The constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P-GAP), which is one of the benchmark promoters of Pichia pastoris, was analyzed in terms of putative transcription factor binding sites. We constructed a synthetic library with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected transcription factor (TF) genes were overexpressed or deleted to understand their roles on heterologous protein production. Using enhanced green fluorescent protein, an expression strength in a range between 0.35- and 3.10-fold of the wild-type P-GAP was obtained. Another model protein, recombinant human growth hormone was produced under control of selected promoter variants and 1.6- to 2.4-fold higher product titers were reached compared to wild-type P-GAP. In addition, a GAL4-like TF was found to be a crucial factor for the regulation of P-GAP, and its overexpression enhanced the heterologous protein production considerably (up to 2.2-fold compared to the parental strain). The synthetic P-GAP library generated enabled us to investigate the different putative transcription factors which are responsible for the regulation of P-GAP under different growth conditions, ergo recombinant protein production under P-GAP. Biotechnol. Bioeng. 2017;114: 2319-2327. (c) 2017 Wiley Periodicals, Inc.