US 2013 03375.11A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0337511 A1 Gak et al. (43) Pub. Date: Dec. 19, 2013
(54) METHOD FOR PRODUCING (30) Foreign Application Priority Data PYRROLOQUINOLINE QUINONE USINGA BACTERIUM OF THE GENUS Mar. 3, 2011 (RU) ...... 2O11108,196 METHYLOBACTERIUM OR HYPHOMICROBUM Publication Classification (71) Applicant: AJINOMOTO CO., INC., Tokyo (JP) (51) Int. Cl. (72) Inventors: Evgeniy Rodionovich Gak. Moscow CI2P 17/18 (2006.01) (RU); Natalya Vasilievna Gorshkova, (52) U.S. Cl.
Moscow (RU); Irina L'vovna CPC ...... CI2P 17/182 (2013.01) Tokmakova, Moscow region (RU) USPC ...... 435/119 (73) Assignee: AJINOMOTO CO., INC., Tokyo (JP) (21) Appl. No.: 14/010,101 (57) ABSTRACT (22) Filed: Aug. 26, 2013 The present invention provides a method for producing PQQ O O using a bacterium belonging to the genera Methylobacterium Related U.S. Application Data or Hyphomicrobium, which has been modified to enhance the (63) Continuation of application No. PCT/JP2012/055727, expression of pcq gene cluster and/or gene(s) encoding a filed on Feb. 28, 2012. precursor for PQQ biosynthesis. Patent Application Publication Dec. 19, 2013 Sheet 1 of 10 US 2013/03375.11 A1
Fig. 1
Patent Application Publication Dec. 19, 2013 Sheet 2 of 10 US 2013/03375.11 A1
Fig. 2
Q488A 4 ------MWKKFEEMRSFEWLY ESNR 4E O ------CWTKPTFIDMRISLEVFLYISNR Q4KEK3 ------MWKBAYE) RESFEWM 4KAV2 TWSKATERFEW \ 8SB KWWAPAYCOLRECSFEWAYVR E2EVO AWOKPEATEDT, RF gFE,TEMY FANR BYSS. MVLAMTPRAPAPT.H PHQHTL.ASLTRTANPIEGEL.MTWTTPAACDF RFGFEITMYIAAR E234 ------MNWTPAYTELREGFEMANR Q608 PA ------RWEKSYNEMREFEMENR C&WTXO. AATEMRFGFEWNYWWNK LEDHPO ------MWAAERFGFEWWINK R. GX88 G.Y.GNHGRGTCAASEHKRREEMWKBESTEMRFGFEMWCNR 491. 28 ------vK-WAAPWSECWGIwWSYESAE 3y ------K-WAAPWSECGIEWSESA, ELW85 ------vK-WAARWSECWGMEWSESAE C5AY 4 ------K-WSAPWAECGIESESA, CRCK2 ------AK-WSAPWAEICWGMEWSESAE {5AY5 ------vK-WSAPWVAECWGMEWSYESAE D8JXU ------MESSYRFGGTTMKIWTKPAVREQEWGLEVISYLPAE D8JQF5 ------IMKTWTKPAVREOEWGLEW; SYLPAE AAYZ.3 ------A-WKAPKWEWPWGIENNIAAA AAYZ28 ------A-WKTEPKIWEWPWGMENMACAA AAYNW ------MS-WAPK WEWPWGMEENMYACAS Q9I3B4 ------A-WTPKWTEIPGAESWCGE A8LN54 ------AA-WKPREECGMEENMYGPS k k s * : . *k
488 A 4 ------AFEO ------AKEK3 ------AK4 V2 ------O8 SE35 ------B2 EVO ------Y S ------B239A ------6884 ------6WXO ------Di ------3. GX88 ------49.48 E-- - --
3.85
CK2 CEAQYS D8JQF5 - - - -- AAYZY 3 RK------AAY228 RK------A. YNW. RKAERRS 9,334 KK------Patent Application Publication Dec. 19, 2013 Sheet 3 of 10 US 2013/03375.11 A1
Fig. 3
Psti (1445) - poq
pHT1 pgqB A 26405bp Y-mxbM
- BamHI (8817) Patent Application Publication Dec. 19, 2013 Sheet 4 of 10 US 2013/03375.11 A1
Fig. 4
\ Pst (805) K PrxfaF f / Bamh (1197) pCM11.0 Ecli36 (1205)
tetAs Patent Application Publication Dec. 19, 2013 Sheet 5 of 10 US 2013/03375.11 A1
Fig. 5
pCMpmxafABCDE 12005 bp
Patent Application Publication Dec. 19, 2013 Sheet 6 of 10 US 2013/03375.11 A1
Fig. 6
| FpqqA2A3
| Prmxaf
v.| . pqGA N -/ pcqB Patent Application Publication Dec. 19, 2013 Sheet 7 of 10 US 2013/03375.11 A1
N. v X. x
------x - pdcA3
pqcE pagD, EG574 pagA2, EG570 | ------paqA2DE
D fpqqC EG573 - pogo r EG572 pcqE r. paqB pqGA - Ie - II- - pcqA2BCDE Patent Application Publication Dec. 19, 2013 Sheet 8 of 10 US 2013/03375.11 A1
Fig. 8
a's oriV ress Psti (805) / s- K ^ Prmxfar | a pCM11.0 yerBamHt (1197)(1205) A - l 5834 bp
------pqq) is paqC spacA tetA EGS73- i pogoE ^,\ , fpqqB 'paqA EG572 ------paqABCDEHypho
Patent Application Publication Dec. 19, 2013 Sheet 9 of 10 US 2013/03375.11 A1
Fig. 9
Patent Application Publication Dec. 19, 2013 Sheet 10 of 10 US 2013/03375.11 A1
Fig. 10
- SwapoqA3 tetA, pHABCDEA3A2. TpcqA2 Bamh r W pqqA tetr N pqcB { Y-- . --- - Sac-paqe vis. k - pcq) US 2013/03375 11 A1 Dec. 19, 2013
METHOD FOR PRODUCING with the radical SAM enzyme PaqE has been demonstrated in PYRROLOQUINOLINE QUINONE USINGA K. pneumoniae (Wecksler S R et al. Chem Commun 2010 BACTERIUM OF THE GENUS Oct. 7:46(37):7031-3). METHYLOBACTERIUM OR 0009 Based on sequence analysis and homology models, HYPHOMICROBUM it is Supposed that PaqE recognizes the PdqA protein and forms a bond between the C atoms from the glutammic acid 0001. This application is a Continuation of, and claims and tyrosine in Paq.A., and, therefore, enables recognition of priority under 35 U.S.C. S 120 to, International Application the modified Paq.A by PaqF protein. In the next stage, PaqF is No. PCT/JP2012/055727, filed Feb. 28, 2012, and claims Suggested to catalyze cutting of the generated glutammic priority therethrough under 35 U.S.C. S 119 to Russian Patent acid-tyrosine pair out of Paq.A protein (Puehringeretal. BMC Application No. 2011108196, filed Mar. 3, 2011, the entire Biochemistry 2008, 9:8 doi:10.1186/1471-2091-9-8). ties of which are incorporated by reference herein. Also, the 0010. It was revealed that PQQ biosynthesis in Escheri Sequence Listing filed electronically herewith is hereby chia coli, which does not possess an ability to produce PQQ. incorporated by reference (File name: 2013-08-26. US-460 can be achieved through the expression of pcq gene clusters Seq List: File size: 68 KB: Date recorded: Aug. 26, 2013). of A. calcoaceticus (Goosen N. et al. J Bacteriol (1989) 171: 447-455), K. pneumoniae (Meulenberg J J M et al. FEMS BACKGROUND OF THE INVENTION Microbiol Lett (1990) 71:337-344), and G. Oxydans (Yang et 0002 1. Field of the Invention al. Journal of Industrial Microbiology&Biotechnology 0003. The present invention relates to the microbiological (2010), 37(6), 575-580). Also, the positive effect of copies of industry, and specifically to a method for producing pyrrolo Some paq genes on PQQ production in Methylobacterium quinoline quinine (POO) using a bacterium of the genus extorquens AM1 was described (Wu, Bo: Zhao, Yong-fang; Methylobacterium or Hyphomicrobium which has been Wang, Yin-shan. Wuhan Daxue Xuebao, Ziran Kexueban modified to enhance the expression of the paq gene cluster (1999) 45(6), 869-872). Deletion of mxbM gene and and/or gene(s) encoding a precursor for POO biosynthesis. pqqABC/DE gene cluster in M. extorquens AM1 led to absence of PQQproduction. The deletion mutant transformed 0004 2. Brief Description of the Related Art by a plasmid harboring mXbM gene and pdqABC/DE gene 0005 Pyrroloquinoline quinine (PQQ) is the cofactor for cluster produced PQQ in larger amounts than the wild-type, several bacterial dehydrogenases including glucose dehydro presumably as a result of the higher copy number of pdq genase and methanol dehydrogenase located in the periplasm genes. (Toyama H. and Lidstorm M. E. Microbiology (1998), of Gram-negative bacteria and may be accumulated extracel 144, 183-191). lularly by cultivating these bacteria. All of the PQQ-produc 0011. But, currently, there have been no reports of enhanc ing strains have PQQ-dependent quinoproteins. ing expression of the paq gene cluster in a bacterium, belong 0006. A method for the preparation of PQQ using bacteria ing to the genus Hyphomicrobium, and enhancing expression belonging to the genera Achronobacter; Methylobacillus, of the additional pdqA gene(s) encoding a precursor for PQQ Methylomonas, Methanomonas, Protaminobacter, Methylo biosynthesis in a bacterium of the genera Methylobacterium bacterium, Protomonas, Mycoplana, Ancyclobacter; Micro or Hyphomicrobium for the purpose of producing PQQ. cyclus, Hyphomicrobium, Xanthobacter. Thiobacillus, Alteromonas, Methylophaga and some species of the genus SUMMARY OF THE INVENTION Pseudomonas, which are cultivated in a medium containing methanol and/or methylamine as a carbon Source, is disclosed 0012 Aspects of the present invention include providing methods for producing PQQ using a bacterium belonging to (EP0206471 B1). the genera Methylobacterium or Hyphomicrobium. 0007 Genes involved in PQQ biosynthesis have been characterized for several bacteria, including Klebsiella pneu 0013 The above aspects were achieved by finding that moniae, Acinetobacter calcoaceticus, Methylobacterium enhancing expression of the pdq gene cluster and gene(s) extorquens, and Gluconobacter Oxydans. Six genes and seven encoding PaqA homologous proteins can enhance produc genes are required in K. pneumoniae and M. extorquens tion of PQQ. (AM1), respectively, and only four genes are required in A. 0014. It is an aspect of the present invention to provide a calcoaceticus for PQQ biosynthesis. The pdqA genes from method for producing PQQ comprising cultivating a bacte different species encode small peptides of 23 to 29 amino rium belonging to the genus Hyphomicrobium, and collecting acids which contain conserved glutamic acid and tyrosine PQQ from the culture medium, wherein the bacterium has residues. PQQ is derived from the two amino acidsglutammic been modified to enhance expression of pdq gene cluster. acid and tyrosine encoded in the precursor peptide Paq.A. 0015. It is a further aspect of the present invention to Presumably, five reactions are necessary to form PQQ (Pue provide the method as described above, wherein said pqq hiringer et al. BMC Biochemistry 2008, 9:8 doi:10.11867 gene cluster is the pdqABC/DE operon from Methylobacte 1471-2091-9-8). Some of the proteins involved in PQQ bio rium extorquens. synthesis have been functionally characterized. 0016. It is a further aspect of the present invention to 0008. The PaqB protein is supposed to be involved in provide the method as described above, wherein said transport of PQQ into the periplasm. It has been reported that pqqABC/DE operon comprises DNA fragments of SEQ ID a knock out of PaqB produces small amounts of PQQ in the NO: 1, SEQID NO:3, SEQID NO:5, and SEQID NO: 7, or cytosol and that no PQQ is secreted into the periplasm (Velt variants thereof. erop et al. Journal of bacteriology (1995) 177(17):5088 0017. It is a further aspect of the present invention to 5098). The PaqC protein is an oxidase which catalyzes the provide the method as described above, wherein said pqq final step in PQQ formation. The functions of Paq.D protein gene cluster is the pdqABCDE cluster from Hyphomicrobium are still unknown. Recently, the interaction of Paq) protein denitrificans. US 2013/03375 11 A1 Dec. 19, 2013
