DEHYDROGENASE-CATALYSED PRODUCTION OF FDCA
20180030488 ยท 2018-02-01
Assignee
Inventors
Cpc classification
C12P17/04
CHEMISTRY; METALLURGY
C12N9/0008
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a cell expressing a polypeptide having 5-hydroxymethyl-2-furancarboxylic acid dehydrogenase activity, as well as to a cell expressing a polypeptide having furanic compound transport capabilities. The invention also relates to a process for the production of 2,5-furan-dicarboxylic acid (FDCA) wherein the cells of the invention are used for oxidation of a furanic precursors of FDCA.
Claims
1.-15. (canceled)
16. A process for oxidizing 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to 5-formyl-2-furoic acid (FFA), the process comprising incubating a cell in the presence of HMFCA, wherein the cell comprises an expression construct for expression of a nucleotide sequence encoding an HMFCA dehydrogenase having an amino acid sequence with at least 45% identity with any one of the amino acid sequence of SEQ ID NO: 1 to 11, and wherein, the expression construct is expressible in the cell and expression of the dehydrogenase confers to or increases in the cell the ability to oxidize HMFCA to FFA, as compared to a corresponding wild type cell lacking the expression construct.
17. The process according to claim 16, wherein the incubating is under conditions conducive to the oxidation of HMFCA by the cell.
18. A process for producing FDCA, comprising incubating a cell in a medium comprising one or more furanic precursors of FDCA, and, optionally recovery of the FDCA, wherein the cell comprises an expression construct for expression of a nucleotide sequence encoding an HMFCA dehydrogenase having an amino acid sequence with at least 45% identity with any one of the amino acid sequence of SEQ ID NO: 1 to 11, wherein the expression construct is expressible in the cell and expression of the HMFCA dehydrogenase confers to or increases in the cell the ability to oxidize HMFCA to FFA, as compared to a corresponding wild type cell lacking the expression construct.
19. The process according to claim 18, wherein the incubating is under conditions conducive to the oxidation of furanic precursors of FDCA by the cell to FDCA.
20. The process according to claim 18, wherein at least one furanic precursor of FDCA is selected from the group consisting of HMF, 2,5-dihydroxymethyl furan (DHF), HMFCA, FFA and 2,5-diformyl furan (DFF).
21. The process according to claim 20, wherein at least one furanic precursor of FDCA is HMF.
22. The process according to claim 18, wherein the furanic precursors of FDCA are obtained from one or more hexose sugars, optionally by acid-catalyzed dehydration.
23. The process according to claim 18, comprising recovering the FDCA from the medium by acid or salt precipitation followed by cooling crystallization and/or solvent extraction.
24. A process for producing a polymer from one or more FDCA monomers, comprising: (a) preparing a FDCA monomer in a process according to claim 18; and, (b) producing a polymer from the FDCA monomer obtained in (a).
25. A method of biotransformation of one or more furanic precursors to FDCA, comprising expressing in a cell an expression construct for expression of a nucleotide sequence encoding an HMFCA dehydrogenase having an amino acid sequence with at least 45% identity with any one of the amino acid sequence of SEQ ID NO: 1 to 11, wherein, the expression of the HMFCA dehydrogenase confers to or increases in the cell the ability to oxidize HMFCA to FFA, as compared to a corresponding wild type cell lacking the expression construct.
26. The method according to claim 25, wherein at least one furanic precursor of FDCA is selected from the group consisting of HMF, DHF, HMFCA, FFA and DFF.
27. A cell comprising an expression construct for expression of a nucleotide sequence encoding an dehydrogenase having an amino acid sequence with at least 81.65% identity with the amino acid sequence of SEQ ID NO: 1, wherein, the expression construct is expressible in the cell and expression of the HMFCA dehydrogenase confers to or increases in the cell the ability to oxidize HMFCA to FFA, as compared to a corresponding wild type cell lacking the expression construct.
28. A cell according to claim 27, wherein the cell further comprises at least one of: (a) an aldehyde dehydrogenase activity that oxidizes furanic aldehydes to the corresponding furanic carboxylic acids, and, (b) the ability of transporting furanic compounds into and/or out of the cell.
29. The cell according to claim 28, further comprising a second expression construct for expression of a nucleotide sequence encoding an aldehyde dehydrogenase comprising an amino acid sequence with at least 45% identity with any one of the amino acid sequence SEQ ID NO: 24, 25, 26, 27, 28, 29 and 30, wherein, the second expression construct is expressible in the cell and expression of the aldehyde dehydrogenase confers to or increases in the cell at least one of the abilities of i) oxidizing 5-hydroxymethylfurfural (HMF) to HMFCA, ii) oxidizing DFF to FFA, and iii) oxidizing FFA into FDCA, as compared to a corresponding wild type cell lacking the second expression construct.
30. The cell according to claim 28, further comprising a third expression construct for expression of a nucleotide sequence encoding a polypeptide having the ability to transport at least HMFCA into the cell, which polypeptide comprises an amino acid sequence with at least 45% identity with any one of the amino acid sequence SEQ ID NO's: 17, 31, 32, 33 and 34, wherein, the third expression construct is expressible in the cell and expression of the polypeptide confers to or increases in the cell the ability to transport at least HMFCA into the cell, as compared to a corresponding wild type cell lacking the third expression construct.
31. The cell according to claim 27, wherein the cell is a microbial cell.
32. The cell according to claim 31, wherein the microbial cell is a yeast or filamentous fungal cell selected from a genus from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Agaricus, Aspergillus, Aureobasidium, Myceliophthora, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
33. The cell according to claim 32, wherein the yeast or filamentous fungal cell is selected from a species from the group consisting of Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Pichia pastoris, Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae, Myceliophthora thermophila, Trichoderma reesei and Penicillium chrysogenum.
34. The cell according to claim 31, wherein the microbial cell is a bacterial cell selected from a genus from the group consisting of Escherichia, Anabaena, Aeribacillus, Aneurinibacillus, Burkholderia, Bradyrhizobium, Caulobacter, Cupriavidus, Desulfotomaculum, Desulfurispora, Gluconobacter, Rhodobacter, Pelotomaculum, Pseudomonas, Paracoccus, Bacillus, Geobacillus, Brevibacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Ralstonia, Rhodopseudomonas, Staphylococcus and Streptomyces.
35. The cell according to claim 34, wherein the bacterial cell is selected from a species from the group consisting of A. pallidus, A. terranovensis, B. subtilis, B. amyloliquefaciens, B. coagulans, B. kribbensis, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, B. thermoruber, B. panacihumi, C. basilensis, D. kuznetsovii, D. thermophila, G. kaustophilus, Gluconobacter oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pelotomaculum thermopropionicum, Pseudomonas zeaxanthinifaciens, Pseudomonas putida, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium meliotiand Rhizobium radiobacter.
