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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/18—Erwinia
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/847—Erwinia

Definitions

• This invention relates to the field of lignin degradation. More specifically the invention relates to methods for selecting and isolating microorganisms from 5 nature that are capable of degrading lignin, processes for cloning a gene segment from such an organism, and methods of using the enzyme product of the gene segment to provide valuable chemical feedstocks, methanol and the like from a lignin source material.
• _ Q Lignin is an abundant phenolic biopoly er, comprising approximately one quarter of the weight of dry wood. The structure of lignin is quite complex and insights into its structure have only recently been elucidated.
• lignin does not possess any repeating units nor are its chemical bonds particularly easy to hydrolyze. Ether bonds are often prevalent, providing a variety of linkages to the numerous aromatic residues. A portion of 2o a typical structure is illustrated in Figure 1.
• Lignin is synthesized non-enzymatically by the coupling of three types of phenylpropane units (coumarylalcohol, coniferylalcohol, and sinapylalcohol) to form dilignols, trilignols and oligolignols.
• the lignin product is a copolycondensate, insoluble in water and possessing a molecular weight of tens of thousands.
• This invention provides a biologically pure culture of a lignin-degrading microorganism of the genus Erwinia having the , identifying characteristics of ATCC 39873.
• this invention provides a method for the isolation of lignin-degrading microorganisms from a sample containing lignin-degrading and lignin non-degrading microorganisms comprising: enriching the sample for lignin-degrading microorganisms by incubating the sample in an enriching medium comprising methanol at an alkaline pH; incubating the enriched sample in a selection medium comprising yeast extract lignin as a carbon source at an alkaline pH; isolating lignin-degrading microorganism by recovering those microorganisms capable of forming colonies on a solid medium.
• this invention provides a DNA segment coding for an aryl etherase protein or its single or multiple base substitutions, deletions, insertions or inversions, wherein said DNA segment is derived from a lignin-degrading microorganism, and is capable, when correctly combined with a cleaved vector, of expressing a non-native protein having aryl etherase enzyme activity upon transformation of a host by said vector.
• this invention provides a vector comprising a DNA segment which codes for an aryl etherase protein, the segment being oriented within said vector such that in a host said segment is expressed to produce a non-native aryl etherase protein.
• this invention provides a host organism transformed by a vector comprising -a DNA segment which codes for an aryl etherase protein.
• this invention provides a process for the degradation of lignin comprising contacting a sample of the lignin to be degraded under lignin-degrading conditions with a host organism transformed by a vector comprising a DNA segment which codes for an aryl etherase protein, the segment being oriented within said vector such that said segment is expressed to produce said aryl etherase.
• this invention provides a process for producing methanol from lignin comprising culturing a host organism transformed with a vector comprising a DNA segment which codes for an aryl etherase protein said segment being oriented within said vector such that said segment is expressed to produce said aryl etherase, under methanol forming culture conditions.
• this invention provides a process for the production of chemical feed stocks from lignin comprising culturing a host organism transformed with a vector comprising a DNA segment which codes for an aryl etherase protein, said segment being oriented within said vector such that said segment is expressed to
• TUTE SHEET produce said aryl etherase, under chemical feedstock forming conditions.
• Figure 1 illustrates the molecular structures of various compounds referred to in the specification.
• Figure 2a illustrates the restriction map of plasmid pNCl.
• Figure 2b illustrates the hybridization of Erwinia genomic DNA with 32p-labelled probe DNA from pNCl.
• Lane 1 pNCl 810 bp Bam HI fragment (0.1 ug) ; lanes 2-6, Erwinia DNA digested with Bam HI + Hind III,
• Figure 3 demonstrates the activity of the cloned aryletherase on Kraft lignin as analyzed by thin layer chromatography.
• Lane 1 E. Coli Cs412 (control); lane 2 " , Cs412 pNCl transformant: lane 3, Cs412 pNC2 transformant; lane 4, Kraft lignin standard.
• Figure 4 illustrates the separation of lignin metabolites after 2, 4, 8, and 20 hrs of incubation with the isolated bacterium by thin layer chromatography.
• Solvent Benzene, 2-propanol, NH.OH (4:1:1).
• L lignin
• C control (cells incubated for 20 hrs)
• L.st(20) lignin incubated with the boiled cells for 20 hrs
• L.st lignin standard.