0018. It is a further aspect of the present invention to 0034 a DNA fragment that encodes a protein comprising provide the method as described above, wherein said pqqAB the amino acid sequence of SEQID NO: 13, but that contains CDE cluster comprises DNA fragments of SEQID NO: 14, Substitutions, deletions, insertions, additions or inversions of SEQID NO: 16, SEQID NO: 18, SEQID NO: 20, and SEQ one or several amino acid residues, and said protein has the ID NO: 22, or variants thereof. function of a PQQ precursor: 0019. It is a further aspect of the present invention to 0035 a DNA fragment that encodes a protein comprising provide the method as described above, wherein the expres the amino acid sequence of SEQID No. 15: sion of said gene cluster is enhanced by increasing said gene 0036 a DNA fragment that encodes a protein comprising cluster copy number. the amino acid sequence of SEQID NO: 15, but that contains 0020. It is a further aspect of the present invention to Substitutions, deletions, insertions, additions or inversions of provide the method as described above, wherein the increas one or several amino acid residues, and said protein has the ing said gene cluster copy number is achieved by introducing function of a PQQ precursor: into the bacterium a vector containing said gene cluster. 0037 a DNA fragment that encodes a protein comprising 0021. It is a further aspect of the present invention to the amino acid sequence of SEQID No. 26: provide the method as described above, wherein said bacte 0038 a DNA fragment that encodes a protein comprising rium is Hyphomicrobium denitrificans. the amino acid sequence of SEQID NO: 26, but that contains 0022. It is a further aspect of the present invention to Substitutions, deletions, insertions, additions or inversions of provide the method as described above, wherein said culture one or several amino acid residues, and said protein has the medium contains methanol as a carbon source. function of a PQQ precursor: 0023. It is an aspect of the present invention to provide a 0039 a DNA fragment that encodes a protein comprising method for producing PQQ comprising cultivating a bacte the amino acid sequence of SEQID No. 28; rium belonging to the genera Methylobacterium or Hyphomi 0040 a DNA fragment that encodes a protein comprising crobium, having enhanced expression of pcq gene cluster, the amino acid sequence of SEQID NO: 28, but that contains and collecting PQQ from the culture medium, wherein said Substitutions, deletions, insertions, additions or inversions of bacterium has been further modified to enhance expression one or several amino acid residues, and said protein has the pqqA-like gene(s). function of a PQQ precursor; and 0024. It is a further aspect of the present invention to 0041 combinations thereof. provide the method as described above, wherein said pqq 0042. It is a further aspect of the present invention to gene cluster is the pdqABC/DE operon from Methylobacte provide the method as described above, wherein the expres rium extorquens. sion of said gene cluster or gene(s) is/are enhanced by 0025. It is a further aspect of the present invention to increasing said gene cluster or gene(s) copy number. provide the method as described above, wherein said 0043. It is a further aspect of the present invention to pqqABC/DE operon comprises DNA fragments of SEQ ID provide the method as described above, wherein the increas NO: 1, SEQID NO:3, SEQID NO:5, and SEQID NO: 7, or ing said gene cluster or gene(s) copy number is/are achieved variants thereof. by introducing into the bacterium a vector containing said 0026. It is a further aspect of the present invention to gene cluster or gene(s) copy number. provide the method as described above, wherein said pqq 0044. It is a further aspect of the present invention to gene cluster is the pdqABCDE cluster from Hyphomicrobium provide the method as described above, wherein said bacte denitrificans. rium is Hyphomicrobium denitrificans. 0027. It is a further aspect of the present invention to 0045. It is a further aspect of the present invention to provide the method as described above, wherein said pqqAB provide the method as described above, wherein said bacte CDE cluster comprises DNA fragments of SEQID NO: 14, rium is Methylobacterium extorquens. SEQID NO: 16, SEQID NO: 18, SEQID NO: 20, and SEQ 0046. It is a further aspect of the present invention to ID NO: 22, or variants thereof. provide the method as described above, wherein said culture 0028. It is a further aspect of the present invention to medium comprises methanol as a carbon source. provide the method as described above, wherein said pdqA like gene is selected from the group consisting of BRIEF DESCRIPTION OF THE DRAWINGS 0029 a DNA fragment that encodes a protein comprising 0047 FIG. 1 shows PQQ biosynthesis gene clusters in the amino acid sequence of SEQID No. 2; various strains. 0030 a DNA fragment that encodes a protein comprising 0048 FIG.2 shows an alignment of the Paq Ahomologous the amino acid sequence SEQ ID NO: 2, but that contains proteins from various microorganisms. Substitutions, deletions, insertions, additions or inversions of Q491.48—encoded by pdqA gene from Methylobacterium one or several amino acid residues, and said protein has the extorquens AM1 (SEQID No. 2) function of a PQQ precursor: C5AQY4—encoded by pdqA2 gene from Methylobacterium 0031 a DNA fragment that encodes a protein comprising extorquens AM1 (SEQID No. 11) the amino acid sequence of SEQID No. 11; C5AQY5—encoded by pdqA3 gene from Methylobacterium 0032 a DNA fragment that encodes a protein comprising extorquens AM1 (SEQID No: 13) the amino acid sequence of SEQID NO: 11, but that contains C7C9M1—encoded by gene locus from Methylobacterium Substitutions, deletions, insertions, additions or inversions of extorquens DM4 (SEQID No. 2) one or several amino acid residues, and having the function of C7CLK2—encoded by gene locus from Methylobacterium a POQ precursor: extorquens DM4 (SEQID No. 11) 0033 a DNA fragment that encodes a protein comprising D8JXU1—encoded by pdqA gene from Hyphomicrobium the amino acid sequence of SEQID No:13; denitrificans ATCC51888 (SEQID No: 15) US 2013/03375 11 A1 Dec. 19, 2013
D8JSB5—encoded by pdqA2 gene from Hyphomicrobium DESCRIPTION OF THE PREFERRED denitrificans ATCC51888 (SEQ ID No. 26) EMBODIMENTS D8JQF5—encoded by pcqA3 gene from Hyphomicrobium 0057 The present invention is described in detail below. denitrificans ATCC51888 (SEQ ID No. 28) 0058 1. Bacterium Q4KEK3 encoded by gene locus PFL 2223 (NCBI Entrez 0059. The bacterium according to the presently disclosed Gene) from Pseudomonas fluorescens Pf-5 (strain: Pf-5) Subject matter can be a POQ-producing bacterium, wherein (SEQ ID No. 37) the bacterium has been modified to have enhanced expression Q4K4V2 encoded by gene locus PFL 5673 (NCBI Entrez of the genes involved in the pyrroloquinoline quinone biosyn Gene) from Pseudomonas fluorescens Pf-5 (strain: Pf-5) thesis. (SEQ ID No. 38) 0060. The term “PQQ-producing bacterium can mean a B2UEVO encoded by genelocus Rpic 0286 (NCBI Entrez bacterium which has an ability to produce and excrete a PQQ Gene) from Ralstonia pickettii 12J (SEQID No:39) into a medium, when the bacterium is cultured in the medium. B2U914 encoded by gene locus Rpic 2488 (NCBI Entrez The term “PQQ-producing bacterium also can mean a bac Gene) from Ralstonia pickettii 12J (SEQID No: 40) terium which is able to produce and cause accumulation of C6WTX0 encoded by gene loci Mmol 0459, Mmol PQQ in a culture medium in an amount larger thana wild-type 0021, Mmol 0993, or Mmol 0794 (NCBI Entrez Gene) or parental strain, for example, Methylobacterium, Such as the from Methylotenera mobilis JLW8 (SEQ ID No.: 41) M. extorquens strain AM1, or Hyphomicrobium, Such as the A4YZY3—encoded by gene locus BRAD05793 (NCBI H. denitrificans strain ATCC51888. The term “PQQ-produc Entrez, Gene) from Bradyrhizobium sp. ORS278 (SEQ ID ing bacterium' can also mean that the microorganism is able No: 42) to cause accumulation in a medium of an amount not less than A4YZ28—encoded by gene locus BRAD05478 (NCBI 0.3 mg/L, and in another example, not less than 1.0 mg/L, of Entrez, Gene) from Bradyrhizobium sp. ORS278 (SEQ ID PQQ. No:43) 0061 The phrase “a bacterium belonging to the genus Methylobacterium' can mean that the bacterium is classified A4YNW1—encoded by gene locus BRADO1710 (NCBI as the genus Methylobacterium according to the classification Entrez, Gene) from Bradyrhizobium sp. ORS278 (SEQ ID known to a person skilled in the art of microbiology. Specifi No:44) cally, those classified into the group Methylobacterium Q608P4 encoded by gene locus MCA1445.1 (NCBI Entrez according to the taxonomy used by the NCBI (National Cen Gene) from Methylococcus capsulatus Bath (SEQID No.: 45) ter for Biotechnology Information) database (http://www. Q488A4 encoded by gene locus CPS 0862 (NCBI Entrez incbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) can be Gene) from Colwellia psychrerythraea 34H (SEQID No:46) used. Examples of the bacterium belonging to the genus Q9L3B4 encoded by gene locus GOX0987 (NCBI Entrez Methylobacterium include, but are not limited to, Methylo Gene) from Gluconobacter oxydans 621H (SEQ ID No.: 47) bacterium extorquens (M. extorquens), Methylobacterium Q1GX88 encoded by gene locus Mfla 0021 (NCBI Entrez chloromethanicum, Methylobacterium mesophilicum, Gene) from Methylobacillus flagellatus KT (SEQID No.: 48) Methylobacterium nodulans, Methylobacterium organophi A8LN54 encoded by gene locus Dshi 0450 (NCBI Entrez lum, Methylobacterium Oryzae, Methylobacterium Gene) from Dinoroseobacter shibae DFL 12 (SEQ ID No: podarium, Methylobacterium populi, Methylobacterium 49) radiotolerans, Methylobacterium rhodesianum, Methylobac B1 LV85 encoded by gene locus Mrad2831 0519 (NCBI terium variabile, Methylobacterium sp., etc. According to the Entrez, Gene) from Methylobacterium radiotolerans JCM present taxonomy of the genus, Protomonas is reclassified 2831 (SEQID No:2) into the genus Methylobacterium (Bousfield U and Green P D41FL0-encoded by gene locus EAM 0512A(NCBI N. Int J Syst Bacteriol 35 (1985), 209). The species Entrez Gene) from Erwinia amylovora ATCC 49946 (SEQID Pseudomonas rhodos, Pseudomonas radiora, and No. 50) Pseudomonas mesophilica, were also transferred to the genus D7DHP0 encoded by gene loci M301 0054, M301 Methylobacterium (Green P N and Bousfield U. Int J Syst 2651, M301 1191, or M301 2123 (NCBI Entrez Gene) Bacteriol 33 (1983), 875-877). from Methylotenera sp. 301 (SEQID No.: 41) 0062. The phrase “bacterium belonging to the genus B1Y7S1-encoded by gene locus Lcho 0244 (NCBI Entrez Hyphomicrobium' can mean that the bacterium is classified Gene) from Leptothrix cholodnii (strain ATCC 51168, LMG into the genus Hyphomicrobium according to the classifica 8142, SP-6) (Leptothrix discophora SP-6 (SEQID NO: 51) tion known to a person skilled in the art of microbiology, 0049 FIG.3 shows a map of plasmid pHT1. although the bacterium is not particularly limited. Specifi cally, those classified into the group Hyphomicrobium 0050 FIG. 4 shows a map of plasmid pCM110. according to the taxonomy used by the NCBI (National Cen 0051 FIG. 5 shows a map of plasmid pPQQ1 (pCMp ter for Biotechnology Information) database (http://www. a?ABCDE). incbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) can be 0052 FIG. 6 shows a map of plasmid pPQQ1 pdqA2A3. used. Examples of the bacterium belonging to the genus 0053 FIG. 7 shows genetic organization of the paq genes Hyphomicrobium include, but are not limited to, Hyphomi on the H. denitrificans chromosome. The positions of the crobium denitrificans (H. denitrificans), Hyphomicrobium primers used for PCR-amplification are shown by the arrows. aestuarii, Hyphomicrobium chloromethanicum, Hyphomi 0054 FIG. 8 shows construction of plasmid pHABCDE. crobium denitrificans, Hyphomicrobium facile, Hyphomicro 0055 FIG. 9 shows construction of plasmid pHAB bium methylovorum, Hyphomicrobium vulgare, Hyphomi CDEA3. crobium sp., etc. 0056 FIG. 10 shows construction of plasmid 0063 Examples of PQQ-producing bacteria and parent pHABCDEA3A2. strains which can be used to derive PQQ-producing bacteria US 2013/03375 11 A1 Dec. 19, 2013