36. A nucleic acid vector molecule comprising at least one of: (a) a nucleotide sequence encoding the polypeptide having HMFCA dehydrogenase activity, which polypeptide comprises an amino acid sequence that has at least 81.65% sequence identity with the amino acid sequence of SEQ ID NO: 1; (b) a nucleotide sequence as set out in SEQ ID NO: 12 or 13; (c) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of (b) or (c) due to the degeneracy of the genetic code; and, (d) a nucleotide sequence which is the reverse complement of a nucleotide sequence as defined in (a) to (d).
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
General Methodology
Strains and Plasmids
[0142] Pseudomonas putida S12gcd or P. putida KT2440gcd (glucose-dehydrogenase deficient mutants of P. putida S12 (ATCC 700801), resp., P. putida KT2440 (DSM6125)), or wild type P. putida S12, were used as the host for expression of the yiaY gene from Aeribacillus pallidus strain CA1828 (see below). Escherichia coli strain TG90 was used for general cloning purposes.
[0143] For episomal expression of the A. pallidus gene the pBBR1MCS-derived pBTmcs (Koopman et al., 2010a, Biores Technol 101: 6291-6196) was used. In pBTmcs the expression of the target gene is driven from the constitutive tac promoter.
Media & Culture Conditions
[0144] Mesophile mineral salts medium (MMM) contained the following (per liter of demineralized water): 15.52 g of K.sub.2HPO.sub.4, 6.52 g of NaH.sub.2PO.sub.4, 2.0 g of (NH.sub.4).sub.2SO.sub.4, 0.1 g of MgCl.sub.2.6H.sub.20, 10 mg of EDTA, 2 mg of ZnSO.sub.4.7H.sub.20, 1 mg of CaCl.sub.2.2H.sub.20, 5 mg of FeSO.sub.4.7H.sub.20, 0.2 mg of Na.sub.2MoO.sub.4.2H.sub.20, 0.2 mg of CuSO.sub.4.5H.sub.20, 0.4 mg of CoCl.sub.2.6H.sub.20, and 1 mg of MnCl.sub.2.2H.sub.20, supplemented with a carbon source as specified.
[0145] Thermophile mineral salts medium (TMM) contained the following (per liter of demineralized water): 10 g of Bis-Tris, 10 M FeSO.sub.4.7H2O, 4 mM tricine, 1.32 mM K2HPO4, 9.53 mM NH4Cl, 0.2 g yeast extract, 5 g of NaCl, 1.47 g of Na2SO4, 0.08 g of NaHCO3, 0.25 g of KCl, 1.87 g of MgCl2.6H2O, 0.41 g of CaCl2.2H2O, 0.008 g of SrCl2.6H2O, 0.008 g of H3BO3, 0.90 g of NaNO3, and 1 ml of vitamin solution (Thiamine, 0.1 g/L; Riboflavin, 0.1 g/L; Nicotinic acid, 0.5 g/L, Panthothenic acid, 0.1 g/L; Pyridoxamine-HCl, 0.5 g/L; Pyridoxal-HCl, 0.5 g/L; D-Biotin, 0.1 g/L; Folic acid, 0.1 g/L; p-Aminobenzoic acid, 0.1 g/L; Cobalamin, 0.1 g/L). Carbon sources were supplemented as specified.
[0146] As complete medium for propagation of mesophiles, Luria-Bertani (LB) broth was used: 10 g/l Bacto trypton (Difco), 5 g/l yeast extract (Difco), 10 g/l NaCl. For plate culturing, LB was solidified with 1.5% (w/v) of agar (Difco). For selection of either E. coli, P. putida S12 or P. putida KT2440 transformants carrying pBTmcs-derived plasmids, 50 g/ml of kanamycin (Km) was added to the media. Antibiotics were purchased from Sigma-Aldrich. P. putida was cultured at 30 C.; E. coli was cultured at 37 C.
[0147] As complete medium for propagation of thermophiles, TGP broth was used: 17 g/L trypton, 3 g/L soy pepton, 5 g/L NaCl, 2.5 g/L K2HPO4, 4 g/L glycerol and 4 g/L Na-pyruvate (pH7). For plate culturing, TGP was solidified with 1.5% (w/v) of agar (Difco). Aeribacillus pallidus was cultivated at 60 C.
Assays & Analytical Methods
Cell Dry Weight (CDW) Measurement:
[0148] CDW content of bacterial cultures was determined by measuring optical density at 600 nm (OD.sub.600) using a Biowave Cell Density Meter (WPA Ltd) or a Quant MQX200 universal microplate spectrophotometer (Biotek), using flat-bottom 96-well microplates (Greiner). An OD.sub.600 of 1.0 corresponds to 0.56 g CDW/L (Biowave) or 1.4 g CDW/L (Quant) for P. putida.
HPLC Analyses:
[0149] Furan compounds (FDCA, HMF, HMF-alcohol, HMFCA and FFA) were analyzed by RP-HPLC as described by Koopman et al. (2010a, Biores Technol 101: 6291-6196).
Chemicals
[0150] 5-Hydroxymethylfurfural (HMF) was purchased at Eurolabs Ltd (Poynton, UK). Analytical standards of FDCA and 5-hydroxymethyl-furoic acid (HMFCA) were purchased from Immunosource B.V. (Halle-Zoersel, Belgium), respectively, Matrix Scientific (Columbia S.C., USA). All other chemicals were purchased from Sigma-Aldrich Chemie B.V. (Zwijndrecht, The Netherlands).
Molecular and Genetic Techniques:
[0151] Genomic DNA was isolated from A. pallidus CA1828 using the MasterPure Gram Positive DNA Purification Kit (Epicentre). Plasmid DNA was isolated with the JETSTAR Maxi Plasmid Purification Kit (GENOMED, ITK diagnostics). Agarose-trapped DNA fragments were isolated with the
[0152] DNA Clean & Concentrator (Zymo research). PCR reactions were performed with Phusion Flash PCR Master Mix (Thermo Scientific) according to the manufacturer's instructions. Oligonucleotide primers (specified in the examples) were synthesized by Sigma-Aldrich. Plasmid DNA was introduced into electrocompetent cells using a Gene Pulser electroporation device (BioRad). Other standard molecular biology techniques were performed according to Sambrook and Russell (2001, supra).
Example I: Isolation of HMF Metabolizing Aeribacillus pallidus Strains
[0153] Compost (15 g) was mixed with 15 ml of 0.9% (w/v) NaCl solution and incubated for 40 min at 750 rpm and 80 C. The resulting compost slurry was incubated in TMM supplemented with 0.65 g/L of HMF in shake flasks at 60 C. and 180 rpm for 3 days. The culture was transferred at regular intervals to fresh TMM-HMF and plated on solid TMM-HMF. Single colonies were re-streaked on TMM-HMF and TGP plates, and reassessed for their ability to metabolize HMF and also FDCA. Two isolates that metabolized both HMF and FDCA (strain CA1809 and CA1828) were identified as Aeribacillus pallidus by 16S rDNA sequencing and selected for further study.
