CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production.

Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis. This study further enhanced its cellulase production through the disruption of seb1 gene, which encodes Seb1, a transcription factor that binds to the stress response element (STRE) and regulates a variety of cellular processes. Gene seb1 was cloned from strain T. pinophilus EMU and disrupted using CRISPR-Cas9 technology. The seb1-disruptants (TpΔseb1 strains) showed distinct morphology from its parent strain. They presented a hyphal branching phenotype with decreased transcription levels of rhoA and ras1 genes involved in hyphal branching. Furthermore, TpΔseb1 strains displayed lower cell biomass, higher specific protein content, and 20%-40% enhancement in filter paper cellulase (FPase) activity, however, insignificant changes in the transcription levels of cbh1 and bgl1 genes involved in cellulase production. Through this study, we confirmed that seb1 gene disruption in T. pinophilus EMU caused more hyphal branching, reduced cell growth, increased protein secretion, and enhanced cellulase production. In addition, we successfully established the CRISPR-Cas9 genome-editing platform in T. pinophilus EMU.

High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration.

CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S. cerevisiae, in vivo DNA assembly, CRISPR Cas9, and delta integration. Enhanced green fluorescent protein was used as a marker to confirm large DNA construct in vivo assembly and genome integration. Based on this platform, an efficient 2,3- BDO producing yeast strain was rapidly constructed with up to 25-copy genome integration of 2,3-BDO biosynthesis pathway. Further strain engineering was conducted by multiplex disruption of ADH1, PDC1, PDC5 and MTH1 using a 2µ-based replicative CRISPR Cas9 plasmid containing donor DNAs. As a result, the 2,3-BDO titer was improved by 3.9 folds compared to that obtained by the initially engineered yeast and 50.5 g/L 2,3-BDO was produced by the final engineered yeast strain 36aS5-CFBDO in fed-batch fermentation without strain evolution and process optimization. This study demonstrated that the new replicative and integrative CRISPR Cas9 genome-editing platform was promising in generating an efficient 2,3-BDO-producing S. cerevisiae strain.

Enhanced cellulase and reducing sugar production by a new mutant strain Trichoderma harzianum EUA20.

Trichoderma harzianum EU2-77 was a mutant strain of the wild-type strain T. harzianum NP13a isolated in Singapore. A multi-mutagenesis one-screening (MMOS) method was developed to further improve strain EU2-77 and a new mutant EUA20 was obtained. It exhibited filter paper cellulase (FPase) activity up to 14.79 IU/mL within 6 days shake flask cultivation. Activities of FPase, endoglucanase, ß-glucosidase, and xylanase, and protein content by EUA20 were respectively increased to 5.73, 4.35, 7.34, 1.80 and 2.70 folds. Using pretreated oil palm empty fruit bunch (OPEFB) and corncob powder as the substrates, strain EUA20 presented approximate 6.52 and 8.80 IU/ml FPase activity. Reducing sugar yield of 615.8 and 636.8 mg/g biomass were respectively obtained for OPEFB and corncob powder using cellulolytic enzymes of strain EUA20. Our results demonstrated that mutant strain EUA20 had great potential in on-site cellulase production for effective biomass bioconversion.

Enzyme production and oil palm empty fruit bunch bioconversion to ethanol using a hybrid yeast strain.

Oil palm empty fruit bunch (OPEFB) is a lignocellulosic biomass generated in palm oil mills. It is a sustainable resource for fuels and chemicals. In this study, OPEFB was converted to ethanol by an integrative OPEFB conversion process including dilute alkaline pretreatment, cellulolytic enzyme production, separate OPEFB hydrolysis, and cofermentation using a hybrid xylose-fermenting yeast. OPEFB was pretreated using 1% (w/v) NaOH solution followed by 1% (v/v) H2 O2 . Further, cellulolytic enzymes were produced by submerged fermentation using Trichoderma reesei Rut C30 and used for OPEFB hydrolysis. The filter paper cellulase activity of the crude cellulolytic enzymes was 15.1 IU/mL, which was higher than those obtained by reported Trichoderma strains under laboratory conditions. Glucose and xylose yields reached 66.9% and 74.2%, respectively, at 30 filter paper unit (FPU)/g-biomass enzyme dosage and 10% (w/v) biomass loading. The hybrid yeast strain ScF2 was previously constructed through recursive genome shuffling of Pichia stipitis and Saccharomyces cerevisiae and was used in OPEFB hydrolysate fermentation. About 16.9 g/L ethanol was produced with an ethanol yield of 0.34 g/g sugars, which was 67% of theoretical ethanol yield.

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain.

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly.

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

A novel homodimer laccase from Cerrena unicolor BBP6: Purification, characterization, and potential in dye decolorization and denim bleaching.

