Metabolic Engineering of Pediococcus acidilactici BD16 for Heterologous Expression of Synthetic alaD 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 Pediococcus acidilactici BD16 (alaD+). Computational biology tools assisted the designing of a synthetic alaD 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 P. acidilactici BD16 (alaD+) strain was carried out using fed-batch fermentation under oxygen depression conditions, which significantly enhanced L-alanine levels. The recombinant strain expressing the synthetic alaD 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 P. acidilactici BD16 (alaD+) is suggested as the best alternative to chemical-based commercial synthesis of L-alanine for potential industrial applications.

Protein Engineering of a Pyridoxal-5′-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for β-Alanine Production

In the present study, a pyridoxal-5′-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce β-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of β-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Physiological and biochemical effects of morphactin IT 3233 on callus and tumour tissues of Nicotiana tabacum L. cultured in vitro III. Transamination processes catalysed by aminotransferase L-alanine: 2-oxoglutarate

An active alanine transaminase was found both in callus and tumour tissues of tobacco. The enzyme is more active in the latter tissue, and the reaction balance is strongly shifted towards alanine production, while in callus tissue towards glutamic acid formation. Morphactin applied to the tissue cultures stimulates markedly the enzyme activity only in callus. A negative correlation was observed between the intensity of transamination processes and enhanced synthesis of proteins in the tissues studied. Morphactin disturbs nitrogen metabolism in the callus tissue. Tumour tissue is more resistant to the action of this substance. The different hormonal activities in these tissues may be the cause of the different effects of morphactin.