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.
Highlights
The global market value of amino acids, especially the nonessential ones, has enhanced significantly over the years owing to their humungous industrial applications [1]
The most preferred route for microbial L-alanine synthesis from pyruvate via glycolytic pathway is facilitated by the alanine dehydrogenase enzyme (AlaDH), which catalyzes the reductive amination of pyruvate into L-alanine
L-alanine production using recombinant strains remained challenging due to the requirement of long fermentation hours, lesser stability, poor yield, less purity and co-product formation. This has led to the implementation of advanced strain improvement approaches, such as metabolic engineering, for the development of highly stable and robust L-alanine producing microbial strains capable of withstanding the fermentation pressures of large scale industrial fermenters
Summary
The global market value of amino acids, especially the nonessential ones, has enhanced significantly over the years owing to their humungous industrial applications [1]. L-Alanine, though a non-essential amino acid, has extensive food, pharmaceutical and veterinary applications [2,3] It is an FDA-approved food additive and nutritional supplement, mainly used as a low-calorie sweetener, a fat substitute and as an ingredient of therapeutic formulations for treating hypoglycemia, liver diseases, prostate hypertrophy and urea cycle disorders [4,5,6]. It is widely used as a hair and skin conditioning agent and as an ingredient in cosmetics and several personal care products [7,8]. The production of L-alanine in most of the previously reported native and recombinant microbial strains suffered from slow fermentation productivities, lesser purity and poor yield, due to the formation of racemic mixtures and co-products
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