Example II: Identification of a Novel, Dehydrogenase-Catalysed HMF Catabolic Pathway in HMF Degrading A. pallidus Isolates
[0154] The genomes of A. pallidus strains CA1809 and CA1828 were sequenced through PacBio sequencing, and automated ORF calling and annotation was performed. In the annotated genomes, homologues were identified of the hmfABCDE genes of Cupriavidus basilensis HMF14 which constitute the furoic acid degradation cluster (Koopman et al., 2010, Proc Nat Acad Sci USA 107: 4919-4924).
[0155] Considering the ability of strains CA1809 and CA1828 to metabolize FDCA in addition to HMF strongly suggested that HMF was metabolized via FDCA as in C. basilensis HMF14. However, unexpectedly no homologue of the hmfFGH cluster of C. basilensis HMF14 was found which constitutes the degradation pathway from HMF to furoic acid via FDCA. This result suggested that an alternative pathway for the oxidation of HMF to FDCA, and possibly the subsequent decarboxylation to furoic acid, existed in the A. pallidus isolates. Mining the genomes for gene clusters that comprised genes encoding both oxidizing and decarboxylating activities resulted in the identification of a putative HMF degradation cluster, comprising genes encoding an alcohol dehydrogenase, an aldehyde dehydrogenase, and two decarboxylases (Tables 1 A and B). Together, these genes encode a putative pathway for the oxidation of HMF to FDCA, via hydroxymethylfuroic acid (HMFCA), as in C. basilensis HMF14 but involving an alcohol dehydrogenase activity for the oxidation of HMFCA to formylfuroic acid (FFA) rather than an oxidase activity.
TABLE-US-00001 TABLE 1 A Putative HMF degradation cluster of A. pallidus CA1809 Corresponding locus in locus % identity % similarity putative C. basilensis % identity % similarity ID best BLAST hit (x AA/y AA) (x AA/y AA) function HMF14 (x AA/y AA) (x AA/y AA) 03430 MFS transporter 86 93 MFS transporter hmfT1 60 75 [Geobacillus (384/446) (418/446) (270/450) (341/450) kaustophilus] Sequence ID: ref WP_011229501.1 03431 4-hydroxybenzoate 87 94 FDCA hmfF 52 68 decarboxylase (405/466) (442/466) decarboxylase (240/462) (315/462) [Geobacillus subunit kaustophilus] Sequence ID: ref| WP_011229502.1 03432 alcohol 82 92 HMFCA dehydrogenase (320/392) (363/392) dehydrogenase [Geobacillus kaustophilus] Sequence ID: ref| WP_011229504.1 03433 aldehyde 88 95 HMF/FFA aldh 37 55 dehydrogenase (429/488) (466/488) dehydrogenase (174/470) (261/470) [Geobacillus kaustophilus] Sequence ID: ref| WP_011229505.1 03434 phenolic acid 91 96 FDCA hmfG 54 74 decarboxylase (162/179) (172/179) decarboxylase (99/183) (136/183) subunit B subunit [Geobacillus kaustophilus] Sequence ID: ref| WP_011229508.1|
TABLE-US-00002 TABLE 1 B Putative HMF degradation cluster of A. pallidus CA1828 Corresponding locus in locus % identity % similarity putative C. basilensis % identity % similarity ID best BLAST hit (x AA/y AA) (x AA/y AA) function HMF14 (x AA/y AA) (x AA/y AA) 03227 MFS transporter 86 93 MFS transporter hmfT1 61 76 [Geobacillus (384/446) (418/446) (232/383) (293/383) kaustophilus] Sequence ID: ref| WP_011229501.1 03228 4-hydroxybenzoate 87 94 FDCA hmfF 52 68 decarboxylase (405/466) (442/466) decarboxylase (240/462) (315/462) [Geobacillus subunit kaustophilus] Sequence ID: ref| WP_011229502.1 03229 alcohol 82 92 HMFCA dehydrogenase (320/392) (363/392) dehydrogenase [Geobacillus kaustophilus] Sequence ID: ref| WP_011229504.1 03230 aldehyde 88 95 HMF/FFA aldh 37 55 dehydrogenase (429/488) (466/488) dehydrogenase (174/470) (261/470) [Geobacillus kaustophilus] Sequence ID: ref| WP_011229505.1 03231 phenolic acid 91 96 FDCA hmfG 54 74 decarboxylase (162/179) (172/179) decarboxylase (99/183) (136/183) subunit B subunit [Geobacillus kaustophilus] Sequence ID: ref| WP_011229508.1|
Example III: Expression of YiaY from A. pallidus in P. putida S12 Confers the Ability to Oxidize HMF to FDCA
[0156] The yiaY gene was cloned as a 1988-bp synthetic XbaI-SalI fragment (SEQ ID NO: 15), including the PldhL1 promoter region from B. coagulans DSM1, in pBTmcs yielding plasmid pKW007. Plasmid pKW007 was introduced into P. putida KT2440gcd (CA1877), yielding P. putida CA2101. P. putida KT2440gcd carrying pBTmcs (strain CA2046) was tested as an empty vector control.
[0157] P. putida strains CA2101 and CA2046 were grown in 100-ml shake flasks containing 10 ml of MM+80 mM glycerol and 2 mM glucose supplemented with 50 mg/L kanamycin. Cells were harvested at the end of the log phase (OD6004), washed and resuspended in MM supplemented with 19.4 g/L of K2HPO4, 8.15 g/L of NaH2PO4, 80 mM glycerol and 50 mg/L kanamycin. Aliquots (10 ml) of washed cell suspensions (at an OD600 of 1-2) were incubated with HMF in 100-ml Erlenmeyer flasks and samples were drawn at regular intervals for analysis of FDCA.
Example IV: Optimized Oxidation of HMF to FDCA Through Coexpression of YiaY from A. pallidus and Aldh and HmfT1 from C. basilensis HMF14
[0158] The yiaY gene of A. pallidus CA1828 was synthesized including the ribosome binding site TAGGAAAGGAAGATTAACCC (SEQ ID NO: 21). The yiaY fragment (SEQ ID NO: 16) was digested with KpnI and XbaI to replace the hmfH gene in pBThmfH-adh (WO2012064195) yielding plasmid pKW010. Plasmid pKW010 was introduced into pJNNhmfT1(t) (WO2012064195)-harbouring P. putida S12gcd yielding P. putida CA2111, and into P. putida KT2440gcd (also harbouring pJNNhmfT1(t)) yielding P. putida CA2112. Thus, the HMFCA oxidizing alcohol dehydrogenase encoded by yiaY could be co-expressed with the HMF dehydrogenase and the HMFCA transporter from C. basilensis HMF14 to eliminate the bottlenecks of HMF oxidation to HMFCA and HMFCA uptake.
