Patent Application
20180346942
The invention relates to an engineered eukaryotic microorganism wherein genes encoding an acyl-CoA dehydrogenase are introduced into the microorganism and a method for producing methacrylic acid esters and precursors thereof using the microorganism.
Methyl methacrylate (MMA) is a critical raw material in the production of acrylic polymers. MMA is traditionally produced from non-sustainable, hazardous raw materials, such as acetone and hydrogen cyanide. Accordingly, there is a need in the art for MMA production methods that are sustainable and rely on non-toxic production methods.
As such a production method, methods are proposed wherein 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid to be precursors of methacrylic acids are produced from natural products such as saccharides using naturally occurring microorganisms (see Patent Literatures 1 and 2 and Non Patent Literature 1). However, these methods still rely on chemical techniques in the step for producing a methacrylic acid by dehydrating the precursor.
Additionally, there are methods proposed wherein a methacrylic acid is produced from glucose using a not naturally occurring engineered microorganism into which a plurality of enzyme genes are introduced, but these are the combination of a known enzyme reaction and a hypothetical enzyme reaction analogized therefrom and not verified (see Patent Literatures 3 to 5). Particularly, Patent Literature 5 presents examples of a wide variety of biocatalysts (hydrolase, wax ester synthase, alcohol acetyltransferase) which have typical ester-producing activity but fails to state that those biocatalysts have the synthesis activity of methacrylic acid esters.
The inventors have reported a production method of a methacrylic acid ester from methacrylyl-CoA by biocatalysts (Patent Literature 6) and a production method of a methacrylic acid ester from biomass in the presence of an alcohol by adding the action of AAT to a microorganism which has a methacrylic acid producing ability (Patent Literature 7). Additionally, the inventors have reported a method for synthesizing methacrylyl-CoA from 3-hydroxyisobutyryl-CoA using E. coli into which a dehydratase gene is introduced (Patent Literature 8).
It is an object of the invention to provide a novel biological production method of methacrylic acid esters such as MMA.
Hosts for industrial use such as E. coli do not produce MAA-CoA even when exogenous genes related to the biosynthesis of methacrylyl-CoA (MAA-CoA) are introduced thereinto. This is presumed due to the absence of the electron acceptor for acyl-CoA dehydrogenase, which is one of the enzymes related to the biosynthesis of MAA-CoA, in E. coli. The inventors succeeded in the biological production of MAA-CoA by introducing an exogenous acyl-CoA dehydrogenase gene into a yeast cell.
More specifically, the invention relates to the following (1) to (10).
(1) A eukaryotic microorganism into which a gene encoding an acyl-CoA dehydrogenase (isobutyryl-CoA dehydrogenase) is introduced.
(2) The eukaryotic microorganism according to (1), wherein the gene encoding an acyl-CoA dehydrogenase is derived from at least one selected from genus Pseudomonas, genus Bacillus, genus Sphingobacterium, genus Comamonas, genus Brevundimonas, genus Sphingomonas, genus Ochrobactrum, genus Pedobacter, genus Paenibacillus, genus Achromobacter, genus Acinetobacter, genus Shewanella, genus Listonella, genus Agrobacterium, genus Mesorhizobium, genus Rhizobium, genus Paracoccus, genus Xanthobacter, genus Streptomyces, genus Geobacillus, genus Rhodococcus, genus Saccharomyces, genus Candida and genus Aspergillus.
(3) The eukaryotic microorganism according to (1) or (2), wherein a signal sequence-added acyl-CoA dehydrogenase gene is introduced in such a way that the acyl-CoA dehydrogenase expresses a function in a mitochondrion.
(4) The eukaryotic microorganism according to (3), wherein the signal sequence comprises the sequence as set forth in SEQ ID NO: 1.
(5) The eukaryotic microorganism according to any one of (1) to (4), wherein the eukaryotic microorganism is a yeast.
(6) The eukaryotic microorganism according to any one of (1) to (4), further comprising at least one exogenous gene selected from genes encoding branched-chain keto acid dehydrogenase, genes encoding enoyl-CoA hydratase, genes encoding hydroxyacyl-CoA hydrolase, genes encoding thioesterase, and genes encoding alcohol acyl transferase.
(7) A method for producing methacrylyl-CoA from valine using the eukaryotic microorganism according to (6).
(8) A method for producing 3-hydroxyisobutyryl-CoA from valine using the eukaryotic microorganism according to (6).
(9) A method for producing 3-hydroxyisobutyric acid from valine using the eukaryotic microorganism according to (6).
(10) A method for producing a methacrylic acid ester from valine using the eukaryotic microorganism according to (6).
Provided herein are novel methods for the biological production of MMA and MMA precursors in engineered microorganisms, wherein one or more enzymes which enable MMA or MMA precursor production is introduced into the microorganism. Herein, one or more enzyme genes including an acyl-CoA dehydrogenase gene, which enables the production of MMA or an MMA. Optionally, the one or more enzymes is targeted to the mitochondria. Mitochondrial targeting of enzymes enables the biosynthetic processes which produce MMA or MMA precursors to take advantage of the favorable energetic, enzymatic, and other characteristics of the mitochondrial niche, which greatly enhances yield. In one implementation of the invention, various enzymes involved in valine biosynthesis and degradation are engineered into a host eukaryotic microorganism wherein, through a series of reactions, they produce MMA precursors or MMA from valine, the one or more enzymes being optionally targeted to the mitochondria. The scope of the invention encompasses novel gene vectors for the transformation of hosts, novel microorganism strains expressing enzymes which enable the synthesis of MMA precursors or MMA, and MMA precursors and MMA produced by the engineered microorganisms of the invention.
According to the invention, the biological production of methacrylic acid ester precursors such as methacrylyl-CoA (MAA-CoA) is provided. The MAA precursor is biologically converted to methacrylic acid esters such as MAA to begin with, thereby enabling the bio-integrated production of methacrylic acid esters. The method of the invention is a biological production which uses an organic raw material such as biomass and thus can produce methacrylic acid esters non-toxically without destroying environments. Additionally, methacrylic acid esters to be obtained are water-insoluble, thereby being collected and purified easily from a fermented product, whereby low-cost production of methacrylic acid esters is enabled.
The invention encompasses an engineered microorganism which produces methacrylic acid esters such as methyl methacrylate (MMA) or precursors thereof such as methacrylyl-CoA (MAA-CoA) and 3-hydroxyisobutyric acid (3-HIB) by the various biosynthesis pathways. Collectively, methacrylic acid esters and precursors thereof (for example, MMA and ester thereof) will be referred to herein as “MMA end-products.” The above biosynthesis pathways comprise a number of enzymatic steps that are accomplished in microorganisms engineered to possess the appropriate set of enzymatic capabilities, as described below.
In the invention, the “methacrylic acid” (IUPAC name: 2-methyl-2-propenoic acid) includes any salts or ionized forms thereof. Examples of the salt of methacrylic acid include sodium salts, potassium salts, calcium salts and magnesium salts.
The “methacrylic acid ester” is a compound represented by formula 1. In the formula 1, R represents a linear or branched hydrocarbon group having 1 to 20 carbon atoms. The hydrocarbon group may be saturated or unsaturated, acyclic or cyclic. Linear or branched-chain unsubstituted alkyl groups, aralkyl groups or aryl groups having 1 to 10 carbon atoms are preferable. Particularly preferable are alkyl groups, benzyl groups or phenyl groups having 1 to 8 carbon atoms of methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, tert-pentyl group, n-hexyl group, isohexyl group, 2-hexyl group, dimethyl butyl group, ethyl butyl group, heptyl group, octyl group and 2-ethylhexyl group.
CH2═C(CH3)COO—R (Formula 1)
Examples of the “methacrylic acid ester precursor” include methacrylyl-CoA, 3-hydroxyisobutyryl-CoA, 3-hydroxyisobutyric acid and isobutyryl-CoA.
The invention encompasses various engineered eukaryotic microorganisms which produce methacrylic acid esters and precursors thereof as end-products. Alternatively, the engineered eukaryotic microorganisms of the invention may comprise eukaryotic microorganisms which produce methacrylic acid esters.
The engineered eukaryotic microorganism of the invention comprises cells which will be referred to as “hosts” or “host cells,” as they serve as hosts for the various Enzymatic Capabilities described below. A host cell can be of any species. Exemplary classes of hosts include yeasts, filamentous fungi and algae. Examples of the species which may be provided as the host include Saccharomyces cerevisiae, Pichia pastoris and Aspergillus niger.
Preferable microorganism (host cell) is yeasts. Exemplary species which may serve as hosts include microorganisms belonging to genus Saccharomyces such as Saccharomyces cerevisiae, microorganisms belonging to genus Schizosaccharomyces, microorganisms belonging to genus Pichia such as Pichia pastoris, microorganisms belonging to genus Candida such as Candida tropicalis, Candida lipolytica, Candida utilis and Candida sake, microorganisms belonging to genus Kluyveromyces, microorganisms belonging to genus Williopsis, microorganisms belonging to genus Debaryomyces, microorganisms belonging to genus Galactomyces, microorganisms belonging to genus Torulaspora, microorganisms belonging to genus Rhodotorula, microorganisms belonging to genus Yarrowia, and microorganisms belonging to genus Zygosaccharomyces. Of these, microorganisms belonging to genus Saccharomyces are preferable, and Saccharomyces cerevisiae is more preferable.
Preferred host species are those which are amenable to cell culture or which are otherwise suitable for bioproduction, wherein end-products can be synthesized in large scale production and can be inexpensively harvested and separated from the cells in which they are produced.
The host cell of the invention may be transformed to express any number of different enzymatic proteins, regulatory sequences, and other genes and gene products. Transformation may be accomplished by any means known in the art which is amenable to the selected host microorganism. In yeast, for example, alkaline cationic transformation protocols, such as lithium acetate in combination with single-stranded carrier DNA and polyethylene glycol may be used. Examples of other transformation techniques adaptable for the host cell transformation include chemical transformation methods known in the art (e.g., DEAE-dextran, polyethyleneimine, dendrimer, polybrene, calcium phosphate, lipofectin, DOTOP, Lipofectamine or CTAB/DOPE, and DOTMA); and physical transformation methods (e.g., injections, gene shock or laser-guided transduction, fine needles, and gene guns). The engineered microorganism of the invention may be transiently transformed or stably transformed.
The enzymatic gene and/or accompanying regulatory sequence introduced into the host microorganism can be configured in various ways. It will be understood that heterologous expression of a foreign gene in a host microorganism may require that codon optimizations and other sequence modifications be made in order that the gene be properly transcribed and translated in the host, as known in the art. It will also be understood that localization signals, promoters, and other elements which may be combined with the enzymatic gene sequence must be selected or altered so as to be effective in the host microorganisms, as known in the art.
Enzymatic gene introduced into the host microorganism will typically be operably linked to the downstream of a promoter sequence. In some embodiments, a constitutive promoter is utilized with the enzymatic gene to ensure constant and high level expression. Exemplary constitutive promoters include the TEF1, and GDS promoters. The genes coding for the enzyme may alternatively be placed under the control of an inducible promoter. For example, in some embodiments, one or more enzymatic capabilities of the engineered microorganism may be placed under the control of the inducible promoter known in the art and compatible with the host microorganism in order to allow for timed staging of enzymatic steps in the production of MMA end-products. For example, the GAL 10 and GAL 1 galactose inducible promoters may be used in yeast.
Further, the enzymatic gene introduced into host microorganisms may also comprise a trafficking signal that directs the localization of the expression product to a specific cellular compartment. For example, a signal which localizes enzymes to the mitochondrial compartments, the cell membrane, or the chloroplast (in plant cells) may be operably linked to gene sequences coding for enzymatic proteins. The scope of the invention encompasses an engineered host wherein one or more of the enzymes introduced into the host are targeted to the mitochondria, for example, the matrix of the mitochondria. Further, the enzyme gene introduced into the host microorganism may comprise a trafficking signal which commands the localization to a specific cell compartment. For example, the matrix of the mitochondria. Examples of the signal sequence which can achieve such a targeting in a yeast include the presequence of subunit 9 of the yeast mitochondrial ATPase (Su9)(SEQ ID NO: 1) or the presequence of subunit IV of the yeast cytochrome C oxidase (Cox1) (Literature [Avalos et al., Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnol. 2013, pr; 31 (4):335-41. 2013, pr; 31(4):335-41]. Additionally, exemplary mitochondrial targeting sequences also include the presequence (residues 19-40) of the human NAOH dehydrogenase (ubiquinone) flavoprotein 2 (NOUFV2); the presequence (residues 1-20) of influenza virus protein (PB2), the presequence of yeast tryptophanyl tRNA-synthase (MSW), and the sequences described in Literature [Omura, “Mitochondria-Targeting Sequence, a Multi-Role Sorting Sequence Recognized at All Steps of Protein Import into Mitochondria,” Journal of Biochemistry. 1998, Vol. 123 Issue 6, p 1010-1016. 7p]. In some embodiments, the enzymatic gene is intended to operate in the cytosol and no trafficking moieties are utilized. In another embodiment, one or more of the expressed enzymatic proteins is designed to be secreted from the cell and gene sequences coding for such proteins are operably linked to secretion signals with direct the translated protein out of the cells.
In some embodiments, a termination sequence is utilized. For example, in yeast, CYCT transcription termination sequence may be operably linked to the enzymatic protein coding sequences to enhance proper expression in yeast cells. Other exemplary regulatory elements that can be used include the rrnB (T1) element from E. coli, the AdhT element from yeast, and the TEF1 element from yeast.
It will be understood that the enzymatic capabilities described below may be introduced by the transformation of the host microorganism with various nucleic acid constructs. Exemplary nucleic acid constructs of the invention comprise plasmids and linear nucleic acid constructs, which may comprise cloning vectors, expression cassettes, and other DNA constructs known in the art.
For a given host microorganism being transformed to express two or more enzymatic genes, such two or more enzymatic genes may be introduced into the microorganism as a single nucleic acid construct, or may be introduced in separate nucleic acid constructs. When two or more separate nucleic acid constructs are utilized, the transformation of the microorganism may occur as a single transformation event employing multiple nucleic acid constructs, or as a series of sequential transformation events to introduce the two or more genes in stages.
It will be understood that genes introduced into the host microorganism may be under the control of different promoters. For example, where the relative ratios of enzymes need to be tuned, some may be under the control of a weak promoter and some may be under the control of a stronger promoter, in order to create the desired balance of enzymatic activity in the cell. Likewise, enzymatic activity can be balanced by varying the copy number of the various enzymatic genes introduced into the host microorganism. For example a gene may be introduced as a single copy or as two, three, four or more copies, for example as end-to-end copies on a single nucleic acid construct.
