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Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.

upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs. Keywords: gene expression/genome analysis/mRNA/ Saccharomyces cerevisiae/stress responses IntroductionThe availability of the complete genome sequence of the eukaryotic microorganism, Saccharomyces cerevisiae (Goffeau et al., 1996) has allowed researchers to monitor gene transcription on a global (or genome-wide) scale for the ®rst time. The resulting pro®les de®ne the complete set of mRNA molecules (the transcriptome; Velculescu et al., 1997) present in the yeast cell under a given set of physiological or developmental conditions (Oliver, 1997). Massively parallel analytical procedures are used in transcriptome analysis that involve the hybridization of labelled mRNA or cDNA molecules to arrays of`target' molecules representing all of the~6000 protein-encoding genes de®ned by the yeast genome (Mewes et al., 1997). These targets may be either oligonucleotides (Wodicka et al., 1997) or PCR products (Lashkari et al., 1997;Hauser et al., 1998) fabricated in either`micro' (on glass slides or chips; Lashkari et al., 1997;Wodicka et al., 1997) or`macro' (on nylon or polypropylene membranes; Hauser et al., 1998) formats. The mRNA or cDNA hybridization probes may be labelled either radioactively (usually with 33 P; Hauser et al., 1998) or¯uorescently (usually with Cy5 or Cy3; Winzeler et al., 1999). Whatever the experimental protocol employed, all transcriptome analyses using hybridization arrays have in common that they produce massive amounts of data that have to be`mined', using computational techniques, in order to extract meaningful biological information. A number of algorithms have been developed Brown et al., 2000;Kell and King, 2000) to permit the comparison of the transcription patterns of all 6000 protein-encoding genes in different physiological conditions or throughout a time course of development (Cho et al., 1998;Chu, 1998;Spellman et al., 1998) or physiological adaptation . While these algorithms are effective in clustering together genes that show similar patterns of regulation, it is clear that the composition of any particular cluster is enormously sensitive to the thresholds set either for transcript detection or for a signi®cant level of regulation, and thus to the way in which the data have been normalized or otherwise processed.Because of these concerns about data processing, it is important that we make use of existing biological knowledge in mining hybridization array data. This may be done in two ways, either empirically (e.g. by adjusting threshold levels until genes already known to be co-regulated are clustered together) or, more formally, by using supervised methods of machine learning (Brown et al., 2000;Kell and King, 2000). Whatever approach is used, there is the problem that the prior knowledge has been gained using a di...

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A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.

BackgroundCold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.ResultsA gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures.ConclusionBy taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.

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