The white-rot fungus Cerrena unicolor BBP6 produced up to 243.4 U mL-1 laccase. A novel laccase isoform LacA was purified; LacA is a homodimer with an apparent molecular mass of 55 kDa and an isoelectric point of 4.7. Its optimal pH was 2.5, 4.0, and 5.5 when 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP) were used as the substrates, respectively. The optimal temperature was 60°C for ABTS and 80°C for both guaiacol and 2, 6-DMP. LacA retained 82-92% activity when pH was greater than 4 and 42%-92% activity at or below 50°C. LacA was completely inhibited by 0.1 mM L-cysteine, 1 mM Dithiothreitol, and 10 mM metal ions, Ca2+, Mg2+ and Co2+. LacA had good affinity for ABTS, with a Km of 49.1 µM and a kcat of 3078.9 s-1. It decolorized synthetic dyes at 32.3-87.1%. In the presence of 1-hydroxybenzotriazole (HBT), LacA decolorized recalcitrant dyes such as Safranine (97.1%), Methylene Blue (98.9%), Azure Blue (96.6%) and simulated textile effluent (84.6%). With supplemented manganese peroxidase (MnP), Mn2+ and HBT, the purified LacA and BBP6 fermentation broth showed great potential in denim bleaching, with an up to 5-fold increase in reflectance values.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

BACKGROUND: Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation. RESULTS: Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene. CONCLUSIONS: This study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.

Polyhydroxybutyrate production from oil palm empty fruit bunch using Bacillus megaterium R11.

Oil palm empty fruit bunch (OPEFB), contains abundant cellulose and hemicelluloses and can be used as a renewable resource for fuel and chemical production. This study, as the first attempt, aims to convert OPEFB derived sugars to polyhydroxybutyrate (PHB). OPEFB collected from a Malaysia palm oil refinery plant was chemically pretreated and enzymatically hydrolyzed by an in-house prepared cellulase cocktail. The PHB producer, Bacillus megaterium R11, was isolated in Singapore and could accumulate PHB up to 51.3% of its cell dry weight (CDW) from both glucose and xylose. Tryptone was identified as its best nitrogen source. PHB content and production reached 58.5% and 9.32 g/L, respectively, for an overall OPEFB sugar concentration of 45 g/L. These respectively reached 51.6% and 12.48 g/L for OPEFB hydrolysate containing 60 g/L sugar with a productivity of 0.260 g/L/h.

Effects of lignin-derived phenolic compounds on xylitol production and key enzyme activities by a xylose utilizing yeast Candida athensensis SB18.

Candida athensensis SB18 is potential xylitol producing yeast isolated in Singapore. It has excellent xylose tolerance and is able to produce xylitol in high titer and yield. However, by-products, such as phenolic compounds, derived in lignocellulosic biomass hydrolysate might negatively influence the performance of this strain for xylitol production. In this work, four potential phenolic inhibitors, such as vanillin, syringaldehyde, 4-hydroxybenzaldehyde and phenol, were evaluated for their inhibitory effects on xylitol production by C. athensensis SB18. Phenol was shown to be the most toxic molecule on this microorganism followed by syringaldehyde. Vanillin and 4-hydroxylbenzaldehyde was less toxic than phenol and syringaldehyde, with vanillin being the least toxic. Inhibition was insignificant when the total content of inhibitors was below 1.0 g/L. The presence of phenolic compounds affected the activity of xylose reductase, however not on that of xylitol dehydrogenase. C. athensensis SB18 is therefore a potential xylitol producer from hemicellulosic hydrolysate due to its assimilation of such phenolic inhibitors.

Improved ethanol production by a xylose-fermenting recombinant yeast strain constructed through a modified genome shuffling method.

BACKGROUND: Xylose is the second most abundant carbohydrate in the lignocellulosic biomass hydrolysate. The fermentation of xylose is essential for the bioconversion of lignocelluloses to fuels and chemicals. However the wild-type strains of Saccharomyces cerevisiae are unable to utilize xylose. Many efforts have been made to construct recombinant yeast strains to enhance xylose fermentation over the past few decades. Xylose fermentation remains challenging due to the complexity of lignocellulosic biomass hydrolysate. In this study, a modified genome shuffling method was developed to improve xylose fermentation by S. cerevisiae. Recombinant yeast strains were constructed by recursive DNA shuffling with the recombination of entire genome of P. stipitis with that of S. cerevisiae. RESULTS: After two rounds of genome shuffling and screening, one potential recombinant yeast strain ScF2 was obtained. It was able to utilize high concentration of xylose (100 g/L to 250 g/L xylose) and produced ethanol. The recombinant yeast ScF2 produced ethanol more rapidly than the naturally occurring xylose-fermenting yeast, P. stipitis, with improved ethanol titre and much more enhanced xylose tolerance. CONCLUSION: The modified genome shuffling method developed in this study was more effective and easier to operate than the traditional protoplast-fusion-based method. Recombinant yeast strain ScF2 obtained in this study was a promising candidate for industrial cellulosic ethanol production. In order to further enhance its xylose fermentation performance, ScF2 needs to be additionally improved by metabolic engineering and directed evolution.

