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

Abstract

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.

Ascertaining the Influence of Lacto-Fermentation on Changes in Bovine Colostrum Amino and Fatty Acid Profiles

“…Alanine has extensive applications [63,[94][95][96][97][98][99] and can be synthesized by various microorganisms [94,[100][101][102][103][103][104][105]. However, some of the LAB strains can metabolize alanine to volatile compounds, and these reactions are initiated mainly by oxidative deamination and decarboxylation [106].…”

Phenolic Biotransformations in Wheatgrass Juice after Primary and Secondary Fermentation

“…Moreover, interesting exogenous genetic attributes can be added by employing advanced genetic engineering approaches, such as metabolic engineering or enzyme engineering, to further widen their prospects for food utilization [ 12 , 13 ]. Keeping this in mind, a natural chilly pickle isolate, namely Pediococcus acidilactici BD16 (MTCC 10973), was chosen for the expression of synthetic alaD gene cassette encoding alanine dehydrogenase enzyme (AlaDH) using pLES003 shuttle vector [ 14 , 15 ]. AlaDH is capable of catalysing reductive amination of pyruvate into L-alanine, which is an important food additive with distinct biomedical activities, such as anti-bacterial, anti-cancer and anti-urolithiatic [ 16 ].…”

Construction of a Temperature-Sensitive Shuttle Vector between Lactic Acid Bacteria and <i>Escherichia coli</i> and Its Application to a Tn<i>10</i>-Based Random Mutagenesis Tool in Lactic Acid Bacteria

“…To study the mechanisms of those desirable effects genetically, we have constructed a shuttle vector, named pLES003, that is replicable in both Escherichia coli and several LAB hosts, such as lactobacilli, enterococci, and pediococci, and carries an erythromycin resistance gene (ery) as a selective marker in those hosts. [8][9][10][11] This shuttle vector contains a theta (θ)-type replication system, which is preferable for use in a plasmid development because theta-type plasmids are more stably harbored than those constructed based on another type of replication system, the rolling circle replicating mechanism. 12) In a previous study, the shuttle vector pLES003 and its derivative pLES003-b, 13) the replication origin region of which is reversed from that of pLES003, have been used to analyze the functional gene and metabolic engineering.…”

“…12) In a previous study, the shuttle vector pLES003 and its derivative pLES003-b, 13) the replication origin region of which is reversed from that of pLES003, have been used to analyze the functional gene and metabolic engineering. 10,11,13) For a new application of our shuttle vector pLES003-b, the aim of the present study is to develop a temperature-sensitive mutant of this vector through the error-prone PCR technique. Furthermore, we have constructed a plasmid vector for a genetic engineering tool used in random mutagenesis on LABs using the thermo-sensitive mutation and transposon Tn10.…”