[0159] P. putida CA2111 and CA2112 were grown in 100-ml shake flasks containing 10 ml of MM+80 mM glycerol and 2 mM glucose supplemented with 50 mg/L kanamycin, 30 mg/L of gentamicin and 100 M of salycilic acid. Cells were harvested at the end of the log phase (OD6004), washed and resuspended in MM 50 mg/L kanamycin, 30 mg/L gentamicin and 10 M of salicylic acid. Aliquots (10 ml) of washed cell suspensions (at an OD600 of 1-2) were incubated with HMF in 100-ml Erlenmeyer flasks and samples were drawn at regular intervals for analysis of FDCA.
Example V: Construction of Optimized Strains for Oxidation of HMF to FDCA Through Coexpression of Mesophilic HMFCA Alcohol Dehydrogenases and Aldh and HmfT1 from C. basilensis HMF14
[0160] The yiaY homologue of Bacillus kribbensis DSM17871, Brevibacillus thermoruber 423, Bacillus sp. FJAT-14578, and Bacillus sp. L1(2012) were synthesized including a ribosome binding site containing spacer TAGGAAAGGAAGATTAACCC (SEQ ID NO: 21) as well as recognition sites for restriction enzymes (KpnI, resp., NheI; compatible with XbaI)) for cloning (SEQ ID NO.'s: 19, 36, 38 and 39).
[0161] The yiaY homologues of Aneurinibacillus terranovensis DSM18919 and Brevibacillus panacihumi W25 were synthesized including a ribosome binding site containing spacer GAATTCCACATGACAAGGGGAGACCGC (SEQ ID NO: 40) as well as recognition sites for restriction enzymes (KpnI, resp., XbaI) for cloning (SEQ ID NO.'s: 35 and 37). The coding nucleotide sequence for the B. kribbensis enzyme (SEQ ID NO: 19), the B. thermoruber enzyme (SEQ ID NO: 36) and both Bacillus sp. enzymes (SEQ ID NO: 38 and 39) were obtained via reverse translation of the amino acid sequences (http://www.bioinformatics.org/sms2/rev_trans.html) using the P. putida codon usage table of http://www.kazusa.or.jp/codon/. The coding nucleotide sequence for the A. terranova and B. panacihumi enzyme was obtained via reverse translation of the amino-acid sequences using the E. coli sequence optimization tool of GeneArt (https://www.thermofisher.com/nl/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html).
[0162] The yiaY-homologue fragments of B. kribbensis, B. thermoruber, Bacillus sp. FJAT-14578, and Bacillus sp. L1(2012) were digested with KpnI and NheI (compatible with XbaI in pBThmfH-adh) to replace the hmfH gene in pBThmfH-adh (WO2012064195) yielding plasmids pKW2210, pKW2212, pKW2214, and pKW2215. The yiaY-homologue fragments of A. terranovensis and B. panacihumi were digested with KpnI and XbaI to replace the hmfH gene in pBThmfH-adh (WO2012064195) yielding plasmids pKW2211 and pKW2213.
[0163] Plasmids pKW2210, pKW2211, pKW2212, pKW2213, pKW2214 and pKW2215 were introduced into P. putida KT2440gcd_pJNNhmfT1 (CA1965), yielding, respectively, P. putida CA21780, CA21781, CA21782, CA21783, CA21784 and CA21785 for expression of the YiaY homologues in an optimized host background including aldh and hmfT1. For performance evaluation, P. putida strains CA21780, CA21781, CA21782, CA21783, CA21784 and CA21785 were grown in 100-ml shake flasks containing 10 ml of MM+80 mM glycerol and 2 mM glucose supplemented with 50 mg/L kanamycin, 30 mg/L of gentamicin and 100 M of salicylic acid. Cells were harvested at the end of the log phase (OD6004), washed and resuspended in MM 50 mg/L kanamycin, 30 mg/L gentamicin and 10 M of salicylic acid. Aliquots (10 ml) of washed cell suspensions (OD600 of 1-2) were incubated with HMF in 100-ml Erlenmeyer flasks and samples were drawn at regular intervals for analysis of FDCA. The results for P. putida CA21780, CA21781 and CA21783 are shown in
Example VI: Construction of a P. putida Strain Expressing the Aeribacillus pallidus proP Encoded HMFCA Transporter
[0164] The proP gene (SEQ ID NO: 18) was amplified from genomic DNA of Aeribacillus pallidus CA1828 by PCR using primers proP(f) (gccgaattcATGAAGAATATCGCTAATACG; SEQ ID NO: 22) and proP(r) (gccgctagcTTATTTGAGGTTTCCTTTTGTTTCC; SEQ ID NO: 23). The PCR product was introduced as a 1350-bp EcoRI-NheI fragment (SEQ ID NO: 20) in pJNNmcs(t) yielding pJNNproP(t). Plasmids pBThmfH aldh and pJNNproP(t) were successively introduced into P. putida KT2440gcd (CA1877), yielding P. putida CA21783. P. putida CA21783 was cultured in 100-ml shake flasks containing 10 ml of MM+80 mM glycerol and 2 mM glucose supplemented with 50 mg/L kanamycin, 30 mg/L of gentamicin and 100 M of salycilic acid. Cells were harvested at the end of the log phase (OD600=4), washed and resuspended in MM 50 mg/L kanamycin, 30 mg/L gentamicin and 10 M of salicylic acid. Aliquots (10 ml) of washed cell suspensions (OD600 of 1-2) were incubated with HMF in 100-ml Erlenmeyer flasks and samples were drawn at regular intervals for analysis of FDCA. It is clear that expression of the proP-encoded HMFCA transporter considerably accelerates the oxidation of HMF to FDCA as compared to a corresponding control strain that does not express proP.