Reference to various enzymatic genes, proteins, and other genetic/protein elements is made herein. It will be understood by one of skill in the art that equivalents may be used in place of the gene and protein sequences enumerated or referenced herein. For example, variants of cited sequences may be used, including nucleic acid sequence variants and peptide sequence variants.
For example, the listed sequences may have nucleotides and/or amino acids comprising 1 or several, for example, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 1 or 2 substitutions, additions, insertions or deletions, introduced thereinto.
Alternatively, genes and proteins having nucleic acid sequences or amino acid sequences having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, to the sequences mentioned are included.
Such alterations of the original sequences may be neutral or may alter (e.g. enhance) the desired enzymatic activity of the target gene. It will also be understood that gene equivalents include orthologs, paralogs, and homologs of the enumerated sequence from other species. In one embodiment, the equivalent genes of the invention comprise genes having at least 90 percent sequence identity or similarity between the wild type sequence and the variant. Further, it will be understood that functional equivalents of enumerated gene sequences may be used, such functional equivalents performing identical enzymatic functions to the enzymes or regulatory signals, while not necessarily having sequence similarity, identity or homology.
According to the present application, the engineered microorganism of the invention comprises specific “enzymatic capabilities.” Having specific enzymatic capabilities means that the microorganism has an ability to produce a specific product from a specific reactant and such an ability may be enabled by the expression of one or more enzymes which are commanded to convert the reactant to the product.
In some embodiments, one or more enzymatic capabilities possessed by the microorganism are the results of the microorganism having been transformed to express the requisite protein or multiple proteins (e.g. as in the multimeric BCKAD enzyme comprising Enzymatic Capability 2, as describe below) for such enzymatic capability. In some embodiments, all of the enzymatic capabilities possessed by an engineered microorganism of the invention are imparted by introduction of the requisite proteins by genetic transformation. In some embodiments, one or more of the enzymatic capabilities possessed by the engineered microorganism of the invention comprises a native enzymatic capability, i.e. the host microorganism's native enzymes are capable of imparting the activity without supplementation by introduced genes. For example, in some microorganisms, Enzymatic Capability 2 is possessed by the microorganism through the action of native BCKAD genes.
In one embodiment, one or more proteins introduced by the transformation are targeted to a cell compartment or organelle. In an embodiment, one or more or all proteins introduced into the microorganism by the transformation are targeted to mitochondria. In one embodiment, the mitochondrial-targeted proteins are targeted to the mitochondrial matrix. In one embodiment, the host cell is a yeast cell and the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 mitochondrial-targeted sequence.
The pathways for forming the MMA end-products are largely overlapping, as depicted in
For example, three enzymatic steps convert valine to MAA-CoA, which can be harvested and further processed ex-vivo to create MMA. Alternatively, an MAA-CoA producing microorganism may comprise additional enzymatic capabilities that allow the MAA-CoA to be converted to 3-HIB or MMA directly. 3-HIB formed by the microorganism can be harvested and processed ex-vivo to form MMA.
While the efficient formation of MMA end-products is not found in natural microorganisms, the pathways which allow the engineered microorganism to form such products are based on valine synthetic and catabolic pathways that are common across a wide range of microorganisms. Accordingly, there is a diverse set of enzymes available to the skilled practitioner for imparting the desired enzymatic
capabilities. The specific enzymatic capabilities which allow formation of MAACoA, 3-HIB, and MMA are described hereinafter in detail.
Enzymatic Capability 1: Production of 2-Oxoisovaleric Acid from Valine
A first enzymatic step in the conversion of valine to am MMA precursor is the formation of 2-oxoisovalerate from valine. This may be accomplished by the action of a branched chain aminotransferase enzyme (BCAT). In some cases, native BCAT activity or equivalent enzymatic activity is present and sufficient to produce adequate 2-oxoisovalerate from valine for the synthesis of an MMA precursor. Alternatively, host microorganisms may be transformed to express one or more BCAT or equivalent genes. Exemplary BCAT genes include the BAT1 and BAT2 genes of Saccharomyces cerevisiae. Additional BCAT genes that may be used include those listed in Table 1.
The second step in the bioproduction of MAA precursors from valine is the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. This step may be accomplished by the branched-chain α-keto acid dehydrogenase (BCKAD) complex. The BCKAD complex comprises four subunits, the E1 subunit, the E2 subunit, the E3 subunit, and a dihydrolipoamide dehydrogenase.
In some cases, sufficient BCKAD or equivalent activity is present in the host microorganism to efficiently convert 2-oxoisovalerate to isobutyryl-CoA. For example, as described in Example 1, engineered yeast cells produced ample MMA precursors without the introduction of BCKAD genes, demonstrating that sufficient native activity may be present in the host.
Alternatively, the host may be transformed to express one or more enzymes that perform the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. For example, the host microorganism may be engineered to express the four constituents of the BCKAD complex. For example, the four genes encoding the Pseudomonas aeruginosa-BCKAD complex may be introduced into the host to impart or augment native BCKAD activity. These genes include: the bkdA1 gene, encoding E1 (Genbank Accession No. NP250937), bkdA2 gene, encoding E3 (Genbank Accession No. NP_250938), bkdB gene, encoding E2 (Genbank Accession No. NP250939) and IpdV gene, encoding dihydrolipoamide dehydrogenase (Genbank Accession No. NP250940). Alternatively, the host may be transformed to express a bkdA1 gene selected from Table 2, a bkdA2 gene selected from Table 3, a bkdB gene selected from Table 4, and an IpdV gene selected from Table 5.
Stigmatella aurantiaca DW4/3-1
Glaciecola nitratireducens FR1064
Corallococcus coralloides DSM 2259
Sinorhizobium fredii HH103
Alteromonas macleodi ATCC 27126
Pseudomonas fluorescens F113
Sinorhizobium meliloti SM11
Brucella suis VBI22
Shewanella baltica BA175
Brucella melitensis M5-90
Alteromonas macleodi str. ‘English
Marinobacter hydrocarbonoclasticus
Pseudomonas aeruginosa NCGM2.S1
Pseudomonas fluorescens A506
Alteromonas macleodi str. ‘Balearic
Sinorhizobium meliloti BL225C
Thermus thermophilus JL-18
Shewanella baltica OS117
Brucella melitensis NI
Brucella canis HSK A52141
Pseudomonas aeruginosa M18
Pseudomonas pulida S16
Shewanella baltica OS678
Alteromonas macleodi str. ‘Black Sea
Marinobacter adhaerens HP15
Burkholderia cepacia GG4
Brucella melitensis M28
Sphingobium sp. SYK-6
Pseudomonas pulida DOT-T1E
Burkholderia pseudomallei 1026b
Oceanimonas sp. GK1
Shewanella putrefaciens 200
Brucella pinnipedialis B2/94
Pelaglbacterium halotolerans B2
Sinorhizobium fredii USDA 257
Shewanella woodyi ATCC 51908
Shewanella denitrificans OS217
Shewanella oneidensis MR-1
Shewanella halifaxensis HAW-EB4
Anaeromyxobacter dehalogenans 2CP-C
Parachlamydia acanthamoebae UV7
Agrobacterium tumefaciens str. C58
Pseudoalteromonas atlantica T6c
Pseudomonas fluorescens SBW25
Sorangium cellulosum ‘So ce 56’
Pseudomonas fluorescens Pf-5
Oceanithermus profundus DSM 14977
Bradyrhizobium japonicum USDA 110
Brucella melitensis ATCC 23457
Deinococcus deserti VCD115
Pseudomonas putida KT2440
Shewanelle loihica PV-4
Shewanelle baltica OS185
Thermus thermophilus HB8
Brucella suis 1330
Burkholderia pseudomallei 1106a
Mesorhizobium ciceri biovar biserrulae
Polaromonas sp. JS666
Burkholderia glumae BGR1
Alicycliphilus denitrificans BC
Brucella abortus A13334
Alicycliphilus denitrificans K601
Burkholderia pseudomallei 668
Burkholderia gladioli BSR3
Sphingomonas wittichii RW1
Pseudoalteromonas sp. SM9913
Brucella suis ATCC 23445
Myxococcus xanthus DK 1622
Shewanella sediminis HAW-EB3
Pseudomonas putida F1
Brucella abortus bv. 1 str. 9-941
Burkholderia mallei SAVP1
Pseudomonas aeruginosa DK2
Brevundimonas subvibrioides ATCC
Sphingobium chlorophenolicum L-1
Shewanella sp. MR-7
Hahella chejuensis KCTC 2396
Shewanella violacea DSS12
Novosphingobium aromaticivorans
Pseudomonas putida ND6
Thermus thermophilus HB27
Burkholderia ambifaria MC40-6
Burkholderia pseudomallei 1710b
Shewanella frigidimarina NCIMB 400
Hirschia baltica ATCC 49814
Thermoplasma acidophilum DSM 1728
Mesorhizobium opportunistum
Sinorhizobium meliloti 1021
Burkholderia mallei NCTC 10247
Bdellovibrio bacteriovorus HD100
Shewanella baltica OS155
Bordetella petrii DSM 12804
Brucella canis ATCC 23365
Brucella ovis ATCC 25840
Colwellia psychrerythraea 234H
Mesorhizobium loti MAFF303099
Pseudomonas putida BIRD-1
Stigmatella aurantiaca DW4/3-1
Pseudomonas aeruginosa PA7
Anaeromyxobacter sp. K
Maricaulls maris MCS10
Anaeromyxobacter dehalogenans 2CP-1
Burkholderia cenocepacia J2315
Pseudomonas brassicacearum subsp.
brassicacearum NFM421
Brucella melitensis bv. 1 str. 16M
Shewanella baltica OS195
Burkholderia sp. 383
Haliangium ochraceum DSM 14365
Variovorax paradoxus S110
Achromobacter xylosoxidans A8
Ruegeria sp. TM1040
Pseudoalteromonas haloplanktis
Pseudomonas fluorescens Pf0-1
Sphingobium japonicum UT26S
Shewanella sp. W3-18-1
Waddlia chondrophila WSU 86-1044
Shewanella baltica OS223
Rubrobacter xylanophilus DSM 9941
Idiomarina loihiensis L2TR
Caulobacter sp. K31
Shewanelle pealeana ATCC 700345
Agrobacterium sp. H13-3
Shewanella amazonensis SB2B
Brucella abortus S19
Novosphingobium sp. PP1Y
Burkholderia mallei NCTC 10229
Burkholderia thailandensis E264
Halomonas elongata DSM 2581
Pseudomonas aeruginosa LESB58
Sinorhizobium fredii NGR234
Alteromonas macleodii str. ‘Deep
Brucella microti CCM 4915
Sphingopyxis alaskensis RB2256
Shewanella sp. ANA-3
Shewanella putrefaciens CN-32
Burkholderia pseudomallei K96243
Burkholderia mallei ATCC 23344
Burkholderia cenocepacia AU 1054
Pseudomonas aeruginosa UCBPP-
Thermoplasma volcanium GSS1
Pseudomonas entomophila L48
Burkholderia cenocepacia MC0-3
Burkholderia cenocepacia HI2424
Alteromonas sp. SN2
Shewanella sp. MR-4
Brucella melitensis biovar Abortus
Ramlibacter tataouinensis TTB310
Ochrobactrum anthropi ATCC 49188
Erythrobacter litoralis HTCC2594
Pseudomonas putida GB-1
Burkholderia ambifaria AMMD
Sinorhizobium medicae WSM419
Anaeromyxobacter sp. Fw109-5
Pusillimonas sp.T7-7
Shewanella piezotolerans WP3
Rhodoferax ferrireducens T118
Glaciecola sp. 4H-3-7 + YE-5
Marinobacter aquaeolei VT8
Ferrimonas balearica DSM 9799
Sinorhizobium meliloti AK83
Pseudomonas putida W619
Arthrobacter sp. FB24
Stigmatella aurantiaca DW4/3-1
Chlamydia trachomatis G/9301
Glaciecola nitratireducens
Chlamydia psittaci NJ1
Listeria monocytogenes
Pseudonocardia dioxanivorans
Belliella baltica DSM 15883
Staphylococcus aureus subsp.
aureus T0131
Mycoplasma bovis HB0801
Listeria ivanovii subsp. ivanovii
Muricauda ruestrlngensls DSM
Lactococcus garvieae ATCC
Salinibacter ruber M8
Chlamydia trachomatis F/SW5
Listeria monocytogenes M7
Bacillus subtilis subsp. subtilis str. RO-NN-1
Listeria monocytogenes
Sinorhizobium fredii HH103
Alteromonas macleodii ATCC
Staphylococcus aureus subsp.
aureus M013
Bacteroides fragilis 638R
Mycobacterium chubuense NBB4
Enterococcus hirae ATCC 9790
Streptomyces bingchenggensis
Pseudomonas fluorescens F113
Staphylococcus aureus subsp.
aureus 11819-97
Staphylococcus aureus subsp.
aureus LGA251
Staphylococcus aureus subsp.
aureus VC40
Streptomyces sp. SirexAA-E
Staphylococcus aureus subsp.
aureus FD133
Mycoplasma bovis Hubel-1
Sinorhizobium meliloti SM11
Granulicella mallensis
Lactococcus garvieae Lg2
Chlamydia trachomatis Sweden2
Paenibacillus polymyxa M1
Bacillus amyloliquefaciens LL3
Chlamydia psittaci CP3
Listeria monocytogenes J0161
Lactobacillus casei BD-II
Listeria monocytogenes
Melissococcus plutonius DAT561
Pyrobaculum oguniense TE7
Chlamydia trachomatis E/SW3
Nocardiopsis alba ATCC BAA-
Chlamydia trachomatis F/SW4
Listeria monocytogenes
Staphylococcus aureus subsp.
aureus 71193
Brucella suis VBI22
Synechocystis sp. PCC 6803
Mycobacterium massiliense str.
Listeria monocytogenes L99
Exiguobacterium antarcticum B7
Listeria monocytogenes
Ignavibacterium album JCM
Staphylococcus lugdunensis
Bacillus amyloliquefaciens subsp.
plantarum YAU B9601-Y2
Chlamydia trachomatis G/11074
Corynebacterium variabile DSM
Niastella koreensis GR20-10
Bacteriovorax marinus SJ
Mycoplasma pneumoniae FH
Lactobacillus rhamnosus GG
Shewanelle baltica BA175
Acidithiobacillus caldus SM-1
Streptomyces cattleya NRRL
Brucella melitensis M5-90
Chlamydia trachomatis D-EC
Staphylococcus
pseudintermedius ED99
Lactobacillus casei LC2W
Bacillus amyloliquefaciens subsp.
plantarum CAU B946
Alteromonas macleodii str.
