# Patent

## Title
Alpha (1,2) fucosyltransferases suitable for use in the production of fucosylated oligosaccharides

## Abstract

The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.

## Background
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named “37847-510F01US_ST25.txt”, which was created on Aug. 20, 2012 and is 71.1 KB in size, are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The invention provides compositions and methods for producing purified oligosaccharides, in particular certain fucosylated oligosaccharides that are typically found in human milk.
BACKGROUND OF THE INVENTION
Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides. More than 130 different complex oligosaccharides have been identified in human milk, and their structural diversity and abundance is unique to humans. Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome, in the prevention of disease, and in immune function. Prior to the invention described herein, the ability to produce human milk oligosaccharides (HMOS) inexpensively was problematic. For example, their production through chemical synthesis was limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost. As such, there is a pressing need for new strategies to inexpensively manufacture large quantities of HMOS.
SUMMARY OF THE INVENTION
The invention features an efficient and economical method for producing fucosylated oligosaccharides. Such production of a fucosylated oligosaccharide is accomplished using an isolated nucleic acid comprising a sequence encoding a lactose-utilizing α(1,2) fucosyltransferase gene product (e.g., polypeptide or protein), which is operably linked to one or more heterologous control sequences that direct the production of the recombinant fucosyltransferase gene product in a bacterium such as Escherichia coli (E. coli). In one example, the bacterium is an enteric bacterium. The amino acid sequence of the lactose-accepting α(1,2)fucosyltransferase gene product is preferably at least 10% and less than 40% identical to FutC (SEQ ID NO:2).
Also within the invention is a nucleic acid construct comprising an isolated nucleic acid encoding a lactose-accepting α(1,2)fucosyltransferase enzyme, said nucleic acid being operably linked to one or more heterologous control sequences that direct the production of the enzyme in a host bacteria production strain, wherein the amino acid sequence of the gene product (enzyme) encoded by the nucleic acid comprises about 70% identity to SEQ ID NO:2. For example, the construct comprises SEQ ID NO: 7, which encodes a FutL protein. By “heterologous” is meant that the control sequence and protein-encoding sequence originate from different bacterial strains. A suitable production host bacterial strain is one that is not the same bacterial strain as the source bacterial strain from which the fucosyltransferase-encoding nucleic acid sequence was identified.
A method for producing a fucosylated oligosaccharide, e.g., an HMOS, in a bacterium is carried out by providing a bacterium such as a production host strain, Escherichia coli (E. coli), that is characterized by a reduced level of β-galactosidase activity, a defective colonic acid synthesis pathway, a mutation in an ATP-dependent intracellular protease, a mutation in a lacA gene and an exogenous α(1,2)fucosyltransferase gene. Preferably, a mutation in a thyA gene in the host bacterium allows for the maintenance of plasmids that carry thyA as a selectable marker gene. Exemplary alternative selectable markers include antibiotic resistance genes such as BLA (beta-lactamase), or proBA genes (to complement a proAB host strain proline auxotropy) or purC (to complement a purC host strain adenine auxotrophy). The bacterium comprising these characteristics is cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved from the bacterium or from a culture supernatant of the bacterium. In some cases, the method further comprises culturing the bacterium in the presence of tryptophan and in the absence of thymidine. In preferred embodiments, the production host strain comprises E. coli K12. Other production host organisms are listed below.
The invention provides a purified fucosylated oligosaccharide produced by the methods described herein. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacterium is used directly in such products. The fucosylated oligosaccharide produced by the engineered bacterium is 2′-fucosyllactose (2′-FL) or lactodifucotetraose (LDFT). The new alpha 1,2-fucosyltransferases are also useful to synthesize HMOS of larger molecular weight bearing alpha 1,2 fucose moieties, e.g., lacto-N-fucopentaose (LNF I) and lacto-N-difucohexaose (LDFH I).
