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Applied Microbiology and Biotechnology (v.63, #5)

Biotechnology in the wood industry by C. Mai; U. Kües; H. Militz (pp. 477-494).

Wood is a natural, biodegradable and renewable raw material, used in construction and as a feedstock in the paper and wood product industries and in fuel production. Traditionally, biotechnology found little attention in the wood product industries, apart from in paper manufacture. Now, due to growing environmental concern and increasing scientific knowledge, legal restrictions to conventional processes have altered the situation. Biotechnological approaches in the area of wood protection aim at enhancing the treatability of wood with preservatives and replacing chemicals with biological control agents. The substitution of conventional chemical glues in the manufacturing of board materials is achieved through the application of fungal cultures and isolated fungal enzymes. Moreover, biotechnology plays an important role in the waste remediation of preservative-treated waste wood.

Metabolic engineering for improved fermentation of pentoses by yeasts by T. W. Jeffries; Y.-S. Jin (pp. 495-509).

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g⁻¹ sugar consumed, so commercialization seems feasible for some applications.

Lincomycin, cultivation of producing strains and biosynthesis by J. Spížek; T. Řezanka (pp. 510-519).

Lincomycin and its derivatives are antibiotics exhibiting biological activity against Gram-positive bacteria. The semi-synthetic chlorinated lincomycin derivative is used in clinical practice. The chemical structure of lincosamide antibiotics, cultivation of producing strains and analytical procedures used for separation and isolation of these compounds are described in this review. Biosynthesis of lincomycin and related compounds and its genetic control are briefly discussed.

Collagenolytic proteases from bacteria by K. Watanabe (pp. 520-526).

Collagen degradation occurs during various physiological and pathological conditions. However, only a limited number of proteases with unique characteristics can trigger collagen degradation. Until recently, practical applications of collagenolytic proteases from bacteria had not been considered because their functions in bacteria are closely related to pathogenicity. However, bacterial collagenolytic proteases have many interesting and useful features. This review focuses on the collagenolytic proteases from bacteria, in particular their molecular properties and practical applications.

Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells by Y. H. Sung; S. W. Lim; J. Y. Chung; G. M. Lee (pp. 527-536).

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l^–1 YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 μg ml^–1, which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q _hTPO (the specific rate of hTPO production). The supplementation of YH in SFM increased q _hTPO by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.

Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process by S. Alfenore; X. Cameleyre; L. Benbadis; C. Bideaux; J.-L. Uribelarrea; G. Goma; C. Molina-Jouve; S. E. Guillouet (pp. 537-542).

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l⁻¹), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l⁻¹ in 45 h) and average productivity (3.3 vs 2.6 g l⁻¹ h⁻¹) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l⁻¹. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l⁻¹ in 45 h) in order to achieve a highly competitive dynamic process.

Aerobic and anaerobic ethanol production by Mucor circinelloides during submerged growth by T. L. Lübbehüsen; J. Nielsen; M. McIntyre (pp. 543-548).

The dimorphic organism Mucor circinelloides is currently being investigated as a potential host for heterologous protein production. The production of ethanol on pentose and hexose sugars was studied in submerged batch cultivations to further the general knowledge of Mucor physiology, with a view to the minimisation or elimination of the by-product ethanol for future process design. Large amounts of ethanol were produced during aerobic growth on glucose under non-oxygen limiting conditions, which is indicative of M. circinelloides being a Crabtree-positive organism. Ethanol production on galactose or xylose was less significant. The response of the organism to increased ethanol concentrations, both as the sole carbon source and in the presence of a sugar, was investigated in terms of biomass formation and morphology.

Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli by D.-H. Lee; M.-D. Kim; W.-H. Lee; D.-H. Kweon; J.-H. Seo (pp. 549-552).

The cyclohexanone monooxygenase (CHMO) gene of Acinetobacter sp. NCIMB 9871 was simultaneously expressed with the genes encoding molecular chaperones and foldases in Escherichia coli. While the expression of the CHMO gene alone resulted in the formation of inclusion bodies, coexpression of the chaperone or foldase genes remarkably increased the production of soluble CHMO enzyme in recombinant E. coli. Furthermore, it was found that molecular chaperones were more beneficial than foldases for enhancing active CHMO enzyme production. The recombinant E. coli strain simultaneously expressing the genes for CHMO, GroEL/GroES and DnaK/DnaJ/GrpE showed a specific CHMO activity of 111 units g⁻¹ cell protein, corresponding to a 38-fold enhancement in CHMO activity compared with the control E. coli strain expressing the CHMO gene alone.

Purification and characterization of arylsulfatase from Sphingomonas sp. AS6330 by J.-H. Kim; D.-S. Byun; J. S. Godber; J.-S. Choi; W.-C. Choi; H.-R. Kim (pp. 553-559).

