Transfer of plasmid into the pentose-fermenting yeast Pachysolen tannophilus.

The pentose-fermenting yeast Pachysolen tannophilus can convert glucose and xylose in lignocellulosic hydrolysates to ethanol. However, it performs poorly in industrially relevant lignocellulosic hydrolysates containing mixed sugars and inhibitors. Efforts have been directed at improving the performance of this yeast to enable efficient lignocellulosic biomass conversion. While some successes have been reported using random mutagenesis and/or hybridization-based approaches, further genetic improvement of this yeast is hampered by the lack of efficient gene transfer methods as well as limited genetic information to guide further construction of robust strains of P. tannophilus. In this study, we aimed to address this short-coming by establishing the optimal conditions needed for efficient gene transfer into P. tannophilus. We ascertained that plasmids can be transferred into P. tannophilus through trans-kingdom conjugation or lithium acetate (LiAc) transformation. The efficiency of plasmid YEp13 (2-micron, LEU2) transferred into a P. tannophilus leucine auxotroph (Leu-) reached as high as 1.93 × 10-2 transconjugants per input recipient and 3.25 × 104 transformants per µg plasmid DNA through trans-kingdom conjugation and transformation, respectively. In trans-kingdom conjugation, the number of recipient P. tannophilus cells played an important role, while the ratio of donor (Escherichia coli) to recipient cells was less important. For efficient transformation in P. tannophilus, the use of PEG 3350 was essential, as no transformants were obtained in its absence. The transformation efficiency increased with the addition of single-stranded carrier DNA and incubation at 30 °C for >60 min. Plasmids with different replication origins or 2-micron plasmids with different CUG codon-optimized antibiotic resistance markers were unable to transform P. tannophilus under our experimental conditions. The results are of interest in the genetic manipulation and improvement of P. tannophilus.

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response.

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to the hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.

Neuroendocrine modulation sustains the C. elegans forward motor state.

Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.

Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains.

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70-0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp-1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.

Deletion of hxk1 gene results in derepression of xylose utilization in Scheffersomyces stipitis.

A major problem in fermenting xylose in lignocellulosic substrates is the presence of glucose and mannose which inhibit xylose utilization. Previous studies showed that catabolite repression in some yeasts is associated with hexokinases and that deletion of one of these gene(s) could result in derepressed mutant strain(s). In this study, the hxk1 encoding hexokinase 1 in Scheffersomyces stipitis was disrupted. The ∆hxk1 SS6 strain retained the ability to utilize the main hexoses and pentoses commonly found in lignocellulosic hydrolysates as efficiently as the wild-type (WT) strain. SS6 also fermented the dominant sugars to ethanol; however, on xylose, the ∆hxk1 strain produced more xylitol and less ethanol than the WT. On mixed sugars, as expected the WT utilized glucose ahead of xylose and xylose utilization did not commence until all the glucose was consumed. In contrast, the ∆hxk1 mutant showed derepression in that it started to utilize xylose even when considerable glucose (about 1.72%, w/v) remained in the medium. Similarly, mannose did not repress xylose utilization by the ∆hxk1 mutant and xylose and mannose were simultaneously utilized. The results are of interest in efforts to engineer yeast strains capable of efficiently utilizing glucose and xylose simultaneously for lignocellulosic biomass conversion.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Comparative analysis of genes regulated by Dzip1/iguana and hedgehog in zebrafish.

BACKGROUND: The zebrafish genetic mutant iguana (igu) has defects in the ciliary basal body protein Dzip1, causing improper cilia formation. Dzip1 also interacts with the downstream transcriptional activators of Hedgehog (Hh), the Gli proteins, and Hh signaling is disrupted in igu mutants. Hh governs a wide range of developmental processes, including stabilizing developing blood vessels to prevent hemorrhage. Using igu mutant embryos and embryos treated with the Hh pathway antagonist cyclopamine, we conducted a microarray to determine genes involved in Hh signaling mediating vascular stability. RESULTS: We identified 40 genes with significantly altered expression in both igu mutants and cyclopamine-treated embryos. For a subset of these, we used in situ hybridization to determine localization during embryonic development and confirm the expression changes seen on the array. CONCLUSIONS: Through comparing gene expression changes in a genetic model of vascular instability with a chemical inhibition of Hh signaling, we identified a set of 40 differentially expressed genes with potential roles in vascular stabilization.