include, but are not limited to, bacterial strains which inher 0067. The genes encoding enzymes involving in PQQ bio ently have the ability to produce PQQ. For example, parent synthesis in M. extorquens are known. M. extorquens contains strains which can be used to derive PQQ-producing bacteria apdqABC/DE operon in which the pdqC and pqqD genes are can include, but are not limited to, strains belonging to the fused, while the pdqFG genes forman operon with three other genus Methylobacterium, Such as M. mesophilicum ATCC genes (Zhang et al. Microbiology 149:1033-1040 (2003)). 29983, a pink-pigmented bacterium that utilizes L(-)-arabi The major pcqABC/DE operon is expressed under the control nose, D(-)-galalctose, D(-)-glucose, glycerol, Sodium cit of one promoter located upstream of pdqA gene. rate, sodium glutamate, sodium malate, Sodium malonate, 0068. The term “pdqABC/DE operon from Methylobac Sodium pyruvate, and Sodium Succinate as sole carbon terium extorquens' can mean a DNA that has a nucleotide sources for energy (Austin and Goodfellow. Int. J. Syst. Bac sequence which includes the following structural genes: teriol. 29: 373-378, 1979), M. extorquens AM1 (ATCC pqqA. pgqB, pdqCD, and pdqE genes, which encode a pre 43645, DSM 1337, JCM 2802, ATCC 14718, DSM 1338, cursor and enzymes for PQQ biosynthesis. JCM 2805) (Urakami and Komagata. (1984) Int. J. Syst. 0069. The pdqA gene encodes a precursor for PQQ bio Bacteriol. 34 (2): 188-201): M. rhodesianum JCM 2808 synthesis PaqA protein. The pdqA gene (nucleotides comple (ATCC 21612), and M. rhodesianum JCM 2809 (ATCC mentary to nucleotides in positions from 1825146 to 21613) (Green, Bousfield, Hood. Int. J. Syst. Bacteriol 0.38, 1825235; GenBank accession no. NC 012808.1; gi: 124-127, 1988). Examples of parent strains belonging to the 240 136783) is located between the mxbM gene and the pcqB genus Hyphomicrobium can include the Strains H. variable gene on the chromosome of M. extorquens strain AM1. The NCIB 10517 (US3989594 (A)); H. vulgare NCIB 9698 nucleotide sequence of the pdqA gene of the Strain AM1 and (Skerman et al. Int. J. Syst. Bacteriol. 30: 225-420, 1980), H. the amino acid sequence of the PaqA protein encoded by the methylovorum IFO 14180 (ATCC 35216) (Izumi et al. J. pqqA gene are shown in SEQID NO: 1 and SEQID NO: 2, Ferment. Technol. 60,371-375, 1982); and Hyphomicrobium respectively. sp. DSM 1869 (ATCC 51888) that utilizes methanol, monom 0070 The pcqB gene encodes a PQQ synthesis protein ethylamine, dimethylamine, trimethylamine, pectin, acetate PaqB with an unknown function. The pdqB gene (nucleotides as Sole carbon Sources, and possess the possibility to utilize complementary to nucleotides in positions from 1824199 to formate and ethanol (Urakami et al. Int. J. Syst. Bacteriol. 1824891: GenBank accession no. NC 012808.1; gi: 1995, 45,528-532). 240 136783), is located between the pdqA gene and the 0064. Above-mentioned strains are available from ATCC pqqCD gene on the chromosome of M. extorquens strain (American Type Culture Collection, 12301 Parklawn Drive, AM1. The nucleotide sequence of the pdqB gene of the strain Rockville, Md. 20852, P.O. Box 1549, Manassas, Va. 20108, AM 1 and the amino acid sequence of the PaqB protein United States of America), DSMZ (German Collection of encoded by the pdqB gene are shown in SEQID NO:3 and Microorganisms and Cell Cultures, Inhoffenstrase 7B, SEQ ID NO: 4, respectively. 3.8124, Braunschweig, Germany), JCM (Japan Collection of 0071. The pdqCD gene encodes PQQ synthesis protein Microorganisms (RIKEN BioResource Center, 2-1 Hiro PaqCD. The pdqCD gene (nucleotides complementary to sawa, Wako, Saitama 351-0198, Japan), or NCIMB (National nucleotides in positions from 1822914 to 1824032: GenBank Collections of Industrial and Marine Bacteria, Tony Research accession no. NC 012808.1; gi: 240136783) is located Station 135, Abbey Road, Aberdeen AB98DG, United King between the pdqB gene and the pdqE gene on the chromo dom). Some of M. extorquens Strain AM1. The nucleotide sequence of the pdqCD gene of the strain AM1 and the amino acid 0065. The phrase "enhancing the expression of the gene sequence of the fused PaqCD protein encoded by the pdqCD cluster and/or gene(s) can mean that the expression of the gene are shown in SEQID NO:5 and SEQID NO: 6, respec gene cluster and/or gene(s) is higher than that of a non tively. modified Strain, for example, a wild-type strain. Examples of 0072 The pdqE encodes PQQ synthesis protein Paq. Such modifications can include increasing the copy number of with an unknown function. The pdqE gene (nucleotides the expressed gene cluster or gene per cell, increasing the complementary to nucleotides in positions from 1821763 to expression level of the gene cluster or gene, and so forth. The 1822887: GenBank accession no. NC 012808.1; gi: copy number of an expressed gene cluster or gene is mea 240 136783) is located between the pdqCD gene and the pdqE sured, for example, by restricting the chromosomal DNA gene on the chromosome of the M. extorquens strain AM1. followed by Southern blotting using a probe based on the The nucleotide sequence of the pdqE gene of the strain AM1 gene sequence, fluorescence in situ hybridization (FISH), and and the amino acid sequence of the PaqB protein encoded by the like. The level of gene cluster or gene expression can be the pdqE gene are shown in SEQID NO: 7 and SEQID NO: measured by various known methods including Northern 8, respectively. blotting, quantitative RT-PCR, and the like. Furthermore, 0073. The nucleotide sequence of the pdqABC/DE operon wild-type non-modified strains can include, for example, M. from M. extorquens AM1 is shown in SEQID NO: 9. extorquens AM1, or H. denitrificans ATCC51888. 0074 The complete genome sequences of several methy 0066. The term “paq gene cluster can mean a gene cluster lotrophic bacteria have been published recently. Analysis of or operon that encodes proteins involved in the PQQ biosyn the available genomes revealed the presence of additional thesis. At present, for example, the sequences of the pdqAD copies of pcqA outside of the pdqABCDE cluster. In M. CDEF operon from Klebsiella pneumoniae, the pdqADCDE extorquens AM1, second and third copies located in tandem cluster from Acinetobacter calcoaceticus, the pdqADC/DE and presumably expressed under the control of one promoter, and pqqFG cluster from Methylobacterium extorquens, and have been annotated. According to the present invention, one the pdqADCDEF cluster from Gluconobacter oxydans have of the pdqA gene copies which is located between the serS been disclosed. The schematic structures of pdq gene clusters gene, oriented in the opposite direction to the pdqA gene from various microorganisms are shown in FIG. 1. copies, and the other pdqA gene copy on the chromosome of US 2013/03375 11 A1 Dec. 19, 2013
M. extorquens AM1 (nucleotides in positions from 4753498 0078. The nucleotide sequence of the pdqABCDE gene to 4753587: GenBank accession no. NC 012808.1; gi: cluster from H. denitrificans ATCC51888 is shown in SEQID 240136783), was given the name pdqA2 gene. The nucleotide NO: 24. sequence of the pdqA2 gene of the strain AM1 and the amino 007.9 The pdqA2 gene (synonym: Haen 0553) encodes a acid sequence of the PQQA2 protein encoded by the pdqA2 predicted PaqA protein. The pdqA2 gene (nucleotides gene are shown in SEQ ID NO: 10 and SEQ ID NO: 11, complementary to nucleotides in positions from 585973 to respectively. Further according to the present invention, the 58.6080: GenBank accession no. NC 014313.1; gi: other pdqA gene copy which is located between the pdqA2 300021538) is located between the Haen 0552 gene and the gene and the MexAM1—METAlp4630 gene, oriented in the Hden 0554 gene on the chromosome of the H. denitrificans opposite direction, on the chromosome of M. extorquens strain ATCC51888. The nucleotide sequence of the pdqA2 AM1 (nucleotides in positions from 4753650 to 4753739: gene of the strain ATCC51888 and the amino acid sequence of GenBank accession no. NC 012808.1; gi: 240136783), was the putative PdqA2 protein encoded by the pdqA2 gene are given the name pdqA3 gene. The nucleotide sequence of the shown in SEQID NO: 25 and SEQID NO:26, respectively. pqqA3 gene of the strain AM1 and the amino acid sequence of 0080. The pdqA3 gene (synonym: Haen 2110) encodes a the PQQA3 protein encoded by the pdqA3 gene are shown in predicted PaqA protein. The pdqA3 gene (nucleotides SEQ ID NO: 12 and SEQID NO: 13, respectively. complementary to nucleotides in positions from 2160533 to 0075. The homology level between the Paq.A, PaqA2 and 2160432; GenBank accession no. NC 014313.1; gi: PaqA3 proteins from M. extorquens AM1 is very high (see 300021538) is located between the Haen 2109 gene and the FIG. 2). Furthermore, inventors of the presently disclosed Hden 21 11 gene, both oriented in opposite directions, on the Subject matter have examined available genome sequences of chromosome of H. denitrificans strain ATCC51888. The five strains of genera Methylobacterium and found that M. nucleotide sequence of the pdqA3 gene of the strain chloromethanicum CM4, M. populi BJ001, M. radiotolerans ATCC51888 and the amino acid sequence of the putative JCM2831 have the same organization of pdqA locus as M. PaqA3 protein encoded by the pdqA3 gene are shown in SEQ extorquens AM1. M. extorquens DM4 has one additional ID NO: 27 and SEQID NO: 28, respectively. copy of the pdqA gene located beyond the pdqABC/DE I0081. The term “pdqA-like gene' can mean a gene that is operon. Methylobacterium sp. 4-46 and M. nodulans located in a bacterial genome and encodes a precursor for ORS2060 have two copies of the pdqA gene located in the PQQ biosynthesis. According to the present invention, each beginning of pdqABCDE cluster. of the above-described pdqA, pdqA2, and pqqA3 genes from 0076 Computer analysis of the Hyphomicrobium denitri M. extorquens AM1, also as pqqA, pdqA2, and pdqA3 genes ficans ATCC51888 genome revealed the presence of three from H. denitrificans ATCC51888, can be named “pdqA-like gene copies of the pdqA gene which are located in different gene'. The presence of a pdqA-like gene in a bacterial loci: the first pdqA gene copy (ORF Haen 1488), named genome can be determined by analysis of the genome according to the present invention the pdqA gene, is con sequence, in addition to databases containing information tained in a predicted pdqABCDE gene cluster, the second one about annotated pdqA genes from various microorganisms. (ORF Haen 0553), named according to the present inven pqqA-like genes from a wide range of microorganisms can be tion the pdqA2 gene, is contained in a predicted pdqADE used. Examples of the bacterium containing in-genome gene cluster, and the third one (ORF Haen 1488), named pqqA-like gene are not limited to the above-mentioned according to the present invention the pdqA3 gene, is located methylotrophs. Examples of the bacterium containing in separately (see FIG. 7). genome pdqA-like genes can also include, Methylococcus 0077. The term “pdqABCDE gene cluster from Hyphomi capsulatus Bath, Colwellia psychrerythraea 34H. Glucono crobium denitrificans' can mean a nucleotide sequence bacter oxydans 621H, Methylobacillus flagellatus KT, Dino which includes the following ORFs (ORF open reading roseobacter Shibae DFL 12, Leptothrix cholodni SP-6, frame) encoding a precursor and putative PQQ biosynthesis Erwinia amylovora ATCC 49946, Acinetobacter sp. enzymes: putative pdqA gene (Hden 1488), putative pdqB RUH2624, Saccharopolyspora erythraea NRRL23338. gene (Hden 1487), putative pdqC gene (Hden 1486), puta Bradyrhizobium sp. ORS278, Bradyrhizobium sp. BTAi1/ tive pdqD gene (Hden 1485), and putative pdqE gene ATCC BAA-1182, Ralstonia picketti 12.J., Pseudomonas (Hden 1484). The nucleotide sequence of the pdqA gene of fluorescensa, etc. Some of the bacteria have multiple copies H. denitrificans and the amino acid sequence of the putative of pdqA-like gene in-genome, for instance, Methylotenera PaqA protein encoded by the pdqA gene are shown in SEQID mobilis JLW8, Methylovorus sp. SIP3-4, Methylobacterium NO: 14 and SEQ ID NO: 15, respectively. The nucleotide extorquens DM4, and Methylobacterium sp. 4-46. sequence of the pdqB gene of H. denitrificans and the amino I0082 Since there may be some differences in DNA acid sequence of the putative PaqB protein encoded by the sequences between the genera, species or strains, the gene pqqB gene are shown in SEQID NO: 16 and SEQID NO: 17, cluster and the gene(s) which expression is/are enhanced are respectively. The nucleotide sequence of the pdqC gene of H. not limited to the genes shown in SEQID No. 1, SEQID No: denitrificans and the amino acid sequence of the putative 3, SEQID No. 5, SEQID No. 7, SEQID No: 10, SEQID No: PaqC protein encoded by the pdqC gene are shown in SEQID 12, SEQID No: 14, SEQID No: 16, SEQID No: 18, SEQID NO: 18 and SEQ ID NO: 19, respectively. The nucleotide No: 20, SEQID No. 22, SEQID No. 25, SEQID No. 27, but sequence of the pdqD gene of H. denitrificans and the amino can include genes homologous to SEQID No: 1, SEQID No: acid sequence of the putative Paq) protein encoded by the 3, SEQID No. 5, SEQID No. 7, SEQID No: 10, SEQID No: pqqD gene are shown in SEQID NO:20 and SEQID NO:21, 12, SEQID No: 14, SEQID No: 16, SEQID No: 18, SEQID respectively. The nucleotide sequence of the pdqE gene of H. No: 20, SEQ ID No. 22, SEQ ID No. 25, SEQ ID No. 27. denitrificans and the amino acid sequence of the putative Therefore, the protein variants encoded by the genes can have Paq protein encoded by the pdqE gene are shown in SEQID exemplary homology of not less than 80%, not less than 90%, NO: 22 and SEQID NO. 23, respectively. or not less than 95%, with respect to the entire amino acid US 2013/03375 11 A1 Dec. 19, 2013