Arylsulfatase was purified from Sphingomonas sp. AS6330 through ionic exchange, hydrophobic- and gel-chromatographies. The purity increased 12,800-fold with approximately 19.1% yield against cell homogenate. The enzyme was a monomeric protein with apparent molecular weight of 62 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 41 kDa as determined by gel filtration. The enzyme had optimum reaction conditions for hydrolysis of sulfate ester bonds in agar and p-nitrophenyl sulfate (NPS) at pH 7.0 and 45°C, with a specific activity of 3.93 and 97.2 U, respectively. The enzyme showed higher activity towards agar than other sulfated marine polysaccharides such as porphyran, fucoidan and carrageenan. The K _m and V _max of the enzyme for hydrolysis of NPS were 54.9 μM and 113 mM/min, respectively. With reaction of 200 g agar with 100 U arylsulfatase for 8 h at 45°C, gel strength increased 2.44-fold, and 97.7% of the sulfate in the agar was hydrolyzed.

Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme by P. Baldrian (pp. 560-563).

The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was bellow 2.0 for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K _m=38 μM), 4.0 for 2,6-dimethoxyphenol (K _m=48 μM), 4.5 for guaiacol (K _m=93 μM) and 7.0 for syringaldazine (K _m=131 μM). The temperature optimum was between 60 and 70 °C depending on the pH and buffer used. The enzyme was stable up to 45 °C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu²⁺ and inhibited by Mn²⁺, sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.

Conversion of adamsite (phenarsarzin chloride) by fungal manganese peroxidase by R. Haas; O. Tsivunchyk; K. Steinbach; E. v. Löw; K. Scheibner; M. Hofrichter (pp. 564-566).

Fungal manganese peroxidase was found to convert the persistent chemical warfare agent adamsite (phenarsarzin chloride) in a cell-free reaction mixture containing sodium malonate, Mn²⁺ ions, and reduced glutathione. The organo-arsenical compound disappeared completely within 48 h accompanied by the formation of a more polar metabolite with a clearly modified UV spectrum. Thus, As(III) in the adamsite molecule was oxidized by manganese peroxidase to As(V) which added dioxygen and released chloride.

Identification of a type-D feruloyl esterase from Neurospora crassa by V. F. Crepin; C. B. Faulds; I. F. Connerton (pp. 567-570).

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri–food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.

A derivative of the menaquinone precursor 1,4-dihydroxy-2-naphthoate is involved in the reductive transformation of carbon tetrachloride by aerobically grown Shewanella oneidensis MR-1 by M. J. Ward; Q. S. Fu; K. R. Rhoads; C. H. J. Yeung; A. M. Spormann; C. S. Criddle (pp. 571-577).

Transformation of carbon tetrachloride (CT) by Shewanella oneidensis MR-1 has been proposed to involve the anaerobic respiratory-chain component menaquinone. To investigate this hypothesis a series of menaquinone mutants were constructed. The menF mutant is blocked at the start of the menaquinone biosynthetic pathway. The menB, menA and menG mutants are all blocked towards the end of the pathway, being unable to produce 1,4-dihydroxy-2-naphthoic acid (DHNA), demethyl-menaquinone and menaquinone , respectively. Aerobically grown mutants unable to produce the menaquinone precursor DHNA (menF and menB mutants) showed a distinctly different CT transformation profile than mutants able to produce DHNA but unable to produce menaquinone (menA and menG mutants). While DHNA did not reduce CT in an abiotic assay, the addition of DHNA to the menF and menB mutants restored normal CT transformation activity. We conclude that a derivative of DHNA, that is distinct from menaquinone, is involved in the reduction of CT by aerobically grown S. oneidensis MR-1. When cells were grown anaerobically with trimethylamine-N-oxide as the terminal electron acceptor, all the menaquinone mutants showed wild-type levels of CT reduction. We conclude that S. oneidensis MR-1 produces two different factors capable of dehalogenating CT. The factor produced under anaerobic growth conditions is not a product of the menaquinone biosynthetic pathway.

Engineering of carbon catabolite repression in recombinant xylose fermenting Saccharomyces cerevisiae by C. Roca; M. B. Haack; L. Olsson (pp. 578-583).

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (Δmig1, XYL1, XYL2, XKS1) and CPB.MBH2 (Δmig1, Δmig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l⁻¹ glucose and 50 g l⁻¹ xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.

Growth characteristics and oxidative capacity of Acetobacter aceti IFO 3281: implications for l-ribulose production by A. K. Kylmä; T. Granström; M. Leisola (pp. 584-591).

