Heterologous Expression of Inulinase Gene in Bacillus licheniformis 24 for 2,3-Butanediol Production from Inulin

Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-fi-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli Stellar (TM) and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme's function. During flaskbatch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the monoand disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF).