Fig. 1 Electrophoretogram of PCR products used for the construction of hexokinase（HxkA）and pyruvate kinase（PkiA）expressed A. niger strains A:PCR products of DNA sequences of hexokinase gene HxkA and pyruvate kinase gene PkiA. M:Trans 8K DNA marker. 1:HxkA gene. 2:PkiA gene. B:Genome PCR verification of recombinant expressing A. niger strain AnG1-HxkA and AnG1-PkiA. M:Trans 8K DNA marker. NC:Negative control. 1-5:The genome PCR verification of the transformants of AnG1-HxkA. 6-10:The genome PCR verification of the transformants of AnG1-PkiA

Fig.2 SDS-PAGE of the recombinant GST-fused HxkA and PkiA proteins before and after protein purification by affinity chromatography A:SDS-PAGE of GST-HxkA before and after affinity chromatography. M:Protein marker. 1:SDS-PAGE of GST-HxkA before affinity chromatography. 2:SDS-PAGE of recombinant GST-HxkA after affinity chromatography. B:SDS-PAGE of GST-PkiA before and after affinity chromatography. M:Protein marker. 1:SDS-PAGE of GST-PkiA before affinity chromatography. 2:SDS-PAGE of GST-PkiA after affinity chromatography

Fig. 3 Effects of metabolites on the activities of HxkA（A）and PkiA（B）in A. niger * P<0.05,**P<0.01

Fig.4 Effects of key metabolites under different concent-rations on the activities of HxkA（A）and PkiA（B）in A. niger The dotted lines refer to changes in enzyme activity in the presence of small molecule metabolites with activation at different concentrations. Solid lines refer to changes in enzyme activity in the presence of small molecule metabolites with inhibition at different concentrations

Fig.5 Multiple sequence alignment of HxkA and PkiA from A. niger A:Multiple sequence alignment of HxkA from A. niger with hexokinases from different species,the substrate binding sites of hexokinases are labeled with red stars.B:Multiple sequence alignment of PkiA from A. niger with pyruvate kinases from different species,the catalytic active sites of pyruvate kinases are labeled with red stars

Fig.6 Simulated 3D structure results of HxkA（A）and PkiA（B）from A. niger based on homologous modeling A:Protein structures of 2 sub-units of HxkA in A. niger shown in blue and green;the catalytic active sites of HxkA were labeled in red. B:Protein structures of 4 sub-units of PkiA shown in blue,green,purple and yellow;the catalytic active sites of PkiA are labeled in red

Fig.7 Molecular docking simulation of HxkA with key metabolites A-D:Molecular docking simulation of HxkA with fructose 6-phosphate,PEP,ADP,and ATP,respectively. The hydrogen bonds between the amino acids and key metabolites are represented as yellow dash lines,the hydrogen bond distances are represented in red numbers

Fig.8 Molecular docking simulation of PkiA and key metabolites A-D:Molecular docking simulation of PkiA with ADP,fructose 1,6-diphosphate,malic acid,and fumaric acid,respectively. The hydrogen bonds between the amino acids and key metabolites are represented as yellow dash lines;the hydrogen bond distances are represented in red numbers.