• Products were detected under a short-wave ultraviolet lamp (UVS.12, Ultraviolet Products INC.) .
• Lignin has great potential as a renewable resource for the production of chemical feedstocks; however, it is relatively resistant to microbial degradation. Lignin also represents a major component of
• This invention provides methods for the isolation of a lignin-degrading microorganisms and the cloning of a gene encoding an enzyme capable of breaking arylether linkages into E. Coli plasmid pBR322.
• the cloned aryletherase degrades lignin from Kraft black liquor and also several lignin model compounds.
• this invention provides a method for the selection of lignin-degrading microorganisms. Historically, two basic methods for recovery of desirable microbial variants have been employed, screening and selection.
• SHEET for example, from a mutagenesis treatment; thus each isolate must be examined individually to identify the desired characteristic. Since tens of millions of isolates must be examined, this approach can be highly labor intensive. This is particularly true if the characteristic is an intracellular one. In such a situation since the cells must be disrupted in order to identify the desired characteristic, back-up cultures of each isolate must be maintained, necessitating at least 0 twice the number of cultures.
• the experimental conditions are chosen so as to establish a growth differential between the rare strains possessing the desired characteristic and all other strains which do not j z possess said trait.
• the selected strain will not grow under the conditions ' of the experiment while the non-selected strains will grow.
• the size of the remaining population of o cells to be examined is dramatically reduced.
• conditions may be established such that the selected strain will grow while the non-selected strains are inhibited, here again effectively reducing the population to be examined and enriching the remaining 5 population with the variant of interest.
• SHEET j_ likely be a degradation product of lignin metabolism and hence microbes capable of lignin degradation would likely be tolerant to the presence of methanol. Further, the ability of lignin degraders to use lignin as a carbon c source provides a further rational basis for the selection system employed herein.
• the selection scheme was used to isolate a lignin degrading microorganism from nature, the method is useful for 0 selection regardless of the source of the variant strain.
• the strain may be naturally occurring, an induced mutation, or a recombinant resulting from sexual, asexual or genetic engineering processes.
• the method of selection described herein is particularly useful in ] _c- permitting the experi entor to recover the desired variant strain from among the population of non-lignin degrading strains from which it arose.
• a particularly useful lignin-degrading bacterium tentatively identified as a saprophytic
• the sample was first enriched for potential lignin degrades by culturing in a medium comprising methanol between about 0.2% and about 5% v/v, with about 1% being preferred. Since many lignin-degrading strains
• SHEET described by R. Summers et al. (Appl. Environ. Microbiol. 38: (1) 66-71 (1979)), yeast extract, and Kraft lignin at a pH 9.0. Following a series of transfers through a medium comprising glucose, a prototroph able to grow on lignin as the sole carbon source was recovered. As mentioned above, one such isolate has been tentatively identified as Erwinia sp. The metabolic products resulting from the incubation of this isolate with lignin were resolved by the layer chroma ography ( Figure 4) .
• PNPG p-Nitrophenyl-? -D-glycopyranoside
• the enzyme was partially purified from sonicated cell-free extracts by polyethylenimine treatment, ammonium sulfate fractionation, and DEAE cellulose ion exchange chromatography resulting in a 33% yield.
• the enzyme is active over a wide pH range (4.5-9.5), with an optimum of pH 6.0-7.0. Heat inactivation studies showed that enzyme activity was readily lost at temperature above 50°C.
• Bam HI digested Erwinia DNA was ligated into the Bam HI site of pBR322 and used to transform E__ Coli strain Cs412 (McLaughlin, J.R. , et al. , Nucleic Acid Res 10: 3905-3919 (1982)) by the methods of Hanahan (J Mol. Biol. 166: 447-580 (1983)). Transformants were plated on M9 minimal medium (Maniatis, T. et al. Ins "Molecular Cloning" Cold Spring Harbor Laboratory pp. 68-69 (1982)) containing salicin as sole carbon source and ampicillin (40 ug/ml) as the other selective agent.
• Salicin (Fig. 1) , which contains a pseudoether linkage between glucose and an aromatic nucleus, supported the growth of the Erwinia isolate but not the E. coli host strain Cs412.
• PNPG chromogenic substrate.
• Three isolates were obtained with the capability of releasing yellow p-nitrophenol from PNPG.
