Metabolic Engineering of BD16 for Heterologous Expression of Synthetic Gene Cassette and L-Alanine Production in the Recombinant Strain Using Fed-Batch Fermentation

Metabolic engineering substantially aims at the development of more efficient, robust and industrially competitive microbial strains for the potential applications in food, fermentation and pharmaceutical industries. An efficient lab scale bioprocess was developed for high level fermentative production of L-alanine using metabolically engineered BD16 (). Computational biology tools assisted the designing of a synthetic gene cassette, which was further cloned in shuttle vector pLES003 and expressed using an auto-inducible P289 promoter. Further, L-alanine production in the recombinant BD16 strain was carried out using fed-batch fermentation under oxygen depression conditions, which significantly enhanced L-alanine levels. The recombinant strain expressing the synthetic gene produced 229.12 g/L of L-alanine after 42 h of fed-batch fermentation, which is the second highest microbial L-alanine titer reported so far. After extraction and crystallization, 95% crystal L-alanine (217.54 g/L) was recovered from the culture broth with an enantiomeric purity of 97%. The developed bioprocess using recombinant BD16 is suggested as the best alternative to chemical-based commercial synthesis of L-alanine for potential industrial applications.