Staphylococcus aureus subsp.
aureus ECT-R 2
Mycoplasma genitalium M2288
Aequorivita sublithincola DSM
Streptomyces hygroscopicus
Actinoplanes missouriensis 431
Chlamydia trachomatis A2497
Thermus sp. CCB_US3_UF1
Listeria monocytogenes serotype
Staphylococcus aureus subsp.
aureus TCH60
Lactobacillus rhamnosus ATCC
Marinobacter
hydrocarbonoclasticus ATCC
Pseudomonas aeruginosa
Bacillus amyloliquefaciens TA208
Pseudomonas fluorescens A506
Alteromonas macleodii str.
Sinorhizobium meliloti BL225C
Mycoplasma hyopneumoniae 168
Thermus thermophilus JL-18
Listeria monocytogenes
Listeria monocytogenes Finland
Leuconostoc sp. C2
Shewanella baltica OS117
Brucella melitensis NI
Lactobacillus buchneri CD034
Brucella canis HSK A52141
Bacillus amyloliquefaciens XH7
Pseudomonas aeruginosa M18
Enterococcus faecium Aus0004
Tetragenococcus halophilus
Terriglobus roseus DSM 18391
Terriglobus roseus DSM 18391
Deinococcus gobiensis I-0
Mycoplasma genitalium M6320
Staphylococcus aureus 04-02981
Saprospira grandis str. Lewin
Chlamydia trachomatis A2497
Staphylococcus aureus subsp.
aureus HO 5096 0412
Paenibacillus mucilaginosus 3016
Pseudomonas putida S16
Shewanella baltica OS678
Chlamydia trachomatis E/150
Mycoplasma genitallum M6282
Alteromonas macleodii str. ‘Black
Arthrobacter sp. Rue61a
Chlamydia trachomatis G/9768
Lactobacillus salivarius CECT
Thermus thermophilus
Chlamydla trachomatis G/11222
Marinobacter adhaerens HP15
Burkholderia cepacia GG4
Synechocystis sp. PCC 6803
Streptomyces flavogriseus ATCC
Actinoplanes sp. SE50/110
Synechocystis sp. PCC 6803
Brucella melitensis M28
Listeria monocytogenes 10403S
Sphingobium sp. SYK-6
Enterococcus faecium DO
Paenibacillus mucilaginosus K02
Enterococcus faecalis 62
Listeria monocytogenes ATCC
Listeria monocytogenes FSL R2-
Streptomyces violaceusniger Tu
Pseudomonas putida DOT-T1E
Burkholderia pseudomallei 1026b
Staphylococcus aureus subsp.
aureus str. JKD6008
Listeria monocytogenes
Paenibacillus terrae HPL-003
Mycoplasma pneumoniae 309
Staphylococcus aureus subsp.
aureus S0385
Staphylococcus aureus subsp.
aureus TW20
Oceanimonas sp. GK1
Shewanella putrefaciens 200
Brucella pinnipedialis B2/94
Chlamydia trachomatls D-LC
Kitasatospora setae KM-6054
Pelagibacterium halotolerans B2
Sinorhizobium fredii USDA257
Enterococcus faecalis D32
Bacillus amyloliquefaciens Y2
Chlamydia trachomatis L2c
Leuconostoc mesenteroides
Chlamydia trachomatis E/11023
Melioribacter roseus P3M
Mycoplasma genitalium M2321
Akkermansia muciniphila ATCC
Mycoplasma crocodyli MP145
Acidobacterium sp. MP5ACTX9
Shewanella woodyi ATCC 51908
Shewanella denitrificans OS217
Nitratifractor salsuginis DSM
Prochlorococcus marinus subsp.
marinus str. CCMP1375
Shewanella oneidensis MR-1
Lactobacillus sakei subsp. sakei
Shewanella halifaxensis HAW-
Anaeromyxobacter dehalogenans
Parachlamydia acanthamoebae
Rhodococcus erythropolis PR4
Paenibacillus sp. JDR-2
Listeria seeligeri serovar 1/2b str.
Agrobacterium tumefaciens str.
Pseudoalteromonas atlantica T6c
Chlamydia muridarum Nigg
Pseudomonas fluorescens
Sorangium cellulosum ‘So ce 56’
Melissococcus plutonius ATCC
Pseudomonas fluorescens Pf-5
Bacillus subtilis subsp. spizizenii
Oceanithermus profundus DSM
Bradyrhizoblum Japonicum USDA
Lactobacillus salivarius UCC118
Paenibacillus mucilaginosus
Croceibacter atlanticus
Brucella melitensis ATCC 23457
Gramella forsetii KT0803
Arthrobacter arilaitensis Re117
Candidatus Koribacter versatilis
Deinococcus deserti VCD115
Chloroflexus sp. Y-400-fl
Stackebrandtia nassauensis DSM
Pseudomonas putida KT2440
Synechococcus sp. JA-2-3B′a(2-
Shewanella loihica PV-4
Synechococcus sp. JA-3-3Ab
Shewanella baltica OS185
Enterococcus faecalis V583
Staphylococcus epidermidis
Staphylococcus aureus subsp.
aureus JH9
Mycoplasma pneumoniae M129
Staphylococcus aureus subsp.
aureus COL
Staphylococcus epidermidis
Thermus thermophilus HB8
Brucella suis 1330
Aeropyrum pernix K1
Microbacterium testaceum
Marinomonas sp. MWYL1
Renibacterium salmoninarum
Beutenbergia cavernae DSM
Burkholderia pseudomallei 1106a
Mesorhizobium ciceri biovar
Arthrobacter phenanthrenivorans
Streptomyces scabiei 87.22
Exiguobacterium sp. AT1b
Deinococcus proteolyticus MRP
Polaromonas sp. JS666
Streptomyces cattleya NRRL
Mycoplasma conjunctivae
Burkholderia glumae BGR1
Alicycliphilus denitrificans BC
Staphylococcus aureus subsp.
aureus USA300_TCH1516
Brucella abortus A13334
Alicycliphilus denitrificans K601
Chlamydia trachomatis 434/Bu
Burkholderia pseudomallei 668
Burkholderia gladioli BSR3
Staphylococcus aureus subsp.
aureus MSSA476
Sphingomonas wittichii RW1
Staphylococcus lugdunensis
Pseudoalteromonas sp. SM9913
Brucella suis ATCC 23445
Truepera radiovictrix DSM 17093
Mycoplasma pulmonis UAB CTIP
Micromonospora aurantiaca
Prochlorococcus marinus str. MIT
Shewanella sediminis HAW-EB3
Listeria innocua Clip11262
Chitinophaga pinensis DSM 2588
Mycoplasma hyopneumoniae
Pseudomonas putida F1
Brucella abortus bv. 1 str. 9-941
Burkholderia mallei SAVP1
Pseudomonas aeruginosa DK2
Frankia alni ACN14a
Brevundimonas subvibrioides
Sphingobium chlorophenolicum L-1
Shewanella sp. MR-7
Hahella chejuensis KCTC 2396
Shewanella violacea DSS12
Staphylococcus camosus subsp.
carnosus TM300
Bacteroides fragilis NCTC 9343
Novosphingobium
aromaticivorans DSM 12444
Deinococcus geothermalis DSM
Cyanothece sp. ATCC 51142
Pseudomonas putida ND6
Leuconostoc gasicomitatum LMG
Intrasporangium calvum DSM
Thermus thermophilus HB27
Burkholderia ambifaria MC40-6
Chloroflexus aggregans DSM
Burkholderia pseudomallei 1710b
Mycoplasma agalactiae
Shewanella frigidimarina NCIMB
Staphylococcus aureus subsp.
aureus MSHR1132
Staphylococcus aureus subsp.
aureus ED98
Hirschia baltica ATCC 49814
Thermoplasma acidophilum DSM
Mesorhizobium opportunistum
Sinorhizobium meliloti 1021
Salinispora tropica CNB-440
Burkholderia mallei NCTC 10247
Bdellovibrio bacteriovorus HD100
Shewanella baltica OS155
Candidatus Phytoplasma mali
glomerata
Candidatus Phytoplasma
australiense
Staphylococcus
pseudintermedius HKU10-03
Bordetella petrii DSM 12804
Deinococcus maricopensis DSM
Listeria monocytogenes L312
Verrucosispora maris AB-18-032
Brucella canis ATCC 23365
Brucella ovis ATCC 25840
Colwellia psychrerythraea 34H
Mesorhizobium loti MAFF303099
Cellulomonas fimi ATCC 484
Pseudomonas putida BIRD-1
Maribacter sp. HTCC2170
Pyrobaculum calidifontis JCM
Stigmatella aurantiaca DW4/3-1
Staphylococcus aureus subsp.
aureus MRSA252
Mycoplasma agalactiae PG2
Pseudomonas aeruginosa PA7
Lactobacillus buchneri NRRL B-
Photorhabdus asymbiotica
Staphylococcus aureus subsp.
aureus Mu3
Staphylococcus aureus subsp.
aureus JKD6159
Bacillus amyloliquefaciens DSM 7
Anaeromyxobacter sp. K
Maricaulis maris MCS10
Anaeromyxobacter dehalogenans
Burkholderia cenocepacia J2315
Pseudomonas brassicacearum
Staphylococcus aureus subsp.
aureus Mu50
Lactobacillus rhamnosus Lc 705
Rhodococcus opacus B4
Leuconostoc kimchil IMSNU
Brucella melitensis bv. 1 str. 16M
Shewanella baltica OS195
Staphylococcus aureus subsp.
aureus NCTC 8325
Cellulophaga algicola DSM
Leuconostoc citreum KM20
Burkholderia sp. 383
Marivirga tractuosa DSM 4126
Haliangium ochraceum DSM
Synechocystis sp. PCC 6803
Variovorax paradoxus S110
Achromobacter xylosoxidans A8
Ruegeria sp. TM1040
Arthrobacter chlorophenolicus A6
Chloroflexus aurantiacus J-10-fl
Haliscomenobacter hydrossis
Pseudoalteromonas haloplanktis
Thermoproteus uzoniensis 768-
Pseudomonas fluorescens Pf0-1
Lactobacillus casei ATCC 334
Lactobacillus casei str. Zhang
Sphingobium japonicum UT26S
Cyanothece sp. PCC 8801
Shewanella sp. W3-18-1
Propionibacterium freudenreichii
Bacillus subtilis subsp. subtilis str.
Nocardiopsis dassonvillei subsp.
dassonvillei DSM 43111
Waddlia chondrophila WSU 86-
Shewanella baltica OS223
Frankia sp. Ccl3
Bacillus licheniformis ATCC
Idiomarina loihiensis L2TR
Cellulomonas flavigena DSM
Bacillus clausii KSM-K16
Caulobacter sp. K31
Enterococcus faecalis OG1RF
Thermobaculum terrenum ATCC
Roseiflexus castenholzii DSM
Prochlorococcus marinus str. MIT
Shewanella pealeana ATCC
Agrobacterium sp. H13-3
Rhodococcus equi 103S
Shewanella amazonensis SB2B
Bacillus cellulosilyticus DSM
Brucella abortus S19
Novosphingobium sp. PP1Y
Listeria monocytogenes HCC23
Leuconostoc mesenteroides
Thermobifida fusca YX
Deinococcus radiodurans R1
Burkholderia mallei NCTC 10229
Burkholderia thailandensis E264
Pedobacter heparinus DSM 2366
Halomonas elongata DSM 2581
Pseudomonas aeruginosa
Sinorhizobium fredii NGR234
Alteromonas macleodii str. ‘Deep
Brucella microti CCM 4915
Meiothermus ruber DSM 1279
Sphingopyxis alaskensis RB2256
Chlamydia trachomatis
Chlamydia trachomatis D/UW-
Listeria monocytogenes serotype
Mycoplasma genitalium G37
Shewanella sp. ANA-3
Shewanella putrefaciens CN-32
Burkholderia pseudomallei
Synechococcus sp. PCC 7002
Mycobacterium ulcerans Agy99
Arthrobacter aurescens TC1
Burkholderia mallei ATCC 23344
Saccharomonospora viridis DSM
Mycobacterium sp. MCS
Staphylococcus aureus subsp.
aureus JH1
Burkholderia cenocepacia AU
Meiothermus silvanus DSM 9946
Chlamydia trachomatis L2b/UCH-
Parabacteroides distasonis ATCC
Staphylococcus haemolyticus
Lactobacillus fermentum IFO
Bacillus amyloliquefaciens FZB42
Staphylococcus aureus subsp.
aureus MW2
Frankia sp. EAN1pec
Pseudomonas aeruginosa
Thermoplasma volcanium GSS1
Paenibacillus polymyxa E681
Lactobacillus casei BL23
Pseudomonas entomophila L48
Terriglobus saanensis SP1PR4
Staphylococcus aureus subsp.
aureus USA300_FPR3757
Lactobacillus rhamnosus GG
Staphylococcus aureus RF122
Burkholderia cenocepacia MC0-3
Thermus scotoductus SA-01
Paracoccus denitrificans PD1222
Thermomonospora curvata DSM
Burkholderia cenocepacia HI2424
Oenococcus oeni PSU-1
Roseiflexus sp. RS-1
Listeria monocytogenes 08-5578
Alteromonas sp. SN2
Shewanella sp. MR-4
Streptomyces avermitilis MA-
Staphylococcus aureus subsp.
aureus N315
Bacteroides fragilis YCH46
Thermobispora bispora DSM
Paenibacillus sp. Y412MC10
Erysipelothrix rhusiopathiae str.
Pyrobaculum arsenaticum DSM
Mycoplasma bovis PG45
Brucella melitensis biovar Abortus
Rhodococcus jostii RHA1
Ramlibacter tataouinensis
Ochrobactrum anthropi ATCC
Erythrobacter litoralis HTCC2594
Bacillus subtilis BSn5
Pseudomonas putida GB-1
Actinosynncma mirum DSM
Cyanothece sp. PCC 7822
Mycobacterium sp. KMS
Burkholderia ambifaria AMMD
Sinorhizobium medicae WSM419
Mycobacterium vanbaalenii PYR-1
Chloroherpeton thalassium ATCC
Lysinibacillus sphaericus C3-41
Salinibacter ruber DSM 13855
Bacteroides thetaiotaomicron VPI-
Chlamydia trachomatis A/HAR-13
Anaeromyxobacter sp. Fw109-5
Chlamydia trachomatis
Simkania negevensis Z
Acidobacterium capsulatum
Micromonospora sp. L5
Listeria monocytogenes EGD-e
Cyanothece sp. PCC 8802
Prochlorococcus marinus str. MIT
Carnobacterium sp. 17-4
Saccharopolyspora erythraea
Pusillimonas sp. T7-7
Synechococcus elongatus PCC
Shewanella piezotolerans WP3
Streptosporangium roseum DSM
Paenibacillus polymyxa SC2
Rhodoferax ferrireducens T118
Streptomyces griseus subsp.
griseus NBRC 13350
Pyrobaculum aerophilum str. IM2
Mycobacterium abscessus ATCC
Salinispora arenicola CNS-205
Listeria monocytogenes 08-5923
Sphaerobacter thermophilus
Mycoplasma hyopneumoniae 232
Marinithermus hydrothermalis
Herpetosiphon aurantiacus ATCC
Listeria weishimeri serovar 6b str.
