RECOMBINANT MICROORGANISMS THAT CATABOLIZE ACETOVANILLONE AND METHODS OF USING SAME
20260043053 ยท 2026-02-12
Assignee
Inventors
- Timothy Donohue (Middleton, WI, US)
- Benjamin Hall (Waukesha, WI, US)
- Daniel Noguera (Madison, WI)
- Jeanette Neri (Bull Valley, IL, US)
Cpc classification
C12N9/1205
CHEMISTRY; METALLURGY
International classification
Abstract
Recombinant microorganisms that catabolize lignin aromatics, such as acetovanillone, and methods of using same to catabolize the lignin aromatics.
Claims
1. A recombinant microorganism comprising a recombinant aromatic kinase gene, wherein the recombinant aromatic kinase gene encodes an aromatic kinase protein, wherein the aromatic kinase protein is a homolog of Saro_1862 of Novosphingobium aromaticivorans DSM12444 comprising amino acid sequence comprising a lysine at a position corresponding to position 16 of SEQ ID NO:4.
2. The recombinant microorganism of claim 1, wherein the amino acid sequence is at least 95% or at least 99% identical to SEQ ID NO:4 or any sequence identified in Table 7.
3. The recombinant microorganism of claim 1, wherein the amino acid sequence is at least 80% identical to SEQ ID NO:4.
4. The recombinant microorganism of claim 1, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:4.
5. The recombinant microorganism of claim 1, wherein the recombinant microorganism is a bacterium.
6. The recombinant microorganism of claim 1, wherein the recombinant microorganism is a member of Actinomycetota, Alphaproteobacteria, Aquificae, Bacillota, Bacteroidota, Betaproteobacteria, Chlorobiota, Chloroflexota, Desulfuromonadia, Epsilonproteobacteria, Gammaproteobacteria, Nitrospiria, Planctomycetota, Thermomicrobiota, or Verrucomicrobiota.
7. The recombinant microorganism of claim 1, wherein the recombinant microorganism is an Alphaproteobacterium.
8. The recombinant microorganism of claim 1, wherein the recombinant microorganism is from an order selected from the group consisting of Sphingomonadales, Actinomyces, Gammaproteobacteria, Betaproteobacteria, and Bacilli.
9. The recombinant microorganism of claim 1, wherein the recombinant microorganism is from family Erythrobacteraceae.
10. The recombinant microorganism of claim 1, wherein the recombinant microorganism is from genus Novosphingobium.
11. The recombinant microorganism of claim 1, wherein the recombinant microorganism is capable of growing on acetovanillone, guaiacylpropanone, or p-OH acetophenone as a sole carbon source.
12. The recombinant microorganism of claim 1, wherein: the amino acid sequence is at least 95% identical to SEQ ID NO:4; and the recombinant microorganism is from genus Novosphingobium.
13. The recombinant microorganism of claim 12, wherein the recombinant microorganism is capable of growing on acetovanillone, guaiacylpropanone, or p-OH acetophenone as a sole carbon source.
14. The recombinant microorganism of claim 12, wherein the recombinant microorganism is capable of growing on acetovanillone as a sole carbon source.
15. A method of catabolizing a lignin aromatic, the method comprising culturing the recombinant microorganism of claim 1 in a medium comprising the lignin aromatic to thereby catabolize the lignin aromatic.
16. The method of claim 15, wherein the lignin aromatic comprises one or more of acetovanillone, guaiacylpropanone, and p-OH acetophenone.
17. The method of claim 15, wherein the lignin aromatic comprises acetovanillone.
18. A recombinant aromatic kinase protein comprising an amino acid sequence at least 95% identical to SEQ ID NO:4, wherein the amino acid sequence comprises a lysine at a position corresponding to position 16 of SEQ ID NO:4.
19. The recombinant aromatic kinase protein of claim 18, wherein the amino acid sequence is at least 99% identical to SEQ ID NO:4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0051] The recombinant microorganisms of the invention can comprise a recombinant aromatic kinase gene encoding an aromatic kinase protein. The aromatic kinase proteins of the invention are enzymes capable of phosphorylating a lignin aromatic. In preferred versions of the invention, the aromatic kinase protein is a homolog of Saro_1862 of Novosphingobium aromaticivorans DSM12444 comprising amino acid sequence comprising a lysine at a position corresponding to position 16 of SEQ ID NO:4.
[0052] Saro_1862 of Novosphingobium aromaticivorans DSM12444 has a coding sequence of SEQ ID NO:1 and a protein sequence of SEQ ID NO:2.
[0053] The homologs of Saro_1862 of Novosphingobium aromaticivorans DSM12444 can comprise proteins comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:4, orthologs of Saro_1862 of Novosphingobium aromaticivorans DSM12444, and recombinant variants of the orthologs of Saro_1862 of Novosphingobium aromaticivorans DSM12444. Each of these homologs can be modified to comprise a lysine at a position corresponding to position 16 of SEQ ID NO:4. An exemplary coding sequence of SEQ ID NO:4 is SEQ ID NO:3. Exemplary homologs of Saro_1862 of Novosphingobium aromaticivorans DSM12444 comprise an amino acid sequence of SEQ ID NO: 4, any amino acid sequence identified in Table 6 (e.g., by accession numbers from NCBI available on Sep. 12, 2023), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4 or any sequence identified in Table 6.
[0054] The aromatic kinase protein of the invention is preferably capable of catalyzing the phosphorylation of a lignin aromatic, such as acetovanillone, guaiacylpropanone, p-OH acetophenone, or the lignin aromatics shown in
[0055] The recombinant microorganisms of the invention may comprise any type of microorganism. The microorganism may be prokaryotic or eukaryotic. Suitable prokaryotes include bacteria and archaea. Suitable types of bacteria include - and -proteobacteria, gram-positive bacteria, gram-negative bacteria, ungrouped bacteria, phototrophs, lithotrophs, and organotrophs. Suitable eukaryotes include yeast and other fungi. The microorganism in some versions can be from an order selected from the group consisting of Sphingomonadales and Pseudomonadales. The microorganism in some versions can be from a family selected from the group consisting of Sphingomonadaceae and Pseudomonadaceae. The microorganism in some versions can be from a genus selected from the group consisting of Sphingomonas, Sphingobium, Sphingosinicella, Sphingopyxis, Novosphingobium, Pseudomonas, Erythrobacter (e.g., sp. SG61-1L), and Altererythrobacter. The microorganism in some versions can be a member of Actinomycetota, Alphaproteobacteria, Aquificae, Bacillota, Bacteroidota, Betaproteobacteria, Chlorobiota, Chloroflexota, Desulfuromonadia, Epsilonproteobacteria, Gammaproteobacteria, Nitrospiria, Planctomycetota, Thermomicrobiota, or Verrucomicrobiota. The microorganism in some versions can be an Alphaproteobacterium. The microorganism in some versions can be from an order selected from the group consisting of Sphingomonadales, Actinomyces, Gammaproteobacteria, Betaproteobacteria, and Bacilli. The microorganism in some versions can be from family Erythrobacteraceae. The microorganism in some versions can be from genus Novosphingobium. The microorganism in some versions can be an Novosphingobium aromaticivorans, such as Novosphingobium aromaticivorans DSM12444.
[0056] The recombinant genes of the invention can be configured to be expressed or overexpressed in the microorganism. If a microorganism endogenously comprises a particular gene, the gene may be modified to exchange or optimize promoters, exchange or optimize enhancers, or exchange or optimize any other genetic element to result in increased expression of the gene. Alternatively, one or more additional copies of the gene or coding sequence thereof may be introduced to the cell for enhanced expression of the gene product. If a microorganism does not endogenously comprise a particular gene, the gene or coding sequence thereof may be introduced to the microorganism for heterologous expression of the gene product. The gene or coding sequence may be incorporated into the genome of the microorganism or may be contained on an extra-chromosomal plasmid. The gene or coding sequence may be introduced to the microorganism individually or may be included on an operon. Techniques for genetic manipulation are described in further detail below. The recombinant microorganisms of the invention may be genetically altered to express or overexpress any of the specific genes or gene products explicitly described herein or homologs thereof. Various promoters capable of driving overexpression of a gene are well known in the art. See, e.g., U.S. Pat. No. 9,708,630, which is incorporated herein by reference in its entirety.
[0057] In some versions, the recombinant genes of the invention comprise a coding sequence operably linked to a heterologous genetic element. In some versions, the heterologous genetic element is a heterologous promoter.
[0058] Homolog as used herein with respect to a proteins and/or protein sequences refers to a protein and/or protein sequence derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, homolog as used herein with respect to nucleic acids and/or nucleic acid sequences refers to nucleic acids and/or nucleic acid sequences derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Nucleic acid or gene product (amino acid) sequences of any known gene, including the genes or gene products described herein, can be determined by searching any sequence databases known in the art using the gene name or accession number as a search term. Common sequence databases include GenBank (www.ncbi.nlm.nih.gov), ExPASy (expasy.org), KEGG (www.genomc.jp), among others. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity (e.g., identity) over 50, 100, 150 or more residues (nucleotides or amino acids) is routinely used to establish homology (e.g., over the full length of the two sequences to be compared). Higher levels of sequence similarity (e.g., identity), e.g., 30%, 35% 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, can also be used to establish homology. Accordingly, homologs of the genes or gene products described herein include genes or gene products having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the genes or gene products described herein. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. The homologous proteins should demonstrate comparable activities and, if an enzyme, participate in the same or analogous pathways. Homologs include orthologs and paralogs. Orthologs are genes and products thereof in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same or similar function in the course of evolution. Paralogs are genes and products thereof related by duplication within a genome. As used herein, orthologs and paralogs are included in the term homologs.
[0059] For sequence comparison and homology determination, one sequence typically acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters. A typical reference sequence of the invention is a nucleic acid or amino acid sequence corresponding to the genes or gene products described herein.
[0060] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2008)).
[0061] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity for purposes of defining homologs is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
[0062] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. The above-described techniques are useful in identifying homologous sequences for use in the methods described herein.
[0063] The term encode, such as in the phrase the recombinant aromatic kinase gene encodes an aromatic kinase protein, is used herein in an open-ended sense, such that a gene that encodes a specified protein or amino acid sequence must code for at least the specified protein or amino acid sequence but may also code for further proteins, domains, moieties, or sequences within the same reading frame as the specified protein or amino acid sequence. Thus, in some versions,
[0064] The terms identical or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described above (or other algorithms available to persons of skill) or by visual inspection.
[0065] The phrase substantially identical in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90, about 95%, about 98%, or about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such substantially identical sequences are typically considered to be homologous, without reference to actual ancestry. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, at least about 250 residues, or over the full length of the two sequences to be compared.
[0066] The terms corresponds to and corresponding to used with reference to an amino acid residue or position refer to an amino acid residue or position in a first protein sequence being positionally equivalent to an amino acid residue or position in a second reference protein sequence by virtue of the fact that the residue or position in the first protein sequence aligns to the residue or position in the reference sequence using bioinformatic techniques, for example, using the methods described herein for preparing a sequence alignment. The corresponding residue in the first protein sequence is then assigned the position number in the second reference protein sequence.
[0067] Derived: When used with reference to a nucleic acid or protein, derived means that the nucleic acid or polypeptide is isolated from a described source or is at least 70%, 80%, 90%, 95%, 99%, or more identical to a nucleic acid or polypeptide included in the described source.
[0068] Endogenous: As used herein with reference to a nucleic acid molecule, genetic element (e.g., gene, promoter, etc.), or polypeptide in a particular cell, endogenous refers to a nucleic acid molecule, genetic element, or polypeptide that is in the cell and was not introduced into the cell or transferred within the genome of the cell using recombinant engineering techniques. For example, an endogenous genetic element is a genetic element that was present in a cell in its particular locus in the genome when the cell was originally isolated from nature.
[0069] Exogenous: As used herein with reference to a nucleic acid molecule, genetic element (e.g., gene, promoter, etc.), or polypeptide in a particular cell, exogenous refers to any nucleic acid molecule, genetic element, or polypeptide that was introduced into the cell or transferred within the genome of the cell using recombinant engineering techniques. For example, an exogenous genetic element is a genetic element that was not present in its particular locus in the genome when the cell was originally isolated from nature.
[0070] Expression: The process by which a gene's coded information is converted into the structures and functions of a cell, such as a protein, transfer RNA, or ribosomal RNA. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs).
[0071] Introduce: When used with reference to genetic material, such as a nucleic acid, and a cell, introduce refers to the delivery of the genetic material to the cell in a manner such that the genetic material is capable of being expressed within the cell. Introduction of genetic material includes both transformation and transfection. Transformation encompasses techniques by which a nucleic acid molecule can be introduced into cells such as prokaryotic cells or non-animal eukaryotic cells. Transfection encompasses techniques by which a nucleic acid molecule can be introduced into cells such as animal cells. These techniques include but are not limited to introduction of a nucleic acid via conjugation, electroporation, lipofection, infection, and particle gun acceleration.
[0072] Isolated: An isolated biological component (such as a nucleic acid molecule, polypeptide, or cell) has been substantially separated or purified away from other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA and proteins. Nucleic acid molecules and polypeptides that have been isolated include nucleic acid molecules and polypeptides purified by standard purification methods. The term also includes nucleic acid molecules and polypeptides prepared by recombinant expression in a cell as well as chemically synthesized nucleic acid molecules and polypeptides. In one example, isolated refers to a naturally occurring nucleic acid molecule that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5 end and one on the 3 end) in the naturally-occurring genome of the organism from which it is derived.
[0073] Gene: Genes minmally include a promoter operably linked to a coding sequence, and can include other elements that facilitate or regulate the transcription and/or translation of the coding sequence.
[0074] Heterologous: The term heterologous refers to an element in an arrangement with another element that does not occur in nature. For example, a gene or protein that is heterologous to a given cell is a gene or protein that does not occur in the cell in nature. A promoter that is heterologous to a given coding sequence is a promoter that is not operably linked to the coding sequence in nature.
[0075] Nucleic acid: Encompasses both RNA and DNA molecules including, without limitation, cDNA, genomic DNA, and mRNA. Nucleic acids also include synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand, the antisense strand, or both. In addition, the nucleic acid can be circular or linear.
[0076] Operably linked: A first element is operably linked with a second element when the first element is placed in a functional relationship with the second element. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. A secretion signal sequence is operably linked to a protein (such as an enzyme) when the secretion signal sequence affects secretion of the protein from a cell. Operably linked and operationally linked are used interchangeably.
[0077] Overexpress: When a gene is caused to be transcribed at an elevated rate compared to the endogenous or basal transcription rate for that gene. In some examples, overexpression additionally includes an elevated rate of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using RT-PCR and protein levels can be assessed using SDS-PAGE gel analysis.
[0078] Recombinant: A recombinant nucleic acid or polypeptide is one comprising a sequence that is not naturally occurring. A recombinant gene is a gene that comprises a recombinant nucleic acid sequence, is present within a cell in which it does not naturally occur, and/or is present in a different locus (e.g., genetic locus or on an extrachromosomal plasmid) within a particular cell than in a corresponding native cell. A recombinant cell (such as a recombinant microorganism) is one that comprises a recombinant nucleic acid, a recombinant gene, or a recombinant polypeptide. An example of a recombinant gene is a gene that has a coding sequence operably linked to a heterologous promoter.
[0079] Recombinant variant: Used with reference to an ortholog, recombinant variant refers to a variant of the ortholog that comprises one or more modifications to amino acid sequence of the ortholog. Exemplary modifications include substitutions, deletions, and insertions. The recombinant variant preferably comprises an amino acid sequence at least 95% or at least 99% identical to the amino acid sequence of the ortholog.
[0080] Another aspect of the invention is directed to methods of catabolizing a lignin aromatic. The methods can comprise culturing the recombinant microorganism of the invention in a medium comprising the lignin aromatic to thereby catabolize the lignin aromatic.
[0081] Lignin aromatic as used herein refers to an aromatic present in or derived from lignin. The lignin aromatics can be a monomer, a dimer, an oligomer, or a polymer. The lignin aromatics can comprise syringyl aromatics, guaiacyl aromatics, p-hydroxyphenyl aromatics, or any combinations thereof. Syringyl, guaiacyl, and p-hydroxyphenyl aromatics differ in their degree of methoxilation of the aromatic ring. Syringyl aromatics comprise methoxy groups at the 3 and 5 positions of the aromatic ring. Guaiacyl aromatics comprise a methoxy group on only one of the 3 and 5 positions on the aromatic ring. p-Hydroxyphenyl aromatics are devoid of methoxy groups on either of the 3 and 5 positions of the aromatic ring.
[0082] Some aspects of the invention are directed to methods of catabolizing a lignin aromatic. The methods can comprise culturing a recombinant microorganism of the invention in a medium comprising the lignin aromatic to thereby catabolize the lignin aromatic.
[0083] In some versions, the lignin aromatic is derived from, isolated from, or provided in the form of depolymerized lignin. In some versions, the medium comprises depolymerized lignin. Methods of depolymerizing lignin are well known in the art. See Pandey et al. 2010 (Pandey M P, Kim C S. Lignin Depolymerization and Conversion: A Review of Thermochemical Methods. Chemical & Engineering Technology, 2010, Vol. 34, Issue 1, pp. 3-145) and Wang et al. 2013 (Wang H, Tucker M, Ji Y. Recent Development in Chemical Depolymerization of Lignin: A Review. Journal of Applied Chemistry, 2013, Volume 2013, Article ID 838645).
[0084] The depolymerized lignin can be derived from pretreated lignocellulosic biomass. Methods of pretreating lignocellulosic biomass are well known in the art. See Kumar et al. 2017 (Kumar AK and Sharma S. Recent Updates on Different Methods of Pretreatment of Lignocellulosic Feedstocks: A Review. Bioresour. Bioprocess. (2017) 4:7); Kumar et al. 2009 (Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P., Methods for Pretreatment of lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research 2009, 48, (8), 3713-3729); Wang et al. 2013 (Wang H, Tucker M, Ji Y. Recent Development in Chemical Depolymerization of Lignin: A Review. (2013) Journal of Applied Chemistry. 2013:1-9), and Karlen et al. 2020 (Karlen S D, Fasahati P, Mazaheri M, Serate J, Smith R A, Sirobhushanam S, Chen M, Tymkhin V I, Cass C L, Liu S, Padmakshan D, Xic D, Zhang Y, McGee M A, Russell J D, Coon J J, Kaeppler H F, de Leon N, Maravelias C T, Runge, Kaeppler S M, Sedbrook J C, Ralph J. Assessing the viability of recovering hydroxycinnamic acids from lignocellulosic biorefinery alkaline pretreatment waste streams. ChemSusChem. 2020 Jan. 26). Examples include chipping, grinding, milling, steam pretreatment, ammonia fiber expansion (AFEX, also referred to as ammonia fiber explosion), ammonia recycle percolation (ARP), CO.sub.2 explosion, steam explosion, ozonolysis, wet oxidation, acid hydrolysis, dilute-acid hydrolysis, alkaline hydrolysis, organosolv, ionic liquids, gamma-valerolactone, enzymatic pretreatment, biological pretreatment, and pulsed electrical field treatment, among others.
[0085] The lignocellulosic biomass can be derived from any source, such as corn cobs, corn stover, cotton seed hairs, grasses, hardwood stems, leaves, newspaper, nut shells, paper, softwood stems, sorghum, switchgrass, waste papers from chemical pulps, wheat straw, wood, woody residues, mixed biomass species such as those produced by native prairie, and other sources. Sources that maintain -5 bonds in lignin are preferred.
[0086] Another aspect of the invention is directed to recombinant aromatic kinase proteins. In some versions, the recombinant aromatic kinase proteins comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4, wherein the amino acid sequence comprises a lysine at a position corresponding to position 16 of SEQ ID NO:4.
[0087] Another aspect of the invention is directed to phosphorylating a lignin aromatic. In some versions, the methods comprise contacting a lignin aromatic with a recombinant aromatic kinase protein of the invention to thereby phosphorylate the lignin aromatic. The lignin aromatic can comprise any lignin aromatic described herein as being phosphorylated by the recombinant aromatic kinase protein having a sequence of SEQ ID NO:4. Exemplary lignin aromatics include those shown as being phosphorylated by the recombinant aromatic kinase proteins of the invention in
[0088] Another aspect of the invention is directed to recombinant genes. The recombinant genes can comprise any recombinant gene of the invention and/or encode any aromatic kinase protein of the invention.
[0089] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.
[0090] The elements and method steps described herein can be used in any combination whether explicitly described or not.
[0091] All combinations of method steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
[0092] As used herein, the singular forms a, an, and the include plural referents unless the content clearly dictates otherwise.
[0093] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0094] All patents, patent publications, and peer-reviewed publications (i.e., references) cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated as being incorporated by reference. In case of conflict between the present disclosure and the incorporated references, the present disclosure controls.
[0095] It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the claims.
EXAMPLES
A Newly Discovered Kinase from Novosphingobium Aromaticivorans Involved in Catabolism of Aromatic Monomers
SUMMARY
[0096] The plant polymer lignin is an abundant renewable source of aromatics that could be used for production of various chemicals. Oxidative lignin depolymerization methods generate a variety of products that includes acetovanillone, an aromatic monomer that is a derivative of vanillin and contains an acetyl side chain on the ring. The Alphaproteobacterium Novosphingobium aromaticivorans strain DSM12444 can convert a variety of lignin-derived aromatic compounds into valuable products, but it cannot metabolize acetovanillone. In this work, we used adaptive laboratory evolution to identify a mutation in the previously uncharacterized gene product Saro_1862 that is both necessary and sufficient for growth of N. aromaticivorans in the presence of acetovanillone as a sole organic carbon source. We show that the resulting glutamate (E) to lysine (K) substitution at amino acid residue 16 allows a recombinant form of Saro_1862 to phosphorylate acetovanillone in the presence of ATP. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro, and we provide genetic evidence that activity of Saro_1862 is required for metabolism of several other aromatic compounds in vivo. Structural predictions suggest that the E16K amino acid substitution in Saro_1862 enhances ATP binding in this newly discovered aromatic kinase. A search for homologs of Saro_1862 and other genes that are required for acetovanillone metabolism suggests that homologs of this newly discovered kinase could be involved in aromatic metabolism in organisms across the bacterial phylogeny.
INTRODUCTION
[0097] Among the challenges facing society is the industrial production of valuable chemicals from renewable raw materials. As the second most abundant biopolymer on Earth, lignin is a potential raw material for producing chemicals from renewable sources. While lignin oxidation is an efficient means of lignin depolymerization, it can generate modified aromatics, such as acetovanillone, that are unable to be metabolized by Novosphingobium aromaticivorans and other microbes that are being developed as hosts for creating valuable products from lignin. This work used adaptive laboratory evolution to identify a previously uncharacterized N. aromaticivorans kinase that phosphorylates acetovanillone and other aromatics. Discovery of this kinase expands our understanding of the enzymes involved in aromatic metabolism and provides new routes for engineering metabolism of acetovanillone and other lignin-derived aromatics into products by this and other microbes.
[0098] Strains of Novosphingobium aromaticivorans DSM12444 that convert aromatic monomers and other components of deconstructed biomass into 2-pyrone-4,6-dicarboxylic acid (PDC) (15), cis, cis-muconic acid (16), or other products (17) were developed. However, previous analysis showed that N. aromaticivorans DSM12444 was incapable of metabolizing acetovanillone (14). Because acetovanillone is structurally similar to vanillin and other aromatics that N. aromaticivorans DSM12444 can metabolize (18,19), we hypothesized that adaptive laboratory evolution could be used to yield strains able to metabolize this chemically modified product of lignin deconstruction.
[0099] In support of this hypothesis, studies in other bacteria have recently identified an acetovanillone metabolic pathway encoded by acvABCDEF and vceAB in Sphingobium sp. SYK-6 (20) or the homologs hpeHICBADEF in Rhodococcus rhodochrous GD02 (21), which converts acetovanillone to vanilloyl acetic acid, followed by further transformation to vanillic acid via a Coenzyme A (22) intermediate (
[0100] Here we report the use of adaptive laboratory evolution to identify mutations that allow growth of N. aromaticivorans in the presence of acetovanillone as a sole organic carbon source. By analyzing genome sequence changes in a set of evolved strains, we find that they each contain a mutation that generates a glutamate to lysine amino acid substitution at residue 16 of a previously uncharacterized gene product, Saro_1862. We show that this mutation is sufficient for acetovanillone metabolism when it is generated in the parent N. aromaticivorans strain, and that the resulting E16K amino acid change in Saro_1862 allows a recombinant form of this protein to phosphorylate acetovanillone in the presence of ATP. Purified recombinant Saro_1862 E16K also phosphorylates a variety of aromatic monomers in vitro, and we provide evidence that this newly discovered kinase is required for metabolism of selected compounds in vivo. Structural predictions suggest that the E16K substitution in Saro_1862 allows kinase activity by generating a functional ATP binding site. By searching for homologs of Saro_1862 and other genes reported to be required for acetovanillone metabolism in genome sequences of other bacteria, we find that homologs of the protein are present across the bacterial phylogeny where they could be involved in metabolism of acetovanillone and other aromatic compounds.