Sequence diversity and gene expression analyses of expansin-related proteins in the white-rot basidiomycete, Phanerochaete carnosa.

Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8-9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12-18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes.

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions) and site multiplicity (backup and status quo) might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P) N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A)-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A) content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A) is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A)-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have contributed to the A+T → G+C shift in genome-wide nucleotide composition during metazoan radiation.

Transcriptional profiling of Saccharomyces cerevisiae T2 cells upon exposure to hardwood spent sulphite liquor: comparison to acetic acid, furfural and hydroxymethylfurfural.

Global gene expression was analyzed in Saccharomyces cerevisiae T2 cells grown in the presence of hardwood spent sulphite liquor (HW SSL) and each of the three main inhibitors in HW SSL, acetic acid, hydroxymethyfurfural (HMF) and furfural, using a S. cerevisiae DNA oligonucleotide microarray. The objective was to compare the gene expression profiles of T2 cells in response to the individual inhibitors against that elicited in response to HW SSL. Acetic acid mainly affected the expression of genes related to the uptake systems of the yeast as well as energy generation and metabolism. Furfural and HMF mainly affected the transcription of genes involved in the redox balance of the cell. On the other hand, the effect of HW SSL on S. cerevisiae T2 cells was distinct and considerably more diverse as compared to the effect of individual inhibitors found in lignocellulosic hydrolysates. This is not surprising as HW SSL contains a complex mixture of inhibitors which may act synergistically. HW SSL elicited significant changes in expression of genes involved in diverse and multiple effects on several aspects of the cellular structure and function. A notable response to HW SSL was decreased expression of the ribosomal protein genes in T2 cells. In addition, HW SSL decreased the expression of genes functioning in the synthesis and transport of proteins as well as metabolism of carbohydrates, lipids, vitamins and vacuolar proteins. Furthermore, the expression of genes involved in multidrug resistance, iron transport and pheromone response was increased, suggesting that T2 cells grown in the presence of HW SSL may have activated pheromone response and/or activated pleiotropic drug response. Some of the largest changes in gene expression were observed in the presence of HW SSL and the affected genes are involved in mating, iron transport, stress response and phospholipid metabolism. A total of 59 out of the 400 genes differentially expressed in the presence of HW SSL, acetic acid, HMF and furfural, belonged to the category of poorly characterized genes. The results indicate that transcriptional responses to individual lignocellulosic inhibitors gave a different picture and may not be representative of how the cells would respond to the presence of all the inhibitors in lignocellulosic hydrolysates such as HW SSL.

Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

BACKGROUND: Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base) unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. CONCLUSIONS/SIGNIFICANCE: These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification.

Reverse transcription PCR (RT-PCR) is prone to false positives when contaminating DNA molecules are present at the start of a reaction. Contaminants that derive from earlier work using a given primer pair (carryover PCR products) are of particular concern when those primers are used routinely, as in clinical diagnostics or environmental monitoring. In addition, contamination by genomic DNA can significantly interfere with quantitative and qualitative analysis of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a method that can be used to neutralize carryover and genomic DNA contamination in RT-PCR studies. Restriction enzymes (REs) added to the amplification cocktail cleave contaminant DNA molecules while sparing the intended target nucleic acid. Restriction, reverse transcription, and amplification steps all take place in the same sealed vessel, thus avoiding any danger of recontamination. ConR eliminates carryover contamination in PCR without compromising target sequence amplification. Because the method is effective against both genomic and carryover contamination, it can be employed routinely in one-step RT-PCR, whatever the RNA target or the nature of the potential DNA contaminant. A variation of this decontamination method, amplicon primer site restriction (APSR), is effective specifically against carryover contamination. APSR, unlike ConR, can be applied during PCR-based amplification of DNA target molecules.