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Background  State-of-the-art strain engineering techniques for the host  Pichia pastoris  (syn.  Komagataella  spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in  P. pastoris . However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for  P. pastoris .    Methods  With the Golden Pi CS system, we provide a flexible modular system for advanced strain engineering in  P. pastoris  based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes).    Results  All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2–192% of the glyceraldehyde-3-phosphate dehydrogenase promoter  P    GAP  ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger “loop-out” of vector DNA from the  P. pastoris  genome.    Conclusions  Golden Pi CS, a modular Golden Gate-derived  P. pastoris  cloning system, is very flexible and efficient and can be used for strain engineering of  P. pastoris  to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. Golden Pi CS vectors are available at Addgene.    Electronic supplementary material  The online version of this article (10.1186/s12918-017-0492-3) contains supplementary material, which is available to authorized users.

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris