Enhancement of amino acid production and secretion by Lactococcus lactis using a droplet-based biosensing and selection system

• Published in: Metabolic engineering communications Vol. 11; p. e00133
• Main Authors: Hernandez-Valdes, Jhonatan A., aan de Stegge, Myrthe, Hermans, Jos, Teunis, Johan, van Tatenhove-Pel, Rinke J., Teusink, Bas, Bachmann, Herwig, Kuipers, Oscar P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2020; Elsevier
• Subjects: Amino acids; Biosensor; Droplet-technology; EMS; FACS; Full Length; Lactococcus lactis; Lactococcus lactis; Amino acids; Droplet-technology; EMS; FACS; Biosensor
• ISSN: 2214-0301; 2214-0301
• DOI: 10.1016/j.mec.2020.e00133

Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5–10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.
The development of a fluorescent sensor cell, able to grow only in the presence of essential amino acids, allows selection of strains with an improved capacity of amino acid production and secretion, by a droplet-based screening platform. [Display omitted]
•An amino acid-responsive biosensor was developed for fluorescent-based screening.•Production of amino acids in wild-type Lactococcus lactis strains was improved after selection of EMS-mutants using high-throughput droplet-based screening technology.•Mutations in genes encoding proteins of the proteolytic system of L. lactis improve amino acid secretion. For instance glutamate secretion was enhanced 10-fold and leucine up to 30-fold.•The identification of target genes is important to improve amino acid production in other lactic acid bacteria (LAB).