Ramón I. Santamaría

Myxococcus xanthus induces actinorhodin overproduction and aerial mycelium formation by Streptomyces coelicolor

Interaction of the predatory myxobacterium Myxococcus xanthus with the non‐motile, antibiotic producer Streptomyces coelicolor was examined using a variety of experimental approaches. Myxococcus xanthus cells prey on S. coelicolor, forming streams of ordered cells that lyse the S. coelicolor hyphae in the contact area between the two colonies. The interaction increases actinorhodin production by S. coelicolor up to 20‐fold and triggers aerial mycelium production. Other bacteria are also able to induce these processes in S. coelicolor though to a lesser extent. These studies offer new clues about the expression of genes that remain silent or are expressed at low level in axenic cultures and open the possibility of overproducing compounds of biotechnological interest by using potent inducers synthesized by other bacteria.

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.

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.

Promicromonospora kroppenstedtii sp. nov., isolated from sandy soil.

A new actinobacterial strain, RS16(T), was isolated from sandy soil collected in Zamora, Spain, and was studied to determine its taxonomic position. A neighbour-joining tree based on 16S rRNA gene sequence analysis revealed that the novel isolate formed an independent branch between Promicromonospora sukumoe DSM 44121(T) and Promicromonospora citrea DSM 43110(T). Levels of 16S rRNA gene sequence similarity between the novel isolate and its phylogenetic neighbours ranged from 98.7 to 98.9 %. Chemotaxonomic properties, such as the predominant menaquinone and polar lipids, supported the assignment of the novel isolate to the genus Promicromonospora, however, a significant number of physiological differences were found between the novel isolate and the other recognized species of the genus Promicromonospora. On the basis of these results, it is proposed that strain RS16(T) represents a novel species of the genus Promicromonospora, for which the name Promicromonospora kroppenstedtii sp. nov. is proposed. The type strain is RS16(T) (=DSM 19349(T)=LMG 24382(T)).

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.

Preparation of a robust immobilized biocatalyst of β-1,4-endoxylanase by surface coating with polymers for production of xylooligosaccharides from different xylan sources

Xylooligosaccharides display interesting prebiotic effects on human health. The endoxylanase Xys1Δ, from Streptomyces halstedii JM8, was immobilized and stabilized on glyoxyl-agarose beads by multipoint covalent attachment using a novel strategy based on surface coating with a multilayer of polymers. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymers and a layer of polyethylenimine. The optimized biocatalyst was 550-fold more stable than one-point covalent immobilized Xys1Δ (at 70u202f°C, pH 7). This biocatalyst was tested for the production of xylooligosaccharides from soluble xylans from various sources. Hydrolysis of beechwood, wheat straw and corncob xylans was 93% in 4u202fh, 44% in 5u202fh and 100% in 1u202fh, respectively. Maximum values of xylooligosaccharides were found for beechwood at 20.6u202fmg/mL, wheat at 12.5u202fmg/mL and corncob at 30.4u202fmg/mL. The optimized biocatalyst was reused for 15 reaction cycles without affecting its catalytic activity.

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.