sequence shown in SEQID No: 1, SEQID No. 3, SEQID No: a probe which can be prepared from the nucleotide sequence, 5, SEQID No. 7, SEQID No: 10, SEQID No: 12, SEQ ID provided that it encodes a functional protein. “Stringent con No: 14, SEQID No: 16, SEQID No: 18, SEQID No: 20, SEQ ditions' can include those under which a specific hybrid, for ID No. 22, SEQ ID No. 25, SEQ ID No. 27, as long as the example, a hybrid having homology of not less than 60%, is activity or function of the corresponding protein is main formed and a non-specific hybrid, for example, a hybrid hav tained. The term “homology” may also be used to refer to ing homology lower than the above, is not formed. Other “identity”. The phrase “protein variant', as used in the pres exemplary homologies can include not less than 70%, not less ently disclosed subject matter, means proteins which have than 80%, not less than 90%, not less than 95%, and not less changes in the sequences, whether they are deletions, inser than 98%. For example, stringent conditions are exemplified tions, additions, or Substitutions of amino acids. The number by washing one time or more, such as two or three times, at a of changes in the variant proteins can depend on the position salt concentration of 1 xSSC, 0.1% SDS. Another exemplary in the three dimensional structure of the proteins or the type of salt concentration can include 0.1xSSC, 0.1% SDS at 60° C. amino acid residues. Exemplary embodiments can be 1 to 30, Duration of washing depends on the type of membrane used 1 to 15, 1 to 5, or 1 to 3 in SEQID NO:2, SEQID NO:4, SEQ for blotting and, as a rule, should be what is recommended by ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, the manufacturer. For example, the recommended duration of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID washing for the HybondTMN+ nylon membrane (Amersham) NO:21, SEQID NO:23, SEQID NO:26, or SEQID NO:28. understringent conditions is 15 minutes. By way of example, These changes in the variants can occur in regions of the washing can be performed 2 to 3 times. The length of the protein which are not critical for the three dimensional struc probe can be suitably selected depending on the hybridization ture of the protein. This is because Some amino acids have conditions, and can be 100 by to 1 kbp. for example. More high homology to one another so the three dimensional struc over, codons in the gene sequences may be replaced with ture is not affected by Such a change. An alignment of the other equivalent codons which are easily used in the host into Paq.A homologues proteins from different microorganisms is which the genes are introduced. shown on FIG. 2. It is known that PaqA peptides from various I0087 Methods which can be used to enhance gene cluster microorganisms include two conserved glutamate and and/or gene(s) expression include increasing the gene cluster tyrosine residues, which are separated by three amino acid and/or the gene(s) copy number, and introducing a gene clus residues (sequence motif -E-X-X-X-Y, SEQ ID NO: 52). ter and/or a gene into a vector that is able to increase the copy Changes in the protein variants can occur in non-conservative number of the gene cluster and/or the gene in a bacterium of regions. the Methylobacterium or Hyphomicrobium genera. Examples I0083. The term “a protein having the function of PQQ of vectors include but are not limited to broad-host-range precursor can mean that the protein can be involved in PQQ vectors such as pCM11.0, pRK310, pVK101, pBBR122, biosynthesis as a precursor for PQQ, specifically, the protein pBHR1, and the like. can have a three dimensional structure which is sufficient to be recognized and used as a substrate by PQQ biosynthesis I0088. Enhancement of the gene cluster and/or gene enzymes with Subsequent conversion into PQQ. expression can also be achieved by introducing multiple cop ies of the gene cluster and/or the gene into a bacterial chro 0084 Homology between two amino acid sequences can mosome by, for example, homologous recombination, Mu be determined using the well-known methods, for example, integration, or the like. For example, one act of Mu integra the computer program BLAST 2.0, which calculates three tion allows for the introduction of up to 3 copies of the gene parameters: score, identity and similarity. into a bacterial chromosome. 0085. The substitution, deletion, insertion or addition of one or several amino acid residues should be conservative I0089. The copy number of gene cluster and/or gene can mutation(s) so that the activity or the function is maintained. also be increased by introducing multiple copies of the gene The representative conservative mutation is a conservative into the chromosomal DNA of the bacterium. In order to Substitution. Examples of conservative Substitutions can introduce multiple copies of the gene cluster and/or the gene include substitution of Seror Thr for Ala, substitution of Gln, into the bacterial chromosome, homologous recombination His or Lys for Arg, substitution of Glu, Gln, Lys, H is or Asp can be carried out using a sequence with multiple copies of for ASn, substitution of Asn. Glu or Gln for Asp, substitution the sequence in the chromosomal DNA. Sequences with mul of Ser or Ala for Cys, substitution of Asn. Glu, Lys, His, Asp tiple copies in the chromosomal DNA include, but are not or Arg for Gln, substitution of Asn., Gln, Lys or Asp for Glu, limited to, repetitive DNA, or inverted repeats present at the substitution of Pro for Gly, substitution of ASn, Lys, Gln, Arg end of a transposable element. Also, it is possible to incorpo or Tyr for His, substitution of Leu, Met, Val or Phe for Be, rate the gene cluster and/or the gene into a transposon, and substitution of Ile, Met, Val or Phe for Leu, substitution of allow it to be transferred to introduce multiple copies of the ASn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val gene cluster and/or the gene into the chromosomal DNA. or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for 0090. Enhancing of the gene cluster and/or gene expres Phe, substitution of Thr or Ala for Ser, substitution of Ser or sion may also be achieved by placing the DNAs under the Ala for Thr, substitution of Phe or Tyr for Trp, substitution of control of a potent promoter. For example, the lac promoter, His, Phe or Trp for Tyr, and substitution of Met, Be or Leu for the trp promoter, the trc promoter, the P, or the P. promoters Val. oflambda phage are all known to be potent promoters. Potent I0086. Therefore, the gene cluster and gene(s) can be a promoters providing a high level of gene expression in a variant(s) which hybridizes under stringent conditions with bacterium belonging to the genus Hyphomicrobium or Methy the nucleotide sequence shown in SEQID No: 1, SEQID No: lobacterium can be used. Especially, methanol dehydroge 3, SEQID No. 5, SEQID No. 7, SEQID No: 10, SEQID No: nase promoter P is known as a strong methanol-inducible 12, SEQID No: 14, SEQID No: 16, SEQID No: 18, SEQID promoter in Methylobacterium. The use of a potent promoter No: 20, SEQID No. 22, SEQID No. 25, SEQID No. 27, or can be combined with multiplication of gene copies. US 2013/03375 11 A1 Dec. 19, 2013
0091 Alternatively, the effect of a promoter can be EXAMPLES enhanced by, for example, introducing a mutation into the 0100. The present invention will be more concretely promoter to increase the transcription level of the gene cluster explained below with reference to the following non-limiting and/or the gene located downstream of the promoter. Further examples. more, it is known that substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the Example 1 start codon, profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, Construction of Plasmids Containing PQQ depending on the nature of the three nucleotides preceding Biosynthesis Genes from M. extorquens the start codon (Gold et al., Annu. Rev. Microbiol. 35, 365 0101 Construction of the Plasmid pPQQ1 403, 1981; Hui et al., EMBO J., 3, 623-629, 1984). 0102 The plasmid pPQQ1 was constructed for overex 0092. Moreover, it is also possible to introduce a nucle pression of pcqABC/DE operon from M. extorquens AM1 otide Substitution into the promoter region of the gene cluster (FIG. 5). For this purpose, plasmid pHT1 (FIG. 3; Toyama H, and/or the gene on the bacterial chromosome, which results in Lidstrom M. E. Microbiology. 1998 January: 144 (Pt 1): 183 a stronger promoter function. 91)) was treated with restrictases BamHI and XhoI followed 0093 Methods for preparation of plasmid DNA, diges by treatment with a Klenow fragment of DNA-polymerase I. tion, and ligation of DNA, transformation, selection of an After that, the 6.2 kb blunted BamHI-XhoI fragment contain oligonucleotide as a primer, and the like may be ordinary ing pdqABC/DE operon and gene mxbM encoding the posi methods well-known to one skilled in the art. These methods tive regulator of the pdqgenes from M. extorquens was cloned are described, for instance, in Sambrook, J., Fritsch, E. F., and into the region between BamHI-Ec1 136II sites of vector Maniatis, T., “Molecular Cloning: A Laboratory Manual, pCM110 (FIG. 4, the GenBank accession number for this Second Edition’, Cold Spring Harbor Laboratory Press cloning vector is AF327718) to produce plasmid pOQ1 (FIG. (1989). 