Margarita Díaz

The Schizosaccharomyces pombe cwg2+ gene codes for the beta subunit of a geranylgeranyltransferase type I required for beta-glucan synthesis.

The product of the Schizosaccharomyces pombe cwg2+ gene is involved in the biosynthesis of beta‐D‐glucan. When grown at the non‐permissive temperature, cwg2‐1 mutant cells lyse in the absence of an osmotic stabilizer and display a reduced (1‐3) beta‐D‐glucan content and (1‐3) beta‐D‐glucan synthase activity. The cwg2+ gene was cloned by the rescue of the cwg2‐1 mutant phenotype using an S. pombe genomic library and subsequently verified by integration of the appropriate insert into the S. pombe genome. Determination of the nucleotide sequence of this gene revealed a putative open reading frame of 1065 bp encoding a polypeptide of 355 amino acids with a calculated M(r) of 40,019. The cwg2+ DNA hybridizes to a main transcript, the 5′ end of which maps to a position 469 bp upstream of the predicted start of translation. The sequence between the transcription and the translation start sites is unusually long and has several short open reading frames which suggest a translational control of the gene expression. Comparative analysis of the predicted amino acid sequence shows that it possesses significant similarity to three Saccharomyces cerevisiae proteins, encoded by the DPR1/RAM1, CDC43/CAL1 and ORF2/BET2 genes respectively, which are beta subunits of different prenyltransferases. When grown at 37 degrees C, cwg2‐1 mutant extracts were specifically deficient in geranylgeranyltransferase type I activity, as measured in vitro. Multiple copies of the CDC43 gene can partially suppress the growth and (1‐3) beta‐D‐glucan synthase defect of the cwg2‐1 mutant at the restrictive temperature. In a similar manner, the cwg2+ gene can partially suppress the cdc43‐2 growth defect. These results indicate that cwg2+ is the structural gene for the beta subunit of geranylgeranyltransferase type I in S. pombe and that this enzyme is required for (1‐3) beta‐D‐glucan synthase activity. The functional homology of Cwg2 with Cdc43, which has been implicated in the control of cell polarity, suggests a link between two morphogenetic events such as establishment of cell polarity and cell wall biosynthesis.

Expression of the genes coding for the xylanase Xys1 and the cellulase Cel1 from the straw-decomposing Streptomyces halstedii JM8 cloned into the amino-acid producer Brevibacterium lactofermentum ATCC13869.

Abstract. The xylanase (xysA) and the cellulase (celA1) genes from Streptomyces halstedii JM8 were cloned into Escherichia coli/Brevibacterium lactofermentum shuttle vectors and successfully expressed in both hosts when placed downstream from the kanamycin resistance promoter (Pkan) from Tn5 but not when under the control of their own promoters. Xylanase was secreted into the culture media of B. lactofermentum by removal of the same leader peptide as is removed in S. halstedii. The main difference between the production of xylanase by Streptomyces and corynebacteria was the low level of processing of the mature extracellular xylanase by B. lactofermentum, probably due to the lack of protease activity in this microorganism.

Posttranslational processing of the xylanase Xys1L from Streptomyces halstedii JM8 is carried out by secreted serine proteases.

The xylanase Xys1L from Streptomyces halstedii JM8 is known to be processed extracellularly, to produce a protein of 33.7 kDa, Xys1S, that retains catalytic activity but not its cellulose-binding capacity. This paper demonstrates that at least five serine proteases isolated from Streptomyces spp. have the ability to process the xylanase Xys1L. The genes of two of these extracellular serine proteases, denominated SpB and SpC, were cloned from Streptomyces lividans 66 (a strain commonly used as a host for protein secretion), sequenced, and overexpressed in S. lividans; both purified proteases were able to process Xys1L in vitro. Three other previously reported purified Streptomyces serine proteases, SAM-P20, SAM-P26 and SAM-P45, also processed Xys1L in vitro. The involvement of serine proteases in xylanase processing-degradation in vivo was demonstrated by co-expression of the xylanase gene (xysA) and the gene encoding the serine protease inhibitor (SLPI) from S. lividans. Co-expression prevented processing and degradation of Xys1L and resulted in a threefold increase in the xylanase activity present in the culture supernatant. SpB and SpC also have the capacity to process other secreted proteins such as p40, a cellulose-binding protein from S. halstedii JM8, but do not have any clear effect on other secreted proteins such as amylase (Amy) from Streptomyces griseus and xylanase Xyl30 from Streptomyces avermitilis.

Expression of the pstS gene of Streptomyces lividans is regulated by the carbon source and is partially independent of the PhoP regulator.

BackgroundPstS is a phosphate-binding lipoprotein that is part of the high-affinity phosphate transport system. Streptomyces lividans accumulates high amounts of the PstS protein in the supernatant of liquid cultures grown in the presence of different carbon sources, such as fructose or mannose, but not in the presence of glucose or in basal complex medium.ResultsFunctionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell. Regulation of the pstS promoter was studied with Northern blot experiments, and protein levels were detected by Western blot analysis. We observed that the pstS gene was expressed in cultures containing glucose or fructose, but not in complex basal medium. Northern blot analyses revealed that the pst operon (pstSCAB) was transcribed as a whole, although higher transcript levels of pstS relative to those of the other genes of the operon (pstC, pstA and pstB) were observed. Deletion of the -329/-144 fragment of the pstS promoter, including eight degenerated repeats of a sequence of 12 nucleotides, resulted in a two-fold increase in the expression of this promoter, suggesting a regulatory role for this region. Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive pstS expression that was independent of this regulator. Thus, the PhoP-independent expression of the pstS gene makes this system different from all those studied previously.Conclusion1.- In S. lividans, only the PstS protein bound to the cell has the capacity to bind phosphate and transfer it there, whereas the PstS form accumulated in the supernatant lacks this capacity. 2.- The stretch of eight degenerated repeats present in the pstS promoter may act as a binding site for a repressor. 3.- There is a basal expression of the pstS gene that is not controlled by the main regulator: PhoP.

