be swapped among homologous proteins to create new, chimeric proteins. These “blocks” are encoded by elements of sequence that are not necessarily contiguous along the polypeptide chain. We used NCR to design a library in which blocks of structure from Hypocrea jecorina cellobiohydrolase I (Cel7A) and its two thermostable homologues from Talaromyces emersonii and Chaetomium thermophilum are shuffled to create 531,438 possible chimeric enzymes. We constructed a maximally informative subset of 35 chimeras to analyze this library and found that the blocks contribute additively to the stability of a chimera. Within two highly stabilizing blocks, we uncovered six single amino acid substitutions that each improve the stability of H. jecorina cellobiohydrolase I by 1–3 °C. The small number of measurements required to find these mutations demonstrates that noncontiguous recombination is an efficient strategy for identifying stabilizing mutations.

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution

“…Although these studies demonstrate that GH7 enzyme thermal stability can be improved through a variety of protein engineering approaches, we do not yet understand why these mutations, either singular or multiple, result in improved stability. Moreover, there has only been one report of improved thermal stability of H. jecorina Cel7A in the scientific literature (41).…”

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

“…This suggests that each sequence/structural block behaves differently in different contexts. For certain soluble protein properties (e.g., thermostability), it has been shown that block contributions are additive, that is, context independent, and that chimera stability can be predicted using linear regression (28,29,49,50). Our data suggest that ChR localization and photocurrent properties, however, require a more complex model to account for the nonlinear dependence of function on block sequence.…”

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

“…StickWRLD has been used previously to detect interpositional dependencies (IPDs) between residues in both DNA 3 and protein [15][16][17] alignments.…”

“…It has long been known that not all point mutations toward consensus provide improved stability or activity. More recently, proteins designed to take advantage of known IPDs in their sequence have been shown to result in greater activity than the same protein designed strictly from consensus 1,2 (manuscript in preparation), similar to the idea of stabilizing point mutations 3 .…”

“…Systems based on Markov Random Fields, such as GREMLIN 7 , attempt to overcome these problems. Additionally while sophisticated biological/biochemical techniques such as noncontiguous recombination 3,8 can be used to identify essential protein elements by region, they require considerable time and bench work for single-base-pair precision to be achieved. .…”