Glaciecola sp. 4H-3-7 + YE-5
Cellulophaga lytica DSM 7489
Mycoplasma hyopneumoniae J
Marinobacter aquaeolei VT8
Mycobacterium sp. JLS
Picrophilus torridus DSM 9790
Staphylococcus aureus subsp.
aureus str. Newman
Ferrimonas balearica DSM 9799
Listeria monocytogenes serotype
Sinorhizobium meliloti AK83
Pseudomonas putida W619
Stigmatella aurantiaca
Mycoplasma putrefaciens
Spirochaeta thermophila
Glaciecola nitratireducens
Chlamydia psittaci NJ1
Listeria monocytogenes
Bacillus coagulans 36D1
Desulfosporosinus orientis
Streptococcus suis D9
Lactococcus lactis subsp.
cremoris NZ9000
Staphylococcus aureus
Sulfobacillus acidophilus
Listeria ivanovii subsp.
ivanovii PAM 55
Corallococcus coralloides
Streptococcus suis ST1
Mycoplasma hyorhinis
Listeria monocytogenes M7
Zymomonas mobilis subsp.
mobilis ATCC 10988
Bacillus subtilis subsp.
subtilis str. RO-NN-1
Listeria monocytogenes
Sinorhizobium fredii HH103
Alteromonas macleodli
Sulfobacillus acidophilus
Staphylococcus aureus
Mycoplasma gallisepticum
Bacteroides fragilis 638R
Chlamydophila psittaci
Haloferax mediterranei
Chlamydophila psittaci
Mycobacterium canettii CIPT
Mycoplasma hyorhinis GDL-1
Mycoplasma leachii
Streptomyces
bingchenggensis BCW-1
Mycobacterium tuberculosis
Pseudomonas fluorescens
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Streptomyces sp. SirexAA-E
Staphylococcus aureus
Cardinium endosymbiont
pergandiella
Flavobacterium
branchiophilum FL-15
Methanocella conradii
Sinorhizobium meliloti SM11
Pyrobaculum sp. 1860
Acetobacter pasteurianus
Paenibacillus polymyxa M1
Bacillus amyloliquefaciens
Chlamydia psittaci CP3
Mycobacterium tuberculosis
Listeria monocytogenes
Bacillus megaterium WSH-
Lactobacillus casei BD-II
Acetobacter pasteurianus
Listeria monocytogenes
Pyrobaculum oguniense TE7
Amycolatopsis mediterranei
Thermococcus sp. 4557
Streptococcus macedonicus
Nocardiopsis alba ATCC
Bacillus cereus NC7401
Listeria monocytogenes
Staphylococcus aureus
Brucella suis VBI22
Mycobacterium tuberculosis
Mycobacterium massiliense
Listeria monocytogenes L99
Exiguobacterium
antarcticum B7
Rhodothermus marinus
Listeria monocytogenes
Leptospira interrogans
Acetobacter pasteurianus
Mycobacterium tuberculosis
Staphylococcus lugdunensis
Bacillus amyloliquefaciens
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Mycobacterium bovis BCG
Lactobacillus rhamnosus
Shewanella baltica BA175
Streptococcus infantarius
Streptomyces cattleya NRRL
Streptococcus suis GZ1
Geobacillus
thermoleovorans
Brucella melitensis M5-90
Staphylococcus
pseudintermedius ED99
Lactobacillus casei LC2W
Bacillus amyloliquefaciens
Alteromonas macleodii str.
Chlamydophila psittaci
Staphylococcus aureus
Streptococcus agalactiae
Streptococcus thermophilus
Streptococcus gallolyticus
Streptomyces hygroscopicus
Thermus sp.
Listeria monocytogenes
Streptococcus suis S735
Staphylococcus aureus
Lactobacillus rhamnosus
Pseudomonas aeruginosa
Bacillus anthracis str. H9401
Bacillus amyloliquefaciens
Pseudomonas fluorescens
Chlamydophila psittaci 6BC
Alteromonas macleodii str.
Sinorhizobium meliloti
Bacillus sp. JS
Mycoplasma
hyopneumoniae 168
Thermus thermophilus JL-18
Listeria monocytogenes
Listeria monocytogenes
Acetobacter pasteurianus
Leuconostoc sp. C2
Shewanella baltica OS117
Brucella melitensis NI
Oscillibacter valericigenes
Lactococcus lactis subsp.
cremoris A76
Mycoplasma gallisepticum
Mycobacterium africanum
Alicyclobacillus
acidocaldarius subsp.
acidocaldarius Tc-4-1
Lactobacillus buchneri
Brucella canis HSK A52141
Bacillus amyloliquefaciens
Pseudomonas aeruginosa
halophilic archaeon DL31
Deinococcus gobiensis I-0
Acetobacter pasteurianus
Mycoplasma genitalium
Staphylococcus aureus 04-
Mycoplasma gallisepticum
Mycoplasma gallisepticum
Staphylococcus aureus
Paenibacillus mucilaginosus
Pseudomonas putida S16
Amycolatopsis mediterranei
Shewanella baltica OS678
Streptococcus suis A7
Micavibrio aeruginosavorus
Alteromonas macleodii str.
Arthrobacter sp. Rue61a
Lactobacillus salivarius
Thermus thermophilus
Marinobacter adhaerens
Mycoplasma gallisepticum
Burkholderia cepacia GG4
Mycobacterium tuberculosis
Streptomyces flavogriseus
Brucella melitensis M28
Listeria monocytogenes
Sphingobium sp. SYK-6
Zymomonas mobilis subsp.
mobilis ATCC 29191
Mycoplasma gallisepticum
Bacillus thuringiensis
Bacillus cereus F837/76
Paenibacillus mucilaginosus
Mycobacterium tuberculosis
Listeria monocytogenes
Listeria monocytogenes FSL
Natrinema sp. J7-2
Streptomyces violaceusniger
Pseudomonas putida DOT-
Burkholderia pseudomallei
Staphylococcus aureus
Listeria monocytogenes
Solibacillus silvestris
Paenibacillus terrae HPL-
Mycoplasma pneumoniae
Streptococcus suis D12
Streptococcus suis JS14
Staphylococcus aureus
Staphylococcus aureus
Oceanimonas sp. GK1
Shewanella putrefaciens
Brucella pinnipedialis B2/94
Streptococcus thermophilus
Kitasatospora setae KM-
Pelagibacterium
halotolerans B2
Mycobacterium tuberculosis
Sinorhizobium fredii USDA
Enterococcus faecalis D32
Acetobacter pasteurianus
Bacillus amyloliquefaciens
Mycobacterium tuberculosis
Leuconostoc mesenteroides
Solitalea canadensis DSM
Mycoplasma gallisepticum
Mycoplasma genitalium
Caldicellulosiruptor
saccharolyticus DSM 8903
Mycoplasma crocodyli
Shewanella woodyi ATCC
Streptomyces coelicolor
Shewanella denitrificans
Prochlorococcus marinus
Streptococcus suis BM407
Shewanella oneidensis MR-1
Bacillus anthracis str. CDC
Lactobacillus sakei subsp.
sakei 23K
Shewanella halifaxensis
Anaeromyxobacter
dehalogenans 2CP-C
Parachlamydia
acanthamoebae UV7
Rhodococcus erythropolis
Bacillus halodurans C-125
Paenibacillus sp. JDR-2
Listeria seeligeri serovar
Agrobacterium tumefaciens
Pseudoalteromonas
atlantica T6c
Chlamydia muridarum Nigg
Pseudomonas fluorescens
Sorangium cellulosum ‘Soce
Halopiger xanaduensis SH-6
Melissococcus plutonius
Synechococcus sp. CC9605
Streptococcus suis
Pseudomonas fluorescens
Bacillus subtilis subsp.
spizizenii str. W23
Rhodospirillum centenum
Bradyrhizobium japonicum
Lactobacillus salivarius
Culex quinquefasciatus Pel
Paenibacillus mucilaginosus
Haloferax volcanii DS2
Brucella melitensis ATCC
Lactobacillus reuteri DSM
Bacillus pumilus SAFR-032
Bacillus cereus G9842
Streptococcus agalactiae
Chloroflexus sp. Y-400-fl
Pseudomonas putida
Synechococcus sp. JA-2-
Haloterrigena turkmenica
Shewanella lolhica PV-4
Mycobacterium tuberculosis
Synechococcus sp. JA-3-
Shewanella baltica OS185
Enterococcus faecalis V583
Staphylococcus epidermidis
Staphylococcus aureus
Mycoplasma pneumoniae
Staphylococcus aureus
Staphylococcus epidermidis
Thermus thermophilus HB8
Brucella suis 1330
Aeropyrum pernix K1
Microbacterium testaceum
Gemmatimonas aurantiaca T
Bacillus cereus AH820
Thermaerobacter
marianensis DSM 12885
Mycoplasma leachii PG50
Burkholderia pseudomallei
Leifsonia xyli subsp. xyli str.
Treponema azotonutricium
Geobacillus sp. Y412MC61
Mesorhizobium ciceri biovar
Arthrobacter
phenanthrenivorans Sphe3
Streptomyces scabiei 87.22
Exiguobacterium sp. AT1b
Lactobacillus plantarum
Ilyobacter polytropus DSM
Bacillus anthracis str. Ames
Polaromonas sp. JS666
Streptomyces cattleya NRRL
Halobacterium sp. NRC-1
Chlamydophila psittaci RD1
Mycoplasma conjunctivae
Bacillus cereus E33L
Coprothermobacter
proteolyticus DSM 5265
Mycobacterium tuberculosis
Geobacillus
thermodenitrificans NG80-2
Burkholderia glumae BGR1
Alicycliphilus denitrificans
Staphylococcus aureus
Bacillus thuringiensis str. Al
Bacillus thuringiensis
Brucella abortus A13334
Bacillus thuringiensis
Fluviicola taffensis DSM
Alicycliphilus denitrificans
Geobacillus
thermoglucosidasius C56-
Alicyclobacillus
acidocaldarius subsp.
acidocaldarius DSM 446
Prochlorococcus marinus
Streptococcus suis
Anaerolinea thermophila
Bartonella tribocorum CIP
Burkholderia pseudomallei
Burkholderia gladioli BSR3
Zymomonas mobilis subsp.
mobilis ZM4
Staphylococcus aureus
Sphingomonas wittichii RW1
Staphylococcus lugdunensis
Chlamydophila pecorum
Staphylococcus
saprophyticus subsp.
saprophyticus ATCC 15305
Natrialba magadii ATCC
Pseudoalteromonas sp.
Nautilia profundicola AmH
Brucella suis ATCC 23445
Prochlorococcus marinus
Mycoplasma pulmonis UAB
Micromonospora aurantiaca
Prochlorococcus marinus
Shewanella sediminis HAW-
Listeria innocua Clip11262
Mycoplasma synovlae 53
Bacillus cereus ATCC 14579
Mycoplasma
hyopneumoniae 7448
Pseudomonas putida F1
Brucella abortus bv. 1 str. 9-
Burkholderia mallei SAVP1
Mesoplasma florum L1
Pseudomonas aeruginosa
Mycoplasma penetrans HF-2
Frankia alni ACN14a
Brevundimonas
subvibrioides ATCC 15264
Thermococcus sibiricus MM
Myxococcus fulvus HW-1
Modestobacter marinus
Sphingobium
chlorophenolicum L-1
Shewanella sp. MR-7
Hahella chejuensis KCTC
Shewanella violacea DSS12
Mycobacterium bovis
Synechococcus sp. CC9902
Staphylococcus carnosus
Bacteroides fragilis NCTC
Lactobacillus fermentum
Novosphingobium
aromaticivorans DSM 12444
Geobacillus sp. WCH70
Geobacter sp. M18
Deinococcus geothermalis
Streptococcus suis SC84
Bacillus coagulans 2-6
Wolbachia sp. wRi
Streptococcus agalactiae
Cyanothece sp. ATCC 51142
Desulfotalea psychrophila
Desulfotomaculum
carboxydivorans CO 1 SRB
Pseudomonas putida ND6
Methanocella paludicola
Nocardia cyriacigeorgica
Leuconostoc gasicomitatum
Intrasporanglum calvum
Streptococcus pasteurianus
Thermus thermophilus HB27
Streptococcus thermophilus
Mycoplasma mycoides
Burkholderia ambifaria
Burkholderia pseudomallei
Mycobacterium avium 104
Shewanella frigidimarina
Staphylococcus aureus
Natronomonas pharaonis
Staphylococcus aureus
Hirschia baltica ATCC 49814
Streptococcus thermophilus
Thermoplasma acidophilum
Bacillus selenitireducens
Mesorhizobium
opportunistum WSM2075
Sinorhizobium meliloti 1021
Salinispora tropica CNB-440
Burkholderia mallei NCTC
Desulfobulbus propionicus
Bdellovibrio bacteriovorus
Shewanella baltica OS155
Candidatus Phytoplasma
Halobacillus halophilus DSM
Candidatus Phytoplasma
Geobacter bemidjiensis Bem
Staphylococcus
pseudintermedius HKU10-
Mycobacterium bovis BCG
Bordetella petril DSM 12804
Listeria monocytogenes
Verrucosispora maris AB-18-
Brucella canis ATCC 23365
Brucella ovis ATCC 25840
Bacillus anthracis str. ‘Ames
Caldicellulosiruptor
kronotskyensis 2002
Colwellia psychrerythraea
Pseudovibrio sp. FO-BEG1
Acetobacter pasteurianus
Mesorhizobium loti
Propionibacterium acnes
Pseudomonas putida BIRD-1
Cyanothece sp. PCC 7424
Pyrobaculum calidifontis
Stigmatella aurantiaca
Staphylococcus aureus
Sanguibacter keddieli DSM
Pseudomonas aeruginosa
Lactobacillus buchneri
Aerococcus urinae ACS-120-
Bacillus atrophaeus 1942
Staphylococcus aureus
Staphylococcus aureus
Candidatus Sulcia muelleri
Bacillus amyloliquefaciens
Anaeromyxobacter sp. K
Isosphaera pallida ATCC
Maricaulis maris MCS10
Anaeromyxobacter
dehalogenans 2CP-1
Burkholderia cenocepacia
Pseudomonas
brassicacearum subsp.
brassicacearum NFM421
Synechococcus sp. RCC307
Staphylococcus aureus
Streptococcus thermophilus
Lactobacillus rhamnosus Lc
Leuconostoc kimchii IMSNU
Mycoplasma gallisepticum
Spirochaeta coccoides DSM
Brucella melitensis bv. 1 str.