Ethanol production from horticultural waste treated by a modified organosolv method.

In this study, we investigated the use of horticultural waste (HW) collected in Singapore as a renewable raw material for bioethanol production. A modified organosolv method using ethanol cooking under mild conditions followed by H(2)O(2) post-treatment was investigated for HW pretreatment. It was found that the addition of acid catalysts in the pretreatment process was not critical and post-treatment using H(2)O(2) was essential for the enhancement of HW digestibility. Enzymatic hydrolysis of the organosolv pretreated HW with 17.5% solid content, enzyme loading of 20 FPU/g HW of filter paper cellulase, and 80 CBU/g HW of ß-glucosidase resulted in a HW hydrolysate containing 26.9 g/L reducing sugar after 72 h. Fermentation of the above hydrolysate medium produced 11.69 g/L ethanol at 8h using Saccharomyces cerevisiae. It proved that horticultural waste was a potential feedstock for fuel ethanol production and organosolv pretreatment method developed in this study was effective.

Xylitol production from D-xylose and horticultural waste hemicellulosic hydrolysate by a new isolate of Candida athensensis SB18.

This paper describes the production of xylitol from d-xylose and horticultural waste hemicellulosic hydrolysate by a new strain of Candida athensensis SB18. Strain SB18 completely consumed 250 and 300 g L(-1) D-xylose and successful converted it to xylitol in the respective yield of 0.83 and 0.87 g g(-1), resulting in 207.8 and 256.5 g L(-1) of xylitol, respectively. The respective volumetric productivity were 1.15 and 0.97 g L(-1) h(-1). Approximately 100.1 g L(-1) of xylitol was obtained from the bioconversion of detoxified horticultural waste hemicellulosic hydrolysate using strain SB18. The yield and productivity were 0.81 g g(-1) xylose and 0.98 g L(-1) h(-1), respectively. Strain C. athensensis SB18 was able to completely utilize glucose, mannose, xylose and partially arabinose. This work demonstrates that stain C. athensensis SB18 is a promising strain for high-titer and high-yield xylitol production and it has great potential in bioconversion of hemicellulosic hydrolysate.

New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production.

A new Trichoderma viride stain was isolated from Singapore soil samples. Its mutants were developed by using ethyl methyl sulfonate (EMS) treatment and UV-irradiation followed by a semi-quantitative plate clearing assay on phosphoric-acid-swollen cellulose plates. Mutant EU2-77 proved to be the most promising extracellular cellulase producer among 20 mutants in a screening program performed in shake flask fermentation after plate screening. Soluble protein content, filter paper cellulase (FPase) activity, ß-glucosidase activity and endoglucanase (CMCase) activity of the fermentation broths of the mutant strain were increased to 1.67, 2.49, 2.16, and 2.61 folds, respectively, compared with the wild strain. This enzyme complex produced by mutant EU2-77 contained FPase (2.19 IU/ml), CMCase (16.46 IU/ml), ß-glucosidase (4.04 IU/ml), xylanase (42.37 IU/ml), and ß-xylosidase (0.12 IU/ml). The soluble protein concentration in the enzyme complex was 1.69 mg/ml. The hydrolytic capacities of fermentation supernatants of T. reesei Rut-C30, the wild strain T. viride NP13a and mutant T. viride EU2-77 were compared with the commercial enzymes on the hydrolysis of waste newspaper. The crude enzymes prepared by T. viride EU2-77 showed much higher hydrolysis performance than that from the commercial strain Rut-C30 and demonstrated much comparable hydrolytic performances with the commercial enzyme mixtures. T. viride mutant EU2-77 produced high levels of extracellular cellulases as well as ß-glucosidase, rendering the supplementation of ß-glucosidase unnecessary in waste newspaper hydrolysis.

Utilization of horticultural waste for laccase production by Trametes versicolor under solid-state fermentation.

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 µm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.

Enzymatic hydrolysis of sodium dodecyl sulphate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis.

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/beta-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 degrees C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.

Horticultural waste as the substrate for cellulase and hemicellulase production by Trichoderma reesei under solid-state fermentation.

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), beta-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and beta-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes.

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge.

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l(-1) phenol at room temperature (25 degrees C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25 degrees C can be described using the Haldane equation. It has a maximal specific growth rate (mu(max)) of 0.28 h(-1), a half-saturation constant (K(S)) of 1,167.1 mg l(-1), and a substrate inhibition constant (Ki) of 58.5 mg l(-1). Values of yield coefficient (Y(X/S)) are between 0.4 and 0.6 mg dry cell (mg phenol)(-1). Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l(-1)). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.