TABLE-US-00003 TABLE2 YiaYaminoacidsequencealignment Adh_Bp ---------MESPFSFHLPTNVQFGVGSASRLGEMLLSMGVRRVFLVTDQGVRQAGLLDE Adh_Bk ---------MDVEFSFHLPTLIEFGFGKASLLGERLLKLGVGNVFLVSDKGVASAGLLQK Adh_Bt --MSQTVQGTDFAFSFHLPTLIEFGYGRASRLGERLQHLGVTNVFVVTDKGVEAAGLLNG Adh_At --MSPAVKAINFEFSFNLPTLIEFGYGKMEKFGQQLISIGVKRIFMVTDKGVESAGLLAA YiaY MIGNYAKKAIDFEFTFYLPTLIEFGYGKASRMGEMLEQMGIKNVFLVTDKGVEAAGLLAG Adh_Gk MVGHYIQKEVEFEFSFHLPTSIQFGYGKASQLGNQLVDMGIKSAFLVTDRGVEATGLLAG Adh_Bsp ---------MYPSFEFHLPTKIHFGYNTIKQLDH--LPFEIKRAFIVTDQGVLNSGLVEN Adh_BspL1 ------------------------------------------------------------ Adh_Pt ------------------------------------------------------------ Adh_Dk ------------------------------------------------------------ Adh_Dt -----------------MKTTVCFGANIVSSIDDRCRDYNARHVLIVTDQGVEKAGILEK Adh_Bp VIHSLEEKGLHFQIYADVEPDPSLETIQAGAAMFQQQSFDCMVAIGGGSPIDTAKGIRVL Adh_Bk LEQSLQTSDIHFKTYLEVEPDPSLETIDLGARAFNSGKYDCIVAVGGGSAIDTAKGIRVV Adh_Bt LVGSLQSAGIAFDLYTEVEPDPGLETIDRGAAVFRAKPYDCLVAVGGGSPIDAAKGMRVV Adh_At LTDSLQAAAIQFDIYTDVESDPSLETIDRGVEVFQQKPYDCIVAVGGGSPIDTAKGIRVV YiaY IVQSLESSNIRYVIYSDVEPDPSLETIDRGASVFKEQSFDCILAVGGGSPIDTAKGIRVV Adh_Gk IIQSLESSNIQYCVYADVEPDPSLETIDQGAAAFKEQPFDCIVAIGGGSPIDTAKGIRVV Adh_Bsp VTNILKDHQISYVIYSEVEPDPSVETVDKAAQMFQREEADALIAIGGGSPIDTAKGVRVI Adh_BspL1 ---------------------PSVETVDKAAKAFAEAECDLLIAVGGGSPIDTAKGVRVV Adh_Pt -----------------VEPDPGLETVHKAAAFLGRTRPDCLVALGGGSSIDVAKGARVI Adh_Dk --------------------DPGLETIHRCASCFRENKCDLILAVGGGSPIDTAKGARVI Adh_Dt VEKVLSDAGIENVVFDDVEPDPGLETIHRCASCFRENKCDLFLAIGGGSPIDTAKGARII *.:**:..:*::*:******.****:: Adh_Bp AANGGGIGQYAGVNRVPAASAIPLIAIPTTSGTGSEVTIFGVYSDWENHVKITVTSPHMA Adh_Bk AGNGGSIGDFAGVDKIGKAPQIPLIAVPTTSGTGSEVTIFGVYSDWVKNVKVTVTSQYMA Adh_Bt TSCGGSIADYAGVNRVPMAPAVPLVAVPTTSGTGSEVTMFGVYSDWHNHVKVTVTSPHMA Adh_At AANGGNIGHYAGVNQIPVAPTIPLLAIPTTSGTGSEVTNFGVYSDWQNNVKVTVTSQYMA YiaY VTNGGNIGDYAGVNRVAKKSEIPLVAVPTTSGTGSEVTIFGVYSDWENQVKVTVTSPYMA Adh_Gk ATNGGSIGDYAGVNRIKKKSEIPLIALPTTSGTGSEVTIFGVYSDWKNNVKVTVTSPYMA Adh_Bsp AGNGGSIRDYAGVNLIKQKSNIPLTATPTTSGTGSEVTIFAVFSDWEENRKVTVTSPFLA Adh_BspL1 ASNGGSIRNYSGVNLVKEAPSVPLVAIPTTAGTGSEVTIFAVFSDDKENRKVTVTSSHLS Adh_Pt YDNGGKISDYAGVNKVKVKPSLPLMAVPTTAGTGSEVTVFAVLSDWEQNIKITVTSEYLA Adh_Dk VENGGHIRDYAGVNKVPRAPVTPLTATPTTSGTGSEVTTFAVLSDWENRMKITISSPFLA Adh_Dt VDNGGHIRDYAGVNKVPRAPRTPLLAIPTTSGTGSEVTTFAVLSDWENRMKITISSPFLA ***.::**::**:*:***:********.***:.*:*::*.:: Adh_Bp PSTALIDPALTLSLPAKMTAATGIDALAHGIETFFSLRSSPASDALAIHAMKMIAPHLRR Adh_Bk PTIALVDPELTMRLPRKMTAASGIDALAHGIESYFSLRSTSASRALSLEAINIVGNHLRQ Adh_Bt PTIALVDPALTVSLPAKMTAASGIDALAHGIETFFSVRSRPASDALAMEAIAAVNAHLRR Adh_At PTIAWVDPALTMSLPAKMTAASGIDALAHGIETFFSLGSSPASDALAIEAIHTVNRYLSR YiaY PEIALVDPELTMSLPQKMTAASGIDALAHGIETFFSLRSRPASDALAVEAMATVSAYLRR Adh_Gk PEIALVDPKLTMSLPKKITAASGIDALAHGIETFFSLRSQPISDVLAIEAMTTVNRYLRR Adh_Bsp PDISIVDPKMTMTAPPAITAASGFDAFAHGAETFVSRASQPASDVLAFSAMSTVSKYLRR Adh_BspL1 PDVSIIDPKLTLTAPPSITAAAGFDAFAHAARAFVSRISQPPSDALALSAMKTVHTYLRR Adh_Pt PEAAFVDPLAMVSAPPGITAASGIDALSHAVEAYVSRAASPVSDNLALGAVELIGGHLRQ Adh_Dk PEVAVVDPLLTMTAPPSVTAASGIDALSHAIETYVSLKAQPPARALALKAIELIGESLRT Adh_Dt PEVAVVDPILTLTAPPSVTAASGIDALSHAIETYVSLKAQPPARALALKAIELIGESLRA *::**:*:***:*:**::*.*::.*::*:.*::* Adh_Bp AVRDGADMEARIGMSQGSVLAGMAFNNGFLGLAHAIGSALSGHCHVPHGVAIGLLLPHVV Adh_Bk SVANGEDKEARCGMSHGSLLAGMAFNNGFLGLAHAIGSALSGHCHVPHGVAIGLLLPHVV Adh_Bt AVHDGSDVEARIGMSHGSLLAGMAFTNGFLGLAHAIGSALSGHCHVPHGIAIGLLLPHVV Adh_At AVHNGSDMEARIGMSHGSLLAGMAFNNGFLGLAHAIGSALSGHCHVPHGVAIGLLLPKVV YiaY AVEDGTDKEARIGMSQGSLLAGMAFNNGFLGLAHAIGSALSGHCHVSHGVAIGLLLPKVV Adh_Gk AVEDGTNKEARIGMSYGSLLAGMAFNNGFLGLAHAIGSALSGHCHVSHGVAIGLLLPKVV Adh_Bsp AVYNGEDVEARIKMAEASLLAGMAFNQSYLGLTHAIGSALSGHAHVSHGVAIGLLLPGVI Adh_BspL1 AVYNGDDIEARMKMAEASLLAGMAFNQSYLGLAHAIGSAISVHAHVSHGVVIGLLLPKVI Adh_Pt AVANGGDLAARTGAALGSLLAGMAFNNAFLGLTHSIGAALSGHVHVSHGVAVGLLLPYVM Adh_Dk AVADGSDKEARTRMSLGSLLAGMAFNNSLLGLTHSIGAALSGHAHVSHGMAIGLLLPYVM Adh_Dt AVADGSNKEARTKMSLGSLLAGMAFNNSLLGLTHSIGAALSGHAHVSHGMAVGLLLPYVM :*:*:**:.