A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material.

BACKGROUND: Komagataella phaffii (Pichia pastoris) is a methylotrophic 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 in K. phaffii could 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. RESULTS: Here, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. 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 express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed 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, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. CONCLUSIONS: We have added K. phaffii to 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 of K. phaffii without compromising its genome or weakening the resulting strain.

Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Murine Factor H Co-Produced in Yeast With Protein Disulfide Isomerase Ameliorated C3 Dysregulation in Factor H-Deficient Mice.

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome.

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

Interplay of a ligand sensor and an enzyme in controlling expression of the Saccharomyces cerevisiae GAL genes.

The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins--a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)--control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

Immunoaffinity purification of the class V chitin synthase of Wangiella (Exophiala) dermatitidis.

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

Cytolocalization of the class V chitin synthase in the yeast, hyphal and sclerotic morphotypes of Wangiella (Exophiala) dermatitidis.

Wangiella (Exophiala) dermatitidis is a polymorphic fungus that produces polarized yeast and hyphae, as well as a number of non-polarized sclerotic morphotypes. The phenotypic malleability of this agent of human phaeohyphomycosis allows detailed study of its biology, virulence and the regulatory mechanisms responsible for the transitions among the morphotypes. Our prior studies have demonstrated the existence of seven chitin synthase structural genes in W. dermatitidis, each of which encodes an isoenzyme of a different class. Among them, the class V chitin synthase (WdChs5p) is most unique in terms of protein structure, because it has an N-terminal myosin motor-like domain with a P-loop (MMD) fused to its C-terminal chitin synthase catalytic domain (CSCD). However, the exact role played by WdChs5p in the different morphotypes remains undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at 37 degrees C and consequently virulence. This report describes the expression in Escherichia coli of a 12kDa polypeptide (WdMyo12p) of WdChs5p, which was used to raise in rabbits a polyclonal antibody that recognized exclusively its MMD region. Results from the use of the antibody in immunocytolocalization studies supported our previous findings that WdChs5p is critically important at infection temperatures for maintaining the cell wall integrity of developing yeast buds, elongating tips of hyphae, and random sites of expansion in sclerotic forms. The results also suggested that WdChs5p localizes to the regions of cell wall growth in an actin-dependent fashion.

New biosynthetic step in the melanin pathway of Wangiella (Exophiala) dermatitidis: evidence for 2-acetyl-1,3,6,8-Tetrahydroxynaphthalene as a novel precursor.

The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.

Structural relationships among the ribosomal stalk proteins from the three domains of life.

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.

Molecular cloning and characterization of WdTUP1, a gene that encodes a potential transcriptional repressor important for yeast-hyphal transitions in Wangiella (Exophiala) dermatitidis.

The general transcriptional repressor Tup1p is known to influence cell development in many fungi. To determine whether the Tup1p ortholog (WdTup1p) of Wangiella dermatitidis also influences cellular development in this melanized, polymorphic human pathogen, the gene (WdTUP1) that encodes this transcription factor was isolated, sequenced and disrupted. Phylogenetic analysis showed that the WdTup1p sequence was closely related to homologues in other polymorphic, conidiogenous fungi. Disruption of WdTUP1 produced mutants (wdtup1Delta) with pronounced growth and cellular abnormalities, including slow growth on various agar media and exclusively as a filamentous morphotype in liquid media. We concluded that WdTup1p represents an important switch regulator that controls the yeast-to-filamentous growth transition. However, detailed observations of the filamentous growth of the disruption mutant showed that the hyphae produced by the wdtup1Delta mutants, unlike those of the wild-type, were arrested at a stage prior to the formation of true hyphae and subsequent conidia production.

Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins.

The ribosome has a distinct lateral protuberance called the stalk; in eukaryotes it is formed by the acidic ribosomal P-proteins which are organized as a pentameric entity described as P0-(P1-P2)(2). Bilateral interactions between P0 and P1/P2 proteins have been studied extensively, however, the region on P0 responsible for the binding of P1/P2 proteins has not been precisely defined. Here we report a study which takes the current knowledge of the P0 - P1/P2 protein interaction beyond the recently published information. Using truncated forms of P0 protein and several in vitro and in vivo approaches, we have defined the region between positions 199 and 258 as the P0 protein fragment responsible for the binding of P1/P2 proteins in the yeast Saccharomyces cerevisiae. We show two short amino acid regions of P0 protein located at positions 199-230 and 231-258, to be responsible for independent binding of two dimers, P1A-P2B and P1B-P2A respectively. In addition, two elements, the sequence spanning amino acids 199-230 and the P1A-P2B dimer were found to be essential for stalk formation, indicating that this process is dependent on a balance between the P1A-P2B dimer and the P0 protein.

Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans.

In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit-the so-called stalk-structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal P-proteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and two-hybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B-P2A and P1A-P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins.

Non-AUG translation initiation of mRNA encoding acidic ribosomal P2A protein in Candida albicans.

The eukaryotic 60S ribosomal subunit has a set of very acidic proteins (P-proteins), which form a distinct lateral protuberance called the stalk structure. This protein complex is directly involved in the elongation step of polypeptide synthesis. In our study on acidic ribosomal P-proteins from the human opportunistic pathogen Candida albicans, we isolated and characterized one of the genes, called CARP2A, and its product, the P2A protein. The CARP2A gene is intron-less, present in a single copy per haploid genome, and transcriptionally active. The open reading frame of the studied gene contains information for a sequence of 108 amino acids. Based on this, the molecular mass and isoelectric point of the P2A protein were theoretically calculated to be 10.85 kDa and 3.7, respectively. The characteristic feature of the CARP2A gene transcript is the presence of a GUG start codon, which is rare in eukaryotic organisms and not previously reported in yeast. To our knowledge this is the first report showing the presence of a naturally occurring non-AUG start codon on mRNA in yeast species.