Means and methods for lignin pyrolysis
AssigneeUniversitaet des Saarlandes
InventorsChristoph Wittmann, Jost Van Duuren, Michael Kohlstedt, Jessica Stolzenberger, Soeren Starck, Nadja Barton, Mirjam Selzer, Michael Fritz, Paul De Wild, Martin Kuhl, Judith Becker
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Abstract
Conversion of organic educts, in particular biomass feedstock, into useful organic compounds via pyrolysis and bio-catalysts.
Claims53
A method of producing a phenolic compound, preferably catechol comprising the steps of
- i) Subjecting lignin to a fast pyrolysis process;
- ii) Condensing the resulting pyrolysis vapors, optionally directly introducing the resulting pyrolysis vapors into at least one aqueous phase, resulting in at least one acquatic phase comprising a phenolic compound, preferably catechol, and at least one organic phase;
- iii) Separating the resulting at least one acquatic phase comprising a phenolic compound, preferably catechol and the resulting at least one organic phase.
The method according to any one of the preceding claims, wherein the amount of the aqueous phase in which the pyrolysis vapors are introduced is at least 2 g per g lignin subjected to fast pyrolysis.
The method according to any one of the preceding claims, wherein the amount of the aqueous phase in which the pyrolysis vapors are introduced is in a range from 2 to 8 g aqueous phase per g lignin subjected to fast pyrolysis.
The method according to any one of the preceding claims, wherein the aqueous phase comprises at least 90% water.
The method according to any one of the preceding claims, wherein the biocatalyst is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria, algae, and plant cells.
The method according to any one of the preceding claims, wherein the host cell is a bacterial host cell selected from the group consisting of Bacillus bacteria (e.g., B. subtilis, B. megaterium), Acinetobacter bacteria, Nocardia bacteria, Xanthobacter bacteria, Escherichia bacteria (e.g., E. coli (e.g., strains DH10B, Stbl2, DH5-alpha, DB3, DB3.1, DB4, DB5, JDP682 and ccdA-over (e.g., U.S. application Ser. No. 09/518,188))), Streptomyces bacteria, Erwinia bacteria, Klebsiella bacteria, Serratia bacteria (e.g., S. marcescens), Pseudomonas bacteria (e.g., P. aeruginosa, P. putida), Salmonella bacteria (e.g., S. typhimurium, S. typhi), Megasphaera bacteria (e.g., Megasphaera elsdenii), photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema bacteria (e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola)), Pelodictyon bacteria (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium bacteria (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhodospirillum bacteria (e.g., R. rubrum)), Rhodobacter bacteria (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanelfi), Corynebacterium bacteria (e.g. Corynebacterium glutamicum ATCC 13032), Amycolatopsis bacteria (e.g. Amycolatopsis sp. ATCC 39116)
The method of any one of the preceding claims, wherein the host cell is a non-genetically modified host cell.
The method of any of the preceding claims, wherein said host cell comprises at least one (optionally heterologous) gene encoding a polypeptide having catechol 1,2-dioxygenase activity.
The method of any of the preceding claims, wherein said host cell comprises at least one (optionally heterologous) catA gene.
The method of any of the preceding claims, wherein said host cell comprises, operably linked to, e.g. upstream of, the at least one (optionally heterologous) gene, a promoter sequence corresponding to
- vi) SEQ ID No. 5 [Pem7];
- or
- vii) SEQ ID No. 6 [Pem7*];
- or
- viii) SEQ ID No. 7 [Ptuf];
- or
- ix) SEQ ID No. 8 [PrpoD];
- or
- x) SEQ ID No. 9 [Plac];
- or
- xi) SEQ ID No. 10 [PgyrB];
- xii) SEQ ID No. 11;
- or
- xiii) SEQ ID No. 12;
- or
- xiv) SEQ ID No. 13;
- or
- xv) SEQ ID No. 14;
- or
- xvi) SEQ ID No. 15;
- or
- xvii) SEQ ID No. 16;
- or
- xviii) SEQ ID No. 88 [Ptuf_1];
- or
- xix) SEQ ID No. 89 [Ptuf_short];
- or
- xx) SEQ ID No. 90 [Ptuf_s_2];
- or
- xxi) SEQ ID No. 91 [Ptuf_s_3];
- or
- xxii) SEQ ID No. 92 [Ptuf_s_4];
- or
- xxiii) SEQ ID No. 93 [Ptuf_s_5];
- or
- xxiv) SEQ ID No. 94 [Ptuf_s_6];
- or
- xxv) SEQ ID No. 95 [Ptuf_s_7];
- or
- xxvi) SEQ ID No. 96 [Ptuf_s_8];
- or
- xxvii) SEQ ID No. 97 [Ptuf_s_9];
- or
- xxviii) SEQ ID No. 98 [Ptuf_s_10];
- or
- xxix) SEQ ID No. 99 [Ptuf_s_11];
- or
- xxx) SEQ ID No. 100 [Ptuf_s_12];
- or
- xxxi) SEQ ID No. 101 [Pgro];
- or
- xxxii) SEQ ID No. 102 [Pgro_1];
- or
- xxxiii) SEQ ID No. 103 [Pgro_2];
- or
- xxxiv) SEQ ID No. 104 [Pgro_4];
- or
- xxxv) SEQ ID No. 105 [Pgro_5];
- more preferably SEQ ID No. 5 [Pem7].
The method of any of the preceding claims, wherein the at least one (optionally heterologous) gene is constitutively expressed.
The method of any of claims preceding claims, wherein said at least one heterologous gene is derived from Pseudomonas, preferably Pseudomonas putida, more preferably Pseudomonas putida strain KT2440 BN6.
The method of any of claims preceding claims, wherein said host cell is selected from Pseudomonas, preferably Pseudomonas putida, more preferably Pseudomonas putida strain KT2440, strain KT2440 JD2S or strain KT2440 BN6, even more preferably Pseudomonas putida strain KT2440 BN6.
The method of any of the preceding claims, wherein the host cell expresses a functional catA polypeptide and does not express a functional catB polypeptide.
The method any one of the preceding claims, wherein, the cis-cis-muconic acid derived after step iv) or iva) is white in color.
The method of any of the preceding claims, wherein the yield in cis-cis-muconic acid from catechol is greater than 95% w/w, or greater than 99% w/w.
A host cell which expresses a functional catA polypeptide, said catA polypeptide being characterized in that it has
- (a) the amino acid sequence shown in SEQ ID No. 108;
- or
- (b) an amino acid sequence which has at least 40% identity to the amino acid sequence shown in SEQ ID No. 108 and having catechol-1,2-dioxygenase activity;
- and does not express a functional catB polypeptide, said catB polypeptide being characterized in that it has
- (c) the amino acid sequence shown in SEQ ID No. 109;
- or
- (d) an amino acid sequence which has at least 25% identity to the amino acid sequence shown in SEQ ID No. 109 and having muconate cycloisomerase activity.
Corynebacterium glutamicum ATCC13032 cell which has its endogenous catA gene operably linked to a heterologous promoter and which does not express a functional catB polypeptide.
Amycolatopsis sp. ATCC39116 cell expressing its endogenous catA gene and which does not express a functional catB polypeptide.
Dependencies are inferred from the claim text.