A supernumerary synthetic chromosome inKomagataella phaffiias a repository for extraneous genetic material

Abstract

ABSTRACTBackgroundKomagataella phaffii (Pichia pastoris) is a methylotropic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes inK. phaffiicould offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers.ResultsHere, we describe a linear “nano”chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists inK. phaffiiover many generations with a copy number per cell of one, provided non-homologous end joining is compromised (byKU70-knockout). The nanochromosome includes a copy of the centromere fromK. phaffiichromosome 3, aK. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair ofK. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to expressPDI, a gene encoding protein disulfide isomerase. Co-expression withPDIallowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, bothPDIand a gene for GFP-tagged human complement factor H under the control ofPAOX1and demonstrated that the secreted protein was active as a regulator of the complement system.ConclusionsWe have addedK. phaffiito the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering ofK. phaffiiwithout compromising its genome or weakening the resulting strain.