Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of beta-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces

Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources. These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer. The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization. APPL's were shown to be mostly free of nonlignin components. As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups. The yield of APPL from different lignocelluloses correlated with their biodegradability. Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces. In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin. A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate. APPL produced by S. viridosporus was examined for its properties and possible use as an antioxidant. The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL. Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions. As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered. Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation.     

Isolation from haddock tissue of psychrophilic bacteria with maximum growth temperature below 20 degrees C.

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular Enzyme Activities during Lignocellulose Degradation by Streptomyces spp.: A Comparative Study of Wild-Type and Genetically Manipulated Strains

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Isoenzymes were analyzed by polyacrylamide gel electrophoresis and activity staining on the gels. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp.

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of β-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Lignin Degradation by Streptomyces viridosporus: Isolation and Characterization of a New Polymeric Lignin Degradation Intermediate

A new, quantitatively significant intermediate formed during lignin degradation by Streptomyces viridosporus T7A was isolated and characterized. In Streptomyces-inoculated cultures, the intermediate, an acid-precipitable, polyphenolic, polymeric lignin (APPL), accumulated in the growth medium. The APPL was a water-soluble polymer probably consisting of a heterogeneous mixture of molecular weight components of ≥20,000. APPLs were precipitable from culture filtrates after they had been acidified to pH <3 to 5. Noninoculated controls yielded little APPL, but supernatant solutions from inoculated cultures produced quantities of APPL that correlated with the biodegradability of the lignocellulose type. Maximal recovery of APPL was obtained from corn lignocellulose, reaching 30% of the initial lignin present in the substrate. APPLs contained small amounts of carbohydrate, organic nitrogen, and inorganic materials. The lignin origin of APPLs was confirmed by chemical analyses, which included acidolysis, permanganate oxidation, elemental analyses, functional group analyses, nuclear magnetic resonance spectroscopy, and ¹⁴C isotopic techniques. Analyses of APPLs from corn lignocelluloses showed that S. viridosporus-degraded APPLs were lignin derived but significantly different in structure from APPLs derived from uninoculated controls or from a standard corn milled-wood lignin. Degraded APPLs were enriched in phenolic hydroxyl groups and, to a small extent, in carboxyl groups. Degradative changes appeared to be largely oxidative and were thought to involve substantial cleavage of p-hydroxy ether linkages and methoxyl groups in the lignin.     

Metabolism of low molecular weight lignin-related compounds by Streptomyces viridosporus T7A

The ability of the ligninolytic actinomycete Streptomyces viridosporus T7A to degrade selected lignin model compounds, both in the presence and in the absence of lignocellulose, was examined. Compounds studied included benzyl alcohols and aldehydes, plus dimers possessing intermonomeric linkages, which are characteristic of the lignin macromolecule. Oxidation of veratryl alcohol to the corresponding acid was significant only under ligninolytic growth conditions, i.e., in medium containing lignocellulose, while other benzyl alcohols and aldehydes were readily oxidized in its absence. S. viridosporus T7A reduces carbonylic groups of 1,2-diarylethane, but not of 1,2-diarylpropane structures, under both ligninolytic and non-ligninolytic culture conditions. Cleavage of 1,2-diarylpropane (β-1), arylglycerol-β-arylether(β-0-4) and biphenyl structures by this strain could not be detected under either metabolic conditions.     

Activities of cellulase and other extracellular enzymes during lignin solubilization by Streptomyces viridosporus

Summary Two mutant strains of the lignin degrading bacterium Streptomyces viridosporus strain T7A with enhanced abilities to produce a soluble lignin degradation intermediate, acid-precipitable polymeric lignin (APPL) and several mutants derepressed for cellulase production were compared with the wild type to examine the roles of cellulase and selected other extracellular enzymes in lignin solubilization by S. viridosporus. The two APPL-overproducing mutants, T-81 and T-138, had higher cellulase activities than the wild type. Mutants specifically derepressed for cellulase were also isolated and were found to produce more APPL than the wild type. The results are indicative of some involvement of cellulase in the lignin solubilization process. The lignin solubilized from corn (Zea mays) lignocellulose by the mutants was slightly different chemically as compared to wild type solubilized lignin in that it had a higher coumaric acid ester content. The production of extracellular coumarate ester esterase, aromatic aldehyde oxidase, and xylanase was also examined in the mutants. Xylanase and aromatic aldehyde oxidase production did not differ significantly between the mutants and the wild type. Mutant T-81 was found to have a slightly lower activity for esterase as compared with the wild type. It was concluded that xylanase, oxidase and esterase are not the enzymes directly responsible for enhanced lignin solubilization. The results, however, do implicate cellulase in the process.Paper number 86 511 of the Idaho Agricultural Experiment Station     

L-Phenylalanine and L-tyrosine catabolism by selected Streptomyces species.

