Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) is a novel oxygen-independent oxidoreductase. In this work, the PQQGDH gene was integrated into the chromosome of Escherichia coli EcNR2 and then modulated by multiplex-site in situ engineering to achieve improved substrate specificity and thermal stability. After four mutagenesis cycles executed within a single day, six PQQGDH variants were selected and characterized. The variants Ser231Lys/Asn452Thr or Ser231Lys showed higher stability relative to the wild-type enzyme. Additionally, the wild-type PQQGDH could only utilize maltose and lactose at 60% efficiency relative to glucose, while four variants (Ser231Cys/Asn452Thr, Ser231Lys/Asn452Thr, Asn452His, and Asn452Thr) showed significant reductions in their ability to utilize both maltose (30%) and lactose (45%). In particular, Ser231Cys/Asn452Thr and Ser231Lys/Asn452Thr possessed both improved substrate specificity and thermal stability compared to the wild-type PQQGDH. Our study provided a novel and efficient approach to simultaneously introduce multiplex mutations in situ, which is a potential strategy for target enzyme evolution in vivo. (C) 2016 Elsevier Ltd. All rights reserved.