Display of α-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from starch

We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase l-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of l-glutamate. The N terminus of alpha-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of l-glutamate fermentation showed that the soluble starch was utilized to grow and produce l-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 +/- A 2.8 to 4.5 +/- A 0.7 g/l under non-inducing condition and from 50.0 +/- A 2.4 to 12.5 +/- A 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 +/- A 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-gamma-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of l-glutamate without any treatments.