Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.The ability to utilize methanol as a carbon and energy source is limited in eukaryotes to a few yeast species (1,34,57). The metabolic pathway is nearly identical in each species and begins with the oxidation of methanol to formaldehyde, which is catalyzed by the peroxisomal matrix enzyme alcohol oxidase (Aox). A by-product of this reaction is hydrogen peroxide, which is subsequently degraded to water and oxygen by a second peroxisomal enzyme catalase (Cat). The formaldehyde generated by Aox follows one of two paths. A portion leaves the peroxisome and is further oxidized by two cytoplasmic enzymes, formaldehyde dehydrogenase (Fld) and formate dehydrogenase (Fdh), to generate energy for the cell. The remaining formaldehyde is condensed with xylulose-5-phosphate by a third peroxisomal enzyme, dihydroxyacetone synthase (Dhas), to generate two three-carbon molecules that leave the peroxisome and enter a cyclic pathway that regenerates xylulose-5-phosphate and produces one net molecule of glyceraldehyde-3-phosphate for every three turns of this cycle (1, 57).Because three of the methanol pathway enzymes (Aox, Cat, and Dhas) are peroxisomal, the function of this organelle is also essential for methanol growth (21,26,33). This observation has made Pichia pastoris a major model system for the elucidation of peroxisome biogenesis and function (2,40,49). One advantage of P. pastoris for peroxisome studies is that in addition to methanol uti...

No abstract

Strains of the species Komagataella phaffii are the most frequently used “Pichia pastoris” strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76 kbp between two previous gaps on chromosome 1 and another 134 kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5 kbp found in some strains of P. pastoris.

Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5-6% of selected clones and had a proportional increase in recombinant protein.

SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.