TABLE-US-00001 TABLE 1 Percent identity of acetovanillone metabolism proteins encoded by the genes shown in FIG. 1. 1861/ 1860/ 1859/ 1858/ AcvA/ AcvB/ AcvC/ AcvD/ AcvE/ AcvF/ 2625/ 2868/ HpeH HpeI HpeC HpeB HpeA HpeD HpeE HpeF N. aromaticivorans N/A N/A 41% 55% 52% 38% N/A N/A vs Sphingobium sp. N. aromaticivorans N/A N/A 34% 49% 40% 33% 26% 48% vs R. rhodochrous Sphingobium sp. 45% 42% 38% 51% 42% 35% N/A N/A vs R. rhodochrous
Results
Adaptive Laboratory Evolution Generates Acetovanillone-Metabolizing Strains of N. aromaticivorans DSM12444
[0101] While N. aromaticivorans DSM12444 can metabolize a variety of aromatic compounds which share structural similarity with acetovanillone, including vanillin, it is not capable of catabolizing acetovanillone (14). Thus, we hypothesized that adaptive laboratory evolution was a promising strategy for identifying one or more mutations that lead to acetovanillone metabolism in N. aromaticivorans. To test this hypothesis, we grew the parent N. aromaticivorans strain 1244441879 in 3 parallel minimal media cultures with a mixture of acetovanillone and vanillin as the sole carbon sources. We initially grew cultures in media with 0.34 mM acetovanillone and 1.36 mM vanillin, passaged them three times, and gradually increased the acetovanillone: vanillin ratio throughout multiple subsequent passages until the cultures only contained acetovanillone as the sole carbon source (
Identification of a Mutation that is Both Necessary and Sufficient for Acetovanillone Metabolism by N. aromaticivorans
[0102] Analysis of the sequenced genomic DNA revealed one mutation that was shared by all 12 isolated colonies that were generated during adaptive laboratory evolution for growth on acetovanillon. This single nucleotide change is predicted to alter the coding sequence of Saro_1862 and lead to a glutamate (E) to lysine (K) amino acid change at residue 16 (E16K) of this previously uncharacterized protein. To test the role of this mutation in acetovanillone metabolism in the evolved strains, we introduced the same single nucleotide mutation into the genome of the parent strain by homologous recombination to create strain TD606. We found that strain TD606 was able to grow on acetovanillone as a sole carbon source, comparable with evolved strains (
Saro_1862 is Part of a Predicted Acetovanillone Metabolism Operon
[0103] One additional feature of the Saro_1862 mutation that was identified in each of the evolved strains is that this gene is predicted to be part of a transcription unit that encodes homologs of acvCDEFIhpeCBAD genes that were recently reported to be involved in acetovanillone metabolism in Sphingobium sp. SYK-6 and R. rhodochrous GD02, respectively (20,21). Thus, we hypothesized that these homologs of acvCDEFIhpeCBAD might play the same role in acetovanillone metabolism in N. aromaticivorans. To test this hypothesis, we created N. aromaticivorans strains lacking the homologs of acvC/hpeC (Saro_1861) and acvF/hpeD (Saro_1858) in the acetovanillone-metabolizing TD606 strain. Consistent with their predicted role in acetovanillone metabolism, we found that these mutant strains (TD625 lacking Saro_1861 and TD634 lacking Saro_1858) could no longer grow on acetovanillone as a sole carbon source (
Saro_1862 is a Previously Uncharacterized Acetovanillone Kinase
[0104] Two observations provided hints at the function of Saro_1862 in N. aromaticivorans acetovanillone metabolism. First, the predicted N. aromaticivorans Saro_1858-1862 transcription unit lacks genes predicted to encode subunits of the heterodimeric acetovanillone kinases AcvAB/HpcHI. In addition, Saro_1862 is annotated in NCBI to encode a protein with a Tm-1-like ATP binding domain. Thus, we hypothesized that the Saro_1862 E16K protein could be a previously unknown acetovanillone kinase that uses ATP to phosphorylate this aromatic. To test this hypothesis, we expressed and purified the N. aromaticivorans Saro_1862 E16K variant protein and incubated it with acetovanillone and ATP in vitro. HPLC-MS analysis of this reaction mixture showed the disappearance of acetovanillone and the accumulation of a compound with a m/z ratio corresponding to phosphorylated acetovanillone (
[0105] As a control, we expressed and purified the Saro_1862 protein from the wild type N. aromaticivorans DSM12444 strain and detected a greatly reduced level of acetovanillone phosphorylation under identical reaction conditions (
[0106] To make a direct comparison of Saro_1862 E16K acetovanillone kinase activity to the reported activity of the R. rhodochrous GD02 heterodimeric acetovanillone kinase HpeHI, we tested the activity of the variant protein using the spectrophotometric assay that measures conversion of acetovanillone to phospho-acetovanillone described in (21). Under comparable reaction conditions, the Saro_1862 E16K protein phosphorylated acetovanillone at a rate (+SD) of 645 min1 (n=3 preparations of Saro_1862 E16K,
TABLE-US-00002 TABLE 3 Comparison of acetovanillone phosphorylation rates (SD) with WT Saro_1862, Saro_1862-E16K, and HpeHI* Kinase Rate (min.sup.1) Relative WT Saro_1862 0.04 0.01 1 Saro_1862-E16K 64 5 ~1600 HpeHI 4.1 0.4 ~100 *Heterodimeric Rhodococcus rhodocrous acetovanillone kinase (Dexter et al. 2022 (21))
Saro_1862-E16K had 1600 fold higher rate of acetovanillone phosphorylation than wild type Saro_1862. Saro_1862-E16K phosphorylation rate was 16-fold higher than HpeEI from Rhodococcus rhodocrous.
[0107] To investigate the Saro_1862 native oligomeric state, we analyzed pure Saro_1862 E16K samples alongside a protein standard mix using size exclusion chromatography. This analysis resulted in a predicted native molecular weight of 50.3 kDa (
Saro_1862 E16K Phosphorylates a Variety of Aromatic Ketones and Aldehydes In Vitro
[0108] As Saro_1862 represents a previously uncharacterized kinase, we sought to determine if it can phosphorylate other monomers that could be relevant for the metabolism of biomass-derived aromatics by N. aromaticivorans. Using the same reaction conditions as for acetovanillone (
[0109] Based on the results of these in vitro assays, we tested the N. aromaticivorans DSM12444 parent strain and TD606 for growth on some of the aromatics that were phosphorylated by Saro_1862 E16K. This analysis showed that cells containing the Saro_1862 E16K variant (TD606), but not the N. aromaticivorans parent, were able to grow with guaiacylpropanone and p-hydroxyacetophenone as the sole carbon source (
Presence of Saro_1862 Homologs in Bacteria Predicted to Encode Acetovanillone Metabolism Genes
[0110] The discovery of Saro_1862 as a newly identified aromatic kinase prompted us to ask if homologs of this protein are present in other bacteria that are predicted to encode acetovanillone metabolism genes. To answer this question, we searched for bacterial genomes that contained a Saro_1862 homolog as well as homologs of acvCDEF and vceAB or hpeCBAD and hpeEF that are reported to be required for acetovanillone metabolism in Sphingobium sp. SYK-6 and R. rhodochrous GD02, respectively (20,21). We also searched for bacterial genomes that contained acvAB or hpeHI homologs as the predicted acetovanillone kinase along with the other known acetovanillone metabolism genes above. After searching 17,510 genomes in the NCBI representative prokaryotic genome database (downloaded Sep. 12, 2023), we found that more predicted acetovanillone-metabolizing species contain genes encoding AcvAB/HpeHI kinase homologs (n=570) (Table 5, which shows species that encode homologs of AcvAB or HpeHI, AcvC or HpeC, AcvD or HpeB, AcvE or HpeA, AcvF or HpeD, VceA or HpeE, and VceB or HpeF) compared to ones that encode a Saro_1862 kinase homolog (n=266) (Table 4, which shows species that encode homologs of Saro_1862, AcvC or HpeC, AcvD or HpeB, AcvE or HpeA, AcvF or HpeD, VceA or HpeE, and VceB or HpeF). In addition, this analysis predicts that 179 of these species encode homologs of both AcvAB/HpeHI and Saro_1862 (Table 6), suggesting that some bacteria contain two enzymes that could potentially phosphorylate acetovanillone or structurally related substrates. Table 7 shows homologs of Saro_1862 regardless of the predicted acetovanillone-metabolizing ability of the organism containing it. Table 8 shows summaries of the numbers of types of organisms presented in Tables 4 and 5.
TABLE-US-00003 TABLE 8 Summaries of the numbers of types of organisms presented in Tables 4 and 5. From Table 4 From Table 5 Organism Type Number Organism Type Number Actinomycetota 105 Actinomycetota 292 Alphaproteobacteria 84 Alphaproteobacteria 61 Bacillota 28 Aquificae 2 Bacteroidota/ 5 Bacillota 120 Chlorobiota group Betaproteobacteria 6 Bacteroidota/ 8 Chlorobiota group Chloroflexota 1 Betaproteobacteria 15 Desulfuromonadia 1 Desulfuromonadia 1 Gammaproteobacteria 4 Epsilonproteobacteria 1 Planctomycetota 30 Gammaproteobacteria 42 Thermomicrobiota 1 Nitrospiria 1 Verrucomicrobiota 1 Planctomycetota 25 Thermomicrobiota 2
[0111] The results of this analysis also predict that the presence of genes potentially involved in acetovanillone metabolism is not limited to a particular clade of the bacterial phylogeny (
DISCUSSION
[0112] This work sought to expand our knowledge of the aromatic substrate utilization capabilities of N. aromaticivorans DSM12444. Acetovanillone is an aromatic monomer recovered directly or indirectly in significant amounts from several biomass depolymerization methods, but it cannot be used as a sole carbon source by this strain of N. aromaticivorans (14). This work used adaptive laboratory evolution of N. aromaticivorans DSM12444 to generate strains that can metabolize acetovanillone. Genomic analyses identified a single mutation necessary for growth in the presence of acetovanillone in Saro_1862, which led to the biochemical and genetic characterization of this protein as a previously uncharacterized aromatic kinase.
Saro_1862 Represents a Previously Uncharacterized Class of Kinase
[0113] Saro_1862 is annotated in NCBI as a Tm-1-like ATP-binding domain-containing protein. However, biochemical studies of Tm-1 have yet to report kinase activity (23). In addition, the UPF0261 domain within Saro_1862 has no other biochemical characterization other than as a homolog of Tm-1. Saro_1862 is not homologous to either of the subunits of the known heterodimeric acetovanillone kinases AcvAB/HpeHI. In addition, an AlphaFold predicted structure of Saro_1862 (24) does not have significant similarity to other known kinases, according to the DALI Protein Structure Comparison Server (25). Thus, it appears that Saro_1862 represents a previously uncharacterized member of the kinase protein family.