5). The ligated mixture was transformed to E. coli TG1 strain 0094 2. Method (DSM 6056) and plasmid DNA was isolated from the clones 0095. The method for producing PQQ can be a method grown on LB plates with tetracyclin (10 g/ml). The plasmids which includes the steps of cultivating the bacterium accord of the expected structure were selected using restriction ing to the presently disclosed subject matter in a culture analysis. The resulting plasmid was given the name pCO1. E. medium to cause accumulation of PQQ in the medium, and coli TG1 strain is available from DSMZ (German Collection collecting PQQ from the medium. of Microorganisms and Cell Cultures, Inhoffenstrase 7B, 0096. The cultivation, collection, and purification of the 3.8124, Braunschweig, Germany). PQQ from the medium and the like may be performed in a 0103) To check whether the mxbM gene overexpression manner similar to conventional fermentation methods had an affect on PQQ production, the second plasmid pPQQ2 wherein PQQ is produced using a bacterium. without promoter P, was constructed. Deletion of P, 0097. The medium which can be used in the culture can be was obtained as following: the plasmid pPQQ1 was digested either a synthetic or natural medium, so long as the medium with restrictases BamPHI and Pst, blunt-ended, and then self includes a carbon source, a nitrogen source, minerals and, if ligated. The resulting plasmid contained pqqABC/DE operon necessary, appropriate amounts of nutrients which the bacte expressing under the control of the native promoter which is rium may require for growth. The carbon Source can include located on the plasmid downstream of the gene mxbM. The various carbohydrates such as glucose and Sucrose, various construction of strains M. extorquens/ppOQ1 and M. organic acids, alcohol including methanol, ethanol and glyc extorquens/ppOQ2, and assay of the amount of produced erol. Methanol is the most preferable. The nitrogen source can PQQ, were conducted as described in Example 3. The strains include various ammonium salts such as ammonia and M. extorquens AM1/ppOQ1 and M. extorquens AM1/pPQQ2 ammonium Sulphate, other nitrogen compounds such as showed a nearly identical amount of accumulated PQQ (see amines, a natural nitrogen Source Such as peptone, soybean Tablel). Thus, according to the obtained data, enhancing the hydrolysate, and digested fermentative microorganism. The mXbM gene expression does not significantly contribute to Sulfur source can include ammonium Sulphate, magnesium increasing PQQ production by the strain with enhanced Sulphate, ferrous Sulphate, manganese Sulphate, and the like. expression of the pdqgenes. Plasmid pPQQ1 was used for the Minerals can include potassium monophosphate, sodium further experiments. chloride, calcium chloride, and the like. Vitamins can include thiamine, yeast extract, and the like. TABLE 1 0098. The cultivation can be performed under aerobic con Strain PQQ, mg/l (HPLC) ditions such as a shaking culture or a stirring culture with aeration, at a temperature of 20 to 40° C., and in another M. extorquens AM1/pPQQ1 52.50.7 example 25 to 37°C. The pH of the culture is usually between M. extorquens AM1/pPQQ2 53.50.7 5 and 9, and in another example between 6 and 8. The pH of the culture can be adjusted with ammonia, calcium carbonate, 0104 2. Construction of the Plasmid pPQQ1 pcqA2A3 various acids, various bases, and buffers. Usually, a 1 to 5-day 0105. The plasmid pPQQ1 pcqA2A3 was constructed for cultivation period leads to the accumulation of PQQ in the overexpression of pdqABC/DE operon and two pdqA gene liquid medium. copies—the pdqA2 gene and the pdqA3 gene, from M. 0099. After cultivation, solids such as cells can be extorquens AM1. For this purpose, DNA fragment containing removed from the liquid medium by centrifugation or mem the pdqA2 and pqqA3 genes under transcriptional control of brane filtration, and the PQQ can be collected and purified by its own promoter was generated by PCR using primers ion-exchange, concentration, and crystallization methods. EG539 (SEQ ID NO: 29) and EG540 (SEQ ID NO: 30). US 2013/03375 11 A1 Dec. 19, 2013
Chromosome DNA isolated from the M. extorquens AM1 plasmids of the expected Structure have been selected using was used as the template in the reaction. The temperature restriction analysis. The resulting plasmid was given the profile was the following: initial DNA denaturation for 5 min name pHABCDEA3. at 95°C., followed by 30 cycles of denaturation at 95°C. for 0113. 3. Construction of the Plasmid pHABCDEA2A3. 30 sec, annealing at 57°C. for 30 sec, elongation at 72°C. for 0114. The plasmid pHABCDEA2A3 was constructed for 2 min, and the final elongation for 5 min at 72°C. overexpression of the pdqA3 and pqqA2 genes from H. deni 0106 The obtained 1.1 kb fragment was purified followed trificans in addition to pdqABCDE gene cluster. For this by treatment with PstI restrictase, and cloned into Swal-Pst purpose, a DNA fragment containing pdqA2 gene was gen sites of the pPQQ1 plasmid to produce pPQQ1 pcqA2A3 erated by PCR using primers EG570 (SEQID NO: 33) and (FIG. 6). The ligated mixture was transformed to E. coli TG1 EG571 (SEQID NO:34). Chromosome DNA isolated from strain and plasmid DNA was isolated from the clones grown the H. denitrificans ATCC51888 was used as the template in on LB plates with tetracyclin (10 g/ml). The plasmids of the the reaction. The temperature profile was the following: ini expected structure have been selected using restriction analy tial DNA denaturation for 5 min at 95°C., followed by 30 sis. The resulting plasmid was given the name cycles of denaturation at 95°C. for 30 sec, annealing at 57°C. for 30 sec, elongation at 72° C. for 2 min and the final elongation for 5 min at 72°C. Example 2 0115 The obtained 0.36 kb fragment was purified fol lowed by treatment with Xbal and BamHI restrictases, and Construction of Plasmids Containing PQQ cloned between the BamHI and Xbal sites of the plasmid Biosynthesis Genes from Hyphomicrobium pHABCDEA3 to produce pHABCDEA2A3 (FIG. 10). The denitrificans ligated mixture was transformed to E. coli TG1 strain and 0107 Construction of the plasmid pHABCDE. plasmid DNA was isolated from the clones grown on LB 0108. The plasmid pHABCDE was constructed for over plates with tetracyclin (10 ug/ml). The plasmids of the expression of pcqABCDE gene cluster from H. denitrificans expected structure have been selected using restriction analy ATCC51888 (DSM1869) (the strain was obtained from Ger sis. The resulting plasmid was given the name man Collection of Microorganisms and Cell Cultures pHABCDEA2A3. (DSMZ)). For this purpose, DNA fragment containing pqqABCDE gene cluster was generated by PCR using prim Example 3 ers EG572 (SEQID NO:35) and EG573 (SEQID NO:36). Chromosome DNA isolated from the H. denitrificans Production of PQQ by M. extorquens Strains ATCC51888 was used as the template in the reaction. The AM1/ppOQ1 and AM1/pPQQ1pdqA2A3 temperature profile was the following: initial DNA denatur 0116. To test the effect of the enhanced expression of the ation for 5 min at 95°C., followed by 30 cycles of denatur pqqA2 and pqqA3 genes and pdqABC/DE operon from M. ation at 95° C. for 30 sec, annealing at 57° C. for 30 sec, extorquens AM1 on PQQ production, plasmids pPQQ1 and elongation at 72°C. for 2 min and the final elongation for 5 pPQQ1 pcqA2A3 were transferred in PQQ-producing strain min at 72° C. M. extorquens AM1 by biparental mating using E. coli Strain 0109. The obtained 3.7 kb fragment was purified followed S17-1 (ATCC 47055), bearing the respective plasmids, as by treatment with BamHI and SacI restrictases, and ligated described by Abalakina et al. (Abalakina et al. Appl Micro into the pCM110 plasmid which had been previously treated biol Biotechnol. 81(1):191-200 (2008)), with some modifi with the same endonucleases, to produce pHABCDE (FIG. cations: Hypho medium (Harder, Attwood, and Quayle. J. 8). The ligated mixture was transformed to E. coli TG1 strain Gen. Microbiol. 78155-163 (1973)) with 1% of methanoland and plasmid DNA was isolated from the clones grown on LB supplemented with 10 ml/l of vitamin stock solution of fol plates with tetracyclin (10 ug/ml). The plasmids of the lowing composition: biotin 2.0 mg/l, folic acid 2.0 mg/l. expected structure have been selected using restriction analy thiamine-HCl 5.0 mg/l. Ca pantothenate 5.0 mg/l, B 12 0.1 sis. The resulting plasmid was given the name pHABCDE. mg/l, riboflavin 5.0 mg/l, nicotinamide 5.0 mg/l, was used for 0110 2. Construction of Plasmid pHABCDEA3. M. extorquens Strains cultivation. 0111. The plasmid pHABCDEA3 was constructed for 0117 The obtained M. extorquens strains, AM1/PQQ1 overexpression of pdqABCDE gene cluster and pqqA3 gene and AM1/ppOQ1 pcqA2A3, and control strain AM1, were from H. denitrificans ATCC51888. For this purpose, DNA separately cultivated for 60 hours on the plate of supple fragment containing pdqA3 gene was generated by PCR mented Hypho medium (as described above), one loop (from using primers EG568 (SEQID NO:31) and EG569 (SEQID /s plate) of the grown biomass were used as seed cultures, NO:32). Chromosome DNA isolated from the H. denitrifi each of the cultures were inoculated into 5 ml of the supple cans ATCC51888 was used as the template in the reaction. mented Hypho medium, and were each cultivated at 30° C. The temperature profile was the following: initial DNA dena for 72 hours with a rotary shaker at 240 rpm. For maintenance turation for 5 min at 95°C., followed by 30 cycles of dena of the plasmid, 10 ug/ml of tetracycline was Supplemented turation at 95°C. for 30 sec, annealing at 57°C. for 30 sec, during the course of cultivation. elongation at 72°C. for 2 min and the final elongation for 5 0118. After the cultivation, the amount of PQQ which had min at 72° C. accumulated in the medium was determined by HPLC analy 0112 The obtained 0.27 kb fragment was purified fol sis. The analytic conditions for HPLC were the following: lowed by treatment with Xbal restrictase, and cloned between column: Inertsil ODS-3 (4 um, 150 mmx4.6 mm I.D); eluents: the Xbaland Swal sites of the plasmid pHABCDE to produce (A) CHOH, (B) 15 mM TBA solution, A/B-50/50 (V/V) pHABCDEA3 (FIG.9). The ligated mixture was transformed gradient mixer, flow rate: 1.0 ml/min: column temperature: to E. coli TG1 strain and plasmid DNA was isolated from the 40° C.; detection UV250 nm or FL Ex 360 nm Em 455 nm, clones grown on LB plates with tetracyclin (10 ug/ml). The injection vol.: 5ul. TBA solution: Tetra-butylammonium (4.8 US 2013/03375 11 A1 Dec. 19, 2013 g) is solved in water (1 L) and pH is adjusted to 3.5 by the pdqABCDE gene cluster from H. denitrificans demon phosphate. Additionally, an optical density of each culture at strated a higher amount of accumulation of PQQas compared 600 nm (OD 600) was measured. with the parent strain ATCC51888. The strain H. denitrificans 0119 The results of the test tube fermentations are shown ATCC51888/pHABCDEA3 with enhanced expression of the in Table 2 and in Table 3. As it can be seen from Table 2, M. pqqA3 gene from H. denitrificans also demonstrated a higher extorquens strain AM1/ppOQ1 with enhanced expression of amount of accumulation of PQQas compared with the parent the pdqABC/DE operon from M. extorquens AM1 demon strain and the strain ATCC51888/pHABCDE. The strain strated an 8-fold higher amount of accumulation of PQQ as ATCC51888/pHABCDEA2A3 with enhanced expression of compared with the parent strain. As it can be seen from Table the pdqA2 and pdqA3 genes in addition to pdqABCDE gene 3, strain AM1/pFOQ1pdqA2A3 with enhanced expression of the pdqA2 and pdqA3 genes in addition to the pdqABC/DE cluster demonstrated the highest amount of accumulation of operon, demonstrated a doubled amount of accumulation of PQQ as compared with the strain M. extorquens AM1/ pPQQ1. TABLE 4 Strain PQQ, mg/l (HPLC) TABLE 2 H. denitrificans ATCC51888 7.5 + 0.1 Strain OD600mm PQQ, mg/l (HPLC) H. denitrificans ATCC51888/pHABCDE 9.6 0.1 H. denitrificans ATCC51888/pHABCDEA3 10.2O1 M. extorquens AM1 7.9 3.9 H. denitrificans ATCC51888/pHABCDEA2A3 10.9 O1 M. extorquens AM1/pPQQ1 8.6 O.9 31.4 - 1
