Recombinant production of acidophilic L-arabinose isomerase from Lentilactobacillus parakefiri in Bacillus subtilis
Year: 2025
Type: article
Abstract: BACKGROUND: The monosaccharide D-tagatose is a promising alternative to sucrose because of its similar sweetness and lower glycemic index. A novel L-arabinose isomerase (L-AI) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) has been biochemically characterized and used to isomerize D-galactose to D-tagatose in skim milk ultrafiltration permeate at pH 4.5 and 6.5. However, like most L-AIs described in the literature, this enzyme has only been produced recombinantly in Escherichia coli. This study aimed to systematically investigate the intracellular recombinant production of L-AI-Lp in Bacillus subtilis, which has qualified for a presumption of safety (QPS) designation from the European Food Safety Authority. RESULTS: The influence of four promoters on L-AI-Lp production in B. subtilis 007 was investigated in shake flask cultivations. Among these, the PAprE promoter yielded the highest volumetric L-AI activity of 69.2 ± 7.4 µkatGal, 65 °C/LCulture. The production yield was further increased to 147.7 ± 1.0 µkatGal, 65 °C/LCulture by using the nonsporulating, surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF, which was constructed by deleting sigF and sfp in B. subtilis 007. Furthermore, the influence of pH and dissolved oxygen (DO) on bioreactor cultivations of B. subtilis 007 ∆sfp ∆sigF was analyzed. In bioreactor cultivations, the highest L-AI activity of 88.6 ± 2.4 µkatGal, 65 °C/LCulture was measured under unregulated pH and low oxygen conditions (DO ≤ 5%), representing a 3.2-fold increase compared with previous recombinant production in E. coli. The L-AI-Lp was subsequently partially purified by heat treatment and precipitation methods, resulting in a 7.8-fold increase in specific activity to 128.2 nkatGal, 65 °C/mg and a yield of 84%. CONCLUSIONS: The L-AI-Lp was recombinantly produced for the first time in a microbial species with QPS status using the nonsporulating and surfactin-deficient strain B. subtilis 007 ∆sfp ∆sigF. The L-AI-Lp was subsequently partially purified via nonchromatographic methods, providing a basis for a low-cost downstream process. These results represent an important step toward potential industrial application of L-AI-Lp and highlight the potential of B. subtilis 007 ∆sfp ∆sigF as an expression host for the recombinant production of L-AIs compared with previously used hosts from the order Lactobacillales.
Source: Microbial Cell Factories
Authors: Nathanael Weber, Sebastian Götz, Jana Senger, Sabine Lutz-Wahl, Lutz Fischer
Language: English
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,590
Funder: Universität Hohenheim
OpenAlex ID: W7116666485