Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)-l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-d-thioglucopyranoside. Maximum GDP-l-fucose concentration of 38.9 ± 0.6 mg l⁻¹ was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l⁻¹ GDP-l-fucose under the same cultivation condition.     

d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Regio- and Stereo-selective Hydroxylation of Abietic Acid Derivatives by Mucor circinelloides and Mortierella isabellina

Mucor circinelloides and Mortierella isabellina hydroxylated dehydroabietic acid (DehA). DehA was converted regio- and stereo-selectively by whole cells of Mr. circinelloides to give 2α-hydroxydehydroabietic acid in a 75% molar conversion yield (11 mM from 14.7 mM DehA) after 72 h in the cultivation medium containing 3% (v/v) Tween 80. With cells of Ma. isabellina, under the same conditions, 20.5 mM (6.5 g l⁻¹) 2–hydroxydehydroabietic acid (α/β=81/19) was formed from 26.4 mM DehA.     

d-Glucose feeding for improvement of xylitol productivity from d-xylose using Candida tropicalis immobilized on a non-woven fabric

Summary A non-woven fabric was successfully applied for immobilization of Candida tropicalis to produce xylitol from d-xylose. Xylitol productivity was enhanced by feeding of d-glucose (50 g/l·d); 87 g xylitol/L was produced after 64 h cultivation. Non-woven fabric could be used five times for fed-batch cultivation.     

High-resolution studies on vegetation succession, hydrological variations, anthropogenic impact and genesis of a subrecent lake in southern Ecuador

A lake sediment record from Laguna Campana at 2,488 m a.s.l. in the eastern Ecuadorian Andes allows the reconstruction of local environmental conditions over the past ~500 years. A high-resolution multi-proxy approach using pollen, spore, charcoal and XRF analyses provides information about lake genesis, hydrological variations and the development of the surrounding vegetation. Results suggest that Laguna Campana originated from a landslide, which are naturally common and anthropogenically promoted in the study area. Human activities, e.g. deforestation or slash and burn cultivation, impacted the local vegetation development and biodiversity during the recorded period. After a first dense layer of pioneer grasses developed on open soil around the small lake, successional stages of secondary upper mountain rainforest forest mainly composed of Alnus and Weinmannia were observed. The record shows no signs of dense forest regeneration but rather open vegetation with trees and a grassy understory. Especially since ca. a.d. 1980, the proportion of forest in the area was reduced, most probably by fire use for pastures, cultivation and wood extraction. Hydrological variability was derived from differences in minerogenic input and variations in Botryococcus braunii and Sphagnum occurrence. After wettest conditions at the study site, probably triggering the landslide, humid conditions persisted until a time of drier conditions between a.d. 1900 and 1960. A subsequent return to wetter conditions was observed over the last decades. XRF analyses suggest an increase in deposition of atmospherically derived lead since the formation of the lake.     

Regulation of biosynthesis of monensins on chemically defined medium

Addition ofL-valine andDL-isoleucine to the cultivation medium ofStreptomyces cinnamonensis was found to affect the ratio of synthesized monensins A and B. In the presence ofL-valine monensin A is synthesized predominantly, whereas in the presence ofDL-isoleucine the production of monensin B increases.     

Effect of the interrupted adaptation to D-methionine on the efficiency of Rhizobium meliloti strains

Summary R. meliloti strains 107-1, 111 and 152 were adapted to D-methionine in three ways: a) consecutive transfer in the presence of increasing amounts of D-methionine, b) alternate transfer between D- and L-methionine-containing media followed by final cultivation in the presence of each isomer, c) alternate transfer between D-methionine and medium 79 followed by cultivation in medium 79 or in D-methionine-medium. At the end of the experiment efficiency of the strains was ascertained by a plant test.Strain 111 lost efficiency when it was adapted consecutively to 0.125% D-methionine or alternated between D-methionine and either L-methionine or medium 79-Strain 107-1 sucessively adapted to D-methionine lost efficiency within 16 weeks. On adaptation to D-methionine alternated with L-methionine, efficiency was retained in L-methionine medium and lost in D-methionine medium. On alternate adaptation between D-methionine and medium 79, strain 107-1 lost efficiency in the D-methionine-medium but not in medium 79. Efficiency of strain 152 was lost by adaptation to 0.125% D-methionine, but it was maintained on the alternate adaptation between D-methionine and L-methionine or medium 79.     

