Figure 4

NeuNAc synthesis in vivo in the H. jecorina PEC/PSC1 strain. (a) EICs from the HPLC-MS analysis at 222.098 atomic mass units (amu) corresponding to the mass of the [GlcNAc+H]⁺ and [ManNAc +H]⁺ ions. Retention times (RTs) of ManAc (12.288 min) and GlcNAc (12.988 min) are indicated. (1) Chromatogram of the in vitro assay using GST fusion proteins of GlcNAc-2-epimerase and NeuNAc synthase. (2) Chromatogram of the cell-free extract of the PEC/PSC1 strain grown on GlcNAc. (3) Chromatogram of the cell-free extract of the parental strain grown on GlcNAc. (4) Chromatogram of the cell-free extract of the PEC/PSC1 strain grown on chitin. (b) EICs at 310.1134 amu corresponding to the mass of the [NeuNAc+H]⁺ ion. RT of NeuNAc (8.348 min) is indicated. (1) Chromatogram of the in vitro assay using GST fusion proteins of GlcNAc-2-epimerase and NeuNAc synthase. (2) Chromatogram of the cell-free extract of the PEC/PSC1 strain grown on GlcNAc showing 25-fold amplification compared to (1). (3) Chromatogram of the cell-free extract of the parental strain showing a 250-fold amplification compared to (1). (4) Chromatogram of the cell-free extract of the PEC/PSC1 strain after cultivation on chitin for 90 h showing a 25-fold amplification compared to (1). (ad 1 MS) and (ad 4 MS) are the MS spectra of chromatograms 1 and 4, respectively, at RTs of 8.348 min. (ad 4 MS/MS) is the MS/MS spectrum of chromatogram 3 at a RT of 8.348 min.