Directed evolution of bacterial alanine racemases with higher expression level

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme crucial for interconverting L-alanine and D-alanine.
• Alanine racemases are categorized into biosynthetic (low activity) and catabolic (high activity) types, with some reacting with serine.
• Engineering these enzymes can enhance their catalytic properties for biotechnological applications.

Purpose of the Study:

• To improve the catalytic activity and expression of bacterial alanine racemase through DNA shuffling.
• To construct a diverse chimeric gene library of alanine racemase genes from *Escherichia coli* and *Salmonella typhimurium*.
• To screen for engineered racemases with enhanced activity towards both alanine and serine.

Main Methods:

• DNA shuffling was employed to create a chimeric gene library from two biosynthetic alanine racemase genes.
• An *Escherichia coli* serine auxotroph was transformed with the shuffled genes and screened on media with D-serine.
• Recombinant clones were isolated, and the specific activities and expression levels of chimeric racemases were analyzed.

Main Results:

• Three independent clones with enhanced racemase activity were successfully isolated.
• Specific activities of chimeric racemases increased up to threefold compared to parental enzymes.
• The best chimera, Ser15 racemase, exhibited twofold higher protein expression, achieved posttranscriptionally.

Conclusions:

• DNA shuffling is an effective strategy for engineering bacterial alanine racemase with improved catalytic activity and expression.
• The identified chimeric racemases demonstrate potential for enhanced biocatalytic applications.
• Posttranscriptional regulation plays a role in the high protein expression of engineered alanine racemases.