Biology subjects

Lobzaev, E.

Publications and source records attributed to Lobzaev, E..

2 recordsLinked to original sources

bioRxiv2022

Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

synthetic biology↗

bioRxiv2022

Designing human Sphingosine-1-phosphate lyases using a temporal Dirichlet variational autoencoder

Enzymatic deficiencies cause the accumulation of toxic levels of substrates in a cell and are associated with life-threatening pathologies. Restoring physiological enzymes levels by injecting a recombinant version of the defective enzyme could provide a viable therapeutic option. However, these enzyme replacement therapies have had limited success, as the recombinant enzymes are less catalytically active, cause immune response and are difficult to manufacture. Moreover, the vast sequence design space makes finding enzymes with desired therapeutic properties extremely challenging. Here, we present a new enzyme engineering framework, which builds on recent advances in deep learning, variational calculus and natural language processing, to design variants of human enzymes with biochemical features comparable to the wild type protein as a way to rapidly build targeted libraries for downstream screening. We applied our method to design variants of human Sphyngosine-1-phosphate lyase (HsS1PL) as potential therapeutic treatments for nephrotic syndrome type 14 (NPHS14), and characterized their biochemical properties through extensive sequence and molecular dynamics analyses.

bioinformatics↗