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

Abstract

BackgroundState-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.MethodsWith the GoldenPiCS 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).ResultsAll 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.ConclusionsGoldenPiCS, 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. GoldenPiCS vectors are available at Addgene.Electronic supplementary materialThe online version of this article (10.1186/s12918-017-0492-3) contains supplementary material, which is available to authorized users.

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

“…eGFP was used to screen for the AOX1 promoter expression levels. The linearized vector ( Asc I) BB3aN_pAOX1_eGFP_CycTT was transformed into the AOX1 terminator (Prielhofer et al, 2017) and selected on YPD plates with 100 ug/mL nourseothricin. For secreted protein production, the Mut S and Mut − strains were transformed with the pPM2pN21_pAOX1_HSAopt_CycTT vector (Mut − P AOX1 HSA & Mut S P AOX1 HSA), carrying codon‐optimized human serum albumin (HSA) with the native secretion sequence (Puxbaum, Gasser, & Mattanovich, 2016) and the pPM2pZ30_pAOX1_αMF‐vHH_CycTT vector (Mut − P AOX1 vHH and Mut S P AOX1 vHH) carrying a codon‐optimized variable region of a camelid antibody (vHH) with a Saccharomyces cerevisiae α‐mating‐type secretion sequence.…”

A synthetic Calvin cycle enables autotrophic growth in yeast

“…All expression cassettes and sgRNA / hCas9 plasmids were constructed by Golden Gate cloning 22,27,28 . Three native genes of P. pastoris ( AOX1 , DAS1 and DAS2 ) were replaced by the coding sequences of the genes listed in Supplementary Table 4.…”

“…alcohol oxidase 1 (Aox1)). Powerful genetic tools including CRISPR/Cas9 mediated techniques are available for P. pastoris 17,21,22 allowing to re-build and integrate entire metabolic pathways into this host without the use of integrative selection markers. In this work, we use P. pastoris as a chassis cell to de novo engineer a synthetic CBB cycle and to test its capability to promote autotrophic growth on CO 2 .…”

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

“…Golden Gate cloning is also frequently used for assembly of CRISPR gRNA expression arrays for site‐specific mutagenesis in a variety of organisms (McCarty, Shaw, Ellis, & Ledesma‐Amaro, 2019; Vad‐Nielsen, Lin, Bolund, Nielsen, & Luo, 2016). Finally, Golden Gate cloning has led to the development of the modular cloning system MoClo (Engler et al., 2014; Kowarschik, Hoehenwarter, Marillonnet, & Trujillo, 2018; Weber, Engler, et al., 2011; Werner et al., 2012) and of several other related modular cloning systems (Agmon et al., 2015; Andreou & Nakayama, 2018; Binder et al., 2014; Larroude et al., 2019; Lee, DeLoache, Cervantes, & Dueber, 2015; Martella, Matjusaitis, Auxillos, Pollard, & Cai, 2017; Moore et al., 2016; Occhialini et al., 2019; Pollak et al., 2019; Prielhofer et al., 2017; Sarrion‐Perdigones et al., 2011, 2013).…”