F1 6bp

F1,6BP and Antifoam (polypropylene glycol P 2000) were from Sigma-Aldrich Chemie GmbH (Munich, Germany), IPTG was from PEQLAB Biotechnologie GmbH (Erlangen, Germany), Bradford solution was from Biorad and sodium dodecyl sulfate from SERVA Electrophoresis GmbH (Heidelberg, Germany). All other chemical were either from Carl Roth GmbH (Karlsruhe, Germany), Fluka (Taufkirchen, Germany), or Merck KGaA (Darmstadt, Germany) and were of the highest available purity. The crude glycerol was delivered from a biodiesel producer (ADM Hamburg, Germany) and contained 83.20% glycerol, 10.4% water, 6.4% NaCl, with less than 0.01% methanol, and no detectable traces of other organic compounds (according to supplier’s data sheet). All restriction enzymes used were from New England Biolabs (Frankfurt, Germany). The Pwo DNA Polymerase for gene amplification was from Roche Applied Science (Mannheim, Germany), Taq DNA polymerase used for screening-PCR was from Genaxxon Bioscience GmbH (Ulm, Germany).

2 x 10⁷ cells were used per sample. They were centrifuged (1,300 g, 10 min), washed with 1 x PBS and resuspended in 100 μl of SoTE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.15% Triton X-100, protease inhibitor cocktail (Roche)). After 20 min incubation at RT, samples were centrifuged (14,000 g, 10 min, 16° C) and supernatant collected. The reaction mixture containing 20 mM Tris pH 7.8, 10 mM MgCl₂, 1 mM NADP, 1 U PGI (Sigma-Aldrich), 1 U G6PDH, 100 μl cell extract was incubated at 30° C for 5 min prior to activity measurement. The reaction was triggered by addition of 5 mM F1,6bP (Sigma-Aldrich) immediately prior to measurement of NADPH production at 340 nm for 5 min at 30° C using a UV-1601 spectrophotometer (Shimadzu).

ADP, PEP, oxalate (OX), F16BP, F26BP, lactate dehydrogenase (LDH; rabbit muscle), polyethylene glycol (PEG) 8000, antibiotics and buffers were obtained from Sigma-Aldrich. NADH and EDTA-free protease inhibitor mixture tablets were from Roche, glycerol was from BDH Prolabo, IPTG was from Melford and salts were from Fisher Scientific. Restriction enzymes, vector and E. coli competent cells were from Novagen.
N-terminal His₆-tagged TcPYK was prepared as described previously [28 (link)]. Briefly, the expression of His₆-tagged TcPYK was achieved by the T7lac promoter-driven system in E. coli BL21 (DE3) cells after adding IPTG to a final concentration of 1 mM. Pure protein was obtained by immobilized metal ion affinity chromatography (IMAC) followed by gel-filtration chromatography. N-terminally His₆-tagged human pyruvate kinases (M1PYK and M2PYK) were expressed in E. coli BL21(DE3) cells and purified using IMAC and gel-filtration chromatography as described previously [16 (link)].