L-Phenylalanine and L-tyrosine were completely catabolized through homogentisate by Streptomyces setonii 75Vi2 but only partially degraded by Streptomyces badius 252, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. strain V7. Intermediates of catabolism were confirmed by thin-layer, gas, and high-pressure liquid chromatography. Homogentisate 1,2-dioxygenase was present in all cell extracts.     

L-Phenylalanine and L-tyrosine catabolism by selected Streptomyces species

L-Phenylalanine and L-tyrosine were completely catabolized through homogentisate by Streptomyces setonii 75Vi2 but only partially degraded by Streptomyces badius 252, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. strain V7. Intermediates of catabolism were confirmed by thin-layer, gas, and high-pressure liquid chromatography. Homogentisate 1,2-dioxygenase was present in all cell extracts.     

Production of an extracellular polyethylene-degrading enzyme(s) by Streptomyces species.

Extracellular culture concentrates were prepared from Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2 shake flask cultures. Ten-day-heat-treated (70 degrees C) starch-polyethylene degradable plastic films were incubated with shaking with active or inactive enzyme for 3 weeks (37 degrees C). Active enzyme illustrated changes in the films' Fourier transform infrared spectra, mechanical properties, and polyethylene molecular weight distributions.     

Production of an extracellular polyethylene-degrading enzyme(s) by Streptomyces species.

Extracellular culture concentrates were prepared from Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2 shake flask cultures. Ten-day-heat-treated (70 degrees C) starch-polyethylene degradable plastic films were incubated with shaking with active or inactive enzyme for 3 weeks (37 degrees C). Active enzyme illustrated changes in the films' Fourier transform infrared spectra, mechanical properties, and polyethylene molecular weight distributions.     

Catabolic Fate of Streptomyces viridosporus T7A-Produced, Acid-Precipitable Polymeric Lignin upon Incubation with Ligninolytic Streptomyces Species and Phanerochaete chrysosporium

Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii 75Vi2 was followed for 3 weeks in aerated shake flask cultures at 37°C in a yeast extract-glucose medium containing 0.05% (wt/vol) T7A-APPL. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Identified and quantified compounds present in culture media included p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, protocatechuic acid, vanillic acid, and vanillin. The further catabolism of these APPL-derived aromatic compounds varied with the culture examined, and only S. setonii and P. chrysosporium completely degraded all of them. Some new intermediates of APPL metabolism also appeared in culture media, but the patterns were culture specific. Additional evidence from high-pressure liquid chromatography analyses indicated that one strain, S. badius, converted a water-soluble fraction evident by high-pressure liquid chromatography (7 to 10 min retention time range) into new products appearing at shorter retention times. Mineralization of a [¹⁴C-lignin]APPL was also followed. The percent ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-APPL, ¹⁴C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37°C. P. chrysosporium evolved the most ¹⁴CO₂ (10%), and S. viridosporus gave the greatest decrease in recoverable ¹⁴C-APPL (23%). The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities. The significance of these findings relative to the fate of APPLs in nature was discussed.     

Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus

A two-step batch fermentation-bioconversion of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) was developed, utilizing whole cells of Streptomyces viridosporus T7A. In the first step, cells were grown in a yeast extract-vanillin medium under conditions where cells produced an aromatic aldehyde oxidase. In the second step, vanillin was incubated with the active cells and was quantitatively oxidized to vanillic acid which accumulated in the growth medium. Vanillic acid was readily recovered from the spent medium by a combination of acid precipitation and ether extraction at greater than or equal to 96% molar yield and upon recrystallization from glacial acetic acid was obtained in greater than or equal to 99% purity.     

Whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus.

A two-step batch fermentation-bioconversion of vanillin (4-hydroxy-3-methoxybenzaldehyde) to vanillic acid (4-hydroxy-3-methoxybenzoic acid) was developed, utilizing whole cells of Streptomyces viridosporus T7A. In the first step, cells were grown in a yeast extract-vanillin medium under conditions where cells produced an aromatic aldehyde oxidase. In the second step, vanillin was incubated with the active cells and was quantitatively oxidized to vanillic acid which accumulated in the growth medium. Vanillic acid was readily recovered from the spent medium by a combination of acid precipitation and ether extraction at greater than or equal to 96% molar yield and upon recrystallization from glacial acetic acid was obtained in greater than or equal to 99% purity.