Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Enhancement of salinomycin production by ribosome engineering in Streptomyces albus

正Dear Editor,Streptomyces can produce a large variety of secondary metabolites as a major source of anti-infective, antitumor or immune-suppressive agents widely applied in clinical treatment. Antibiotics-resistant bacteria are spreading at alarming rates. Natural active products and their derivatives will play     

Production of alpha-amylase and xylanase by an alkalophilic strain of Penicillium griseoroseum RR-99

An alkalophilic strain of Penicillium sp. RR 99 was isolated that was found to synthesise extra-cellular alpha-amylase and xylanase, when cultivated in presence of starch and xylan respectively. The strain showed maximum alpha-amylolytic activity on 4th day and maximum xylanolytic activity on 6th day of cultivation. The ability of the strain to hydrolyse starchy and hemicellulosic wastes made the strain competent not only for the commercial production of these enzymes but also for successful utilization of wastes.     

链霉菌发酵麦草产木聚糖酶的试验研究

通过正交设计试验 ,找出利用链霉菌和麦草基质发酵生产木聚糖酶的试验条件。培养基 (g/L) :麦草粉 ,4 5 ;(NH4 ) 2 SO4 ,7.5 ;酵母膏 ,8;K2 HPO4 ·3H2 O ,1;MgSO4 ·7H2 O ,0 .5 ;NaCl,0 .3。接种量为 5 .0× 10 8个孢子 / 5 0mL培养基 ,振荡培养 (12 0r/min) 5d     

Production of cellulase and xylanase by a wild strain of Trichoderma viride

Summary The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and -gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of -glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of -glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and -glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.     

代谢工程：一项不断发展的菌株改造技术

对代谢工程的发展进行了简要回顾,分析了代谢工程发展的推动力,重点评述了本期专栏发表的12篇代谢工程和细胞工厂方面的论文。     

Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str(r)), gentamicin, or rifampin (Rif(r)). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str(r) mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif(r) mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase beta subunit). Increased productivity of salinomycin in the Str(r) mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg(2+) concentrations. We conclude that this aberrant protein synthesis ability in the Str(r) mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

Rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces clavuligerus

Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.     

Genome shuffling and ribosome engineering of Streptomyces virginiae for improved virginiamycin production

Bioprocess and Biosystems Engineering - The production of virginiamycin (VGM) from Streptomyces virginiae was improved by genome shuffling and ribosome engineering companied with a high-throughput...     

利用CRISPR-Cas9系统与核糖体工程获得新型可利霉素产生菌

可利霉素(Carrimycin,CAM)是将异戊酰基转移酶基因(Isovaleryltransferase gene,ist)导入到螺旋链霉菌中产生的以异戊酰螺旋霉素(Isovalerylspiramycin,ISP)为主组分的抗生素.原工程菌中的ist基因与螺旋霉素(Spiramycin,SP)生物合成基因簇相距较远...     

Properties of the germination inhibitor of Streptomyces viridochromogenes spores

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium-potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition, ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.     

Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius

Genetic engineering as an important approach to strain optimization has received wide recognition. Recent advances in the studies on the biosynthetic pathways and gene clusters of Streptomyces make stain optimization by genetic alteration possible. Kanamycin B is a key intermediate in the manufacture of the important medicines dibekacin and arbekacin, which belong to a class of antibiotics known as the aminoglycosides. Kanamycin could be prepared by carbamoylkanamycin B hydrolysis. However, carbamoylkanamycin B production in Streptomyces tenebrarius H6 is very low. Therefore, we tried to obtain high kanamycin B-producing strains that produced kanamycin B as a main component. In our work, aprD3 and aprD4 were clarified to be responsible for deoxygenation in apramycin and tobramycin biosynthesis. Based on this information, genes aprD3, aprQ (deduced apramycin biosynthetic gene), and aprD4 were disrupted to optimize the production of carbamoylkanamycin B. Compared with wild-type strain, mutant strain SPU313 (ΔaprD3, ΔaprQ, and ΔaprD4) produced carbamoylkanamycin B as a single antibiotic, whose production increased approximately fivefold. To construct a strain producing kanamycin B instead of carbamoylkanamycin B, the carbamoyl-transfer gene tacA was inactivated in strain SPU313. Mutant strain SPU314 (ΔaprD3, ΔaprQ, ΔaprD4, and ΔtacA) specifically produced kanamycin B, which was proven by LC-MS. This work demonstrated careful genetic engineering could significantly improve production and eliminate undesired products.     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

Production and characterization of xylanase from a Streptomyces species grown on agricultural wastes

Alkali-treated corn stalk gave maximum xylanase production at supporting growth of Streptomyces HM-15. Xylanase was stable for 24 h over a pH range of 5.0 to 7.0, had optimal activity between 50 and 60°C and a halflife of 5 h at 60°C. Xylanase production and activity were inhibited by xylose.The authors are with Department of Biosciences, Sardar Patel University. Vallabh Vidyanagar-388120, Gujarat, India.     

The surface structure of spores and aerial hyphae in Streptomyces viridochromogenes

Summary The fine structure of the surface membrane which covers the cell walls of spores and aerial hyphae of S. viridochromogenes was investigated by means of electron microscopy of air-dried whole mounts, thin sections, negative stainings and freeze-etchings. The delicate sheath of the spores, which is responsible for the surface features, exhibits conspicuous cone shaped protrusions called spines. They are composed of 5–12 rodlike units, each apparently consisting of a bundle of fine fibrils. Non-sporulating hyphae have no spines; their surface membrane shows an irregular pattern consisting of long tubules.     