Shewanella baltica OS195
Staphylococcus aureus
Nitratiruptor sp. SB155-2
Leuconostoc citreum KM20
Burkholderia sp. 383
Bacillus pseudofirmus OF4
Haliangium ochraceum DSM
Variovorax paradoxus S110
Achromobacter xylosoxidans
Ruegeria sp. TM1040
Sulfurovum sp. NBC37-1
Geobacter sulfurreducens
Chloroflexus aurantiacus J-
Pseudoalteromonas
haloplanktis TAC125
Caulobacter crescentus
Thermoproteus uzoniensis
Pseudomonas fluorescens
Lactobacillus casei ATCC
Prochlorococcus marinus
Lactobacillus casei str.
Sphingobium japonicum
Cyanothece sp. PCC 8801
Rhodothermus marinus
Shewanella sp. W3-18-1
Nocardia farcinica IFM
Bacillus subtilis subsp.
subtilis str. 168
Nocardioides sp. JS614
Waddlia chondrophila WSU
Trichodesmium erythraeum
Bacillus tusciae DSM 2912
Shewanella baltica OS223
Frankia sp. Ccl3
Bacillus licheniformis ATCC
Rubrobacter xylanophilus
Conexibacter woesei DSM
Bacillus cereus B4264
Idiomarina loihiensis L2TR
Bacillus clausii KSM-K16
Lactococcus lactis subsp.
cremoris MG1363
Drosophila melanogaster
Caulobacter sp. K31
Bacillus megaterium QM
Bacillus anthracis str. Sterne
Lactobacillus reuteri SD2112
Halobacterium salinarum R1
Streptococcus thermophilus
Caulobacter crescentus
Symbiobacterium
thermophilum IAM 14863
Prochlorococcus marinus
Shewanella pealeana ATCC
Mycobacterium sp. JDM601
Bacillus cereus 03BB102
Agrobacterium sp. H13-3
Bacillus cereus ATCC 10987
Nostoc punctiforme PCC
Bacillus anthracis str. A0248
Mycobacterium smegmatis
Shewanella amazonensis
Bacillus cellulosilyticus DSM
Synechococcus sp. WH
Brucella abortus S19
Acetobacter pasteurianus
Novosphingobium sp. PP1Y
Listeria monocytogenes
Leuconostoc mesenteroides
Bacillus subtilis subsp.
spizizenii TU-B-10
Halomicrobium mukohataei
Thermobifida fusca YX
Streptococcus gallolyticus
Burkholderia mallei NCTC
Prochlorococcus marinus
Spirochaeta thermophila
Streptococcus suis ST3
Synechococcus sp. CC9311
Streptococcus suis SS12
Burkholderia thailandensis
Halomonas elongata DSM
Mycoplasma capricolum
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Alkaliphilus metalliredigens
Sinorhizobium fredii
Alteromonas macleodii str.
Geobacillus sp. C56-T3
Leptospira borgpetersenii
Brucella microti CCM 4915
Meiothermus ruber DSM
Sphingopyxis alaskensis
Haloarcula marismortui
Listeria monocytogenes
Thermosynechococcus
elongatus BP-1
Mycoplasma genitallum G37
Shewanella sp. ANA-3
Bacillus weihenstephanensis
Shewanella putrefaciens CN-
Lactococcus lactis subsp.
cremoris SK11
Burkholderia pseudomallei
Synechococcus sp. PCC
Arthrobacter aurescens TC1
Weissella koreensis KACC
Burkholderia mallei ATCC
Macrococcus caseolyticus
Staphylococcus aureus
Burkholderia cenocepacia
Meiothermus silvanus DSM
Oceanobacillus iheyensis
Lactobacillus plantarum
Parabacteroides distasonis
Staphylococcus
haemolyticus JCSC1435
Lactobacillus fermentum IFO
Candidatus Amoebophilus
Catenulispora acidiphila
Bacillus amyloliquefaciens
Mycobacterium bovis BCG
Staphylococcus aureus
Frankia sp. EAN1pec
Streptococcus equi subsp.
equi 4047
Gloeobacter violaceus PCC
Pseudomonas aeruginosa
Prochlorococcus marinus
Thermoplasma volcanium
Bacillus megaterium DSM
Anabaena variabilis ATCC
Paenlbacillus polymyxa
Chlamydophila abortus
Geobacter uraniireducens
Lactobacillus casei BL23
Streptococcus agalactiae
Pseudomonas entomophila
Halorubrum lacusprofundi
Geobacillus kaustophilus
Staphylococcus aureus
Azospirillum sp. B510
Chlamydophila psittaci
Lactobacillus rhamnosus
Staphylococcus aureus
cyanobacterium UCYN-A
Zymomonas mobilis subsp.
pomaceae ATCC 29192
Burkholderia cenocepacia
Thermus scotoductus SA-01
Candidatus Protochlamydia
Bacillus cereus biovar
Burkholderia cenocepacia
Oenococcus oeni PSU-1
Listeria monocytogenes 08-
Alteromonas sp. SN2
Shewanella sp. MR-4
Streptomyces avermitilis MA-
Mycoplasma hyorhinis HUB-1
Staphylococcus aureus
Prochlorococcus marinus
Bacteroides fragilis YCH46
Thermobispora bispora DSM
Amycolatopsis mediterranei
Paenibacillus sp. Y412MC10
Erysipelothrix rhusiopathiae
Sulfurimonas autotrophica
Acaryochloris marina
Lactobacillus plantarum
Streptococcus equi subsp.
zooepidemicus MGCS10565
Bacillus thuringiensis
Mycobacterium avium
Mycoplasma fermentans
Leptospira interrogans
Brucella melitensis biovar
Rhodococcus jostii RHA1
Ramlibacter tataouinensis
Mycoplasma fermentans
Ochrobactrum anthropi
Erythrobacter litoralis
Bacillus subtilis BSn5
Pseudomonas putida GB-1
Prochlorococcus marinus
Bacillus cereus Q1
Cyanothece sp. PCC 7822
Anoxybacillus flavithermus
Bacillus cereus AH187
Burkholderia ambifaria
Streptococcus uberis 0140J
Sinorhizobium medicae
Halogeometricum
borinquense DSM 11551
Mycobacterium vanbaalenii
Halalkalicoccus jeotgali B3
Lysinibacillus sphaericus C3-
Exiguobacterium sibiricum
Mycoplasma mobile 163K
Streptococcus suis P1/7
Geobacillus sp. Y4.1MC1
Anaeromyxobacter sp.
Simkania negevensis Z
Micromonospora sp. L5
Listeria monocytogenes
Cyanothece sp. PCC 8802
Prochlorococcus marinus
Carnobacterium sp. 17-4
Zymomonas mobilis subsp.
mobilis NCIMB 11163
Leptospira borgpetersenil
Saccharopolyspora
erythraea NRRL 2338
Pusillimonas sp. T7-7
Pediococcus pentosaceus
Shewanella piezotolerans
Streptosporangium roseum
Lactobacillus reuteri JCM
Paenibacillus polymyxa SC2
Rhodoferax ferrireducens
Streptomyces griseus
Pyrobaculum aerophilum str.
Mycobacterium smegmatis
Mycobacterium abscessus
Thermoanaerobacter
tengcongensis MB4
Cyanothece sp. PCC 7425
Brevibacillus brevis NBRC
Salinispora arenicola CNS-
Listeria monocytogenes 08-
Sphaerobacter thermophilus
Mycobacterium tuberculosis
Mycoplasma
hyopneumoniae 232
Marinithermus
hydrothermalis DSM 14884
Mycoplasma mycoides
Listeria welshimeri serovar
Glaciecola sp. 4H-3-7 + YE-5
Geobacter metallireducens
Nostoc sp. PCC 7120
Mycoplasma
hyopneumoniae J
Propionibacterium acnes
Picrophilus torridus DSM
Staphylococcus aureus
Geobacillus sp. Y412MC52
Ferrimonas balearica DSM
Listeria monocytogenes
Sinorhizobium meliloti AK83
Prochlorococcus marinus
Sphingobacterium sp. 21
Pseudomonas putida W619
Arcobacter sp. L
Listeria monocytogenes SLCC2479
Bacillus coagulans 36D1
Lactococcus lactis subsp.
cremoris NZ9000
Staphylococcus aureus subsp.
aureus T0131
Mycoplasma bovis HB0801
Sulfobacillus acidophilus TPY
Listeria ivanovii subsp. ivanovii
Listeria monocytogenes M7
Bacillus subtills subsp. subtills
Listeria monocytogenes
Sinorhizobium fredii HH103
Sulfobacillus acidophilus DSM
Staphylococcus aureus subsp.
aureus M013
Haloferax mediterranel ATCC
Enterococcus hirae ATCC 9790
Pseudomonas fluorescens F113
Staphylococcus aureus subsp.
aureus 11819-97
Staphylococcus aureus subsp.
aureus LGA251
Staphylococcus aureus subsp.
aureus VC40
Haloquadratum walsbyi C23
Staphylococcus aureus subsp.
aureus ED133
Mycoplasma bovis Hubei-1
Methanocella conradii HZ254
Sinorhizobium meliloti SM11
Desulfosporosinus acidiphilus
Pyrobaculum sp. 1860
Paenibacillus polymyxa M1
Bacillus amyloliquefaciens LL3
Listeria monocytogenes J0161
Bacillus megaterium WSH-002
Lactobacillus casei BD-II
Listeria monocytogenes
Melissococcus plutonius DAT56
Pyrobaculum oguniense TE7
Amycolatopsis mediterranei
Bacillus cereus NC7401
Listeria monocytogenes
Staphylococcus aureus subsp.
aureus 71193
Brucella suis VBI22
Listeria monocytogenes L99
Exiguobacterium antarcticum
Sulfolobus solfataricus 98/2
Listeria monocytogenes
Ignavibacterium album JCM
Mycobacterium tuberculosis
Staphylococcus lugdunensis
Bacillus amyloliquefaciens
Corynebacterium variabile
Anaerobaculum mobile DSM
Haloarcula hispanica ATCC
Lactobacillus rhamnosus GG
Geobacillus thermoleovorans
Brucella melitensis M5-90
Staphylococcus
pseudintermedius ED99
Lactobacillus casei LC2W
Bacillus amyloliquefaciens
Staphylococcus aureus subsp.
aureus ECT-R 2
Listeria monocytogenes
Staphylococcus aureus subsp.
aureus TCH60
Lactobacillus rhamnosus ATCC
Pseudomonas aeruginosa
Bacillus anthracis str. H9401
Bacillus amyloliquefaciens
Pseudomonas fluorescens A506
Sinorhizobium meliloti BL225C
Bacillus sp. JS
Listeria monocytogenes
Listeria monocytogenes Finland
Arcobacter butzleri ED-1
Brucella melitensis NI
Brachyspira pilosicoli B2904
Alicyclobacillus acidocaldarius
Thermoanaerobacter wiegelii
Lactobacillus buchneri CD034
Brucella canis HSK A52141
Bacillus amyloliquefaciens XH7
Pseudomonas aeruginosa M18
Enterococcus faecium Aus0004
halophilic archaeon DL31
Tetragenococcus halophilus
Staphylococcus aureus 04-02981
Thermoanaerobacterium
saccharolyticum JW/SL-YS485
Staphylococcus aureus subsp.
aureus HO 5096 0412
Paenibacillus mucilaginosus
Pseudomonas putida S16
Lactobacillus salivarius
Marinobacter adhaerens HP15
Burkholderia cepacia GG4
Brucella melitensis M28
Listeria monocytogenes 10403S
Sphingobium sp. SYK-6
Enterococcus faecium DO
Bacillus thuringiensis serovar
finitimus YBT-020
Bacillus cereus F837/76
Thermoproteus tenax Kra 1
Brachyspira intermedia PWS/A
Paenibacillus mucilaginosus
Enterococcus faecalis 62
Listeria monocytogenes ATCC
Listeria monocytogenes FSL R2-
Natrinema sp. J7-2
Pseudomonas putida DOT-T1E
Burkholderia pseudomallei
Staphylococcus aureus subsp.
aureus str. JKD6008
Listeria monocytogenes
Solibacillus silvestris StLB046
Paenibacillus terrae HPL-003
Staphylococcus aureus subsp.
aureus S0385
Staphylococcus aureus subsp.
aureus TW20
Brucella pinnipedialis B2/94
Streptococcus parasanguinis
Sinorhizobium fredii USDA 257
Enterococcus faecalis D32
Bacillus amyloliquefaciens Y2
Bacillus anthracis str. CDC 684
Lactobacillus sakei subsp. sakei
Bacillus halodurans C-125
Paenibacillus sp. JDR-2
Listeria seeligeri serovar
Pseudomonas fluorescens
Halopiger xanaduensis SH-6
Pseudomonas fluorescens Pf-5
Bacillus subtilis subsp.
spizizenii str. W23
Bradyrhizobium japonicum
Lactobacillus salivarius
Paenibacillus mucilaginosus
Haloferax volcanii DS2
Brucella melitensis
Lactobacillus reuteri
Bacillus pumilus SAFR-032
Bacillus cereus G9842
Arthrobacter arilaitensis Re117
Pseudomonas putida KT2440
Haloterrigena turkmenica DSM
Rothia dentocariosa ATCC
Enterococcus faecalis V583
Staphylococcus epidermidis
Staphylococcus aureus subsp.
aureus JH9
Staphylococcus aureus subsp.
aureus COL
Staphylococcus epidermidis
Buchnera aphidicola (Cinara
tujafilina)
Brucella suis 1330
Bacillus cereus AH820
Thermaerobacter marianensis
Desulfovibrio magneticus RS-1
Burkholderia pseudomallei
Geobacillus sp. Y412MC61
Mesorhizobium ciceri biovar
biserrulae WSM1271
Calditerrivibrio nitroreducens
Wigglesworthia glossinidia
brevialpis
Exiguobacterium sp. AT1b
Flexistipes sinusarabici
Lactobacillus plantarum subsp.
plantarum ST-III
Bacillus anthracis str. Ames
Halobacterium sp. NRC-1
Bacillus cereus E33L
Geobacillus thermodenitrificans
Burkholderia glumae BGR1
Alicycliphilus denitrificans BC
Staphylococcus aureus subsp.
aureus USA300_TCH1516
Bacillus thuringiensis str.