Predicted Mechanism for how the Saro_1862 E16K Amino Acid Change Allows Aromatic Phosphorylation
[0114] The ability of the single E16K amino acid change in Saro_1862 to enable kinase activity suggests this residue plays an important role in catalysis. To investigate a possible role for this residue in enzyme function, we superimposed the AlphaFold (24) predicted structure of Saro_1862 onto the ATPS-bound structure of Tm1 (PDB: 3WRX). This alignment showed that E16 in Saro_1862 corresponds to K20 in Tm1, which makes contacts with the phosphate groups of bound ATPS (
[0115] We also found that the glutamate variant at this position (E16) in N. aromaticivorans DSM12444 is rare, which is consistent with this residue's importance in catalysis. Among all of the Saro_1862 homologs in Novosphingobium species in the NCBI representative prokaryotic genomes database, Saro_1862 from N. aromaticivorans DSM12444 is the only protein that encodes a glutamate (E) at position 16. All other sequenced Novosphingobium spp. are predicted to encode a lysine (K) at the corresponding position of their Saro_1862 homolog, so they would be predicted to have the ability to phosphorylate acetovanillone.
Importance of Saro_1862 in Metabolism of Lignin-Derived Aromatics
[0116] Our results show that Saro_1862 E16K phosphorylates S-, G-, and H-type aromatics in vitro, and it prefers substrates that contain ketone and aldehyde side chains. Our results further show that Saro_1862 E16K is important for metabolism of some of these compounds in vivo, such as acetovanillone, guaiacylpropanone, and p-hydroxyacetophenone (
[0117] We did not detect significant growth of the evolved strains or TD606 in SMB media containing 1 mM acetosyringone as the sole carbon source (
[0118] On the other hand, other aromatics, namely vanillin and p-hydroxybenzaldehyde, were phosphorylated by Saro_1862 E16K in vitro but did not require Saro_1862 E16K for in vivo metabolism (
Phosphorylation of Aromatics by Saro_1862
[0119] Although they are not homologous, our data predict that Saro_1862 and AcvAB/HpeHI play a similar role in acetovanillone phosphorylation. Comparisons of published data on aromatic phosphorylation by AcvAB (20) with our data from Saro_1862 E16K (
[0120] Despite having similar substrate specificities, Saro_1862 E16K phosphorylates acetovanillone at a rate more than an order of magnitude higher than the value reported for the heterodimeric HpeHI protein (21). In addition, the function of Saro_1862 as a single subunit enzyme removes the need to balance levels of multiple gene products for future analyses of aromatic kinase activity. These properties of Saro_1862 predict that heterologous expression of acetovanillone metabolism genes in other bacteria may benefit from using Saro_1862 E16K as the acetovanillone kinase, compared to one of the heterodimeric acetovanillone kinases.
Predicted Acetovanillone Kinases Among Bacteria
[0121] After searching for acetovanillone metabolism genes across the bacterial tree of life (
CONCLUSION
[0122] In this work, we used adaptive laboratory evolution to create strains of N. aromaticivorans DSM12444 capable of metabolizing acetovanillone, an aromatic monomer that results from oxidative lignin depolymerization methods. Genomic analysis of evolved strains identified an E16K amino acid change in Saro_1862 that is both necessary and sufficient for acetovanillone metabolism in N. aromaticivorans. Biochemical and genetic characterization of Saro_1862 reveal it is a previously uncharacterized type of kinase that phosphorylates aromatic monomers, and is required for metabolism of some aromatics with ketone side chains. The single subunit Saro_1862 protein also has some kinetic and biochemical advantages over the unrelated heterodimeric acetovanillone kinases AcvAB/HpeHI, including an order of magnitude greater reaction rate. Homologs of Saro_1862 are found throughout the bacterial phylogeny, highlighting the potential importance of aromatic phosphorylation during metabolism in many organisms.
Materials and Methods
Bacterial Strains and Growth Media
[0123] For all experiments, we used the parent strain N. aromaticivorans 124441879 (33), in which the putative sacB gene has been deleted to allow markerless genomic modification by variants of the pK18mobsacB plasmid (34). N. aromaticivorans cultures were grown in Standard Mineral Base (SMB) (33) at an initial pH of 7.0, supplemented with indicated carbon sources, and shaken at 200 rpm at 30 C. For routine growth and manipulation N. aromaticivorans strains were grown in SMB+10 mM glucose. Where needed to select for the presence or absence of plasmids, media were supplemented with 50 g/mL kanamycin or 10% sucrose (w/v).
[0124] Escherichia coli strains used for cloning and conjugation were grown in lysogeny broth (LB), and shaken at 200 rpm at 37 C. Strain WM6026 was grown in LB supplemented with 300 M diaminopimelic acid. Where needed to select for the presence of plasmids, media were supplemented with 50 g/mL kanamycin.
Adaptive Laboratory Evolution
[0125] Media for the adaptive laboratory evolution experiment was made by mixing SMB-acetovanillone (1.7 mM) with SMB-vanillin (1.8 mM) in different ratios (schematically described in
TABLE-US-00004 TABLE 2 All strains and plasmids used in this study. Name Genotype Description Reference E. coli strains NEB 5-alpha fhuA2(argF- Used for creating and New England lacZ)U169 phoA maintaining plasmids Biolabs, Inc. glnV44 80(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 S17-1 recA pro hsdR RP4- Used for mobilizing plasmids (43) 2-Tc::Mu-Km::Tn7 into N. aromaticivorans via conjugation WM6026 lacI.sup.q, rrnB3, Diaminopimelic acid auxotroph; (44) lacZ4787, used for mobilizing plasmids into hsdR514, N. aromaticivorans via araBAD567, conjugation rhaBAD568, rph-1, att::pAE12(oriR6 K-cat::Frt5), endA::Frt, uidA(MluI)::pir, attHK::pJK1006 (oriR6K-cat::Frt5; trfA::Frt) B834(DE3) F.sup. ompT hsdS.sub.B(r.sub.B.sup. BL21 parent; methionine (45) m.sub.B.sup.) gal dcm met auxotroph used for heterologous (DE3) protein expression TD604 NEB 5-alpha NEB 5-alpha containing This work pK18msB-Saro1862 pK18msB-Saro1862 E16K E16K TD613 NEB 5-alpha NEB 5-alpha containing This work pK18msB- pK18msB-delSaro1861 delSaro1861 TD618 WM6026 pK18msB- WM6026 containing pK18msB- This work delSaro1861 delSaro1861 for conjugation into N. aromaticivorans TD632 NEB 5-alpha NEB 5-alpha containing This work pK18msB- pK18msB-delSaro1858 delSaro1858 TD633 WM6026 pK18msB- WM6026 containing pK18msB- This work delSaro1858 delSaro1858 for conjugation into N. aromaticivorans TD617 B834 pVP68K- B834 carrying a plasmid for This work Saro1862opt E16K expressing an N-terminal His.sub.8- tagged MBP fused with a codon- optimized version of Saro_1862 encoding the E16K substitution TD620 NEB 5-alpha NEB 5-alpha containing This work pVP68K- pVP68K-Saro1862opt E16 Saro1862opt E16 TD627 B834 pVP68K- B834 carrying a plasmid for This work Saro1862opt E16 expressing an N-terminal His.sub.8- tagged MBP fused with a codon- optimized version of Saro_1862 N. aromaticivorans strains 124441879 DSM 12444 Parent strain; putative sacB has (33) Saro_1879 been deleted to allow genomic modifications using a sacB- containing plasmid TD585 DSM 12444 Isolate #1 from acetovanillone- This work Saro_1879, evolved culture A additional mutations TD586 DSM 12444 Isolate #2 from acetovanillone- This work Saro_1879, evolved culture A additional mutations TD587 DSM 12444 Isolate #3 from acetovanillone- This work Saro_1879, evolved culture A additional mutations TD588 DSM 12444 Isolate #4 from acetovanillone- This work Saro_1879, evolved culture A additional mutations TD589 DSM 12444 Isolate #1 from acetovanillone- This work Saro_1879, evolved culture B additional mutations TD590 DSM 12444 Isolate #2 from acetovanillone- This work Saro_1879, evolved culture B additional mutations TD591 DSM 12444 Isolate #3 from acetovanillone- This work Saro_1879, evolved culture B additional mutations TD592 DSM 12444 Isolate #4 from acetovanillone- This work Saro_1879, evolved culture B additional mutations TD593 DSM 12444 Isolate #1 from acetovanillone- This work Saro_1879, evolved culture C additional mutations TD594 DSM 12444 Isolate #2 from acetovanillone- This work Saro_1879, evolved culture C additional mutations TD595 DSM 12444 Isolate #3 from acetovanillone- This work Saro_1879, evolved culture C additional mutations TD596 DSM 12444 Isolate #4 from acetovanillone- This work Saro_1879, evolved culture C additional mutations TD606 DSM 12444 Parent with single nucleotide This work Saro_1879, change encoding an E16K Saro_1862 46G > A substitution in Saro_1862 TD625 DSM 12444 TD606 with deleted Saro_1861 This work Saro_1879, Saro_1862 46G > A, Saro_1861 TD634 DSM 12444 TD606 with deleted Saro_1858 This work Saro_1879, Saro_1862 46G > A, Saro_1858 Plasmids pK18msB-MCS1 pK18mobsacB lacking the (33, 34) multiple cloning site, with a new XbaI site introduced pK18msB- This work Saro1862 E16K pK18msB- pK18msB-MCS1 containing N. This work delSaro1861 aromaticivorans genomic regions that flank Saro_1861 pK18msB- pK18msB-MCS1 containing N. This work delSaro1858 aromaticivorans genomic regions that flank Saro_1858 pVP68K pBR322 origin-containing (36) plasmid for expressing His.sub.8- tagged MBP fusion proteins from a T5 promoter. Encodes kanamycin resistance. pVP68K- pVP68K plasmid containing a This work Saro1862opt E16K codon-optimized version of Saro_1862 with the E16K substitution. The plasmid also encodes an intervening TEV protease site (ENLYFQS) between MBP and Saro_1862 pVP68K- pVP68K plasmid containing a This work Saro1862opt E16 codon-optimized version of wild- type Saro_1862. The plasmid also encodes an intervening TEV protease site (ENLYFQS) between MBP and Saro_1862
Whole Genome Sequencing
[0126] Evolved strains TD561-563 and TD585-596 were grown in 5 mL SMB-acetovanillone, and the parent strain N. aromaticivorans 1244441879 was grown in 5 mL SMB-glucose. Cell pellets were obtained by centrifuging 2.4 mL culture for 2 min at 15,000g. Genomic DNA was isolated using a DNeasy Blood and Tissue Kit (Qiagen), quality checked using a Nanodrop ND1000, and quantified using the Qubit dsDNA BR assay. Genomic DNA (1000 ng) was sheared using a Covaris S220 Focused-ultrasonicator using the manufacturer's protocol for 300 bp target fragment size. DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Prepared sequencing libraries were quantified using the Qubit dsDNA BR assay and fragment sizes were measured using a TapeStation D1000. Pooled libraries were sequenced using paired end 250 bp reads in an Illumina NextSeq 1000 following the manufacturer's protocol.
