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Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids. Conclusion The multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5‐hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis‐folded protein. Additional stress‐related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains. [ABSTRACT FROM AUTHOR]

The Mo K-edge extended X-ray absorption fine structure (EXAFS) of Arabidopsis thaliana was presented in three different forms. These were oxidized as-isolated, reduced and oxidized after catalytic turnover of excess nitrate. The EXAFS Fourier transforms of oxidized as-isolated nitrate reductase and nitrate-oxidized nitrate reductase were then compared with that of oxidized sulfite oxidase. The EXAFS of all three species were dominated by two major interactions as shown by the transforms.