We studied the growth characteristics and oxidative capacities of Acetobacter aceti IFO 3281 in batch and chemostat cultures. In batch culture, glycerol was the best growth substrate and growth on ethanol occurred only after 6 days delay, although ethanol was rapidly oxidized to acetic acid. In continuous culture, both glycerol and ethanol were good growth substrates with similar characteristics. Resting cells in a bioreactor oxidized ribitol to l-ribulose with a maximal specific rate of 1.2 g g⁻¹ h⁻¹). The oxidation of ribitol was inhibited by ethanol but not by glycerol. Biomass yield (Y_SX; C-mmol/C-mmol) on ethanol and glycerol was low (0.21 and 0.17, respectively). In the presence of ribitol the yield was somewhat higher (0.25) with ethanol but lower (0.13) with glycerol, with respectively lower and higher CO₂ production. In chemostat cultures the oxidation rate of ribitol was unaffected by ethanol or glycerol. Cell-free extract oxidized ethanol very slowly but not ribitol; the oxidative activity was located in the cell membrane fraction. Enzymatic activities of some key metabolic enzymes were determined from steady-state chemostat with ethanol, glycerol, or ethanol/glycerol mixture as a growth limiting substrate. Based on the measured enzyme activities, metabolic pathways are proposed for ethanol and glycerol metabolism.

A gene homologous to β-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions by S. Mitsuhashi; J. Ohnishi; M. Hayashi; M. Ikeda (pp. 592-601).

Carbonic anhydrase catalyzes the interconversion of CO₂ and bicarbonate. We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for l-lysine overproduction. A whole-genome sequence revealed two genes encoding putative β-type and γ-type carbonic anhydrases in C. glutamicum. These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively. Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO₂) on both complete and minimal media. The growth defect of the Δbca strain was restored under elevated CO₂ conditions (5% CO₂). Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene (pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO₂. In contrast, the Δgca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO₂ conditions. Attempts to increase the dosage of bca, gca, and pca in the defined l-lysine-producing strain C. glutamicum AHD-2 led to no discernable effects on growth and production. Northern blot analysis indicated that the bca transcript in strain AHD-2 and another l-lysine producer, C. glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases. These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product′s ability to maintain favorable intracellular bicarbonate/CO₂ levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of l-lysine overproduction, both of which are conditions of higher bicarbonate demand.

Changes in structure, activity and metabolism of aerobic granules as a microbial response to high phenol loading by H.-L. Jiang; J.-H. Tay; S. T.-L. Tay (pp. 602-608).

Four column-type sequential aerobic sludge blanket reactors were fed with phenol as the sole carbon and energy source and operated at loading rates of 1.0, 1.5, 2.0 and 2.5 kg phenol m⁻³ day⁻¹. The results indicated that phenol loading exerted a profound influence on the structure, activity and metabolism of the aerobic granules. Compact granules with good settling ability were maintained at loadings up to 2.0 kg phenol m⁻³ day⁻¹, and structurally weakened granules with enhanced production of extracellular polymers and proteins and significantly lower hydrophobicities were observed at the highest loading of 2.5 kg phenol m⁻³ day⁻¹. Specific oxygen uptake rate, catechol 2,3-dioxygenase (C23O) and catechol 1,2-dioxygenase (C12O) activities peaked at a loading of 2.0 kg phenol m⁻³ day⁻¹, and declined thereafter. Granules degraded phenol completely in all four reactors, mainly through the meta cleavage pathway as C23O activities were significantly higher than C12O activities. At the highest loading applied, the anabolism and catabolism of microorganisms were regulated such that phenol degradation proceeded exclusively via the meta pathway, apparently to produce more energy for overstimulation of protein production against phenol toxicity. This work contributes to a better understanding of the ability of aerobic granules to handle high-strength industrial wastewaters containing chemicals that are normally inhibitory to microbial growth.

Complete lab-scale detoxification of groundwater containing 1,2-dichloroethane by S. De Wildeman; G. Linthout; H. Van Langenhove; W. Verstraete (pp. 609-612).

The suspected carcinogenic solvent 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C₂ groundwater pollutant on earth. However, an efficient reductive in situ detoxification technology for this compound is not known. Detoxification results of 1,2-DCA with the recently isolated anaerobic bacterium Desulfitobacterium dichloroeliminans strain DCA1 are presented. First, it was verified that strain DCA1 could compete for nutrients in the presence of fast-growing Enterococcus faecalis; the latter was observed in the enrichment culture from which strain DCA1 was isolated. Subsequently, lab-scale bioaugmentation of the strain to groundwater containing 40 mg 1,2-DCA/l indicated that the bacterium has strong metabolic activity under prevailing environmental conditions, converting the pollutant into ethene. During exponential growth, the maximum 1,2-DCA dechlorination rate exceeded 350 nmol chloride released per min per mg total bacterial protein. Growth and dechlorination within the community with autochthonous bacteria indicated a high competitive strength of strain DCA1. Interestingly this dechlorination process does not produce any toxic byproducts, such as vinyl chloride. Furthermore, complete groundwater detoxification happens within a short time-frame (days) and is robust in terms of bacterial competition, oxygen tolerance, high ionic strength, and pH range.

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Applied Microbiology and Biotechnology (v.63, #5)