Sequence Data Analysis
[0127] Paired end Illumina sequencing reads were processed with Breseq (v0.36.1) (35) using default parameters. Reads were analyzed for genomic differences compared to the N. aromaticivorans DSM 12444 (Assembly Accession Number GCF_000013325.1). The mutations, primarily SNPs and INDELs, identified by Breseq in greater than 80% of sequencing reads were used for further analysis.
Generation of Defined N. aromaticivorans Mutants
[0128] N. aromaticivorans genomic mutations were created by homologous recombination using variants of the pK18mobsacB plasmid (34), with methods described in Supplementary Information. Briefly, plasmids containing regions of homology near the N. aromaticivorans genome modification site were transferred into N. aromaticivorans by conjugation using E. coli S17-1 or WM6026 cells. Transconjugants that had recombined the plasmid into the genome were selected for on plates containing kanamycin. Kanamycin-resistant colonies were streaked onto SMB+10 mM glucose+10% sucrose (w/v) to select for double crossovers that result in the plasmid looping out of the genome. Colonies on sucrose-containing plates were patched onto kanamycin and sucrose plates separately to screen for kanamycin sensitivity and sucrose resistance. Such colonies were then screened by colony PCR to check for deletion of the desired gene, which was confirmed by Sanger sequencing.
Bacterial Growth Curves
[0129] Starter cultures of N. aromaticivorans strains were grown overnight in SMB-vanillate (4 mM). The following morning, these starter cultures were diluted 1:1 in fresh SMB-vanillate (4 mM) and shaken at 30 C. for an additional hour. Then, these cultures were used to inoculate 1.5 mL of SMB media containing a particular aromatic in a 24-well plate at a starting OD.sub.600 of 0.05, as measured in a 1 cm cuvette. Various SMB media containing different aromatics were prepared fresh by adding a 1:100 dilution of 100 aromatic dissolved in methanol to the SMB media. The plates were grown at 30 C., shaking for 1 min every 15 min. Each condition was tested in biological triplicate.
Saro_1862 Expression and Purification
[0130] A gBlock of Saro_1862 was codon optimized for E. coli (Integrated DNA Technologies, Coralville, IA) and cloned into pVP68K (36), which adds an N-terminal Hiss-tagged maltose binding protein (MBP) to the protein coding sequence. Primers were designed to introduce a TEV protease cleavage site (ENLYFQS) directly upstream of the gene encoding Saro_1862. This plasmid was then transformed into in E. coli B834 cells. Protein expression was induced by growth in autoinduction medium ZMS-80155 (37) supplemented with 50 g/mL kanamycin. The ZMS-80155 medium contains 1% N-Z Amine AS, 50 mM phosphate, 20 mM succinate, 0.8% glycerol, 0.015% glucose, 0.5% -lactose, 2 mM MgSO.sub.4, a trace metals mix (37), and a vitamin mix (38). Cultures were grown at room temperature for 24 h (OD.sub.600>10), at which point they were chilled on ice. Cells were pelleted by centrifugation at 6000g for 10 min at 4 C. The cell pellets were then resuspended in 20 mM HEPES pH 7.5 and 300 mM NaCl. Cell suspensions were sonicated using a Branson Sonifier 450 for 30 min at 25% duty cycle on the max power setting for the microtip attachment. Cell debris was separated from the cell lysate by centrifugation at 20,000g for 30 min at 4 C.
[0131] The cell lysate was run through a Cytiva KTA pure 25 chromatography system with a Cytiva HisTrap HP 5 mL column kept at 4 C. for protein purification. The column was pre-equilibrated with 20 mM HEPES pH 7.5, 300 mM NaCl, and 20 mM imidazole, which was also used as the wash buffer following sample injection. Proteins were eluted with a 5 column volume (CV) gradient from 0% to 100% elution buffer, which contained 20 mM HEPES pH 7.5, 300 mM NaCl, and 300 mM imidazole. Elution buffer was held at 100% for 10 CVs until returning to 0%. Eluted protein was concentrated and buffer exchanged using a Amicon Ultra-15 centrifugal filter unit with 20 mM HEPES pH 7.5, 300 mM NaCl. Protein was treated with approximately 0.015 mg/mL TEV protease for 24 h at room temperature. Next, cleaved protein was again run through the Cytiva KTA pure 25 chromatography system with a Cytiva HisTrap HP 5 mL column for separation of cleaved Saro_1862 from the Hiss-MBP. Using the same method, cleaved Saro_1862 eluted at 40% elution buffer while Hiss-MBP cluted at 90% elution buffer, as confirmed by enzyme activity assays and SDS-PAGE. Cleaved Saro_1862 was concentrated and buffer exchanged using a Amicon Ultra-15 centrifugal filter unit with 20 mM HEPES pH 7.5, 300 mM NaCl. Protein concentration was determined using the Bio-Rad Protein Assay, using bovine serum albumin as a standard. Protein purity was assessed by SDS-PAGE.
In Vitro Aromatic Phosphorylation Assays
[0132] To test the ability of Saro_1862 protein to phosphorylate acetovanillone or other substrates, purified recombinant enzyme was mixed with substrate and ATP in a reaction buffer. A typical 100 L reaction contained 20 mM HEPES pH 7.5, 2 mM MgCl.sub.2, 200 M MnCl.sub.2, 1 mM ATP, 200 M aromatic substrate (from a 2 mM stock in water), and 0.01 mg/ml Saro_1862 protein. The reaction was incubated at 30 C. for 1 h before analysis by high pressure liquid chromatography-mass spectrometry (HPLC-MS). Reaction products were separated by reverse-phase HPLC using a Phenomenex Kinetex F5 column (2.6 m pore size, 2.1 mm ID, 150 mm length) attached to a Shimadzu Nexera XR HPLC system. The mobile phase was a binary gradient of solvent A (0.2% formic acid in water) and solvent B (methanol) flowing at 0.4 mL/min. To detect reaction products, absorbance was measured between 190 and 500 nm using a Shimadzu SPD-M20A photodiode array detector. The eluent from the HPLC was also analyzed via mass spectrometry using a Shimadzu triple quadrupole mass spectrometer LCMS-8045. We used negative mode Q3 scans from 100 m/z to 500 m/z around the retention times of HPLC peaks to obtain mass spectra of compounds eluting at such times.