*:******.*.***:*:**:*:******:.:******: Adh_Bp AFNTPVRPEKAELIADVLGSV--QKET----GTAAELVGQLVQDIGLPQRLQEVGVPEAK Adh_Bk EFNSSECPDQAAEIAKILGVK--AEDERQLAEQASHAVGDLVKDIGLPTRLRDMNVPEEK Adh_Bt AFNAPARPDKAAQLARLLGVE--ANPREERGEETSAAVARMVADIGLPTRLRDVGVPEEK Adh_At EFNATVRPDKAAKIAGLMGMK--GEHSEELALQASPAMARLVEDIGLPTRLREVDVTEKK YiaY EFNARVRPEKAAKIAELLGVK--GDREEVLAEQAAPAVASLVKEIGLPTRLRDVDVSEEK Adh_Gk EFNSVVQPEKAAKIAELLGRK--GNQNT-LVQQAALAVASLVKEIGLPTRLRDVDVPKEK Adh_Bsp RYNSISRMDKHIEMAGAFREIDRSLSDWEIIDQLIEDVSRLRDDIGLPQRLQQVGVKEDQ Adh_BspL1 EYNLVAKIDKYAEAGKYIEQSSHGLSNYEAAALFSETVTQLRNDIGLPKQLREVNVKRAQ Adh_Pt EYNLMAKPDKFARLARAMGEVTEGKSLYRAASLAPRAVKAMVKSIGLPVRLKEIGVPEGA Adh_Dk EFNAMARMEKFSKIAVALGEDVKGLSLREAALRSVKAVRELVEDISLPRRLGDVGVTGDM Adh_Dt EFNAMARLEKYGKIAIALGEDVKGLSLREAALRSVKAVRELVEDISLPRRLGEVGVTGDM :*::.:*:.*.**:*::* Adh_Bp LVDIAKDSFKSGMMKWNPRLPTEQEVLELLQKAF Adh_Bk LADIARDSFQSGMMKFNPRRASESEVLELLHRVY Adh_Bt LPAIAKDAFKSGMMTCNPRQPTEQEVRELLRRAF Adh_At LFEIAKDSFKSGMMKFNPRQPSESEVLQLLKEIF YiaY LPDIARDAFKSGMMKFNPRQPSLSEVLTLLQQIY Adh_Gk LPDIAKDSFKSGMMRFNPRQPSEAEVMTLLQQIY Adh_Bsp LKMIAADSVKSGMWKFNPRQASEEEILELLKELY Adh_BspL1 LEAISKDSIKSGMWQFNPRRASEQDVYQMLREML Adh_Pt LAAIAETALKHGMIKFNPRVPSREDILDIVKKAY Adh_Dk IEGMAKDAMGHGMLKFNPRAVTEKDIIAILRKAL Adh_Dt IEGMAKDAMGHGMLKFNPRVVTEKDIMAILQKAL ::::.*****::::::. Adh_Bp = SEQ ID NO: 6 (Brevibacillus panacihumi); Adh_Bk = SEQ ID NO: 2 (Bacillus kribbensis); Adh_Bt = SEQ ID NO: 5 (Brevibacillus thermoruber); Adh_At = SEQ ID NO: 4 (Aneurinibacillus terranovensis); YiaY = SEQ ID NO: 1 (Aeribacillus pallidus); Adh_Gk = SEQ ID NO: 3 (Geobacillus kaustophilus); Adh_Bsp = SEQ ID NO: 7 (Bacillus sp. FJAT-14578); Adh_BspL1 = SEQ ID NO: 10 (Bacillus sp. L1(2012)); Adh_Pt = SEQ ID NO: 11 (Pelotomaculum thermopropionicum); Adh_Dk = SEQ ID NO: 8 (Desulfotomaculum kuznetsovii); and Adh_Dt = SEQ ID NO: 9 (Desulfurispora thermophila). Symbols below the alignment indicate: * = invariant positions; : = strongly conserved positions; . = less strongly conserved positions; no symbol indicated non-conserved positions.
TABLE-US-00004 TABLE3 Aminoacidsequencealignment(ClustalOmega)ofA.pallidusMFStransporter (HMFCAtransporter)with10bestBLASThits Aeribacillustransporter MKNIANTSTERPVNDASVKNRQMVRATIASLIGWSLDLYDLFLLLFVATTIGNLFFPASN gi|499548718|ref|WP_011229501.1|:1-445 MDNITKTNIERPVE-VSIKNSQMVRATIASLIGWALDLYDLFLLLYVATTIGNLFFPASN gi|651977233|ref|WP_026691821.1|:7-435 ------------------NNRQLVSATMASLLGWSFDLYDLFILLYVTPTIGSLFFPSSN gi|654945126|ref|WP_028395291.1|:1-445 MSNVVAT--HSKQESVTVSKREVRSAMVASLLGWSFDLYDLFLLLFVAPTISVLFFPTTN gi|737333963|ref|WP_035316274.1|:3-438 --------SSNQPPKVEISRRQMVNASIASLLGWALDLFDLFVLLYVAPVIGKLFFPTEL gi|558617199|gb|EST53422.1|:11-446 --------SSNQPPKVEISRRQMVNASIASLLGWALDLFDLFVLLYVAPVIGKLFFPTEL gi|737314460|ref|WP_035297308.1|:18-449 ------------RPPAAVGRKQMITAVLASLLGWSLDLYDLFILLYVTPVLGKLFFPADN gi|656061131|ref|WP_029098927.1|:18-449 ------------RPPAAVGRKQMITAVLASLLGWSLDLYDLFILLYVTPVLGKLFFPADN gi|503166469|ref|WP_013401130.1|:2-445 SANMETPVQQASALAAAISRKQMIIAVMASLLGWSLDLYDLFILLYVAPELGKLFFPTDK gi|505187461|ref|WP_015374563.1|:8-445 ------NVQQTSSLTVSISKKQMITAVTASLLGWSLDLYDLFILLYVAPELGKLFFPADK gi|612120256|gb|EZP78263.1|:2-445 SVNTETTVQQASPLTVSISRKQMIIAVMSSLLGWSLDLYDLFILLYVAPELGKLFFPTDK .::*:**:**::**:***:**:*::.