• the recombinant plas ids from 2 of the isolates conferred the ability to utilize salicin to E. coli Cs412 in subsequent transformation studies.
• Plasmid pNCl was shown to contain an 810 bp DNA
• SUBS insert upon digestion with Bam HI.
• the results of further restriction endonuclease mapping are shown in Fig. 2a.
• the insert contains single restriction sites for Sal I, Pst I, and Pvu II but no internal sites for EcoRI, Hind III, Ava I, or Cla I. 32 P-Nick translated
• pNCl was used as a. probe to demonstrate sequence homology between the cloned gene and Erwinia genomic DNA
• Genomic DNA (5 ug) isolated by the method of Saito and Mivra (Biochem. Biophys. Acta. 72: 619-269 1963) was digested to completion with 40 U each of the various restriction endonucleases (BRL), electrophorosed on 0.8% agarose gel, and transferred to a nitrocellulose filter.
• BBL restriction endonucleases
• P-labelled pNCl was prepared and 10 cpm of the probe was hybridized with the filter in 50% formamide, 4X ssc,
• TVTUTE SHEET gene is present as a single copy on the Erwinia sp. chromosome.
• aryletherase gene Cloning of the aryletherase gene into pBR322 allowed its study in the absence of the other enzymes which resulted in ring fission.
• the aryletherase was expressed in E. coli Cs412 and the transformants produced a single, unidentified aromatic compound in 2 hours incubation time with lignin (Fig. 3) .
• Cells of E. coli Cs412 or those transformed with pNCl or pNC2 were grown in 10 ml batch culture, pelleted at 12,000 x g, and resuspended in 1 ml 0.1% Kraft lignin in 10 mM MgCl 2 , 10 mM Tris HC1 pH 7.8. Incubation was at 32°C for 2 hours.
• Solvent system was benzene, 2-propanol, HN.OH (4:1:1). Product and standard were detected with short-wave ultraviolet light. The possibility exists that there may be other lignin degradation products not resolved in the solvent system employed. Vanillin and vanillic acid were demethylated to catechol after incubation with transformed strains. Catechol was identified by TLC after comparison with standards in several solvent systems. TLC analysis also showed that cloned aryletherase was able to degrade p-phenoxyphenol, syringaldehyde, and syringic acid (Fig. 1) . The product
• aryl etherase is capable of cleaving arylether linkages as well as methoxyl groups in aromatic lignin model compounds.
• an enzyme is often referred to by a variety of names, each reflecting a particular substrate specificity of that enzyme.
• aryl etherase is preferred; the enzyme may be referred to as a demethylase, de ethoxylase, aryl, alkyl ether hydrolase, or diaryl etherase each indicating a functional property of the enzyme.
• a deposit of biologically pure culture of the bacterium capable of degrading lignin tentatively identified as Erwinia sp. and a deposit of the plasmid pNCl containing the cloned aryletherase gene transformed into E . coli were made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland on September 20, 1984, the accession numbers ATCC 39873 and ATCC 39874 respectively were assigned and the requisite fees were paid. Access to said cultures will be available during pendency of this patent application to one determined by the Commissioner to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122. All restriction on availability of said culture to the public will be irrevocably removed upon the granting of a patent based upon this application and said cultures will remain permanently available during the term of said patent; and should the cultures become nonviable or be inadvertently
• a host organism transformed by a vector containing an expressible aryl etherase gene is c particularly useful for the production of the aryl etherase protein product.
• the transformed host under appropriate lignin-degrading conditions, can be used in a lignin-degrading process.
• the aryl etherase may be isolated from the host and used in 0 immobilized or free form as lignin-degrading catalyst.
• the transformed host can be used in processes for methanol production as well as chemical feedstock production.

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• 1984
  • 1984-09-27 US US06/655,193 patent/US4713336A/en not_active Expired - Fee Related
• 1985
  • 1985-09-24 FI FI862208A patent/FI862208L/fi not_active IP Right Cessation
  • 1985-09-24 JP JP60504258A patent/JPS62500281A/ja active Pending
  • 1985-09-24 WO PCT/US1985/001832 patent/WO1986002094A1/en not_active Ceased
  • 1985-09-24 DE DE19853590489 patent/DE3590489T1/de not_active Withdrawn
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• 1986
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