Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov'SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside‐the‐box, predictions not found in other state‐of‐the‐art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness.
Fusarium
head blight (FHB), caused by Fusarium graminearum, whose occurrence and prevalence causes 10–70% wheat production
loss, is one of the most destructive diseases influencing the production
of wheat globally. To identify the potential natural products (NPs)
against F. graminearum, we screened
59 Xenorhabdus strains and discovered that the cell-free
supernatant (CFS) of X. budapestensis 14 (XBD14) displays the highest bioactivity. Multiple genetic methods
coupled with HRMS/MS analysis determined the major antifungal NP to
be Fcl-29, a fabclavine derivative. Fcl-29 was found to effectively
control FHB of wheat in the field test and demonstrated broad-spectrum
antifungal activity against important pathogenic fungi. The production
of Fcl-29 was dramatically improved by 33.82-fold with the combinatorial
strategy of genetic engineering (1.66-fold) and fermentation engineering
(20.39-fold). The exploration of a new biofungicide in global plant
protection is now possible.
In
this study, we report the discovery of unexpected mechanistic
intricacies of Baeyer–Villiger monooxygenases (BVMOs) and provide
insights that promise to help in extending their applications in synthetic
organic chemistry and biotechnology. The basic mechanism of BVMOs
as catalysts in the oxidation of unsymmetrical ketones R1–(CO)–R2 is well known, which involves
the intermediacy of short-lived Criegee intermediates. The tendency
of R1 or R2 to migrate preferentially in the
breakdown of the Criegee intermediate follows the traditional requirement
of an antiperiplanar conformation with maximum stabilization of the
incipient positive charge. The challenge of inverting the regioselectivity
of group migration with the formation of abnormal products was recently
met by the semi-rational directed evolution of TmCHMO with the generation
of a quadruple mutant. Although a reasonable model explaining the
mutational effect was suggested, the theoretical analysis did not
include the calculation of both enantiomeric forms of the fleeting
chiral Criegee intermediate in transition states and focused only
on the wild-type enzyme and the quadruple mutant. The present investigation
utilizes complete mutational deconvolution with the experimental construction
of a fitness-pathway landscape comprising 4! = 24 upward climbs. We
were confronted by the discovery that the absolute configuration of
the Criegee intermediate switches from (R) to (S), depending upon the stage of the evolutionary process.
On the basis of X-ray structural data, the physical basis of this
phenomenon was illuminated by quantum chemical analyses performed
on the enzymes at all evolutionary steps of a selected pathway. The
hitherto unexplored role of fleeting chiral intermediates in the mechanism
of other enzyme types deserves increased attention.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.