Bacillus thuringiensis serovar
chinensis CT-43
Brucella abortus A13334
Bacillus thuringiensis serovar
konkukian str. 97-27
Alicycliphilus denitrificans K601
Baumannia cicadellinicola
coagulata)
Geobacillus thermoglucosidasius
Alicyclobacillus acidocaldarius
Rothia mucilaginosa DY-18
Burkholderia pseudomallei 668
Jonesia denitrificans DSM
Burkholderia gladioli BSR3
Staphylococcus aureus subsp.
aureus MSSA476
Sphingomonas wittichii RW1
Staphylococcus lugdunensis
Staphylococcus saprophyticus
Natrialba magadii ATCC 43099
Brucella suis ATCC 23445
Listeria innocua Clip11262
Bacillus cereus ATCC 14579
Pseudomonas putida F1
Brucella abortus bv. 1 str. 9-941
Burkholderia mallei SAVP1
Pseudomonas aeruginosa DK2
Brevundimonas subvibrioides
Sphingobium chlorophenolicum
Thermoanaerobacter sp. X513
Staphylococcus carnosus subsp.
carnosus TM300
Lactobacillus fermentum CECT
Geobacillus sp. WCH70
Bacillus coagulans 2-6
Kocuria rhizophila DC2201
Pseudomonas putida ND6
Methanocella paludicola
Leuconostoc gasicomitatum
Burkholderia ambifaria
Burkholderia pseudomallei
Mycoplasma agalactiae
Staphylococcus aureus subsp.
aureus MSHR1132
Natronomonas pharaonis DSM
Staphylococcus aureus subsp.
aureus ED98
Hirschia baltica ATCC 49814
Thermoplasma acidophilum
Bacillus selenitireducens MLS10
Mesorhizobium opportunistum
Sinorhizobium meliloti 1021
Burkholderia mallei NCTC
Halobacillus halophilus DSM
Staphylococcus
pseudintermedius HKU10-03
Bordetella petrii DSM 12804
Listeria monocytogenes L312
Brucella canis ATCC 23365
Brucella ovis ATCC 25840
Bacillus anthracis str. ‘Ames
Mesorhizobium loti
Pseudomonas putida BIRD-1
Pyrobaculum calidifontis JCM
Candidatus Biochmannia
pennsylvanicus str. BPEN
Staphylococcus aureus subsp.
aureus MRSA252
Mycoplasma agalactiae PG2
Pseudomonas aeruginosa PA7
Lactobacillus buchneri NRRL
Aerococcus urinae ACS-
Bacillus atrophaeus 1942
Thermoanaerobacter sp. X514
Staphylococcus aureus subsp.
aureus Mu3
Staphylococcus aureus subsp.
aureus JKD6159
Bacillus amyloliquefaciens
Maricaulis maris MCS10
Thermoanaerobacter brockii
Burkholderia cenocepacia J2315
Pseudomonas brassicacearum
Staphylococcus aureus subsp.
aureus Mu50
Lactobacillus rhamnosus Lc 705
Spirochaeta coccoides DSM
Brucella melitensis bv.
Staphylococcus aureus subsp.
aureus NCTC 8325
Leuconostoc citreum KM20
Burkholderia sp. 383
Prevotella melaninogenica
Bacillus pseudofirmus OF4
Achromobacter xylosoxidans A8
Ruegeria sp. TM1040
Metallosphaera sedula DSM
Borrelia garinii PBi
Thermoproteus uzoniensis
Pseudomonas fluorescens Pf0-1
Lactobacillus casei ATCC 334
Lactobacillus casei str. Zhang
Bacillus subtilis subsp.
subtilis str. 168
Bacillus tusciae DSM 2912
Bacillus licheniformis ATCC
Borrelia turicatae 91E135
Slackia heliotrinireducens DSM
Acidilobus saccharovorans
Conexibacter woesel DSM
Bacillus cereus B4264
Prevotella denticola F0289
Bacillus clausii KSM-K16
Lactococcus lactis subsp.
cremoris MG1363
Dictyoglomus turgidum DSM
Methanococcus voltae A3
Caulobacter sp. K31
Enterococcus faecalis OG1RF
Bacillus megaterium QM B1551
Bacillus anthracis str. Sterne
Bacillus cytotoxicus NVH
Lactobacillus reuteri SD2112
Halobacterium salinarum R1
Symbiobacterium thermophilum
Chlorobium phaeobacteroides
Bacillus cereus 03BB102
Bacillus cereus ATCC 10987
Vulcanisaeta moutnovskia
Bacillus anthracis str. A0248
Bacillus cellulosilyticus DSM
Brucella abortus S19
Mycobacterium leprae TN
Haloquadratum walsbyi DSM
Listeria monocytogenes HCC23
Bacillus subtilis subsp. spizizenii
Halomicrobium mukohataei
Burkholderia mallei NCTC
Burkholderia thailandensis
Pseudomonas aeruginosa
Sinorhizobium fredii NGR234
Geobaclllus sp. C56-T3
Brucella microti CCM 4915
Haloarcula marismortui ATCC
Listeria monocytogenes serotype
Bacillus weihenstephanensis
Burkholderia pseudomallei
Brachyspira murdochii DSM
Thermoanaerobacterium
thermosaccharolyticum DSM
Mycobacterium ulcerans
Burkholderia mallei
Macrococcus caseolyticus
Desulfovibrio vulgaris str.
Staphylococcus aureus subsp.
aureus JH1
Burkholderia cenocepacia AU
Mycobacterium marinum M
Oceanobacillus iheyensis
Lactobacillus plantarum
Staphylococcus haemolyticus
Lactobacillus fermentum IFO
Bacillus amyloliquefaciens
Staphylococcus aureus subsp.
aureus MW2
Pseudomonas aeruginosa
Thermoplasma volcanium GSS1
Sulfolobus solfataricus P2
Bacillus megaterium DSM 319
Paembacillus polymyxa E681
Lactobacillus casei BL23
Pseudomonas entomophila L48
Halorubrum lacusprofundi
Geobacillus kaustophilus
Staphylococcus aureus subsp.
aureus USA300_FPR3757
actobacillus rhamnosus GG
Staphylococcus aureus RF122
Burkholderia cenocepacla
Thermoanaerobacter
pseudethanolicus ATCC 33223
Bacillus cereus biovar
anthracis str. CI
Burkholderia cenocepacia
Oenococcus oeni PSU-1
Listeria monocytogenes 08-5578
Clostridium tetani E88
Thermoanaerobacterium
xylanolyticum LX-11
Brachyspira pilosicoli
Staphylococcus aureus subsp.
aureus N315
Erysipelothrix rhusiopathiae
Mycobacterium leprae Br4923
Lactobacillus plantarum JDM1
Pyrobaculum arsenaticum DSM
Mycoplasma bovis PG45
Bacillus thuringiensis
Arcobacter butzleri RM4018
Mycobacterium avium subsp.
Brucella melitensis biovar
Ramlibacter tataouinensis
Ochrobactrum anthropi ATCC
Bacillus subtilis BSn5
Pseudomonas putida GB-1
Bacillus cereus Q1
Arcobacter nitrofigilis
Anoxybacillus flavithermus
Lactobacillus brevis ATCC
Bacillus cereus AH187
Burkholderia ambifaria AMMD
Sinorhizobium medicae
Halogeometricum borinquense
Halalkalicoccus jeotgali B3
Lysinibacilius sphaericus C3-41
Exiguobacterium sibiricum
Geobacillus sp. Y4.1MC1
Anaeromyxobacter sp. Fw109-5
Listeria monocytogenes EGD-e
Carnobacterium sp. 17-4
Campylobacter concisus 13826
Pusillimonas sp. T7-7
Sulfolobus acidocaldarius
Pediococcus pentosaceus ATCC
Lactobacillus reuteri JCM
Paenibacillus polymyxa SC2
Pyrobaculum aerophilum str.
Halorhabdus utahensis DSM
Thermoanaerobacter
tengcongensis MB4
Brevibacillus brevis NBRC
Brachyspira hyodysenteriae
Listeria monocytogenes 08-5923
Listeria welshimeri serovar
Picrophilus torridus DSM
Staphylococcus aureus subsp.
aureus str. Newman
Geobacillus sp. Y412MC52
Listeria monocytogenes
Sinorhizobium meliloti AK83
Desulfomicrobium baculatum
Pseudomonas putida W619
A third step in the MMA precursor bioproduction process is the oxidation of isobutyryl-CoA to (methacrylic acid)-coA (MAA-CoA). This step may be performed by a suitable enzyme, such as an isobutryl-coA dehydrogenase enzyme.
The host microorganism may be transformed to express one or more isobutyrylcoA dehydrogenase or equivalent enzymes. For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa ACD1 gene (Genbank Accession Number NP 249437) or an equivalent gene. Other isobutyryl-CoA dehydrogenase enzymes that may be used are listed in Table 6.
Glaciecola nitratireducens
Alcanivorax dieselolei B5
Gordonia
polylsoprenlvorans VH2
Pseudogulbenkiania sp.
Alteromonas macleodii
Phacobactor
gallaeciensis 2.10
Xanthomonas oryzae pv.
oryzicola BLS256
Mycobacterium
chubuense NBB4
Mycobacterium canettii
Pseudoxanthomonas
spadix BD-a59
Mycobacterium
tuberculosis UT205
Collimonas fungivorans
Mycobacterium
tuberculosis CTRI-2
Mycobacterium
intracellulare ATCC
Xanthomonas campestris
Bordetella pertussis CS
Nocardiopsis alba ATCC
Mycobacterium
tuberculosis RGTB327
Mycobacterium
massiliense str. GO 06
Rhodospirillum rubrum
Rhodospirillum
photometricum DSM 122
Mycobacterium
tuberculosis RGTB423
Mycobacterium
tuberculosis H37Rv
Mycobacterium bovis
Shewanella baltica
Streptomyces cattleya
Bradyrhizobium
japonicum USDA 6
Brucella melitensis M5-90
Alteromonas macleodii
Tistrella mobilis
Roseobacter litoralis Och
Streptomyces
hygroscopicus subsp.
jinggangensis 5008
Marinobacter
hydrocarbonoclasticus
Pseudomonas
aeruginosa NCGM2.S1
Alteromonas macleodii
Mycobacterium
intracellulare MOTT-02
Vibrio furnissii NCTC
Shewanella baltica
Mycobacterium rhodesiae
Brucella melitensis NI
Desulfomonile tiedjei
Mycobacterium africanum
Brucella canis HSK
Pseudomonas
aeruginosa M18
Xanthomonas
axonopodis pv. citrumelo
Gordonia sp. KTR9
Shewanella baltica
Alteromonas macleodii
Legionella pneumophila
Marinobacter adhaerens
Mycobacterium
tuberculosis KZN 605
Stenotrophomonas
maltophilia JV3
Legionella pneumophila
Brucella melitensis M28
Bradyrhizobium sp.
Mycobacterium
tuberculosis CCDC5079
Stenotrophomonas
maltophilia D457
Legionella pneumophila
Oceanimonas sp. GK1
Shewanella putrefaciens
Brucella pinnipedialis
Kitasatospora setae KM-
Mycobacterium
tuberculosis CCDC5180
Sinorhizobium fredii
Phaeobacter
gallaeciensis DSM 17395
Mycobacterium
tuberculosis KZN 4207
Legionella pneumophila
Aeromonas salmonicida
Azorhizobium
caulinodans ORS 571
Shewanella woodyi ATCC
Shewanella denitrificans
Rhodomicrobium vannielii
Vibrio vulnificus YJ016
Xanthobacter
autotrophicus Py2
Vibrio sp. Ex25
Shewanella oneidensis
Shewanella halifaxensis
Rhodococcus erythropolis
Pseudoalteromonas
atlantica T6c
Legionella pneumophila
Rhodospirillum centenum
Pseudomonas
mendocina NK-01
Laribacter hongkongensis
Bradyrhizobium
japonicum USDA 110
Rhodobacter sphaeroides
Legionella longbeachae
Brucella melitensis ATCC
Chelativorans sp. BNC1
Arthrobacter arilaitensis
Vibrio vulnificus CMCP6
Aeromonas hydrophila
Polymorphum gilvum
Shewanella loihica PV-4
Mycobacterium
tuberculosis H37Ra
Shewanella baltica
Variovorax paradoxus
Microbacterium
testaceum StLB037
Vibrio parahaemolyticus
Xanthomonas campestris
Rhodopseudomonas
palustris BisB18
Rhodospirillum rubrum
Mesorhizobium ciceri
Ferroglobus placidus
Streptomyces cattleya
Mycobacterium
tuberculosis KZN 1435
Alicycliphilus denitrificans
Brucella abortus A13334
Alicycliphilus denitrificans
Sphingomonas wittichii
Nakamurella multipartita
Psychrobacter sp. PRwf-1
Pseudoalteromonas sp.
Rhodobacter capsulatus
Shewanella sediminis
Acidovorax avenae
Rhodopseudomonas
palustris CGA009
Comamonas testosteroni
Brucella abortus bv. 1 str.
Pseudomonas
aeruginosa DK2
Brevundimonas
subvibrioides ATCC
Sphingobium
chlorophenolicum L-1
Shewanella sp. MR-7
Hahella chejuensis KCTC
Shewanella violacea
Mycobacterium bovis
Novosphingobium
aromaticivorans DSM
Rhodopseudomonas
palustris BisA53
Bordetella bronchiseptica
Nitrobacter winogradskyi
Xanthomonas oryzae pv.
oryzae KACC10331
Bradyrhizobium sp. BTAi1
Nocardia cyriacigeorgica
Xanthomonas
axonopodis pv. citri str.
Mycobacterium avium
Shewanella frigidimarina
Hirschia baltica ATCC
Mesorhizobium
opportunistum WSM2075
Shewanella baltica
Stenotrophomonas
maltophilia K279a
Vibrio harveyi ATCC BAA-
Mycobacterium bovis
Bordetella petrii DSM
Brucella ovis ATCC
Micrococcus luteus
Colwellia psychrerythraea
Pseudovibrio sp. FO-
Mesorhizobium loti
Psychrobacter
cryohalolentis K5
Rhodobacter sphaeroides
Pseudomonas
aeruginosa PA7
Acidianus hospitalis W1
Acidovorax citrulli AAC00-1
Isosphaera pallida ATCC
Maricaulis maris MCS10
Aeromonas veronii B565
Xanthomonas campestris
Methylobacterium sp. 4-
Dinoroseobacter shibae
Bordetella pertussis
Rhodococcus opacus B4
Rhodococcus opacus B4
Brucella melitensis bv. 1
Shewanella baltica
Acidothermus
cellulolyticus 11B
Xanthomonas campestris
Variovorax paradoxus
Ruegeria pomeroyi DSS-3
Achromobacter
xylosoxidans A8
Ruegeria sp. TM1040
Beijerinckia indica subsp.