Example 5 TABLE 3 Strain ODoo PQQ, mg/l (HPLC) Production of PQQ by H. denitrificans Strains M. extorquens AM1/pPQQ1 10.7 SO.O ATCC51888/pCQ1 and ATCC51888/PQQ1A2A3 M. extorquens AM1/pPQQ1pdqA2A3 11.5 O1 114.O 2.8 0.124. To test the effect of the enhanced expression of the pqqA2 and pqqA3 genes, and pqqABC/DE operon from M. Example 4 extorquens AM1, on PQQ production by H. denitrificans, plasmids pPQQ1 and pPQQ1 pcqA2A3 were transferred into Production of PQQ by H. denitrificans Strains H. denitrificans strain ATCC51888 by biparental mating ATCC51888/pHABCDE, using E. coli Strain S 17-1 bearing the respective plasmids as ATCC51888/pHABCDEA3, and described by Abalakina et al. (Abalakina et al. Appl Micro ATCC51888/pHABCDEA2A3 biol Biotechnol. 81(1):191-200 (2008)), with some modifi 0120 To test the effect of the enhanced expression of the cations: B medium (Urakamietal. Applied And Environmen pqqA2 and pqqA3 genes and pqqABCDE gene cluster from tal Microbiology, Vol. 58, No12, p. 3970-3976, 1992) with H. denitrificans on PQQ production, plasmids pHABCDE, 1% of methanol was used for H. denitrificans strains cultiva pHABCDEA3, and pHABCDEA2A3 were transferred into tion. H. denitrificans strain ATCC51888 by biparental mating (0.125 Both H. denitrificans strains, ATCC51888/pOQ1 using E. coli Strain S 17-1 bearing the respective plasmids as and ATCC51888/pOQ1A2A3, were separately cultivated for described by Abalakina et al. (Abalakina et al. Appl Micro 60 hours on a plate of B medium, and one loop (from /s plate) biol Biotechnol. 81 (1):191-200 (2008)), with some modifi of biomass grown on each plate was used as a seed culture. cations: B medium (Urakamietal. Applied And Environmen Each of the cultures were inoculated into 5 ml of the B tal Microbiology, Vol. 58, No12, p. 3970-3976, 1992) with medium, and cultivated at 30° C. for 72 hours with a rotary 1% of methanol was used for H. denitrificans strains cultiva shaker at 240 rpm. For maintenance of the plasmid, 10 g/ml tion. of tetracycline was Supplemented during the course of culti 0121. The obtained H. denitrificans strains, ATCC51888/ Vation. pHABCDE, ATCC51888/pHABCDEA3, ATCC51888/ pHABCDEA2A3, and the control strain ATCC51888, were I0126. After the cultivation, the amount of PQQ which had separately cultivated for 60 hours on the plate of B medium accumulated in the medium was determined by HPLC analy and one loop (from "/s plate) of biomass grown were used as sis, as described above. seed cultures, each of the cultures were inoculated into 5 ml of 0127. The results of the test tube fermentations are shown the B medium, and were each cultivated at 30°C. for 72 hours in Table 5. As it can be seen from Table 5, H. denitrificans with a rotary shaker at 240 rpm. For maintenance of the strain ATCC51888/pCO1 with enhanced expression of the plasmid, 10 ug/ml of tetracycline was Supplemented during pqqABC/DE operon from M. extorquens AM1 demonstrated the course of cultivation. a higher amount of accumulation of PQQ as compared with 0122. After the cultivation, the amount of PQQ which had the parent strain H. denitrificans ATCC51888. The strain H. accumulated in the medium was determined by HPLC analy denitrificans ATCC51888/pOQ1 pcqA2pqqA3 with sis, as described above. enhanced expression of the pdqA2 and pqqA3 genes from M. 0123. The results of the test tube fermentations are shown extorquens AM1 in addition to the pdqABC/DE operon dem in Table 4. As it can be seen from Table 4, H. denitrificans onstrated a higher amount of accumulation of PQQ as com strain ATCC51888/pHABCDE with enhanced expression of pared with the parent strain and the strain ATCC51888/pCO1. US 2013/03375 11 A1 Dec. 19, 2013
TABLE 5 (0145 SEQ ID NO: 16: nucleotide sequence of putative pqqB of H. denitrificans Strain PQQ, mg/l (HPLC) 0146 SEQ ID NO: 17: amino acid sequence of putative H. denitrificans ATCC51888 7.5 + 0.1 PaqB of H. denitrificans H. denitrificans ATCC51888/pPQQ1 8.3 O.1 0147 SEQ ID NO: 18: nucleotide sequence of putative H. denitrificans ATCC51888, pPQQ1 paqA2A3 10.6 - 0.1 pqqC of H. denitrificans 0148 SEQ ID NO: 19: amino acid sequence of putative 0128. While the invention has been described in detail PaqC of H. denitrificans with reference to preferred embodiments thereof, it will be 0149 SEQ ID NO. 20: nucleotide sequence of putative apparent to one skilled in the art that various changes can be pqqD of H. denitrificans made, and equivalents employed, without departing from the (O150 SEQ ID NO: 21: amino acid sequence of putative scope of the invention. All documents cited herein are hereby PaqD of H. denitrificans incorporated by reference. 0151 SEQ ID NO: 22: nucleotide sequence of putative pqqE of H. denitrificans INDUSTRIAL APPLICABILITY 0152 SEQ ID NO: 23: amino acid sequence of putative PaqE of H. denitrificans 0129. According to the present invention, pyrroloquino 0153. SEQ ID NO: 24: nucleotide sequence of pdqAB line quinine is produced by a bacterium belonging to the CDE gene cluster of H. denitrificans ATCC51888 genus Methylobacterium or Hyphomicrobium. 0154 SEQ ID NO: 25: nucleotide sequence of putptive pqqA2 of H. denitrificans strain ATCC51888 Explanation of Sequence Listing (O155 SEQ ID NO: 26: amino acid sequence of putative 0130 SEQID NO: 1: nucleotide sequence of pcqA of M. PdqA2 of H. denitrificans strain ATCC51888 extorquens AM1 0156 SEQ ID NO: 27: nucleotide sequence of putptive 0131 SEQID NO: 2: amino acid sequence of PaqA of M. pqqA3 of H. denitrificans strain ATCC51888 extorquens AM1, C7C9M1, B1 LV85 (O157 SEQ ID NO: 28: amino acid sequence of putative (0132) SEQID NO:3: nucleotide sequence of pdqB of M. PdqA3 of H. denitrificans strain ATCC51888 extorquens AM1 0158 SEQID NO: 29: primer EG539 0.133 SEQID NO: 4: amino acid sequence of PaqB of M. 0159 SEQID NO:30: primer EG540 extorquens AM1 (0160 SEQID NO:31: primer EG568 0134 SEQID NO:5: nucleotide sequence ofpqqCD of M. (0161 SEQID NO:32: primer EG569 extorquens AM1 (0162 SEQID NO:33: primer EG570 0135 SEQID NO: 6: amino acid sequence of PdqCD of (0163 SEQID NO:34: primer EG571 M. extorquens AM1 (0164 SEQID NO:35: primer EG572 0.136 SEQID NO: 7: nucleotide sequence of pdqE of M. (0165 SEQID NO:36: primer EG573 extorquens AM1 (0166 SEQID NO:37: Q4KEK3 0137 SEQID NO: 8:amino acid sequence of PaqB of M. extorquens AM1 (0167 SEQID NO:38: Q4K4V2 0138 SEQID NO: 9: nucleotide sequence ofpqqABC/DE (0168 SEQID NO:39: B2UEVO operon of M. extorquens AM1 (0169 SEQID NO: 40; B2U9I4 0139 SEQID NO: 10: nucleotide sequence of pdqA2 of (0170 SEQID NO:41: C6WTX0, D7DHP0 M. extorquens AM1 (0171 SEQID NO:42: A4YZY3 0140 SEQID NO: 11:amino acid sequence of PQQA2 of (0172 SEQID NO:43: A4YZ28 M. extorquens AM1, C7CLK2 (0173 SEQID NO:44: A4YNW1 0141 SEQID NO: 12: nucleotide sequence of pcqA3 of (0174 SEQID NO. 45: Q608P4 M. extorquens AM1 (0175 SEQID NO: 46: Q488A4 0142 SEQID NO: 13: amino acid sequence of PQQA3 of (0176 SEQID NO:47: Q9L3B4 M. extorquens AM1 (0177 SEQID NO:48: Q1GX88 0143 SEQ ID NO: 14: nucleotide sequence of putative (0178 SEQID NO:49: A8LN54 pqqA of H. denitrificans (0179 SEQID NO:50: D41FLO 0144 SEQ ID NO: 15: amino acid sequence of putative 0180 SEQID NO:51: B1Y7S1 PaqA of H. denitrificans 0181 SEQID NO: 52: sequence motif-E-X-X-X-Y
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 52
<21 Os SEQ ID NO 1 &211s LENGTH: 90 &212s. TYPE: DNA <213> ORGANISM: Methylobacterium extorquens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (90)
US 2013/03375 11 A1 Dec. 19, 2013 12
- Continued Val Gly. Thir Lys Thr Gly Trp Arg Met Gly His Ile Gln Met Asn Gly 1.65 17O 17s gag acc ggc ticg atc gcg tct ctic goc gat atc gag atc ggc cga cqg 576 Glu Thr Gly Ser Ile Ala Ser Lieu Ala Asp Ile Glu Ile Gly Arg Arg 18O 185 19 O gtc titc gtt cac atc aac aac acc aat cog gtc. citg atc gag gat tcg 624 Val Phe Val His Ile Asn Asn Thr Asn Pro Val Lieu. Ile Glu Asp Ser 195 2OO 2O5 tac gag cqc gcg agc gtc gag gCd cqc ggc tigg acc gtc gcc cat gac 672 Tyr Glu Arg Ala Ser Val Glu Ala Arg Gly Trp Thr Val Ala His Asp 21 O 215 22O ggc ctg acc ct c gat Ctc ta 693 Gly Lieu. Thir Lieu. Asp Lieu. 225 23 O
<210s, SEQ ID NO 4 &211s LENGTH: 23 O 212. TYPE: PRT <213> ORGANISM: Methylobacterium extorquens
<4 OOs, SEQUENCE: 4 Met His Pro Arg Glu Gly Lieu. Arg His Ser Pro Ile His Ala Val Lieu. 1. 5 1O 15 Lieu. Thir Asn Gly Asp Val Asp His Val Ala Gly Lieu. Lieu. Thir Lieu. Arg 2O 25 3O Glu Gly Gln Pro Phe Thr Lieu. Tyr Ala Thr Pro Gly Ile Leu Ala Ser 35 4 O 45 Val Ser Asp Asn Arg Val Phe Asp Wal Met Ala Ala Asp Val Val Lys SO 55 6 O Arg Glin Thir Ile Ala Lieu. Asn Glu Thr Phe Glu Pro Val Pro Gly Lieu. 65 70 7s 8O Ser Val Thr Lieu Phe Ser Val Pro Gly Llys Val Pro Leu Trp Lieu. Glu 85 90 95 Asp Ala Ser Met Glu Ile Gly Ala Glu Thr Glu Thir Thr Val Gly Thr 1OO 105 11 O Met Ile Glu Ala Gly Gly Lys Arg Lieu Ala Tyr Ile Pro Gly Cys Ala 115 12 O 125 Arg Val Thr Glu Asp Lieu Lys Ala Arg Ile Ala Gly Ala Asp Ala Lieu 13 O 135 14 O Lieu. Phe Asp Gly Thr Val Lieu. Glu Asp Asp Asp Met Ile Arg Ala Gly 145 150 155 160 Val Gly. Thir Lys Thr Gly Trp Arg Met Gly His Ile Gln Met Asn Gly 1.65 17O 17s Glu Thr Gly Ser Ile Ala Ser Lieu Ala Asp Ile Glu Ile Gly Arg Arg 18O 185 19 O Val Phe Val His Ile Asn Asn Thr Asn Pro Val Lieu. Ile Glu Asp Ser 195 2OO 2O5 Tyr Glu Arg Ala Ser Val Glu Ala Arg Gly Trp Thr Val Ala His Asp 21 O 215 22O Gly Lieu. Thir Lieu. Asp Lieu. 225 23 O
<210s, SEQ ID NO 5 &211s LENGTH: 1119 &212s. TYPE: DNA <213> ORGANISM: Methylobacterium extorquens
US 2013/03375 11 A1 Dec. 19, 2013 14
- Continued gtg ccg cgc ctg. CCC cc ggc gtg cc ctg cgc titc gac gag gtC cc 912 Val Pro Arg Lieu Pro Arg Gly Val Arg Lieu. Arg Phe Asp Glu Val Arg 29 O 295 3 OO aac aag cac gtg Ctg Ctic gcc ccc gag cqc acc titc gac ct c gaC gac 96.O Asn Llys His Val Lieu. Lieu Ala Pro Glu Arg Thr Phe Asp Lieu. Asp Asp 3. OS 310 315 32O aac gcc gtC gog gtc. Ct c aag Ct c gt c gat ggc cq9 aac acg gtt tog 1008 Asn Ala Val Ala Val Lieu Lys Lieu Val Asp Gly Arg Asn. Thr Val Ser 3.25 330 335
Cag at C gcc cag att Ctg ggt cag acc tac gac gcc gaC ccg gCC at C 1056 Glin Ile Ala Glin Ile Lieu. Gly Glin Thr Tyr Asp Ala Asp Pro Ala Ile 34 O 345 35. O atc gala gcc gac atc ctic ccg atg Ctg gcc ggc ctic gcg caa aaa agg 1104 Ile Glu Ala Asp Ile Lieu Pro Met Lieu Ala Gly Lieu Ala Glin Lys Arg 355 360 365 gtt Ctg gag cata 1119 Val Lieu. Glu Arg 37 O
<210s, SEQ ID NO 6 &211s LENGTH: 372 212. TYPE: PRT <213> ORGANISM: Methylobacterium extorquens
<4 OOs, SEQUENCE: 6 Met Thr Ala Glin Phe Pro Pro Pro Val Pro Asp Thr Glu Glin Arg Lieu 1. 5 1O 15 Lieu. Ser His Glu Glu Lieu. Glu Ala Ala Lieu. Arg Asp Ile Gly Ala Arg 2O 25 3O Arg Tyr His Asn Lieu. His Pro Phe His Arg Lieu. Lieu. His Asp Gly Lys 35 4 O 45 Lieu. Ser Lys Asp Glin Val Arg Ala Trp Ala Lieu. Asn Arg Tyr Tyr Tyr SO 55 6 O Glin Ala Met Ile Pro Wall Lys Asp Ala Ala Lieu. Lieu Ala Arg Lieu Pro 65 70 7s 8O Asp Ala Glin Lieu. Arg Arg Ile Trp Arg Glin Arg Ile Val Asp His Asp 85 90 95 Gly Asp His Glu Gly Asp Gly Gly Ile Glu Arg Trp Lieu Lys Lieu Ala 1OO 105 11 O Glu Gly Val Gly Phe Thr Arg Asp Tyr Val Leu Ser Thr Lys Gly Ile 115 12 O 125 Lieu. Ser Ala Thr Arg Phe Ser Val Asp Ala Tyr Val His Phe Val Ser 13 O 135 14 O Glu Arg Ser Lieu. Lieu. Glu Ala Ile Ala Ser Ser Lieu. Thr Glu Met Phe 145 150 155 160 Ser Pro Thr Ile Ile Ser Glu Arg Val Ala Gly Met Leu Lys Asn Tyr 1.65 17O 17s Asp Phe Ile Thir Lys Asp Thir Lieu Ala Tyr Phe Asp Lys Arg Lieu. Thr 18O 185 19 O Glin Ala Pro Arg Asp Ala Asp Phe Ala Lieu. Asp Tyr Val Lys Arg His 195 2OO 2O5 Ala Thir Thr Pro Glu Met Glin Arg Ala Ala Ile Asp Ala Lieu. Thir Phe 21 O 215 22O
Lys Cys Asn Val Lieu. Trp Thr Glin Lieu. Asp Ala Lieu. Tyr Phe Ala Tyr 225 23 O 235 24 O
US 2013/03375 11 A1 Dec. 19, 2013 17
- Continued
