Inspired by the conformational change of the enzyme–substrate
complex in molecular dynamics (MD) simulation with distance restriction,
we propose a strategy for identifying the engineering targets based
on the comparative analysis of enzyme-/substrate- binding modes in
MD simulations with and without distance restriction (prereaction-state
simulation and free-state simulation). Taking the short-chain dehydrogenase/reductase
(SDR) mutant EbSDR8-G94A/S153L (Mu0) with poor activity toward bulky
aryl ketones as an example, H145 and Y188 were identified as the engineering
targets due to the distinct conformation difference in the two simulation
modes. To break the “beam” structure formed by these
residues at the entry of cavity C2 in free-state simulation, the substrate-binding
pocket was reconstructed, and meanwhile the relative size of cavities
C1 and C2 was modulated to improve the enantioselectivity. In this
way, mutants for efficient asymmetric reduction of o-halogenated acetophenones,
propiophenones, aromatic ketoesters, and diaryl ketones were designed,
delivering chiral alcohols with >99% conversion and >98% ee.
The effectiveness of this design strategy was also validated by the
successful redesign of PpYSDR, generating a variant for efficient
reduction of (4-chlorophenyl) 2-pyridyl ketone into the S-product with >99% conversion and 96% ee. MD simulations suggested
a suitable binding pocket with proper energy contribution as the ubiquitous
mechanism for the improvement of activity and enantioselectivity toward
substrates with varied structures. The success in this study provides
hints for the rational design of alcohol dehydrogenases/reductases
with both a broad substrate spectrum and high enantioselectivity.
Isoprene, as a versatile bulk chemical, has wide industrial applications. Here, we attempted to improve isoprene biosynthesis in Saccharomyces cerevisiae by simultaneous strengthening of precursor supply and conversion via a combination of pathway compartmentation and protein engineering. At first, a superior isoprene synthase mutant ISPSLN was created by saturation mutagenesis, leading to almost 4-fold improvement in isoprene production. Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP) pool. To reconstruct metabolic balance between the upstream and downstream flux, additional copies of diphosphomevalonate decarboxylase gene ( MVD1) and isopentenyl-diphosphate delta-isomerase gene ( IDI1) were introduced into the cytoplasmic and mitochondrial engineered strains. Finally, the diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene, the highest ever reported in eukaryotic cells.
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