Changes in cereal cultivation during the Iron Age in southern Sweden: a compilation and interpretation of the archaeobotanical material

Macrofossil data from 73 sites dating to the south Swedish Iron Age (500 b.c.–a.d. 1100) have been compiled and analyzed in order to elucidate long term changes in cereal cultivation. The analyses indicate that “permanent field” agriculture was established at the end of the Bronze Age utilizing Hordeum vulgare var vulgare as a primary crop and Triticum aestivum ssp vulgare/compactum, Triticum spelta/dicoccum/monococcum, Avena sativa and Secale cereale as secondary crops. An observed change towards the end of Roman Iron Age (1–a.d. 400) is the expansion of Secale cereale and Avena sativa cultivation. Evidence also suggests that winter sowing of the former commenced at the latest during the eighth, ninth and tenth centuries a.d. The introduction of winter sowing possibly coincided with the establishment of crop rotation agriculture. During most of the Iron Age southern Sweden displays significant regional variations with regards to cereal cultivation practice. There is however evidence that a more homogenous agriculture appeared across the investigated area from the beginning of the Viking Age (a.d. 800–1100) onwards.     

Selection ofEscherichia coli K12 1EA mutants with increased synthesis of ribitol dehydrogenase

Selection of an interspecific hybridEscherichia coli K 12 1EA in a chemostat on xylitol yielded a stable mutant synthesizing a four-fold amount of ribitol dehydrogenase (EC 1.1.1.56). Subsequent cultivation of the mutant under increased selection pressure resulted in an accumulation of a mutant with 12-fold higher level of ribitol dehydrogenase relative to the parent strain 1EA. A selection during which a UV-mutagenized population of the 1EA mutant was cultivated in a chemostat on xylitol was accompanied by monitoring the activities of ribitol dehydrogenase andD-arabinitol dehydrogenase (EC 1.1.1.11) of two adjacent catabolite operons. A several-fold increase in the activity of the two enzymes was followed by further increase in the activity of ribitol dehydrogenase and a concomitant drop in the activity ofD-arabinitol dehydrogenase. The two hyperproducing strains are compared with the parent mutant as to the rate of synthesis of the two dehydrogenases and growth parameters under the conditions of batch cultivation.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Dynamics of l-valine in relation to the production of cyclosporin A by Tolypocladium inflatum

Summary We report the kinetics of endogenous l-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented l-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular l-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/1 l-valine. The final CyA titre under these conditions was 710 mg/1 compared to only 130 mg/1 attained without l-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in l-valine-supplemented medium. As a result, the conversion yield of CyA on l-valine remained constant during the stationary phase at 0.27 g CyA/g l-valine.     

Is there a relationship between crop farming and the Alnus decline in the eastern Baltic region?

Data from 59 sequences studied through pollen analysis were used to examine the decline in Alnus in Estonia during the Iron Age. Between a.d. 300 and 1300, the Alnus pollen frequency declined markedly in 30 records distributed evenly across the investigated area. The beginning of the decline was time transgressive, coincidental with the start of extensive cultivation, and was frequently connected with the commencement of rye cultivation and the availability of land suitable for cultivation. The greatest reduction in Alnus abundance occurred during the Late Iron Age between a.d. 900 and 1000. This spatially random asynchrony suggests that one or more factors affected Alnus populations across the whole northern region. Human impact is discussed as a plausible cause of the decline. To determine the initiation of extensive crop farming in the eastern Baltic area, pollen diagrams from Latvia, Lithuania and the Novgorod region were also examined.     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine into 2-phenylethanol by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in grape must cultures

A 2³ factorial design was employed to find the best conditions of pH, l-phenylalanine concentration and temperature for the production of 2-phenylethanol by Kluyveromyces marxianus CBS 6556. The cultivation was carried out on grape must, which contains a great amount of nitrogen compounds. Central composite design (CCD) was used for the analysis of treatment combinations. Results showed a second-degree polynomial regression model with good agreement of experimental data, with R ² = 0.92015 (p < 0.05). The maximum production of 2-phenylethanol was found at pH 7.0, temperature of 37 °C, and a concentration of 3.0 g of l-phenylalanine L⁻¹. Further experiments in bioreactors showed that oxygen concentration is also important to 2-phenylethanol production, with best results obtained at oxygen mass transfer rates of 2.0 h⁻¹.     