Pro Thr Ala Arg Pro Asp Ile Val Glu Ile Thr Ala Lys Cys Ala Glu 65 70 7s 8O Lieu. Gly Lieu. Tyr Ser Asn Lieu. Ile Thir Ser Gly Val Gly Gly Ala Lieu. 85 90 95 Ala Lys Lieu. Asp Ala Lieu. Tyr Asp Val Gly Lieu. Asp His Val Glin Lieu 1OO 105 11 O Ser Val Glin Gly Val Asp Ala Ala Asn Ala Glu Lys Ile Gly Gly Lieu. 115 12 O 125 Lys Asn Ala Glin Pro Glin Llys Met Glin Phe Ala Ala Arg Val Thr Glu 13 O 135 14 O Lieu. Gly Lieu Pro Lieu. Thir Lieu. Asn. Ser Val Ile His Arg Gly Asn. Ile 145 150 155 160 His Glu Val Pro Gly Phe Ile Asp Lieu Ala Val Llys Lieu. Gly Ala Lys 1.65 17O 17s Arg Lieu. Glu Val Ala His Thr Glin Tyr Tyr Gly Trp Ala Tyr Val Asn 18O 185 19 O Arg Ala Ala Lieu Met Pro Asp Llys Ser Glin Val Asp Glu Ser Ile Arg 195 2OO 2O5 Ile Val Glu Ala Ala Arg Glu Arg Lieu Lys Gly Glin Lieu Val Ile Asp 21 O 215 22O Lieu Val Val Pro Asp Tyr Tyr Ala Lys Tyr Pro Lys Ala Cys Ala Gly 225 23 O 235 24 O Gly Trp Gly Arg Llys Lieu Met Asn Val Thr Pro Glin Gly Llys Val Lieu. 245 250 255 Pro Cys His Ala Ala Glu Thir Ile Pro Gly Lieu. Glu Phe Trp Tyr Val 26 O 265 27 O Thir Asp His Ala Lieu. Gly Glu Ile Trp Thir Lys Ser Pro Ala Phe Ala 27s 28O 285 Ala Tyr Arg Gly. Thir Ser Trp Met Lys Glu Pro Cys Arg Ser Cys Asp 29 O 295 3 OO Arg Arg Glu Lys Asp Trp Gly Gly Cys Arg Cys Glin Ala Lieu Ala Lieu 3. OS 310 315 32O Thr Gly Asp Ala Ala Asn. Thir Asp Pro Ala Cys Ser Lieu. Ser Pro Lieu. 3.25 330 335 His Ala Lys Met Arg Asp Lieu Ala Lys Glu Glu Ala Ala Glu Thr Pro 34 O 345 35. O Pro Asp Tyr Ile Tyr Arg Ser Ile Gly Thr Asn Val Glin Asn Pro Leu 355 360 365 Ser Glu Lys Ala Pro Lieu. 37 O
<210s, SEQ ID NO 9 &211s LENGTH: 3473 &212s. TYPE: DNA <213> ORGANISM: Methylobacterium extorquens
<4 OOs, SEQUENCE: 9 atgaagtggg Ctgc.ccc.cat cqttt Cogag atctg.cgt.cg gCatggaagt cacgagctac 6 O gagt cq9ccg agat.cgacac Cttcaact aa ggtgatttga gcc.gggttgg ggttgcaggc 12 O atcagcgggt titt cac catg catgtcgtaa t cctgggctic ggctg.cgggc ggcggcgttc 18O
Ctcaatggala Ctgcc.gctgc ticCatctgct C cctggcctg ggcgggcgat tcc.cgcgt.ca 24 O
US 2013/03375 11 A1 Dec. 19, 2013 20
- Continued
1. 5 1O 15 gtc. acc toc tac gag toc got gag at c gac acc titc aac taa 9 O Val Thr Ser Tyr Glu Ser Ala Glu Ile Asp Thr Phe Asn 2O 25
<210s, SEQ ID NO 13 &211s LENGTH: 29 212. TYPE: PRT <213> ORGANISM: Methylobacterium extorquens
<4 OOs, SEQUENCE: 13 Met Lys Trp Ser Ala Pro Val Val Ala Glu Ile Cys Val Gly Met Glu 1. 5 1O 15 Val Thr Ser Tyr Glu Ser Ala Glu Ile Asp Thr Phe Asn 2O 25
<210s, SEQ ID NO 14 &211s LENGTH: 123 &212s. TYPE: DNA <213> ORGANISM: Hyphomicrobium denitrificans 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (123)
<4 OOs, SEQUENCE: 14 atg gala agc agit tac cqc titt gga gga acc acia atg aaa at C tig acg 48 Met Glu Ser Ser Tyr Arg Phe Gly Gly Thr Thr Met Lys Ile Trp Thr 1. 5 1O 15 aag ccc gct gtg cqc gag cag gala gtt ggit cto gaa gtg acg agc tac 96 Llys Pro Ala Val Arg Glu Glin Glu Val Gly Lieu. Glu Val Thir Ser Tyr 2O 25 3O citg ccg gct gala atc gac citc at c toga 123 Lieu Pro Ala Glu Ile Asp Lieu. Ile 35 4 O
<210s, SEQ ID NO 15 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Hyphomicrobium denitrificans
<4 OOs, SEQUENCE: 15 Met Glu Ser Ser Tyr Arg Phe Gly Gly Thr Thr Met Lys Ile Trp Thr 1. 5 1O 15 Llys Pro Ala Val Arg Glu Glin Glu Val Gly Lieu. Glu Val Thir Ser Tyr 2O 25 3O Lieu Pro Ala Glu Ile Asp Lieu. Ile 35 4 O
<210s, SEQ ID NO 16 &211s LENGTH: 933 &212s. TYPE: DNA <213> ORGANISM: Hyphomicrobium denitrificans 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (933)
<4 OOs, SEQUENCE: 16 atg at C at a aaa gtg Ctic ggg to C to C gct ggg ggt gga tt C ccg cag 48 Met Ile Ile Llys Val Lieu. Gly Ser Ser Ala Gly Gly Gly Phe Pro Glin 1. 5 1O 15 tgg aac to aat ggc atg cag tog gca aag gtt cqc to a ggc gct gcc 96 Trp Asn. Cys Asn Gly Met Glin Ser Ala Lys Val Arg Ser Gly Ala Ala
US 2013/03375 11 A1 Dec. 19, 2013 22
- Continued
212. TYPE: PRT <213> ORGANISM: Hyphomicrobium denitrificans
<4 OOs, SEQUENCE: 17 Met Ile Ile Llys Val Lieu. Gly Ser Ser Ala Gly Gly Gly Phe Pro Glin 1. 5 1O 15 Trp Asn. Cys Asn Gly Met Glin Ser Ala Lys Val Arg Ser Gly Ala Ala 2O 25 3O Gly Phe Lys Ala Arg Lieu. Glin Ser Ser Lieu Ala Ala Ser Ser Asp Gly 35 4 O 45 Lys Asn Trp Val Lieu. Lieu. Asn Ala Ser Pro Asp Ile Arg Glin Glin Ile SO 55 6 O Asn Glu Thr Pro Glu Lieu. His Pro Glu Thir Thr Gly Ala Lys Arg Asn 65 70 7s 8O Ser Pro Ile Lys Ala Val Val Val Thr Asn Ala Asp Val Asp His Ile 85 90 95 Ile Gly Lieu. Ile Gly Lieu. Arg Glu Gly Glin Pro Phe Ser Ile Tyr Gly 1OO 105 11 O Ser Asp Lieu Val Lieu Ala Thr Lieu Lys Ala Asn. Ser Val Phe Asn. Wall 115 12 O 125 Cys Asn Pro Glu Ile Val Pro Arg Lieu. Glu Lieu Pro Phe Asp Arg Pro 13 O 135 14 O Thr Glu Lieu. His Gly Ala Gly Val Asp Lieu. Gly Lieu. Thr Val Glu Ala 145 150 155 160 Phe Pro Val Pro Gly Llys Val Ala Lieu. Phe Lieu. Glu Lys Gly Gly Ala 1.65 17O 17s Asn Glu Asn Tyr Gly Ser Arg Asp Gly Asp Thir Ile Gly Lieu Lys Val 18O 185 19 O Thir Asp Arg Lys Ala Gly Llys Ser Phe Phe Tyr Ile Pro Gly Cys Ala 195 2OO 2O5 Glu Val Asp Ala Pro Lieu Ala Asp Arg Ile Arg Gly Ala Asp Val Ile 21 O 215 22O Phe Phe Asp Gly Thr Lieu. Tyr Glu Asp Gly Glu Met Ile Ala Glin Gly 225 23 O 235 24 O Lieu. Lieu. Asn Llys Thr Gly Lys Arg Met Gly His Ile Ser Val Ser Gly 245 250 255 His Glu Gly Ser Ile Ala Ala Lieu. Ser His Lieu. Asn Val Arg Arg Llys 26 O 265 27 O Ile Tyr Val His Ile Asn. Asn. Ser Asn Pro Ile Lieu. Asp Glu Asn. Ser 27s 28O 285 Glu Ala Arg Lys Ala Val Glu Thir Ala Gly Trp Glu Val Gly Phe Asp 29 O 295 3 OO Gly Met Glu Val Arg Lieu. 3. OS 310
<210s, SEQ ID NO 18 211 LENGTH: 777 &212s. TYPE: DNA <213> ORGANISM: Hyphomicrobium denitrificans 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (777)
<4 OOs, SEQUENCE: 18 atgaac ccg gt C agc gtt gga aag aaa tta gca gag ctt cqc ggit gac 48
US 2013/03375 11 A1 Dec. 19, 2013 24
- Continued Glu Ala Pro Met Pro Val Ala Glu Phe Glu Ala Ala Ile Arg Ala Val 2O 25 3O Gly Pro Glu Arg Tyr His Asp Lieu. His Pro Phe His His Met Lieu. His 35 4 O 45 Gly Gly Lys Lieu. Asn Lys Gly Glin Val Glin Ala Trp Ala Lieu. Asn Arg SO 55 6 O Phe Cys Tyr Glin Ser Ala Val Pro Arg Lys Asp Ala Ala Lieu. Ile Ser 65 70 7s 8O Arg Val Tyr Asp Arg Glu Lieu. Arg Arg Glu Trp Thr His Arg Ile Lieu 85 90 95 Asp His Asp Gly Lieu. Lieu Pro Asp Glu Glu Gly Gly Ile Glu Arg Trip 1OO 105 11 O Lieu Val Lieu. Thir Asp Gly Lieu. Gly Lieu. Asp Arg Glu Tyr Val Ile Ser 115 12 O 125 Arg Arg Gly Ala Lieu Pro Ala Thr Val Phe Ala Val Glu Ala Tyr Val 13 O 135 14 O Thr Phe Val Arg Glu Gln Pro Leu. Thir Ile Ala Val Ala Ser Ser Lieu. 145 150 155 160 Thr Glu Lieu. Phe Ala Pro Llys Ile His Lys Glu Arg Ile Ala Gly Met 1.65 17O 17s Lieu. Glu Asn Tyr Asn. Phe Ile Asp Asp Llys Val Met Ala Tyr Phe Lys 18O 185 19 O Arg Arg Lieu. Thr Glin Ala Pro Arg Asp Ala Asp Phe Ala Lieu. ASn Tyr 195 2OO 2O5 Ile Lieu. Glu Asn Ala Arg Thr Arg Asp Glu Glin Glin Ala Cys Ile Asp 21 O 215 22O Ala Val Arg Phe Lys Cys Asn Val Lieu. Trp Val Glin Lieu. Asp Ala Lieu 225 23 O 235 24 O Tyr His Ala Tyr Val Asp Gly His Ile Pro Pro Gly Ala Phe Arg Pro 245 250 255
Glu Asn
<210s, SEQ ID NO 2 O &211s LENGTH: 3O3 &212s. TYPE: DNA <213> ORGANISM: Hyphomicrobium denitrificans 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (303)
<4 OOs, SEQUENCE: 2O atgaac go a gac gca ccg cgt acg aga acg atg gtc gtc. cc.g. gct tcc 48 Met Asn Ala Asp Ala Pro Arg Thr Arg Thr Met Val Val Pro Ala Ser 1. 5 1O 15 aag ccg gga ctg. ccg acg cac at C aag ctg cgc cac gac goc ggc cc 96 Llys Pro Gly Lieu Pro Thr His Ile Llys Lieu. Arg His Asp Ala Gly Arg 2O 25 3O ggc cqa tig cac gitg Ctt gct coc gaa cqc gtg ttic gag cct gaC cct 144 Gly Arg Trp His Val Lieu Ala Pro Glu Arg Val Phe Glu Pro Asp Pro 35 4 O 45 atc gct gtC gag att ttg aag cc tit gat ggc gcg acg agt gtt gag 192 Ile Ala Val Glu Ile Lieu Lys Arg Cys Asp Gly Ala Thir Ser Val Glu SO 55 6 O gaa at C gcc acc gca Ctc gca aaa gag tac aat gca ccg ctg caa gag 24 O Glu Ile Ala Thr Ala Lieu Ala Lys Glu Tyr Asn Ala Pro Lieu. Glin Glu 65 70 7s 8O US 2013/03375 11 A1 Dec. 19, 2013 25
- Continued atc. citc. gcc gac acc ata t cc atgttg cag gala Ctc. tcg gaC aag ggc 288 Ile Luell Ala Asp Thir Ile Ser Met Lieu. Glin Glu Lieu. Ser Asp Llys Gly 85 90 95 gtg gt C a.a.a. gcc tag Wall Wall Ala 1OO
<210s, SEQ ID NO 21 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Hyphomicrobium denitrificans
<4 OOs, SEQUENCE: 21
Met Asn Ala Asp Ala Pro Arg Thir Arg Thir Met Wall Wall Pro Ala Ser 1. 5 1O 15
Lys Pro Gly Luell Pro Thir His Ile Lys Luell Arg His Asp Ala Gly Arg 25 3O
Gly Arg Trp His Wall Lell Ala Pro Glu Arg Wall Phe Glu Pro Asp Pro 35 4 O 45
Ile Ala Wall Glu Ile Lell Lys Arg Asp Gly Ala Thir Ser Wall Glu SO 55 6 O
Glu Ile Ala Thir Ala Lell Ala Glu Tyr ASn Ala Pro Luell Glin Glu 65 70 7s 8O
Ile Luell Ala Asp Thir Ile Ser Met Luell Glin Glu Lell Ser Asp Lys Gly 85 90 95
Wall Wall Ala 1OO
SEQ ID NO 22 LENGTH: 1161. TYPE: DNA ORGANISM: Hyphomicrobium denitrificans FEATURE: NAME/KEY: CDS LOCATION: (1) . . (1161)
<4 OOs, SEQUENCE: 22 atg aac gala at C gca cc.g ttg gag cag titt tog C Ca gac CC a gtg gcg 48 Met Asn Glu Ile Ala Pro Lell Glu Glin Phe Ser Pro Asp Pro Wall Ala 1. 1O 15 gaa gt C tgc gca cgc gcc cc.g gt C ggc Ctg citc. gcg gaa Ctg acg Cat 96 Glu Wall Cys Ala Arg Ala Pro Wall Gly Luell Luell Ala Glu Luell Thir His 2O 25 3O cgc tgc cc.g citt Cag tgt cott tac tgc tog aac cc.g citc. gag citt gac 144 Arg Cys Pro Luell Glin Cys Pro Tyr Cys Ser ASn Pro Lell Glu Luell Asp 35 4 O 45 cgc gt C aac acg gaa ttg acg acg gcc gag tgg Cag gac gtC atg cgt 192 Arg Wall Asn Thir Glu Lell Thir Thir Ala Glu Trp Glin Asp Wall Met Arg SO 55 6 O
Cag gca gcc gala citc. ggc atc. Ctg cag at C CaC citt tcg ggt ggc gag 24 O Glin Ala Ala Glu Lell Gly Ile Luell Glin Ile His Lell Ser Gly Gly Glu 65 70 7s 8O cc.g acg citt cgc aag gat citt gag gat at C gtc gat gtg gcg gcg aag 288 Pro Thir Luell Arg Lys Asp Lell Glu Asp Ile Wall Asp Wall Ala Ala Lys 85 90 95 gct ggc citc. tac acg aac citc. at C acg gcg ggc gtg acg Ctg acc gala 336 Ala Gly Luell Tyr Thir Asn Lell Ile Thir Ala Gly Wall Thir Luell Thir Glu 1OO 105 11 O
US 2013/03375 11 A1 Dec. 19, 2013 27
- Continued
<4 OOs, SEQUENCE: 23 Met Asin Glu Ile Ala Pro Leu Glu Glin Phe Ser Pro Asp Pro Val Ala 1. 5 1O 15 Glu Val Cys Ala Arg Ala Pro Val Gly Lieu. Lieu Ala Glu Lieu. Thir His 2O 25 3O Arg Cys Pro Lieu. Glin Cys Pro Tyr Cys Ser Asn Pro Lieu. Glu Lieu. Asp 35 4 O 45 Arg Val Asn. Thr Glu Lieu. Thir Thr Ala Glu Trp Glin Asp Wal Met Arg SO 55 6 O Glin Ala Ala Glu Lieu. Gly Ile Lieu. Glin Ile His Lieu. Ser Gly Gly Glu 65 70 7s 8O Pro Thir Lieu. Arg Lys Asp Lieu. Glu Asp Ile Val Asp Val Ala Ala Lys 85 90 95 Ala Gly Lieu. Tyr Thr Asn Lieu. Ile Thr Ala Gly Val Thr Lieu. Thr Glu 1OO 105 11 O Asp Arg Lieu Lys Llys Lieu. Glin Asp Lieu. Gly Lieu. Asp His Val Glin Lieu 115 12 O 125 Ser Ile Glin Asp Wall Asp Asp Ala Asn Ala Glu Arg Met Ser Ala Tyr 13 O 135 14 O Lys Gly Gly Lieu Ala Lys Lys Arg Glu Val Gly Llys Trp Val Arg Llys 145 150 155 160 Lieu. Gly Met Pro Lieu. Thir Ile Asn Ala Pro Ile His Arg Phe Asin Ile 1.65 17O 17s Glu Asn Lieu Pro Asn. Ile Ile Asp Phe Ala Val Glu Met Gly Ala Gly 18O 185 19 O Arg Ile Glu Val Ala Asn. Ile Glin Tyr Tyr Ala Trp Ala Lieu Lys Asn 195 2OO 2O5 Arg Ala Ser Lieu Met Pro Thr Arg Ala Glin Val Ile Llys Ser Ala Glu 21 O 215 22O Ile Val Glu Glu Ala Lys Glu Arg Lieu Lys Gly Ile Lieu Val Phe Asp 225 23 O 235 24 O Phe Val Val Pro Asp Tyr Tyr Ala Lys Thr Pro Llys Pro Cys Met Gly 245 250 255 Gly Trp Gly Arg Gly Val Met Asn Val Thr Pro Glin Gly Llys Val Lieu. 26 O 265 27 O Pro Cys His Ala Ser Glu Thir Ile Pro Gly Lieu. Ile Phe Asp Asn Val 27s 28O 285 Lys Asp Arg Arg Lieu Ala Asp Ile Trp Lieu. Asn Gly Glin Ala Phe Glin 29 O 295 3 OO Lys Tyr Arg Gly Thr Ser Trp Met Lys Glu Pro Cys Arg Ser Cys Pro 3. OS 310 315 32O Arg Ala Glu Ile Asp Phe Gly Gly Cys Arg Cys Glin Ala Met Ala Phe 3.25 330 335 Thr Gly Asp Ala Asp Asn. Thir Asp Pro Ala Cys Llys Phe Ser Pro Tyr 34 O 345 35. O
His Ala Ala Phe Val Ser Ala Ala Glu Glin Glu Ser Ala Glu Ala Ala 355 360 365 Pro Pro Pro Phe Val Tyr Arg Arg Met Gly Pro Ile Lys Ala Thir Ser 37 O 375 38O
Asp Asn 385
