Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (P). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (P) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the P has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• P was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by P was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the P for driving antibiotic-resistant genes.