Fig. 1
Construction of glutamate-inducible expression vectors and analysis of GFP expression in shake flask cultures of K. phaffii. A Key features of glutamate- and methanol-inducible expression vectors used in this study. Arrows indicate restriction sites used for linearization of the expression vectors for integration into the K. phaffii genome. Vector maps are provided in Additional file 1: Fig. S1. B Strategy for the generation of recombinant K. phaffii strains expressing GFP from PGDH2, PPEPCK and PAOX1 (Table 1). The gene encoding GFP was cloned downstream of PGDH2, PPEPCK and PAOX1 in expression vectors carrying different selection markers. Recombinant plasmids were linearized with SalI/Pme1 and transformed into K. phaffii GS115 for integration at the HIS4/AOXI locus as indicated. C GFP expression profile from PAOX1, PGDH2 and PPEPCK in cells cultured in YNB containing glycerol (YNBG), methanol (YNBM), glutamate (YNB-Glu) or MSG (YNB-MSG) by live cell confocal imaging. GFP expression was induced for 12 h. D Schematic representation of the strategy for purification of GFP from K. phaffii cell lysates by GST-tagged anti-GFP nanobody-mediated pull-down. E Comparative analysis of GFP expression from PAOX1, PGDH2 and PPEPCK in cells cultured in YNBM (PAOX1) and YNB-MSG (PGDH2 and PPEPCK). GFP was purified from whole cell lysates of cells equivalent to 50 A600 units using glutathione-S-transferase (GST)-tagged anti-GFP nanobodies. GFP bound to GST-tagged anti-GFP nanobodies was subjected to SDS‒PAGE, and proteins were visualized by Coomassie Brilliant Blue R staining (left panel). GFP expression was induced for 24 h. M, protein molecular weight markers (kDa). Right panel, quantitation of GFP bands in the gel by densitometric scanning using ImageJ. MSG-inducible GFP expression from PGDH2 and PPEPCK was normalized to methanol-inducible expression from PAOX1. F Strategy for the integration of pPEPCKA-GFP at the HIS4 locus of KpPPEPCKGFP to generate KpPPEPCKGFP*, in which the PPEPCK-GFP expression cassette is integrated at two genomic loci (PEPCK, HIS4). G Analysis of GFP bound to GST-tagged, anti-GFP nanobodies by SDS‒PAGE. GFP was purified from whole cell lysates of cells equivalent to 20 A600 units. The gel was stained with Coomassie Brilliant Blue R (left panel). GFP expression was induced for 24 h. M, protein molecular weight markers (kDa). Right panel, quantification of GFP in the gel by densitometric scanning using ImageJ. Error bars in the graphs denote the mean ± S.D. from three biological replicates (n = 3), and the p value obtained from Student’s t test is mentioned on the bar of each figure: *P < 0.05; **P < 0.005; ***P < 0.0005; ns, not significant