Identification and characterization of phosphinothricin-tripeptide biosynthetic genes in Streptomyces viridochromogenes.

A 4-kb BamHI fragment of Streptomyces viridochromogenes Tü494 carrying phosphinothricin-tripeptide (PTT) biosynthetic genes has been identified by complementation of a nonproducing mutant which is defective in the tripeptide formation step. Nucleotide sequence analysis revealed one incomplete and three complete genes on the cloned fragment. The incomplete gene ('pms) codes for the C terminus of the phosphinomethylmalic acid synthase as determined by comparison with a region from the bialaphos biosynthetic cluster [Shimotohno et al., Agric. Biol. Chem. 54 (1990) 463-470] and with databases. Subcloning experiments showed that the juxtaposing phsA gene is sufficient to restore productivity of the blocked mutant. Analysis of gene disruption and gene replacement mutants confirmed that phsA specifies an enzyme involved in tripeptide formation. Similarities to peptide synthetases indicate that the condensation step follows a thio-template mechanism. A conserved region located in the C terminus of the PhsA protein showed identity to 4'-phosphopantetheine-binding sites of fatty acid and polyketide synthases. In the N terminus, a typical acyl transfer motif has been identified and this may be involved in transthiolation. A similar motif also appears in the deduced product of the third gene (dea), which probably catalyses the deacetylation of N-acetyl-PTT to PTT. The previously described PTT resistance-encoding gene (pat) was located between the phsA and the dea genes.     

顶头孢霉遗传育种研究进展

顶头孢霉是一类重要的工业微生物,其发酵产物头孢菌素C可用来生产7-ACA,而后者是临床常用抗感染药物头孢类抗生素的重要中间体。头孢菌素C的发酵水平决定了其下游头孢类抗生素的生产水平、产品质量及价格,因此对顶头孢霉的菌种选育工作显得尤其迫切。随着分子生物学的发展,基因工程分子改造在遗传育种领域发挥着越来越重要的作用。文章综述了对头孢菌素C的生物合成以及调控的研究进展,并将国内外对顶头孢霉进行遗传育种的结果进行了归纳总结,提出了可以从提高头孢菌素C发酵水平、延伸代谢途径等不同方面对头孢菌素C生物合成及调控基因,包括外源基因的导入和表达进行改造优化,并对进一步的研究目标进行了展望,认为可以结合比较蛋白质组和基因组改组使遗传育种所获得的工程菌尽快进入产业化。     

Nutritionally defined conditions for germination of Streptomyces viridochromogenes spores.

Spores of Streptomyces viridochromogenes were removed from the surface of solid media with glass beads and suspended in a buffer-detergent solution. Addition of yeast extract and glucose resulted in rapid loss of refractility of the spores. Appearance of germ tubes followed. Germination was accompanied by a decrease in the optical density (OD) of the suspension. The OD decrease was used as an assay for germination. A defined germination medium (DGM) comprised of L-alanine, L-glutamic acid, adenosine, para-aminobenzoic acid, and calcium and magnesium ions provided a germination rate nearly equal to that of complex media. The germination rate was essentially the same if D-alanine and D-glutamate replaced the L-isomers. The optimum pH and temperature for germination were 7.0 and 35 C. Germination was absolutely dependent on the presence of CO2. Spores harvested after growth for longer periods than the usual time (10 days) became less germinable in DGM. The same was observed for spores grown at 37 C as compared with 30 C. Spores incubated in DGM for various time periods before being transferred to a buffer solution did not continue to germinate. Spores harvested after growth of eight species of Streptomyces did not show a decrease in OD when incubated in yeast extract medium. Another strain of S. viridochromogenes did exhibit an OD decrease in the medium. Comparative properties of spores of streptomycetes, fungi, and bacilli are discussed.     

Purification and characterization of a new xylanase (xylanase C) produced by Streptomyces lividans 66

Summary A third extracellular xylanase produced by Streptomyces lividans 66 was isolated from a clone obtained by shotgun cloning through functional complementation of a xylanase- and cellulase-negative mutant using the multicopy vector pIJ702. This enzyme, designated xylanase C, has a relative molecular mass of 22000 and acts on xylan similarly to xylanase B as an endo-type xylanase producing short-chain oligoxylosides. Its specific activity determined at 1100 IU·mg^–1 of protein corresponds on a molecular basis to that of xylanase B and is about three times that of xylanase A. The enzyme shows optimal activity at pH 6.0 and 57°C, values that correspond closely to those observed previously for xylanase A and B. Xylanase C appears not to be glycosylated and has a pI > 10.25. Its K _m and V _max on birchwood xylan are 4.1 mg·ml^–1 and 3.0 mol·min^–1·mg^–1 of enzyme respectively. Whereas specific antibodies raised against xylanase A show no cross-reaction with either xylanase B or with xylanase C, the anti-(xylanase C) antibodies react slightly with xylanase B but not with xylanase A. A comparison of hydrolysis products obtained by reacting individually the three enzymes with birchwood xylan showed characteristic endo-activity patterns for xylanases B and C, whereas xylanase A hydrolysed the substrate preferentially into xylobiose and xylotriose. Sequential xylanase action on the same substrates showed synergistic hydrolysis only when endo-xylanase activity was followed by that of xylanase A.