Optimization of RNA Structure Leads to Increased Biosynthesis of E. coli L-asparaginase when its Gene is Overexpressed

Abstract A highly efficient expression system for recombinant Escherichia coli type II L-asparaginase (EC 3.5.1.1) has been developed based on a synthetic gene optimized for the energy of secondary structure hairpins formation in the 5'-region of mRNA. A producer strain, E. coli BL21[DE3]/pET28a-AsnSYN was created, providing an accumulation of up to 291 ± 9 mg/L of enzymatically active protein (44.5 ± 2.6 mg/(L AU)) when cultivated in stirred flasks, which is 50% higher than the control strain with a natural gene at an induction time of 3 hours. Optimization of the codon composition of the gene made it possible to increase the energy of secondary mRNA structure formation in the 5'-region from –70 to –47 kcal/mol, which, as we assume, contributed to improved translation efficiency. For the developed producer strain, asparaginase that meets pharmacopoeial purity requirements can be obtained with a total yield of ≥25% and a specific activity of >250 IU/mg. The preparation contains no visible impurities according to electrophoresis data and less than 3% multimeric forms according to size exclusion chromatography data. The results obtained demonstrate the promise of using synthetic genes with optimized DNA structure for the industrial production of therapeutic enzymes.