****: Aeribacillustransporter QTLSLAAVYASFAVTLLMRPLGSAIFGIYADKNGRKKAMTVAIIGAGLCTAAFGLLPTIH gi|499548718|ref|WP_011229501.1|:1-445 QTLSLAAVYASFAVTLLMRPLGSAIFGVYADKNGRKKAMTVAIIGAGLSTTAFGLLPTIH gi|651977233|ref|WP_026691821.1|:7-435 PTLSLAAVYASFAVTLLMRPLGSAIFGSYADKNGRKKAMTVAIVGVGVSTAVFGLLPTVP gi|654945126|ref|WP_028395291.1|:1-445 PTLSLAAVYASFAVTLLMRPLGSAIFGSYADKNGRKKAMIVSVVGVGVSTAAFGLLPTVP gi|737333963|ref|WP_035316274.1|:3-438 PTLSLAAVYASFAVTLLMRPIGSALFGSYADRKGRKKAMIVAVIGVGVATALFGALPTVH gi|558617199|gb|EST53422.1|:11-446 PTLSLAAVYASFAVTLLMRPIGSALFGSYADRKGRKKAMIVAVIGVGVATALFGALPTVH gi|737314460|ref|WP_035297308.1|:18-449 PTLSLAAVYASFAVTLLLRPFGSALFGSYADRNGRKRAMVVAVSGVGISTALFGVLPTVA gi|656061131|ref|WP_029098927.1|:18-449 PTLSLAAVYASFAVTLLLRPFGSALFGSYADRNGRKRAMVVAVSGVGISTALFGVLPTVA gi|503166469|ref|WP_013401130.1|:2-445 PTLSLAAVYASFAVTLFMRPLGSLAFGAYADRNGRKRAMVVAVSGVGISTALFGALPTVA gi|505187461|ref|WP_015374563.1|:8-445 PTLSLAAVYASFAVTLFMRPLGSALFGSYADRNGRKRAMVVAVSGVGISTALFGALPTVE gi|612120256|gb|EZP78263.1|:2-445 PTLSLAAVYASFAVTLFMRPLGSALFGTYADRNGRKRAMVVAVSGVGISTALFGALPTVA ****************::**:***:*****::***:***::*.*:.*:*****: Aeribacillustransporter QVGVVAAIAFLILRLVQGVFVGGVVASTHTIGTESASPKYRGFMSGLIGGGGAGLGALFA gi|499548718|ref|WP_011229501.1|:1-445 QVGVAASIAFLILRLVQGIFVGGVVASTHTIGTESASPKYRGLMSGLIGGGGAGLGALFA gi|651977233|ref|WP_026691821.1|:7-435 QIGVFATIIFLVLRLCQGIFVGGVVASSHTIGTESAPPKLRGLMSGLIGGGGAGLGALFA gi|654945126|ref|WP_028395291.1|:1-445 QIGFMASIIFLVLRLVQGIFVGGVVASTHTIGTESAPPKWRGLMSGLIGGGGAGLGALFA gi|737333963|ref|WP_035316274.1|:3-438 IQGVGASIIFLILRLVQGIFVGGVVASTHTIGTESVPPKWRGFMSGFVGGGGAGLGALLA gi|558167199|gb|EST53422.1|:11-446 QIGVGASIIFLILRLVQGIFVGGVVASTHTIGTESVPPKWRGFMSGFVGGGGAGLGALLA gi|737314460|ref|WP_035297308.1|:18-449 HIGAAATILFIILRLIQGVFVGGVVASTHTIGRESVPEKWRGLMSGLVGGGGAGLGALLA gi|656061131|ref|WP_029098927.1|:18-449 HIGAAATILFIILRLIQGVFVGGVVASTHTIGTESVPEKWRGLMSGLVGGGGAGLGALLA gi|503166469|ref|WP_013401130.1|:2-445 QIGAAAAIIFIILRLVQGVFVGGVVASTHTIGTESVPEKWRGLMSGLVGGGGAALGALLA gi|505187461|ref|WP_015374563.1|:8-445 QIGAAAAIIFIILRLIQGVFVGGVVASTHTIGTESVPEKWRGLMSGLVGGGGAALGALLA gi|612120256|gb|EZP78263.1|:2-445 QIGAAAAIIFIVLRLIQGVFVGGVVASTHTIGTESVPEKWRGLMSGLVGGGGAALGALLA ::**:**::*****:********:*******.***:***::*****.****:* Aeribacillustransporter SISYSVVTAIFPGEAFDVWGWRVMFFTGIIGSLFGLFIFRSLEESPLWKQLKEENSKGEV gi|499548718|ref|WP_011229501.1|:1-445 SIAYSIVSAIFPGDAFDTLGWRIMFFTGIIGALFGLFIFRSLDESPLWKQLKEKQSKDKM gi|651977233|ref|WP_026691821.1|:7-435 SIAFTVVSSFFPGEAFSEWGWRVMFFTGILGAIAGLFVFRTLDESPLWKGLQEEKKGKAV gi|654945126|ref|WP_028395291.1|:1-445 SIAFAIISALFPGEAFNEWGWRVLFFTGLLGAGAGLIVFRSLNESPLWAQLHEEKKKTNE gi|737333963|ref|WP_035316274.1|:3-438 SIVYFIVSEAFPGEAFDAWGWRFMFFAGILSAVLGVFVFKSLEESPLWLQAQQKKE---A gi|558617199|gb|EST53422.1|:11-446 SIVYFIVSEAFPGEAFDAWGWRFMFFAGILSAVLGVFVFKSLEESPLWLQAQQKKE---A gi|737314460|ref|WP_035297308.1|:18-449 SIVYFVLSSLFPGEAFSEWGWRFMFFTGILCSVLGLFVFRMLEESPLWVQHKNEQA---A gi|656061131|ref|WP_029098927.1|:18-449 SIVYFVLSSLFPGEAFSEWGWRFMFFTGILCSVLGLFVFRMLEESPLWVQHKNEQA---A gi|503166469|ref|WP_013401130.1|:2-445 SIVYFVLSSVFSGPEFSEWGWRFMFFTGILSSVLGLFVFKKLEESPLWMQHKKKQE---T gi|505187461|ref|WP_015374563.1|:8-445 SIVYFVLSSIFPGPEFSEWGWRFMFFTGILSSVLGLFVFKKLEESPGWVQHKKQVQ---T gi|612120256|gb|EZP78263.1|:2-445 SIVYFVLSNIFSGSEFSEWGWRFMFFTGILCSVLGLFIFKKLEESPLWVQHKKDQE---M **::::***.***.:**:*:::*:::*:*:***:*::: Aeribacillustransporter -SEFQKAPLKTFFTKYYKVLLVNLMIVIGGGSGYYLTSGFIPTFLKVVNKVSASVSSGVL gi|499548718|ref|WP_011229501.1|:1-445 -VEQQKSPFKMFLTKYYKVLFVNLMIVIGGGSGYYLTAGFIPTFLKVVNKVPAAVSSGVL gi|651977233|ref|WP_026691821.1|:7-435 SHTIEQKPVKTLFTTYSKVLLVNLMIVIGGGTGYYLTAGFIPTFLTIINDVSPGTKSGIL gi|654945126|ref|WP_028395291.1|:1-445 EDAVPQSPIKMLFKQYPGVLLVNVMIVMGGGSAYYLTSGFVPTFLKVVNEAPPNVISGVL gi|737333963|ref|WP_035316274.1|:3-438 AKKPEGSPVKMIFTQYRNVLLVNLMLVTGGGTAYYLTSGYLPTFLNVINKVSSGTASLIL gi|558617199|gb|EST53422.1|:11-446 AKKPEGSPVKMIFTQYRNVLLVNLMLVTGGGTAYYLTSGYLPTFLNVINKVSSGTASLIL gi|737314460|ref|WP_035297308.1|:18-449 KPAGQQSPVKMVFTKYLPVLLVNLLIVIGGGSAYYLTSGYLPTFLNVINHVPQTTASMIL gi|656061131|ref|WP_029098927.1|:18-449 KPAGQQSPVKMVFTKYLPVLLVNLLIVIGGGSAYYLTSGYLPTFLNVINHVPQTTSSMIL gi|503166469|ref|WP_013401130.1|:2-445 KPEYQQSPVKMVFTKYLSVLLVNLMIVIGGGSAYYLTCGYLPTFLKVINNIPQTVSSIIL gi|505187461|ref|WP_015374563.1|:8-445 KPENEQSPVKIVFTKYLSVLLINLMIVIGGGSAYYLTCGYLPTFLKVINNIPQTVSSMIL gi|612120256|gb|EZP78263.1|:2-445 KPENQQSPVKMVFSKYLSVLLINLMIVIGGGSAYYLTCGYLPTFLKVINNIPQTVSSMIL *.