Measurement of the Rate of Acetovanillone Phosphorylation by Saro_1862
[0133] The rates of acetovanillone phosphorylation by the Saro_1862 E16K variant enzyme and the Saro_1862 wild type enzyme were measured as described in (21), with two modifications: the reaction buffer used 20 mM Tris pH 8 instead of 20 mM HEPPS pH 8, and the enzyme concentration was approximately 40 nM (Saro_1862 E16K) or 40 M (Saro_1862 wild type) instead of 1 M to help ensure measurements were obtained under initial velocity conditions. Otherwise, the buffer similarly contained 50 M acetovanillone, 2 mM MgCl2, 1 mM MnCl2, 2 mM DTT, and 1 mM ATP. The reaction was monitored by absorbance at 340 nm, and a blank-adjusted reaction rate was calculated based on the difference in extinction coefficients for acetovanillone and phospho-acetovanillone at 340 nm (21) and a measured path length of 0.62 cm based on a 200 L reaction in a 96-well plate (
Size Exclusion Chromatography
[0134] Size exclusion chromatography was used to determine the oligomerization state of native Saro_1862 E16K. A Phenomenex bioZen size exclusion column (SEC-2 1.8 m 150 1504.6 mm) hooked up to a Shimadzu Nexera XR HPLC system set to 0.7 L injections and a 0.3 mL/min flow rate was used for sample separation. The mobile phase was 20 mM HEPES, pH 7.5, containing 300 mM NaCl. Protein retention times were determined by absorbance at 280 nm as measured by a Shimadzu SPD-M20A photodiode array detector. A standard curve was created by plotting log.sub.10 molecular weight versus retention time for standards in the Supelco Protein Standard Mix 15-600 kDa and for bovine serum albumin. The molecular weight of Saro_1862 E16K was determined from the linear fit of this standard curve.
Searching for Genes Encoding Acetovanillone Kinase and Other Acetovanillone Metabolism Proteins in Bacteria
[0135] The conversion of acetovanillone to vanillic acid requires an acetovanillone kinase (Saro_1862 or AcvAB/HpeHI), a carboxylase (AcvCDE/HpeCBA), a phosphatase (AcvF/HpeD), a CoA ligase (VceA or HpeE), and a beta-ketoacyl-CoA hydrolase (VceB or HpeF) (20,21). Thus, we searched NCBI for genomes that contained homologs of Saro_1862 (ABD26302.1), AcvA (WP_014074980.1) or HpcH (UZF45677.1), AcvB (WP_014074979.1) or Hpel (UZF45676.1), AcvC (WP_014074978.1) or HpeC (UZF45675.1), AcvD (WP_014074977.1) or HpeB (UZF45674.1), AcvE (WP_014074976.1) or HpcA (UZF45673.1), AcvF (WP_014074975.1) or HpcD (UZF45672.1), VccA (BAK67171.1) or HpcE (UZF45671.1), and VceB (BAK65920.1) or HpeF (UZF45670.1). We used tBLASTn in BLAST+ version 2.14.1 (39) against the ref_prok_rep_genomes database downloaded from NCBI on Sep. 12, 2023. We used the default tBLASTn parameters, and kept all hits that met the E-value cutoff of 0.01. Next, we used the taxonomy ID of each BLAST hit with TaxonKit software (40) to obtain lineage information for each species. The tBLASTn hits for all proteins, including genomic locations of the hits, can be performed by conduct the tBLASTn search outlined above. Finally, we compared the list of tBLASTn hits for each protein to assemble two separate lists of species with acetovanillone metabolism gene homologs: first, those that encode homologs of Saro_1862, AcvC or HpeC, AcvD or HpeB, AcvE or HpeA, AcvF or HpeD, VceA or HpeE, and VceB or HpeF (Table 3); second, those that encode homologs of AcvA or HpeH, AcvB or Hpel, AcvC or HpeC, AcvD or HpcB, AcvE or HpeA, AcvF or HpeD, VceA or HpeE, and VceB or HpeF (Table 4).
[0136] To display the findings in a tree representing the diversity of bacteria, we started with the phylogenetic tree of all classes of bacteria in the GTDB available in AnnoTree (41). We pruned all unnamed nodes, collapsed some classes into phyla, and pruned any classes that were not present in the NCBI representative prokaryotic genomes database to yield a tree that best matched the subgroups contained in the NCBI representative prokaryotic genomes database. Finally, we used lineage information obtained for each species that contained BLAST hits to label the distribution of our hits in the bacterial phylogenetic tree. Tree manipulations were performed using Tree Viewer (42).
Construction of Plasmids for Generating in-Frame Deletions of Saro_1858 or Saro_1861
[0137] Regions of Novosphingobium aromaticivorans genomic DNA containing 1000 bp upstream and downstream of the genes to be deleted were amplified via PCR. Plasmid pK18msB-MCS1 (a variant of pK18mobsacB (Schfer et al..sup.34) in which the multiple cloning site has been removed, and which contains a gene for kanamycin resistance and sacB for sucrose sensitivity) was linearized via PCR as previously described (Kontur et al..sup.33). The upstream and downstream flanking regions for each gene were combined with lincarized pK18msB-MCS1 using the NEBuilder HiFi Assembly system (New England Biolabs, Ipswich, MA) to produce a plasmid in which the upstream and downstream DNA sequences are adjacent, with no intervening coding region (Table 1). In the case of Saro_1858, the first codon was retained because these nucleotides contained the stop codon of Saro_1859. In the case of Saro_1861, the plasmid was designed to eliminate the entire gene from the start to the stop codon. The plasmids were transformed into NEB 5-alpha competent Escherichia coli cells (New England Biolabs). The transformed E. coli cells were cultured in LB media+kanamycin, the plasmids were purified using a Qiagen Plasmid Maxi Kit (Qiagen, Germany), and DNA sequencing was used to confirm the presence of the desired junction between upstream and downstream fragments.
Construction of Plasmid for Generating the Saro_1862 E16K Mutant in N. aromaticivorans
[0138] Upstream and downstream flanking regions of 1000 bp overlapped at the E16 codon and primers were designed to encode the E16K mutation (from GAG to AAG). These regions were amplified via PCR and cloned into pK18msB-MCS1 as described above.
