The methylotrophic yeast Pichia pastoris is widely used as an expression host for heterologous protein production using methanol as a sole carbon source. Slow growth rate and low specific productivity in methanol are, however, bottlenecks for its utility at industrial scale production. We have, therefore, developed a strategy for overcoming the bottlenecks and improving production of heterologous proteins. Four recombinant P. pastoris strains (Amy-AOX and Phy-AOX that produce alpha-amylase and phytase under only AOX promoter, and Amy-GAP-AOX and Phy-GAP-AOX that produce alpha-amylase and phytase under both AOX and GAP promoters) were generated. This has led to 1.8- and 1.3-fold improvement in alpha-amylase and phytase production in mixed fed batch cultivation as compared to that of Amy-AOX and Phy-AOX. The recombinant strains Phy-GAP-AOX integrated five copies of Phy (3 copies under AOX promoter and two copies under GAP promoter), while Phy-AOX integrated 3 copies of Phy under AOX promoter. On the other hand Amy-GAP-AOX integrated a total of 3 copies of Amy (one copy under AOX promoter and two copies under GAP promoter), while Amy-AOX integrated one copy under AOX promoter. The data suggest that this strategy would be useful in producing high titres of other heterologous proteins too in P. pastoris. (C) 2016 Elsevier Ltd. All rights reserved.