Indica ATCC 9039
Nitrobacter hamburgensis
Pseudoalteromonas
haloplanktis TAC125
Caulobacter crescentus
Stenotrophomonas
maltophilia R551-3
Hyphomonas neptunium
Sphingobium japonicum
Shewanella sp. W3-18-1
Nocardia farcinica IFM
Nocardiopsis dassonvillei
Chromobacterium
violaceum ATCC 12472
Nocardioides sp. JS614
Shewanella baltica
Vibrio sp. EJY3
Roseobacter denitrificans
Mycobacterium sp.
Acidovorax ebreus TPSY
Idlomarina loihiensis
Caulobacter sp. K31
Herbaspirillum
seropedicae SmR1
Caulobacter crescentus
Shewanella pealeana
Mycobacterium sp.
Rhodopseudomonas
palustris TIE-1
Syntrophus aciditrophicus
Rhodococcus equl 103S
Mycobacterium
smegmatis str. MC2 155
Shewanella amazonensis
Brucella abortus S19
Magnetospirillum
magneticum AMB-1
Novosphingobium sp.
Xanthomonas oryzae pv.
oryzae PXO99A
Thermobitida tusca YX
Rhodopseudomonas
palustris HaA2
Halomonas elongata
Pseudomonas
aeruginosa LESB58
Mycobacterium
tuberculosis CDC1551
Bordetella avium 197N
Parvibaculum
lavamentivorans DS-1
Alteromonas macleodii
Bordetella parapertussis
Brucella microti CCM
Gordonia bronchialis
Sphingopyxis alaskensis
Mycobacterium
intracellulare MOTT-64
Xanthomonas oryzae pv.
oryzae MAFF 311018
Rhodopseudomonas
palustris BisB5
Mycobacterium sp. Spyr1
Caulobacter segnis ATCC
Shewanella sp. ANA-3
Shewanella putrefaciens
Rhodobacter sphaeroides
Mycobacterium ulcerans
Mycobacterium sp. MCS
Mycobacterium marinum M
Catenulispora acidiphila
Mycobacterium bovis
Pseudomonas
aeruginosa UCBPP-PA14
Methylobacterium
radiotolerans JCM 2831
Acidovorax sp. JS42
Photobacterium
profundum SS9
Phenylobacterium
zucineum HLK1
Paracoccus denitrificans
Alteromonas sp. SN2
Shewanella sp. MR-4
Streptomyces avermitilis
Rhodopseudomonas
palustris DX-1
Verminephrobacter
eiseniae EF01-2
Vibrio vulnificus MO6-
Mycobacterium avium
Brucella melitensis biovar
Rhodococcus jostii RHA1
Ochrobactrum anthropi
Erythrobacter litoralis
Actinosynnema mirum
Mycobacterium sp. KMS
Amycolicicoccus
subflavus DQS3-9A1
Mycobacterium
vanbaalenii PYR-1
Legionella pneumophila
Legionella pneumophila
Acidiphilium multivorum
Legionella pneumophila
Mycobacterium gilvum
Acidiphilium cryptum JF-5
Xanthomonas campestris
Delftia acidovorans SPH-1
Bradyrhizobium sp.
Psychrobacter arcticus
Methylobacterium
nodulans ORS 2060
Rhodobacter sphaeroides
Carnobacterium sp. 17-4
Alcanivorax borkumensis
Saccharopolyspora
erythraea NRRL 2338
Pusillimonas sp. T7-7
Shewanella piezotolerans
Tsukamurella
paurometabola DSM
Streptomyces griseus
Mycobacterium
smegmatis str. MC2 155
Jannaschia sp. CCS1
Mycobacterium
abscessus ATCC 19977
Mycobacterium
tuberculosis F11
Glaciecola sp. 4H-3-7 + YE5
Marinobacter aquaeolei
Mycobacterium sp. JLS
Pseudomonas
mendocina ymp
Ferrimonas balearica
Sinorhizobium meliloti
In the bioproduction of 3-HIB from valine, one step is the conversion of MAA-CoA to 3-hydroxyisobutyryl-CoA. This may be accomplished by a suitable enzyme, such as an enoyl-CoA hydratase.
The host microorganism may be transformed to express one or more enoyl-coA hydratase (ECH) or equivalent enzymes. For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa echA gene (Genbank Accession Number NP 249436) or an equivalent gene. Alternatively, an ECH gene selected from Table 7 may be used.
In the bioproduction of 3-HIB from valine, the final enzymatic step is the conversion of 3-hydroxyisobutyryl-CoA to 3-HIB by cleaving of the CoA from 3-hydroxyisobutyryl. This may be accomplished by a suitable enzyme, such as a thioesterase.
The host microorganism may be transformed to express one or more enzymes that creave CoA from 3-hydroxyisobutyryl-CoA. For example, the host microorganism may be transformed to express one or more thioesterase enzymes that cleave CoA from 3-hydroxyisobutyryl-CoA (HCH genes). For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa hchA gene (Genbank Accession Number NP 249435) or an equivalent gene. Alternatively, an HCH gene selected from Table 7 may be used.
Desulfosporosinus
orientis DSM 765
Pseudogulbenkiania sp.
Methylophaga sp.
Xanthomonas oryzae
Pseudoxanthomonas
spadix BD-a59
Pseudomonas stutzeri
Pseudomonas stutzeri
Pyrobaculum sp. 1860
Collimonas fungivorans
Bacillus megaterium
Xanthomonas
campestris pv. raphani
Amycolatopsis
mediterranei S699
Leptospira interrogans
Turneriella parva DSM
Shewanella baltica
Ornithobacterium
rhinotracheale DSM
Marinobacter
hydrocarbonoclasticus
Pseudomonas
aeruginosa NCGM2.S1
Vibrio furnissii NCTC
Burkholderia sp. KJ006
Shewanella baltica
Pseudomonas
aeruginosa M18
Xanthomonas
axonopodis pv.
citrumelo F1
Pseudomonas stutzeri
Amycolatopsis
mediterranei S699 V2
Shewanella baltica
Flavobacterium indicum
Marinobacter
adhaerens HP15
Burkholderia cepacia
Stenotrophomonas
maltophilia JV3
Pseudomonas stutzeri
Bacillus cereus F837/76
Stenotrophomonas
maltophilia D457
Burkholderia
pseudomallei 1026b
Owenweeksia
hongkongensis DSM
Leptospirillum
ferrooxidans C2-3
Oceanimonas sp. GK1
Shewanella
putrefaciens 200
Aeromonas salmonicida
Shewanella woodyi
Shewanella
denitrificans OS217
Vibrio vulnificus YJ016
Vibrio sp. Ex25
Shewanella oneidensis
Shewanella halifaxensis
Planctomyces
brasiliensis DSM 5305
Sorangium cellulosum
Cupriavidus
taiwanensis LMG
Pseudomonas
mendocina NK-01
Laribacter
hongkongensis HLHK9
Croceibacter atlanticus
Sideroxydans
lithotrophicus ES-1
Pedobacter saltans
Vibrio vulnificus
Aeromonas hydrophila
Thioalkalivibrio
sulfidophilus HL-EbGr7
Shewanella loihica PV-4
Shewanella baltica
Pseudomonas stutzeri
Ralstonia pickettii 12D
Vibrio parahaemolyticus
Xanthomonas
campestris pv.
campestris str. 8004
Xylella fastidiosa
Burkholderia
pseudomallei 1106a
Leptospira interrogans
Polaromonas sp. JS666
Bacillus cereus E33L
Geobacillus
thermodenitrificans
Burkholderia glumae
Bacillus thuringiensis
Fluviicola taffensis DSM
Pseudomonas fulva 12-X
Burkholderia
pseudomallei 668
Burkholderia gladioli
Azotobacter vinelandii
Psychrobacter sp.
Pseudoalteromonas sp.
Ralstonia eutropha
Shewanella sediminis
Pseudomonas
aeruginosa DK2
Xylella fastidiosa M12
Frankla alnl ACN14a
Burkholderia phymatum
Shewanella sp. MR-7
Hahella chejuensis
Shewanella violacea
Burkholderia sp.
Robiginitalea biformata
Xanthomonas oryzae
Thiobacillus
denitrificans ATCC
Xanthomonas
axonopodis pv. citri str.
Burkholderia ambifaria
Burkholderia
pseudomallei 1710b
Shewanella
frigidimarina NCIMB400
Shewanella baltica
Stenotrophomonas
maltophilia K279a
Vibrio harveyi ATCC
Ruminococcus albus 7
Burkholderia
xenovorans LB400
Burkholderia
multivorans ATCC
Colwellia
psychrerythraea 34H
Maribacter sp.
Psychrobacter
cryohalolentis K5
Ralstonia eutropha H16
Pseudomonas
aeruginosa PA7
Bacillus atrophaeus
Aeromonas veronii
Xanthomonas
campestris pv.
campestris str. ATCC
Burkholderia
cenocepacia J2315
Shewanella baltica
Xanthomonas
campestris pv.
vesicatoria str. 85-10
Burkholderia sp. 383
Bacillus pseudofirmus
Burkholderia
multivorans ATCC
Sulfurovum sp. NBC37-1
Polynucleobacter
necessarius subsp.
necessarius STIR1
Pseudoalteromonas
haloplanktis TAC125
Xylella fastidiosa subsp.
fastidiosa GB514
Stenotrophomonas
maltophilia R551-3
Shewanella sp. W3-18-1
Chromobacterium
violaceum ATCC 12472
Shewanella baltica
Vibrio sp. EJY3
Bacillus megaterium
Herbaspirillum
seropedicae SmR1
Shewanella pealeana
Bacillus cereus
Ralstonia pickettii 12J
Burkholderia
vietnamiensis G4
Mycobacterium
smegmatis str. MC2
Shewanella
amazonensis SB2B
Bacillus cellulosilyticus
Xanthomonas oryzae
Burkholderia
thailandensis E264
Pseudomonas
aeruginosa LESB58
Xanthomonas oryzae
Burkholderia
phytofirmans PsJN
Shewanella sp. ANA-3
Shewanella
putrefaciens CN-32
Burkholderia
pseudomallei K96243
Cupriavidus necator N-1
Burkholderia
cenocepacia AU 1054
Pseudomonas
aeruginosa UCBPP-
Bacillus megaterium
Photobacterium
profundum SS9
Burkholderia
cenocepacia MC0-3
Burkholderia sp.
Burkholderia
cenocepacia HI2424
Xylella fastidiosa M23
Shewanella sp. MR-4
Amycolatopsis
mediterranei U32
Vibrio vulnificus MO6-
Sulfurimonas
autotrophica DSM
Acaryochloris marina
Leptospira interrogans
Leptospira biflexa
Methylotenera sp. 301
Burkholderia ambifaria
Lysinibacills
sphaericus C3-41
Burkholderia sp.
Xanthomonas
campestris pv.
campestris str. B100
Psychrobacter arcticus
Xylella fastidiosa 9a5c
Shewanella
piezotolerans WP3
Mycobacterium
smegmatis str. MC2
Brevibacillus brevis
Herpetosiphon
aurantiacus ATCC
Leptospira biflexa
Marinobacter aquaeolei
Pseudomonas
mendocina ymp
Fenimonas balearica
Sphingobacterium sp.
Enzymatic Capability 6 comprises the ability to convert MAA-CoA to MMA by cleavage of the CoA from MAA-CoA. Such activity may be imparted by an alcohol acyl transferase (AAT) enzyme. In one embodiment, the AAT enzyme is coded by the Malus pumila AAT gene. In one embodiment, the host microorganism is yeast. In another embodiment, the host microorganism is yeast and the AAT gene is a Malus pumila AAT gene codon-optimized for expression in yeast, for example the AAT gene comprising SEQ ID No: 2. Alternatively, an AAT gene selected from Table 9 may be used.
In the invention, examples of the origin of the above enzymes (genes encoding enzymes) include genus Pseudomonas, genus Bacillus, genus Sphingobacterium, genus Comamonas, genus Brevundimonas, genus Sphingomonas, genus Ochrobactrum, genus Pedobacter, genus Paenibacillus, genus Achromobacter, genus Acinetobacter, genus Shewanella, genus Listonella, genus Agrobacterium, genus Mesorhizobium, genus Rhizobium, genus Paracoccus, genus Xanthobacter, genus Streptomyces, genus Geobacillus, genus Rhodococcus, genus Saccharomyces, genus Candida or genus Aspergillus. Of these, genus Pseudomonas and genus Rhodococcus microorganisms are preferable.
Examples of the microorganism classified into genus Pseudomonas include Pseudomonas aeruginosa, Pseudomonas agarici, Pseudomonas alcaligenes, Pseudomonas amygdale, Pseudomonas anguiliseptica, Pseudomonas antimicrobica, Pseudomonas aspleni, Pseudomonas aurantiaca, Pseudomonas aureofaciens, Pseudomonas avellanae, Pseudomonas azotoformans, Pseudomonas balearica, Pseudomonas beijerinckii, Pseudomonas beteli, Pseudomonas boreopolis, Pseudomonas carboxyhydrogena, Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonas cissicola, Pseudomonas citronellolis, Pseudomonas coronafaciens, Pseudomonas corrugate, Pseudomonas doudoroffii, Pseudomonas echinoids, Pseudomonas elongate, Pseudomonas ficuserectae, Pseudomonas flavescens, Pseudomonas flectens, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas fulva, Pseudomonas fuscovaginae, Pseudomonas gelidicola, Pseudomonas geniculata, Pseudomonas glathei, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas huttiensis, Pseudomonas iners, Pseudomonas lancelota, Pseudomonas lemoignei, Pseudomonas lundensis, Pseudomonas luteola, Pseudomonas marginalis, Pseudomonas meliae, Pseudomonas mendocina, Pseudomonas mucidolens, Pseudomonas monteilli, Pseudomonas nautica, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas oryzihabitans, Pseudomonas pertucinogena, Pseudomonas phenazinium, Pseudomonas pictorum, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas pyrrocinia, Pseudomonas resinovorans, Pseudomonas rhodesiae, Pseudomonas saccharophila, Pseudomonas savastanoi, Pseudomonas spinosa, Pseudomonas stanieri, Pseudomonas straminae, Pseudomonas stutzeri, Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas syzygii, Pseudomonas taetrolens, Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas viridiflava, Pseudomonas vulgaris and Pseudomonas wisconsinensis.
Examples of the microorganism classified into genus Rhodococcus include Rhodococcus rhodochrous, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus opacus, Rhodococcus jostii, Rhodococcus pyridinovorans, Rhodococcus rhodnii, Rhodococcus corallinus, Rhodococcus rubropertinctus, Rhodococcus coprophilus, Rhodococcus globerulus, Rhodococcus chlorophenolicus, Rhodococcus luteus, Rhodococcus aichiensis, Rhodococcus chubuensis, Rhodococcus maris and Rhodococcus fascians.
