Pichia pastoris is an established host system for heterologous protein expression. However, the potential productivity of this system can be limited. In this study, the Escherichia coli chaperones (GroES-GroEL) were expressed from the P-GAP promoter and targeted to the secretory pathway through the endoplasmic reticulum (ER). The ability of the ER targeted chaperones to improve production of bacterial protein in P. pastoris was evaluated. The chaperones tagged with alpha-factor secretion- and ER retention-signal sequences were co-expressed with either extracellularly secreted phytase or intracellular D-phenylglycine aminotransferase (D-PhgAT) enzymes. The ER residing GroEL-GroES successfully increased the levels of active phytase extracellularly, 1.5-23-fold higher than the phytase expression alone, but did not enhance the formation of active, intracellular D-PhgAT. These results indicate that the chaperones have the potential to enhance production of active enzymes when present in the same trafficking pathway. This is the first report on the improvement of extracellular bacterial protein production through co-expression with ER residing bacterial chaperones in the Pichia system. The modified P. pastoris expression system may be beneficial for extracellular expression of other prokaryotic proteins. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.