US 2013/03375 11 A1 Dec. 19, 2013 30
- Continued <4 OOs, SEQUENCE: 26 Met Glin Glu Ser Lieu Lleu Lys Glu Pro Glu Ala Lys Thir Trp Val Ala 1. 5 1O 15 Pro Ala Tyr Cys Asp Lieu. Arg Lieu. Gly Phe Glu Val Thr Ala Tyr Ile 2O 25 3O Tyr Val Arg 35
<210s, SEQ ID NO 27 &211s LENGTH: 102 &212s. TYPE: DNA <213> ORGANISM: Hyphomicrobium denitrificans 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (102)
<4 OOs, SEQUENCE: 27 atg gag gac at C atg aag acc tig aca aag cct gcc gtt cqc gag cag 48 Met Glu Asp Ile Met Lys Thir Trp Thr Llys Pro Ala Val Arg Glu Gln 1. 5 1O 15 gaa gtC ggc ct c gala gtt acc tog tac Ctt cog gcc gag at C gaC ct c 96 Glu Val Gly Lieu. Glu Val Thir Ser Tyr Lieu Pro Ala Glu Ile Asp Lieu. 2O 25 3O atc tala Ile
<210s, SEQ ID NO 28 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Hyphomicrobium denitrificans <4 OOs, SEQUENCE: 28 Met Glu Asp Ile Met Lys Thir Trp Thr Llys Pro Ala Val Arg Glu Gln 1. 5 1O 15 Glu Val Gly Lieu. Glu Val Thir Ser Tyr Lieu Pro Ala Glu Ile Asp Lieu. 2O 25 3O
Ile
<210s, SEQ ID NO 29 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG539 <4 OOs, SEQUENCE: 29 tcgc.cgagct caaatgctg
<210s, SEQ ID NO 3 O &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG54o <4 OOs, SEQUENCE: 30 at actgcagc gacgc.cttct tagttgaag 29
<210s, SEQ ID NO 31 &211s LENGTH: 2O &212s. TYPE: DNA US 2013/03375 11 A1 Dec. 19, 2013 31
- Continued <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG568 <4 OOs, SEQUENCE: 31 aatctatogcc gtgtc.gcatc
<210s, SEQ ID NO 32 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG569 <4 OOs, SEQUENCE: 32 at at ct agaic gtcaaatticg gagatgat cq 3 O
<210s, SEQ ID NO 33 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG57o <4 OOs, SEQUENCE: 33 at atctagat gatcqacaaa totgttgcago 3 O
<210s, SEQ ID NO 34 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG571 <4 OOs, SEQUENCE: 34 ataggat.cca Ctgcgctacc gaacgtag 28
<210s, SEQ ID NO 35 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG572 <4 OOs, SEQUENCE: 35 ataggat.cca cacgcagacg tdatagac 28
<210s, SEQ ID NO 36 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer EG573 <4 OOs, SEQUENCE: 36 atagagct c gaagcgatta attgcttgc 29
<210s, SEQ ID NO 37 &211s LENGTH: 23 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens
<4 OO > SEQUENCE: 37 Met Trp His Llys Pro Ala Tyr Thr Asp Lieu. Arg Ile Gly Phe Glu Val US 2013/03375 11 A1 Dec. 19, 2013 32
- Continued
1O 15 Thr Met Tyr Phe Ala Asn Arg 2O
<210s, SEQ ID NO 38 &211s LENGTH: 24 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens
<4 OOs, SEQUENCE: 38 Met Thr Trp Ser Lys Pro Ala Tyr Thr Asp Lieu. Arg Ile Gly Phe Glu 1. 5 1O 15 Val Thr Met Tyr Phe Ala Ser Arg
<210s, SEQ ID NO 39 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Ralstonia pickettii
<4 OOs, SEQUENCE: 39 Met Ala Trp Glin Llys Pro Glu Ala Thr Asp Lieu. Arg Phe Gly Phe Glu 1. 5 1O 15 Ile Thr Met Tyr Ile Ala Asn Arg 2O
<210s, SEQ ID NO 4 O &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Ralstonia pickettii <4 OOs, SEQUENCE: 4 O Met Asn Trp Thir Thr Pro Ala Tyr Thr Glu Lieu. Arg Lieu. Gly Phe Glu 1. 5 1O 15 Ile Thr Met Tyr Ile Ala Asn Arg 2O
<210s, SEQ ID NO 41 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Methylotenera mobilis
<4 OOs, SEQUENCE: 41 Met Trp Thir Thr Pro Ala Ala Thr Glu Met Arg Phe Gly Phe Glu Val 1. 5 1O 15 Thr Met Tyr Val Met Asn Lys 2O
<210s, SEQ ID NO 42 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Bradyrhizobium sp.
<4 OOs, SEQUENCE: 42 Met Ala Trp Lys Ala Pro Lys Ile Val Glu Val Pro Val Gly Met Glu 1. 5 1O 15 Ile Asn Met Tyr Ala Cys Ala Ala Arg Llys 2O 25
<210s, SEQ ID NO 43 US 2013/03375 11 A1 Dec. 19, 2013 33
- Continued
&211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Bradyrhizobium sp.
<4 OOs, SEQUENCE: 43 Met Ala Trp Llys Thr Pro Lys Ile Val Glu Val Pro Val Gly Met Glu 1. 5 1O 15 Ile Asn Met Tyr Ala Cys Ala Ala Arg Llys 2O 25
<210s, SEQ ID NO 44 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Bradyrhizobium sp.
<4 OOs, SEQUENCE: 44 Met Ser Trp Thr Ala Pro Lys Ile Val Glu Val Pro Val Gly Met Glu 1. 5 1O 15 Ile Asn Met Tyr Ala Cys Ala Ser Arg Lys Ala Glu Arg Arg Ser 2O 25 3O
<210s, SEQ ID NO 45 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Methylococcus capsulatus
<4 OOs, SEQUENCE: 45 Met Arg Trp Glu Lys Pro Ser Tyr Asn Asp Met Arg Phe Gly Phe Glu 1. 5 1O 15 Val Thr Met Tyr Ile Tyr Asn Arg 2O
<210s, SEQ ID NO 46 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Colwellia psychrerythraea <4 OOs, SEQUENCE: 46 Met Trp Thr Llys Pro Llys Phe Glu Glu Met Arg Lieu. Gly Phe Glu Val 1. 5 1O 15 Thr Lieu. Tyr Ile Ser Asn Arg 2O
<210s, SEQ ID NO 47 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Gluconobacter oxydans <4 OOs, SEQUENCE: 47 Met Ala Trp Asn Thr Pro Llys Val Thr Glu Ile Pro Leu Gly Ala Glu 1. 5 1O 15 Ile Asin Ser Tyr Val Cys Gly Glu Llys Llys 2O 25
<210s, SEQ ID NO 48 &211s LENGTH: 52 212. TYPE: PRT <213> ORGANISM: Methylobacillus flagellatus
<4 OOs, SEQUENCE: 48 Met Ser Lys Lieu. Gly Lieu. Tyr Asp Gly Asn His Gly Lieu. Arg Gly Thr US 2013/03375 11 A1 Dec. 19, 2013 34
- Continued
1. 5 1O 15 Asp Cys Ala Ala Ser Thr His Lieu Lys Arg Arg Lieu. Ile Met Trp Thr 2O 25 3O Llys Pro Glu Val Thr Glu Met Arg Phe Gly Phe Glu Val Thr Met Tyr 35 4 O 45 Val Cys Asn Arg SO
<210s, SEQ ID NO 49 &211s LENGTH: 32 212. TYPE: PRT <213> ORGANISM: Dinoroseobacter shibae
<4 OOs, SEQUENCE: 49 Met Ala Trp Thr Lys Pro Ile Ile Arg Glu Ile Glu. Cys Gly Met Glu 1. 5 1O 15 Ile Asn Met Tyr Gly Pro Asp Ser Asp Glu Glu Arg Glu Val Lieu. Phe 2O 25 3O
<210s, SEQ ID NO 50 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Erwinia amylovora <4 OOs, SEQUENCE: 50 Met Gln Trp Thr Llys Pro Thr Phe Ile Asp Met Arg Lieu. Gly Lieu. Glu 1. 5 1O 15 Val Thr Lieu. Tyr Ile Ser Asn Arg 2O
<210s, SEQ ID NO 51 &211s LENGTH: 60 212. TYPE: PRT <213> ORGANISM: Leptothrix discophora
<4 OOs, SEQUENCE: 51 Met Val Lieu Ala Met Thr Pro Arg Ala Pro Ala Pro Thr Lieu. His Pro 1. 5 1O 15 His Glin His Thr Lieu. Leu Ala Ser Lieu. Thr Arg Thr Ala Asn Pro Ile 2O 25 3O Glu Gly Asp Leu Met Thr Trp Thir Thr Pro Ala Ala Cys Asp Phe Arg 35 4 O 45 Phe Gly Phe Glu Ile Thr Met Tyr Ile Ala Ala Arg SO 55 6 O
<210s, SEQ ID NO 52 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: conserved sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (4) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<4 OOs, SEQUENCE: 52 Glu Xaa Xala Xala Tyr 1. 5 US 2013/03375 11 A1 Dec. 19, 2013
1. A method for producing pyrroloquinoline quinone a DNA that encodes a protein comprising the amino acid (PQQ) comprising: sequence of SEQID No. 2; A) cultivating in a culture medium a bacterium belonging a DNA that encodes a protein comprising the amino acid to the genus Hyphonicrobium, and sequence of SEQ ID NO: 2, but that contains substitu B) collecting PQQ from the culture medium, tions, deletions, insertions, additions or inversions of wherein the bacterium has been modified to enhance expres one or several amino acid residues, and said protein has Sion of pdq gene cluster. the function of PQQ precursor: 2. The method according to claim 1, wherein said paq gene a DNA that encodes a protein comprising the amino acid cluster is the pdqABC/DE operon from Methylobacterium sequence of SEQID No. 11; extorquens. a DNA that encodes a protein comprising the amino acid 3. The method according to claim 2, wherein said pqqABC/ sequence of SEQID NO: 11, but that contains substitu DE operon comprises DNA comprising the nucleotide tions, deletions, insertions, additions or inversions of sequences of SEQID NO: 1, SEQID NO:3, SEQID NO: 5, one or several amino acid residues, and said protein has and SEQID NO: 7, or variants thereof. the function of PQQ precursor: 4. The method according to claim 1, wherein said paq gene a DNA that encodes a protein comprising the amino acid cluster is the pdqABCDE cluster from Hyphomicrobium sequence of SEQID No:13; denitrificans. a DNA that encodes a protein comprising the amino acid 5. The method according to claim 4, wherein said pdqAB sequence of SEQID NO: 13, but that contains substitu CDE cluster comprises DNA comprising the nucleotide tions, deletions, insertions, additions or inversions of sequences of SEQID NO: 14, SEQID NO: 16, SEQID NO: one or several amino acid residues, and said protein has 18, SEQID NO: 20, and SEQID NO:22, or variants thereof. the function of PQQ precursor: 6. The method according to claim 1, wherein the expres a DNA that encodes a protein comprising the amino acid sion of said gene cluster is enhanced by increasing the copy sequence of SEQID No: 15; number of said pqq gene cluster. a DNA that encodes a protein comprising the amino acid 7. The method according to claim 6, wherein the increasing sequence of SEQID NO: 15, but that contains substitu said copy number is achieved by introducing into the bacte tions, deletions, insertions, additions or inversions of rium a vector comprising said gene cluster. one or several amino acid residues, and said protein has 8. The method according to claim 1, wherein said bacte the function of PQQ precursor: rium is Hyphomicrobium denitrificans. a DNA that encodes a protein comprising the amino acid 9. The method according to claim 1, wherein said culture sequence of SEQID No. 26: medium comprises methanol as a carbon source. a DNA that encodes a protein comprising the amino acid 10. A method for producing pyrroloquinoline quinone sequence SEQ ID NO: 26, but that contains substitu (PQQ) comprising: tions, deletions, insertions, additions or inversions of cultivating in a culture medium a bacterium belonging to one or several amino acid residues, and said protein has the genus Methylobacterium or Hyphomicrobium, the function of PQQ precursor: wherein said bacterium has enhanced expression of pdq a DNA that encodes a protein comprising the amino acid gene cluster, and collecting PQQ from the culture sequence of SEQID No. 28; medium, a DNA that encodes a protein comprising the amino acid wherein said bacterium has been further modified to sequence of SEQID NO: 28, but that contains substitu enhance expression of pdqA-like gene(s). tions, deletions, insertions, additions or inversions of 11. The method according to claim 10, wherein said pqq one or several amino acid residues, and said protein has gene cluster is the pdqABC/DE operon from Methylobacte the function of PQQ precursor, and rium extorquens. combinations thereof. 12. The method according to claim 11, wherein said 16. The method according to claim 10, wherein the expres pqqABC/DE operon comprises DNA comprising the nucle sion of said gene cluster or gene(s) is/are enhanced by otide sequences of SEQID NO: 1, SEQID NO:3, SEQ ID increasing the copy number of said gene cluster or gene(s). NO: 5, and SEQID NO: 7, or variants thereof. 17. The method according to claim 16, wherein the increas 13. The method according to claim 6, wherein said pqq ing the copy number is achieved by introducing into the gene cluster is the pdqABCDE cluster from Hyphomicrobium bacterium a vector comprising said gene cluster or gene(s). denitrificans. 18. The method according to claim 10, wherein said bac 14. The method according to claim 10, wherein said terium is Hyphomicrobium denitrificans. pqqABCDE cluster comprises DNA comprising nucleotide 19. The method according to claim 10, wherein said bac sequences of SEQID NO: 14, SEQID NO: 16, SEQID NO: terium is Methylobacterium extorquens. 18, SEQID NO: 20, and SEQID NO:22, or variants thereof. 20. The method according to claim 10, wherein said culture 15. The method according to claim 11, wherein said pdqA medium comprises methanol as a carbon source. like gene is selected from the group consisting of k k k k k