For the genes encoding the enzyme, RNA is extracted from the above microorganism cell in accordance with a routine method, a primer is designed based on amino acid sequences and gene sequences of the above enzymes (the microorganism or related species thereto) disclosed in the public databases such as NCBI, and PCR can be carried out using the primer to thereby isolate and amplify the gene encoding the enzyme of interest.
The invention encompasses an engineered microorganism capable of producing MAA-CoA from valine. Such an engineered microorganism of the invention comprises the microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, and Enzymatic Capability 3, as described above.
The enzyme may be produced in the cytosol or may be targeted to any other cellular compartment. In one embodiment, the enzyme introduced by transformation and expressed by the MAA-CoA producing microorganism is targeted to the mitochondria. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 presequence. In one embodiment, the engineered microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae.
In one embodiment, the engineered microorganism has been transformed to express one or more acyl-CoA dehydrogenases (isobutyryl-CoA dehydrogenase). For example, the MAA-CoA producing microorganism is transformed to express the Pseudomonas aeruginosa ACD1 gene. In another embodiment, the MAA-coA producing microorganism is transformed to express one or more enzymes which catalyze the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. For example, the MAA-CoA producing microorganism is transformed to express one, some, or all of the components of the BCKAD complex. In yet another embodiment, the MAA-CoA producing microorganism of the invention is transformed to express one or more enzymes which catalyze the formation of 2-oxoisovalerate from valine, for example the BCAT1 or BCAT2 genes from yeast.
The scope of the invention encompasses host microorganisms which have been engineered to produce (S)-3-hydroxyisobutyric acid (3-HIB) from valine. 3-HIB is an MMA precursor which can be readily converted to MMA utilizing various methods known in the art.
The 3-HIB-producing microorganism of the invention is a microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, Enzymatic Capability 3, Enzymatic Capability 4, and Enzymatic Capability 5.
In one embodiment, the 3-HIB-producing microorganism of the invention is a host cell that has been transformed to express an ECH gene, for example, the echA gene from Pseudomonas aeruginosa. In another embodiment, the 3-HIB-producing microorganism of the invention is a host cell that has been transformed to express one or more HCH enzymes that cleave CoA from 3-hydroxyisobutyryl-CoA. For example the 3-HIB producing microorganism may comprise an microorganism which expresses the hchA gene from Pseudomonas aeruginosa. In one embodiment, the engineered microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae. In one embodiment, the enzyme introduced by transformation and expressed by MAA-CoA producing microorganism is targeted to the mitochondria. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 matrix-targeting presequence.
In one embodiment, the engineered microorganism of the invention is capable of producing MMA. An MMA-producing microorganisms of the invention comprises an microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, Enzymatic Capability 3, and Enzymatic Capability 6. In one embodiment, the MMA-producing microorganism of the invention has been transformed to express an alcohol acyl transferase (AAT), for example the AAT gene of Malus pumila. In one embodiment, the AAT gene is encoded by SEQ 1D NO: 2. In one embodiment, the MMA-producing microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 presequence.
In addition to the various combinations of Enzymatic Capabilities described above, the engineered microorganisms of the invention may further comprise genetic modifications to promote MMA end-product formation. For example, the starting material for bioproduction of MMA end-product is valine. Accordingly, in one embodiment, the microorganisms of the invention are engineered to express one or more enzymes which enhance valine formation over wild-type levels. Various genetic modifications are known in the art for enhancing valine formation in various microorganisms, for example as described in Literature [Wada et al., 2008, Enhanced Valine Production in Corynebacterium glutamicum with Defective H+-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase, Bioscience, Biotechnology, and Biochemistry 72 (11):2959-65, 2008; Hasegawa et al., 2013, Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions, Appl Environ Microbiol, 79(4):1250-7; and Park et al., 2007, Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation, PNAS 104 (19) 7799-7802]. The enzyme or regulatory gene inserted to a microorganism to enhance the formation of valine may be under the control of a constitutive promoter or inducible promoter.
Under some culture conditions, such as highly aerated yeast cultures, biosynthetic pathways, such as Erlich pathway reactions, compete with MMA end-product formation. In some embodiments, the engineered microorganism of the invention is further engineered to downregulate or obliterate competing biosynthetic pathway. For example, in one embodiment, the bioengineered microorganisms of the invention is engineered to express miRNA or other sequences that downregulate or otherwise target enzymes which compete with any of Enzymatic Capabilities 1 to 6. For example, in one embodiment, the microorganism of the invention is engineered with an enzymatic or a regulatory gene which reduces Erlich pathway reaction. In another embodiment, the enzymatic or regulatory gene co-introduced into the host species comprise genes which inhibit valine catabolism, for example, the reaction which forms succinyl Co-A from 3-HIB Co-A. Such gene may be placed under the control of an inducible promoter, allowing downregulation to be induced after cultures have been propagated and are being switched to bioproduction mode.
In another embodiment, the engineered microorganism of the invention is further engineered to express an enzyme or a regulatory sequence which enhances the regeneration of co-factor such as NADH or FADH. For example, in one embodiment, the engineered microorganism of the invention is transformed to express a high level of alcohol dehydrogenase, in order to promote NADH formation from NADPH, which such cofactor is necessary for the action of the BCKAD complex. Likewise, genetic modifications may be introduced that increase the rate of regeneration of FAD from FADH2, a cofactor in the action of ACD. Exemplary cofactor manipulation technologies are described in Literatures [Wang et al., “Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl,” Sci Rep. 2013; 3:2643. doi: 10.1 038/srep02643; Nikel et al., “Elimination of D-lactate synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli,” Appl Environ Microbiol. 2010 November; 76 (22):7400-6; Tseng and Prather, “Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways,” Proc Natl Acad Sci USA. 2012 Oct. 30; 109(44):17925-30; and Lopez de Felipe et al., “Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus/actis by Controlled Expression of NADH Oxidase,” J Bacteriol. 1998 August; 180(15): 3804-3808].
The scope of the invention encompasses the engineered microorganism described above, and further encompasses a method of using such an engineered microorganism to produce the end-products such as MMA.
The engineered microorganisms are cultured so as to propagate themselves and to produce the methacrylic acid esters or methacrylic acid ester precursors end-products. The engineered microorganisms may be cultured under continuous culture conditions for the sustained growth of cultures and simultaneous harvest of end-products. Alternatively, the microorganisms may be cultured in batches wherein discreet cultures (e.g. in a single vessel or bioreactor) are used to form end products, and then are processed to recover the end-products.
In some embodiments, the culture of the engineered microorganisms is staged, with a first growth and/or propagation stage followed by a bioproduction stage. The various stages may be implemented by altering culture conditions, wherein the initial growth and propagation stage is fostered by the use of media and/or culture conditions that favor rapid propagation and growth of the engineered microorganism, followed by a change in culture conditions to favor MMA end-product formation.
For example, in one embodiment, the engineered microorganism of the invention is a yeast and is first grown under culture conditions that favor rapid propagation and growth of yeast cultures, for example well aerated conditions with high levels of nutrients in the growth media. This growth stage is followed by a bioproduction stage wherein the cultures are not highly aerated or are not aerated at all and the culture medium lacks one or more nutrients (e.g. micronutrients, or carbon sources that promote growth).
Each condition such as culture temperature or culture time is suitably determined without particular limitation depending on raw materials, microorganisms to be used, and end-products of interest, but the reaction may be usually carried out at 5 to 80° C. for 1 minute to 1 week. The reaction is preferably carried out at 10 to 70° C. for 1 minute to 120 hours, with 10 minutes or more being more preferable. The conditions under which the reaction quenches are preferably selected from these conditions. The pH of the reaction solution is also not particularly limited as long as the reaction proceeds effectively and, for example, pH ranges from 4 to 10, with pH 5.5 to 8.5 being preferable.
For the purpose of effectively progressing the reaction, the culture can also be carried out in a system to which an organic solvent is added in advance. For the organic solvent, for example, linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbons, or saturated or unsaturated aromatic hydrocarbons can be used singly or in combinations of two or more. Specific examples include hydrocarbon solvents (e.g., pentane, hexane, cyclohexane, benzene, toluene and xylene), halogenated hydrocarbon solvents (e.g., methylene chlorides and chloroform), ether solvents (e.g., diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, t-butyl methyl ether and dimethoxyethane) and ester solvents (e.g., methyl formate, methyl acetate, ethyl acetate, butyl acetate and methyl propionate).
In one embodiment, following the growth and propagation stage, the cells are collected and placed in culture vessels having conditions favoring higher bioproduction. For example, yeast cells may be isolated from growth and propagation cultures by centrifugation, followed by rinsing in buffer and resuspension in new culture medium.
In microorganisms wherein one or more of the proteins comprising the Enzymatic Capabilities is under the control of an inducible promoter, the inducing agent may be introduced near or at the beginning of the bioproduction stage. Likewise, in microorganisms which have been engineered for decreased or inhibited activity of metabolic pathways that compete with or otherwise reduce MMA end-product formation, and where such introduced genes are under the control of inducible promoters. The cultured cells may be exposed to the inducing agent at the transition from the growth and propagation stage to the bioproduction stage.
In some embodiments, valine is added to the culture medium to enhance formation of MMA end-products from valine. In another embodiment, valine is added to the culture medium at the transition to a bioproduction culture stage. In another embodiment, valine precursors are included in the growth media to improve valine formation and drive increased rates of MMA end-product formation. Likewise, in microorganisms which have been engineered for increased valine formation by the introduction of genes and where such genes are under the control of inducible promoters, the inducing agent may be introduced at or near the beginning of the bioproduciton stage to increase the amount of precursors for end-product formation. In some embodiments, the engineered microorganisms of the invention are co-cultured with other strains or microorganisms which produce valine, in order to increase the concentration of valine in the culture medium.
During culture, in the case of continuous production systems, or at the completion of the bioproduction stage, in the case of batch cultures, the end-products are recovered from the culture. This may be accomplished by any means. For example in one embodiment, enzymatic and/or physical treatments are applied to lyse cells and to liberate the end-products. The MMA end-products may be isolated from cell cultures by centrifugation, distillation, column separation, chromatography, and other means known in the art. For example, methodologies described in U.S. Pat. No. 8,907,121, by Johnson and Morris, entitled “Methyl methacrylate purification process,” may be employed.
In the case of methacrylic acid ester precursors, the precursor recovered from cultures may be further treated to convert them to a methacrylic acid esters. 3-HIB may be treated enzymatically to convert it to MMA. For example, the dehydration of 3-HIB to MMA may be achieved, for example as described in Literature [Nagai, “New Developments in the Production of Methyl Methacrylate,” Applied Catalysis A: General 221 (2001) 367-377].
The method of the invention can also be used for the production of methacrylic acid esters or precursors thereof from a biomass. A transformant having introduced thereinto the gene encoding an acyl-CoA dehydratase, as well as an enzyme gene group allowing for synthesizing methacrylic acid esters or precursors thereof of interest from a biomass can be used to directly synthesize methacrylic acids or methacrylic acid esters from the biomass in a metabolic engineering (fermentation) approach.
The additional details in regard to the experiment methods described in the invention and Example 1 are presented in
Genes were targeted for expression to the cytoplasm by expressing the heterologous gene, or targeted to the mitochondria by appending the first 69 residues of subunit 9 of the yeast mitochondrial ATPase (Su9) to each gene as encoded by SEQ ID NO: 1.
Expression and correct targeting of these genes was then validated by constructing a C-terminal GFP fusion which was expressed from a GAL1 promoter within the plasmid pYES (leader peptide, plasmid, GAL1 promoter, CYCT terminator and GFP, all provided from the paper: Westermann, B. and Neupert, W. (2000). Yeast 16: 1421-1427). These constructs were transformed in Saccharomyces cerevisiae (CKY263) and grown overnight in glucose supplemented synthetic defined media lacking uracil (SD-CAA). At 24 hour, the cultures were diluted 100 fold and expression induced by growing them in galactose supplemented SO-CAA for 24 hours. The cells were harvested and stained with a red fluorescent mitochondria-selective dye (Mito-IO Red detection kit-Cat# ENZ-51007-500, Enzo Life Sciences, Ann Arbor, Mich.). Fluorescence was then detected using confocal microscopy-only cells with non-diffuse green fluorescence which overlaps with the red dye target gene expression to the mitochondria.
Activity of the first two enzymatic steps (BCKAO and ACO) were confirmed with in vitro assays of crude lysates from cells that expressed GFP-free constructs. MMA enzymes expression cassettes (BCKAO, ACO, ECH, HCH) targeted to either the mitochondria (mtMMA) or cytoplasm (MMA) were cloned from the previous experiment into the pRS series of vectors (pBCKA04-pRS315 backbone (LEU) with bkdA1, bkdA2, bkdB, JpdV; pAC01-pRS316 backbone (URA) with acd1; and pCoA2-pRS314 backbone (TRP) with echA and hchA). These plasmids were transformed into the vacuolar protease-deficient Saccharomyces cerevisiae strain BJ5464. After a 48-hour induction period in galactose supplemented SO-CAA lacking uracil, leucine and tryptophan, cells were harvested, physically disrupted in Tris buffer and the lysate separated by centrifugation. The lysates were then assayed with real-time enzymatic activity assays as depicted below:
100 mM Potassium phosphate pH 8.0
0.4 mM Flavin adenine dinucleotide (FAD)
1.6 mM Phenazine methosulphate (PMS)
Room temperature incubation
Active ACD reduces DCPIP and A600
100 mM Potassium phosphate pH 7.0
0.2 mM Thiamin pyrophosphate (TPP)
4 mM 2-Oxoisovaleric acid
Room temperature incubation
Active BCKAD reduces NAD+ and increases A340
Productivity from the (mt)MMA pathway was assessed using high-pressure liquid chromatography. BJ5464 cells containing mitochondrial or cytoplasmic variants of the pathway were grown overnight under non-inducing conditions. These cultures were then diluted about 100 fold in inducing SD-CAA media and grown for 48 to 72 hours. Supernatant from these cultures were then analyzed on an Agilent 1100 series HPLC with an ICsep USP L-17 using a 0.01 N H2SO4 mobile phase. 3-HIB product was detected at 210 nm with a DAD detector and quantified via a standard curve. These studies suggest production of g/L titers of 3-HIB (5.1±2.9 g/L) in 72 hours with the mitochondrial pathway variant.
All patents, patent applications, and publications cited in this specification are herein incorporated by reference in their entirety to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.
The invention is useful for synthesizing methacrylic acid esters such as MMA to begin with and acrylic resins, which are polymers thereof.
The present application claims priority based on U.S. Provisional Application No. 62/245,980 (filed on Oct. 23, 2015), and the content thereof is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62245980 | Oct 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2016/081345 | Oct 2016 | US |
| Child | 15958859 | US |