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TABLE-US-00005 SEQUENCES NativeSaro_1862codingsequence (SEQIDNO:1) ATGACCGACAAGCCCAGCGTCCTGTTCATCTGCACGCAGGATACC GAGGAAGAGGAAGCCCGCTTCACCCGCGCCGCGCTCGAGGCGGCG GGCGTCGAAGTCGTCCACCTCGATCCCAGTGTCCGCCGCTCGCTC GGCGGGGCGGAAATCTCGCCGGAAATGGTCGCCCAGGCCGGCGGA ATGACCATCGAGGAAGTCCGCGCCCTCGGCCACGAAGGCAAGTGC CAGGACGCGATGATCCGTGGTGCCATCGCCGCCGCGCACGAATGG GACGCCAGACACCCCGTCTCCGGCATTCTCGCGGTCGGCGGCTCG ATGGGCTCGGCGCTTGCCGGTGCGCTCATGCAGAGCTTCCCCTAT GGCCTGCCCAAGCTGATCGTCTCGACCATGGCCTCGGGCTTCACC AAGCCCTACATGGGCGTGAAGGACATCGCGATGATGAACGCGGTG ACCGATATCTCGGGCATCAACACGATCAGCCGCGACGTCTTCCGC AACGCTGCCAACGCCGTTGCCGGAATGGCGAAGGGCTACGACCGC GACAAGGGCCCCGAAAAGCCTCTCGTCCTCATCACCACGCTCGGC ACGACGGAAACCAGCGTGAAACGCATCCGCCAGGCACTGGAAAGC GATGGCTGCGAAGTCATGGTCTTCCATTCCTCCGGCGCGGGCGGC CCCACGCTCGACGGGCTCGCCGCCGACAAGGACGTGGCGCTGGTC CTGGACCTTTCCCCGACCGAGATCCTCGACCACCTCTTCGGCGGC CTGGCTGATGCCGGTCCGGATCGCGGGCGCGCGGCCCTGCGCAAG GGCATCCCGACGATCCTTGCCCCCGGCAATGCCGATTTCATCATC GGCGGTCCGATCGACGCCGCGGAAGCGCAGTTTCCAGGCCGGCGC TACCACCAGCACAACCCGCAGCTCACCGCAGTCCGCACCAACGTC GCGGACCTTCGGAAGCTGGCCGATCACCTTGCCGCCAACGTGCGC GAGGCCAAGGGCCCGGTCCGGGTCTTCACCCCGCTCAAGGGCTTT TCCAGCCACGACAGCGAAACGGGCCACCTGCTCGACCTCTCGGTG CCGGGACCCTTCGCCGAATATCTCGCCAGCGTCATGCCAGGTCAC GTGCCGGTGACCGCCGTGGACGCCCATTTCAACGACGAAGCCTTC TCCAGCGCGGTCATTGCCGCCGCGCGCGAGATGCTTGCCGCAAAG AACTGA NativeSaro_1862proteinsequence (SEQIDNO:2) MTDKPSVLFICTQDTEEEEARFTRAALEAAGVEVVHLDPSVRRSL GGAEISPEMVAQAGGMTIEEVRALGHEGKCQDAMIRGAIAAAHEW DARHPVSGILAVGGSMGSALAGALMQSFPYGLPKLIVSTMASGFT KPYMGVKDIAMMNAVTDISGINTISRDVFRNAANAVAGMAKGYDR DKGPEKPLVLITTLGTTETSVKRIRQALESDGCEVMVFHSSGAGG PTLDGLAADKDVALVLDLSPTEILDHLFGGLADAGPDRGRAALRK GIPTILAPGNADFIIGGPIDAAEAQFPGRRYHQHNPQLTAVRTNV ADLRKLADHLAANVREAKGPVRVFTPLKGFSSHDSETGHLLDLSV PGPFAEYLASVMPGHVPVTAVDAHENDEAFSSAVIAAAREMLAAK N Saro_1862-E16Kcodingsequence (SEQIDNO:3) ATGACTGATAAGCCATCTGTGCTTTTTATTTGCACGCAAGATACT AAAGAGGAGGAAGCACGTTTTACTCGCGCGGCGCTGGAAGCAGCA GGGGTGGAGGTGGTACACCTTGATCCATCCGTCCGTCGCAGTCTG GGGGGTGCCGAGATTTCTCCAGAGATGGTTGCCCAGGCGGGGGGT ATGACCATTGAGGAAGTTCGCGCATTAGGACATGAAGGGAAATGC CAAGACGCGATGATTCGTGGAGCCATTGCTGCCGCGCATGAGTGG GACGCTCGTCATCCTGTTTCCGGAATTCTTGCCGTAGGGGGCTCG ATGGGATCTGCCTTGGCAGGGGCGTTGATGCAGAGTTTCCCCTAC GGCCTGCCCAAGTTGATTGTCTCTACAATGGCTTCGGGGTTTACA AAGCCCTATATGGGTGTAAAAGATATTGCTATGATGAATGCAGTT ACAGATATCTCAGGAATTAATACAATCTCTCGTGACGTATTCCGT AACGCGGCGAATGCAGTGGCGGGGATGGCTAAGGGGTACGACCGT GACAAGGGCCCAGAGAAGCCATTAGTTTTGATTACAACACTGGGA ACCACGGAAACTTCTGTAAAGCGCATCCGCCAAGCCCTGGAGTCC GATGGCTGTGAGGTGATGGTCTTTCACTCTTCTGGGGCAGGGGGA CCTACGTTGGATGGGTTAGCCGCCGATAAGGATGTTGCGTTGGTG TTGGATTTGAGTCCTACAGAGATCCTGGATCATCTGTTTGGGGGG CTTGCTGATGCCGGACCTGACCGCGGACGTGCTGCTTTGCGCAAG GGAATTCCAACAATTTTAGCGCCGGGAAATGCCGATTTTATCATC GGCGGCCCTATTGACGCCGCTGAGGCTCAATTCCCTGGCCGCCGC TATCATCAACATAATCCCCAATTAACAGCAGTCCGTACAAATGTG GCGGATTTGCGCAAGCTTGCCGATCATTTAGCAGCAAATGTTCGC GAAGCGAAGGGTCCAGTGCGTGTATTCACTCCATTGAAGGGGTTT TCAAGCCATGACAGTGAAACTGGGCACTTATTGGATTTAAGCGTG CCTGGGCCGTTCGCCGAATACTTAGCGTCGGTTATGCCCGGTCAC GTTCCCGTTACCGCTGTCGATGCTCACTTCAACGATGAAGCGTTC TCCTCGGCTGTCATTGCAGCCGCGCGCGAGATGTTGGCGGCAAAA AATTAA Saro_1862-E16Kprotein (SEQIDNO:4) MTDKPSVLFICTQDTKEEEARFTRAALEAAGVEVVHLDPSVRRSL GGAEISPEMVAQAGGMTIEEVRALGHEGKCQDAMIRGAIAAAHEW DARHPVSGILAVGGSMGSALAGALMQSFPYGLPKLIVSTMASGFT KPYMGVKDIAMMNAVTDISGINTISRDVFRNAANAVAGMAKGYDR DKGPEKPLVLITTLGTTETSVKRIRQALESDGCEVMVFHSSGAGG PTLDGLAADKDVALVLDLSPTEILDHLFGGLADAGPDRGRAALRK GIPTILAPGNADFIIGGPIDAAEAQFPGRRYHQHNPQLTAVRTNV ADLRKLADHLAANVREAKGPVRVFTPLKGFSSHDSETGHLLDLSV PGPFAEYLASVMPGHVPVTAVDAHENDEAFSSAVIAAAREMLAAK N MBP-Saro_1862-E16Kcodingsequence (SEQIDNO:5) ATGGGACATCACCATCATCACCATCACCATGCATCCAAAATCGAA GAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAAC GGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATT AAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCA CAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCA CACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAA ATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACC TGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATC GCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCG AACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAA CTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAA CCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCG TTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTG GATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTG ATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCA GAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGC CCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGT GTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTC GTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAA GAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAA GGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCG CTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCC GCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATC CCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATC AACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGAC GCGCAGACTTTAATTAACGGCGACGGTGCCGGGCTGGAAGTTCTG TTCCAGGGGCCCGCGATCGCGGAAAACCTGTACTTCCAGTCCACT GATAAGCCATCTGTGCTTTTTATTTGCACGCAAGATACTAAAGAG GAGGAAGCACGTTTTACTCGCGCGGCGCTGGAAGCAGCAGGGGTG GAGGTGGTACACCTTGATCCATCCGTCCGTCGCAGTCTGGGGGGT GCCGAGATTTCTCCAGAGATGGTTGCCCAGGCGGGGGGTATGACC ATTGAGGAAGTTCGCGCATTAGGACATGAAGGGAAATGCCAAGAC GCGATGATTCGTGGAGCCATTGCTGCCGCGCATGAGTGGGACGCT CGTCATCCTGTTTCCGGAATTCTTGCCGTAGGGGGCTCGATGGGA TCTGCCTTGGCAGGGGCGTTGATGCAGAGTTTCCCCTACGGCCTG CCCAAGTTGATTGTCTCTACAATGGCTTCGGGGTTTACAAAGCCC TATATGGGTGTAAAAGATATTGCTATGATGAATGCAGTTACAGAT ATCTCAGGAATTAATACAATCTCTCGTGACGTATTCCGTAACGCG GCGAATGCAGTGGCGGGGATGGCTAAGGGGTACGACCGTGACAAG GGCCCAGAGAAGCCATTAGTTTTGATTACAACACTGGGAACCACG GAAACTTCTGTAAAGCGCATCCGCCAAGCCCTGGAGTCCGATGGC TGTGAGGTGATGGTCTTTCACTCTTCTGGGGCAGGGGGACCTACG TTGGATGGGTTAGCCGCCGATAAGGATGTTGCGTTGGTGTTGGAT TTGAGTCCTACAGAGATCCTGGATCATCTGTTTGGGGGGCTTGCT GATGCCGGACCTGACCGCGGACGTGCTGCTTTGCGCAAGGGAATT CCAACAATTTTAGCGCCGGGAAATGCCGATTTTATCATCGGCGGC CCTATTGACGCCGCTGAGGCTCAATTCCCTGGCCGCCGCTATCAT CAACATAATCCCCAATTAACAGCAGTCCGTACAAATGTGGCGGAT TTGCGCAAGCTTGCCGATCATTTAGCAGCAAATGTTCGCGAAGCG AAGGGTCCAGTGCGTGTATTCACTCCATTGAAGGGGTTTTCAAGC CATGACAGTGAAACTGGGCACTTATTGGATTTAAGCGTGCCTGGG CCGTTCGCCGAATACTTAGCGTCGGTTATGCCCGGTCACGTTCCC GTTACCGCTGTCGATGCTCACTTCAACGATGAAGCGTTCTCCTCG GCTGTCATTGCAGCCGCGCGCGAGATGTTGGCGGCAAAAAATTAA MBP-Saro_1862-E16Kprotein (SEQIDNO:6) MGHHHHHHHHASKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGI KVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAE ITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLP NPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIA EAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPF VGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVA LKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVI NAASGRQTVDEALKDAQTLINGDGAGLEVLFQGPAIAENLYFQST DKPSVLFICTQDTKEEEARFTRAALEAAGVEVVHLDPSVRRSLGG AEISPEMVAQAGGMTIEEVRALGHEGKCQDAMIRGAIAAAHEWDA RHPVSGILAVGGSMGSALAGALMQSFPYGLPKLIVSTMASGFTKP YMGVKDIAMMNAVTDISGINTISRDVFRNAANAVAGMAKGYDRDK GPEKPLVLITTLGTTETSVKRIRQALESDGCEVMVFHSSGAGGPT LDGLAADKDVALVLDLSPTEILDHLFGGLADAGPDRGRAALRKGI PTILAPGNADFIIGGPIDAAEAQFPGRRYHQHNPQLTAVRTNVAD LRKLADHLAANVREAKGPVRVFTPLKGFSSHDSETGHLLDLSVPG PFAEYLASVMPGHVPVTAVDAHENDEAFSSAVIAAAREMLAAKN