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In this review, a comprehensive discussion exclusively on bacterial xylanases; their gene organization; different factors and conditions affecting enzyme yield and activity; and their commercial application have been deliberated in the light of recent research findings and extensive information mining. Improved understanding of biological properties and genetics of bacterial xylanase will enable exploitation of these enzymes for many more ingenious biotechnological and industrial applications.

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K m 263 mM and V max 57 nM/mg/min, and PgAxt1p has K m 0.13 mM and V max 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K m 27 mM and V max 3.8 nM/mg/min, and PgAxt1p has K m 65 mM and V max 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K m 371 mM and V max 341 nM/mg/min for L-arabinose, and K m 25 mM and V max 76 nM/mg/ min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040

Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase. Developments over the past nearly 30 years have produced strains, vectors, and selection mechanisms that have continued to simplify and streamline heterologous protein expression in this fungus. More recent developments in fungal molecular biology have pointed the way toward a fundamental transformation in the ease and efficiency of heterologous protein expression in this important industrial host. Here, 1) we provide a historical perspective on advances in H. jecorina molecular biology, 2) outline host strain engineering, transformation, selection, and expression strategies, 3) detail potential pitfalls when working with this organism, and 4) provide consolidated examples of successful cellulase expression outcomes from our laboratory.

Background The objective of this study was to develop a consortium of effective microorganisms to hasten the composting process and to reduce the composting period. Results An efficient microorganism (EM) consortium was developed using Candida tropicalis (Y6), Phanerochaete chrysosporium (VV18), Streptomyces globisporous (C3), Lactobacillus sp. and enriched photosynthetic bacterial inoculum for rapid composting of paddy straw. Paddy straw was amended with poultry droppings to narrow down its C:N ratio for faster degradation. Composting was carried out in open pits with EM consortium and compared with compost inoculant (CI) consisting of Aspergillus nidulans (ITCC 2011), Trichoderma viride (ITCC 2211), Phanerochaete chrysosporium (NCIM 1073) and A. awamori (F-18). Changes in biochemical and physiochemical parameters like C:N ratio, pH, EC and humus were studied over a period of 60 days to test compost maturity and stability along with microbial and extracellular hydrolytic enzyme activities. Paddy straw amended with EM and CI hasten the composting process by bringing C:N ratio down to 15:1 and achieving a total humus content of 4.82 % within 60 days. High activity of hydrolytic enzyme carboxymethyl cellulase (CMCase) (0.43 IU/g) and microbial activity in terms of dehydrogenase (158.64 lg TPF/g/day) was observed in this treatment. The activity of xylanase was positively correlated (r = 0.987) with alkali-soluble carbon.Conclusion This study illustrates the importance of microbial bioaugmentation to hasten the composting process of paddy straw to produce quality compost.

The 4+ extrachromosomal determinant in the yeast Saccharomyces cerevisiae suppresses certain UAA markers and increases the efficiency of suppression of UAA suppressors and certain frameshift suppressors. Although the exact nature of 4+ determinant is unknown, it is believed to be a self-replicating cytoplasmic factor affecting some component of the translational machinery. In this report we describe growth conditions for efficient mutation or elimination of the 4+ determinant. Incubation of 4+ cultures in hypertonic nutrient medium resulted in rapid conversion to a culture containing predominantly 4-cells during the growth cycle. The kinetics of + to 4-conversion established that the occurrence of 4-cells was due to induction and not to selection of pre-existing 4-cells. The results suggest that the replication of the A+ determinant is sensitive to hypertonic conditions. Media. The routine nutrient medium contained 1% Bactoyeast extract, 2% Bacto-peptone, and 2% dextrose. The hypertonic media were prepared by supplementing the nutrient medium with potassium chloride or ethylene glycol. Glycerol medium consisted of 1% Bacto-yeast extract, 2% Bacto-peptone, and 3% (vol/vol) glycerol. Either 1% or 2% Noble agar was added if solidification was desired.Population Analysis. Freshly grown cells were inoculated into hypertonic media to an initial titer of approximately 106 cells/ml. The cultures were incubated at 300C with vigorous shaking. At various times samples were withdrawn, sonicated to disperse the cell clusters, and plated on nutrient medium after appropriate dilutions. In some experiments the cells were also plated on a nutrient medium containing 10% dextrose, a medium that intensifies the red coloration conferred by the ade2-1 marker (11). The nutrient plates were incubated at 30'C and the number and types of the resulting colonies were scored after 3-5 days. At least 5000-8000 colonies were scored when the 41 cells were relatively rare at early incubation times. Cell densities allowing approximately 200 colonies per plate were used for most experiments, whereas higher densities, allowing colonies per plate, were used for examining the cell types during the first 4 hr of incubation of the cultures in ethylene glycol. The numbers of 0-red colonies could be accurately counted over the high background of white colonies.In some experiments the growth in liquid hypertonic media was monitored by measuring the turbidity with a Klett-Summerson photoelectric colorimeter equipped with a no. 62 light filter.Phenotypes. The degree of suppression in strains carrying various UAA markers can be conveniently estimated from the growth on omission media and from the degree of coloration conferred by the ade2-1 marker. Although these properties can vary with different strains, the patterns usually observed are presented in Table 2