*.:.***::*:::****:.****.*::****.::*..*:* Aeribacillustransporter IATSIMTIVAAVLVGHLSEVIGRKKTFLLIGILCLVGLPYFYLSLANSTTTTGIYLNALG gi|499548718|ref|WP_011229501.1|:1-445 IATSITTILAAIVVGHLSELIGRKKTFMIIGILCVFGLPYFYLSLAHSTTTTSIYLNAIG gi|651977233|ref|WP_026691821.1|:7-435 IASSVVTIISALLVGHLSEIIGRKKTFLAIGVVNIIGLPFFYLSLADAATTPSIYFYTMC gi|654945126|ref|WP_028395291.1|:1-445 IASSIVTIISALLFGHLSELIGRKKVFLLVGVLNIIGLPYFYLALGDSVTTLSIYLNTMG gi|737333963|ref|WP_035316274.1|:3-438 MGASVSAIISAVLFGYLSDVIGRKKTFLLIGFINLILLPVLFIQLGSATSIPMITFYALA gi|558617199|gb|EST53422.1|:11-446 MGASVSAIISAVLFGYLSDVIGRKKTFLLIGFINLILLPVLFIQLGSATSIPMITFYALA gi|737314460|ref|WP_035297308.1|:18-449 AASSIAAIIASVALGHLSTVIGRKKTFVLLGILNLMALPYLYTELAAAQDLSRIALYAMG gi|656061131|ref|WP_029098927.1|:18-449 AASSISAIIASVVLGHLSTIIGRKKTFVLLGILNLMALPYLYTELAAAQDLSRIALYAMG gi|503166469|ref|WP_013401130.1|:2-445 MVSSISAMVAAVVLGHLSTIIGRKKTFILLGIVNFLALPYLYTELADAQDLTMITLYAMG gi|505187461|ref|WP_015374563.1|:8-445 IVSSISAMIAAIALGHLSTIIGRKKTFILLGIVNLIALPYLYTELADAQDMTSITLYAMG gi|612120256|gb|EZP78263.1|:2-445 MVSSISAMIASIVLGHLSTIIGRKKTFILLGTVNLIALPYLYTELAAAQDLTLIILYAMG :*:::::::.*:**:*****.*::*::.**::*.:*::: Aeribacillustransporter LIFLGNAAYAPVLIFLNERFPTSIRSTGTGLSWNMGFAIGGMMPTFVNLASGTVEHIPYT gi|499548718|ref|WP_011229501.1|:1-445 LVFLGNASYAPVLIFLNERFPTEVRSTGRGLSWNVGFAIGGMMPTFVNLASGTVEHIPYT gi|651977233|ref|WP_026691821.1|:7-435 VVFLGNAAYAPVLIFLNERFPTSIRSTGTGISWNMGFAVGGMMPTFVTLASGSVKNIPHT gi|654945126|ref|WP_028395291.1|:1-445 LAFLGNAAYAPVLIFLNERFPTVIRSTGTGLSWNMGFAIGGMMPTFVTLASGKVENIPTT gi|737333963|ref|WP_035316274.1|:3-438 LAFLGNAAYAPILIFLNERFPTSIRSSGTGLSWNMGFAVGGMMPTFVTLASGTTENIPYS gi|558617199|gb|EST53422.1|:11-446 LAFLGNAAYAPILIFLNERFPTSIRSSGTGLSWNMGFAVGGMMPTFVTLASGTTENIPYS gi|737314460|ref|WP_035297308.1|:18-449 LAFLGNASYAPVLIFLNERFPTAIRSTGTGLSWNMGFAIGGMMPTGVTMASGQTSEIPFF gi|656061131|ref|WP_029098927.1|:18-449 LAFLGNASYAPVLIFLNERFPTAIRSTGTGLSWNMGFAIGGMMPTFVTMASGQTSEIPFY gi|503166469|ref|WP_013401130.1|:2-445 LAFLGNGSYAPVLIFLNERFPTSIRSTGTGLSWNMGFAVGGMMPTFVTMASRQTSDIPSS gi|505187461|ref|WP_015374563.1|:8-445 LAFLGNASYAPVLIFLNERFPTTIRSTGTGLSWNMGFAVGGMMPTFVTMASSQTSDIPLS gi|612120256|gb|EZP78263.1|:2-445 LAFLGNGSYAPVLIFLNERFPTAIRSTGTGLSWNMGFAVGGMMPTFVTMTSSQTSDIPLS :****.:***:**********:**:***:***:***:********.::*...** Aeribacillustransporter LMYFTIGIYLVYILGSLIIPETKGNLK gi|499548718|ref|WP_011229501.1|:1-445 LMYFTIVIYLVYILGSFIIPETKGNLK gi|651977233|ref|WP_026691821.1|:7-435 LMYFFIGIFLLYLIGSAVIKETKGNLN gi|654945126|ref|WP_028395291.1|:1-445 LMYFAIGIFLVYIIGSIIVPETKGNLK gi|737333963|ref|WP_035316274.1|:3-438 LMGFSIAVFVVYVIGSLVIPETKGNFE gi|558617199|gb|EST53422.1|:11-446 LMGFSIAVFVVYVIGSLVIPETKGNFE gi|737314460|ref|WP_035297308.1|:18-449 LAYFSIGLFLLYLVGSLIIPETKGNFQ gi|656061131|ref|WP_029098927.1|:18-449 LAYFSIGLFLLYLVGSLIIPETKGNFQ gi|503166469|ref|WP_013401130.1|:2-445 LAYFFIALFLLYLLGSFIIPETKGNFK gi|505187461|ref|WP_015374563.1|:8-445 LTYFSIALFLLYLLGSFIIPETKGNFK gi|612120256|gb|EZP78263.1|:2-445 LAYFSIALFLLYLLGSFIIPETKGNFK ***::::*::**::*****:: Symbols below the alignment indicate: * = invariant positions; : = strongly conserved positions; . = less strongly conserved positions; no symbol indicated non-conserved positions.