Optimization of RNA Structure Enhances Biosynthesis of L-Asparaginase from E. coli During Gene Overexpression

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Abstract

A highly efficient expression system for recombinant type II L-asparaginase from Escherichia coli (EC 3.5.1.1) was developed using a synthetic gene optimized for the folding energy of mRNA secondary structures in the 5'-region. The engineered E. coli BL21[DE3]/pET28a-AsnSYN strain produced up to 291 ± 9 mg/L of enzymatically active protein (44.5 ± 2.6 mg/(L·OD)) in shake-flask cultures, 50% higher than the control strain with the native gene after 3 hours of induction. Codon optimization increased the mRNA secondary structure stability in the 5'-region from –70 to –47 kcal/mol, potentially improving translation efficiency. The purified asparaginase met pharmacopeial standards, with a total yield ≥25%, specific activity >250 IU/mg, and high purity (no detectable impurities by electrophoresis, <3% multimeric forms by gel filtration). These findings highlight the potential of synthetic gene design with DNA structure optimization for industrial-scale production of therapeutic enzymes.

About the authors

R. R. Shaifutdinov

Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Moscow, Russia

N. A. Orlova

Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Moscow, Russia

I. I. Vorobyev

Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: ptichman@gmail.com
Moscow, Russia

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