Structure of xylanase Xys1Δ from Streptomyces halstedii

Xylanases hydrolyze the β-1,4-linked xylose backbone of xylans. They are of increasing interest in the paper and food industries for their pre-bleaching and bio-pulping applications. Such industries demand new xylanases to cover a wider range of cleavage specificity, activity and stability. The catalytic domain of xylanase Xys1 from Streptomyces halstedii JM8 was expressed, purified and crystallized and native data were collected to 1.78u2005A resolution with an Rmerge of 4.4%. The crystals belong to space group P212121, with unit-cell parameters a = 34.05, b = 79.60, c = 87.80u2005A. The structure was solved by the molecular-replacement method using the structure of the homologue Xyl10A from Streptomyces lividans. In a similar manner to other members of its family, Xys1 folds to form a standard (β/α)8 barrel with the two catalytic functions, the acid/base and the nucleophile, at its C-xadterminal side. The overall structure is described and compared with those of related xylanases.

Characterization of cwl1+, a gene from Schizosaccharomyces pombe whose overexpression causes cell lysis

From a Schizosaccharomyces pombe genomic library we have isolated the gene cwl1+ that causes cell lysis when it is overexpressed in the absence of an osmotic stabilizer. Southern hybridization showed that cwl1+ exists as a single copy in the S. pombe genome. The cwl1+ gene nucleotide sequence revealed a putative open reading frame of 924 bp encoding a polypeptide of 308 amino acids with a calculated Mr of 27 000. The cwl1+ DNA hybridizes to a major RNA transcript of 1·5 kb whose 5′ end maps at a position 452 bp upstream from the predicted translation start. Comparison of the amino acid sequence with those included in the current databases, showed no significant similarity to any known sequences. Cells overexpressing the cwl1+ gene under the control of the S. pombe nmt inducible promoter displayed a reduced cell wall content, were unable to separate after division and lysed drastically in the absence of osmotic stabilizer. Disruption of the cwl1+ gene caused no noticeable phenotype. The sequence has been deposited in the EMBL data library under Accession Number X9445.

Xylan-binding xylanase Xyl30 from Streptomyces avermitilis: Cloning, characterization, and overproduction in solid-state fermentation

A DNA fragment from the lignocellulolytic actinomycete Streptomyces avermitilis CECT 3339 was cloned using a DNA probe from the xylanase gene xysA of Streptomyces halstedii. The nucleotide sequence analysis revealed two potential ORFs, xyl30 and hd30, encoding a deduced multimodular F/10 xylanase with a binding domain and a secreted glycoxyl hydrolase, respectively. In Streptomyces lividans carrying the subcloned DNA fragment, two xylanase activity bands with estimated molecular masses of 42.8 and 35 kDa (named Xyl30 forms h and l, respectively), were detected by zymograms and SDS-PAGE. The two xylanases had identical N-terminal sequences, suggesting that Xyl30 l derived from Xyl30 h by C-terminal processing in the culture supernatant. No transcripts of hd30 were detected by RT-PCR. Characterization of the partially purified Xyl30 h confirmed the presence of a modular endoxylanase containing a xylan-binding domain, which after processing in the culture supernatant loses the aforementioned domain and thus its capacity to bind xylan (Xyl30 l). Xyl30 h achieved maximal activity at pH 7.5 and 60 degrees C, retaining more than 50% of its activity from pH 3 to 9 and more than 40% after a 1-h incubation at 70 masculineC. Moreover, in the recombinant host strain up to 400 U xylanase/g medium (dry weight) was produced in solid-state fermentation (SSF) using cereal bran as substrate. The high production yields of this enzyme and its biochemical features make it a good candidate for use in industrial applications.

Morphological and physiological changes in Streptomyces lividans induced by different yeasts.

Abstract. Streptomyces development is a complex process that eventually finishes with the formation of individual unigenomic spores from the aerial hyphae. Intraspecific and interspecific signals must play a key role in triggering or blocking this process. Here we show that interaction between two types of microorganisms, Streptomyces and yeasts, leads to alteration of the Streptomyces developmental program. This alteration is due to the action of invertase produced by the yeast on the sucrose present in the culture media, making glucose and fructose readily available for growth.

Uncovering production of specialized metabolites by Streptomyces argillaceus: Activation of cryptic biosynthesis gene clusters using nutritional and genetic approaches

Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.

Designing continuous flow reaction of xylan hydrolysis for xylooligosaccharides production in packed-bed reactors using xylanase immobilized on methacrylic polymer-based supports

The present study focuses on the development and optimization of a packed-bed reactor (PBR) for continuous production of xylooligosaccharides (XOS) from xylan. For this purpose, three different methacrylic polymer-based supports (Relizyme R403/S, Purolite P8204F and Purolite P8215F) activated with glyoxyl groups were morphologically characterized and screened for the multipoint covalent immobilization of a xylanase. Based on its physical and mechanical properties, maximum protein loading and thermal stability, Relizyme R403/S was selected to set up a PRB for continuous production of XOS from corncob xylan. The specific productivity for XOS at 10u202fmL/min flow rate was 3277 gXOS genzyme-1u202fh-1 with a PBR. This PBR conserved >90% of its initial activity after 120u202fh of continuous operation.