An improved synthetic medium for continuous cultivation of Zymomonas mobilis

Summary A synthetic medium for continuous cultivation of Zymomonas mobilis was developed using the chemostat pulse technique in appropriate experimental designs. Yeast extract could be replaced by a mixture of six mineral salts, Ca-pantothenate, l-as-partate, and l-serine. Kinetic data from continuous cultivations of strains ATCC 10988 and ZM4 are presented and compared with published data.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Biological active metabolite cyclo (l-Trp-l-Phe) produced by South China Sea sponge Holoxea sp. associated fungus Aspergillus versicolor strain TS08

Sponge-associated fungi represent the single most prolific source of novel natural products from marine fungi. Cyclo (l-Trp-l-Phe) exhibits biological functions such as plant growth regulation, moderate cytotoxicity and thus has the application potential in pharmaceutical and agricultural biotechnologies. In this study, a fungal strain TS08 was isolated from sponge Holoxea sp. in the South China Sea and identified as A. versicolor according to its 18S rRNA gene and morphological, physiological, and biochemical characteristics. Meanwhile, cyclo (l-Trp-l-Phe) was found to be produced by A. versicolor strain TS08 mainly in the exponential growth phase. The highest yield of cyclo (l-Trp-l-Phe), 13.24 mg/g (per crude extract of EtOAc), 2.51% of cell dry weigh, was obtained on the tenth day of the fungal cultivation. It was the first time to find the biological active cyclo (l-Trp-l-Phe) in sponge-associated microorganism.     

Expression of <Emphasis Type="Italic">glr</Emphasis> gene encoding glutamate racemase in <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> WX-02 and its regulatory effects on synthesis of poly-γ-glutamic acid

The glr gene, which encodes glutamate racemase involved in the conversion of l-glutamic acid to its D-isomer, was cloned and expressed in Bacillus licheniformis WX-02. Overexpression of the glr gene not only increased the production of poly-γ-glutamic acid (γ-PGA) by 22.5% but also increased the proportion of d-glutamate in γ-PGA from 77 to 85%. The activity of glutamate racemase was higher than in the original strain throughout cultivation. This is the first report that overexpression of the glr gene could enhance the l- and d-glutamate conversion in B. licheniformis WX-02 and increase the proportion of d-glutamate in γ-PGA and the yield of γ-PGA.     

Identification and characterization of Clostridium paraputrificum,a chitinolytic bacterium of human digestive tract

A strictly anaerobic, mesophilic and chitinolytic bacterial strain was isolated from human feces. Based on morphological and physiological properties and 16S rRNA sequence analysis the strain was identified asClostridium paraputrificum. The strain utilized chitin andN-acetyl-d-glucosamine, grew on glucose and hydrolyzed starch. Cultivation of the strain with colloidal chitin as the growth substrate resulted in the production of gas (hydrogen and carbon dioxide) and formation of acetate and lactate (21.6 and 18.9 mmol/L, respectively) and only small quantities of propionate and butyrate (1.7 and 2.6 mmol/L, respectively). In the course of a 10-d cultivation with chitin, the endochitinase activity was detected after 1 d and gradually increased, reaching maximum after 3 d (251 nkat/LN-acetyl-d-glucosamine). The β-N-acetyl-glucosaminidase activity appeared just at the beginning of the cultivation, increased to day 2 and then remained nearly constant. More than 90% of chitin added was degraded within 2 d of cultivation. On the zymogram of the extracellular chitinolytic complex were visible at least 6 isoenzymes with molar mass 43.5–65.0 kDa. The temperature optimum of endochitinase and β-N-acetylglucosaminidase activities was 50°C; the optimum activity of both enzymes was found at pH 4–6.