The bacterium used to produce the oligosaccharides is genetically engineered to comprise an increased intracellular guanosine diphosphate (GDP)-fucose pool (compared to wild type), an increased intracellular lactose pool (compared to wild type), and to comprise fucosyltransferase activity. Accordingly, an endogenous lacZ gene and an endogenous lacI gene of the E. coli are deleted or functionally inactivated to reduce the level of β-galactosidase activity. The bacterium may also comprise a mutation in the lacA gene. The isolated E. coli bacterium also comprises a lacIq gene promoter immediately upstream of a lacY gene. In some cases, the isolated E. coli bacterium comprises a defective colonic acid synthesis pathway due to an endogenous wcaJ gene of the E. coli being deleted or functionally inactivated. The bacterium comprises a mutation in an adenosine-5′-triphosphate (ATP)-dependant intracellular protease. For example, the bacterium comprises a null mutation in a ion gene. The bacterium also comprises a mutation in a thyA gene. Preferably, the bacterium accumulates an increased intracellular lactose pool and an increased intracellular GDP-fucose pool. In one aspect, the E. coli bacterium comprises the genotype ΔampC::PtrpBcI, Δ(lacI-lacZ)::FRT, PlacIqlacY+, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ+), ΔlacA.
The bacterium possesses fucosyl transferase activity. For example, the bacterium comprises an exogenous α(1,2)fucosyltransferase gene. Preferably, the exogenous α(1,2) fucosyltransferase gene comprises at least 10% homology/identity and less than 40% at the amino acid level to Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (futC), e.g., at least 15%, at least 20%, at least 25%, at least 30% identity. In other examples, the sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology/identity to Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (futC). In one example, FutL is 70% identical to FutC at the amino acid level.
The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Percent identity is determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the PSI-BLAST search, the following exemplary parameters are employed: (1) Expect threshold was 10; (2) Gap cost was Existence:11 and Extension:1; (3) The Matrix employed was BLOSUM62; (4) The filter for low complexity regions was “on”. The bacterium expresses a fucosyltransferase gene product encoded by a sequence that is not identical to futC.
Exemplary α(1,2)fucosyltransferase genes include Escherichia coli O126 wbgL, Helicobacter mustelae 12198 (ATCC 43772) alpha-1,2-fucosyltransferase (futL), and Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein (futN). An exogenous α(1,2)fucosyltransferase gene is selected from the group consisting of Escherichia coli O126 wbgL, Helicobacter mustelae 12198 (ATCC 43772) alpha-1,2-fucosyltransferase (futL), Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein (futN), Bacteroides fragilis (NCTC) 9343 fucosyl transferase (bft3/wcfB), Escherichia coli O55:H7 (str. CB9615)fucosyltransferase (wbgN), Helicobacter bilis ATCC 437879 futD, Vibrio cholera O22 wblA, Bacteroides fragilis (NCTC) 9343 alpha-1,2-fucosyltransferase (bft1), Bacteroides ovatus ATCC 8483 futO, and Helicobacter cinaedi CCUG 18818 alpha-1,2-fucosyltransferase (futE).
The invention also features a vector, e.g., a vector containing a nucleic acid. The vector can further include one or more regulatory elements, e.g., a heterologous promoter. The regulatory elements can be operably linked to a gene encoding a protein, a gene construct encoding a fusion protein gene, or a series of genes linked in an operon in order to express the fusion protein. In yet another aspect, the invention comprises an isolated recombinant cell, e.g., a bacterial cell containing an aforementioned nucleic acid molecule or vector. The nucleic acid is optionally integrated into the genome.
Also provided is a nucleic acid construct comprising at least one of a promoter of bacteriophage λ, an E. coli rcsA gene, a bla gene, and a native thyA gene. As an example of such a construct, the plasmid map of pG171 in FIG. 5.
The sequence of pG171 is set forth below with annotations from GenBank regarding specific features (SEQ ID NO: 1):














LOCUS
pEC2-futC-MYC-rcsA-thyA_(pG171)
6244 bp DNA







circular SYN 24-MAY-1995








DEFINITION
Fusion cloning vector pTRXFUS, complete sequence.


ACCESSION
U16857


VERSION
U16857.1 GI:575447


KEYWORDS
thioredoxin gene fusion vector.


SOURCE
Cloning vector pTRXFUS (unknown)


 ORGANISM
Cloning vector pTRXFUS



other sequences; artificial sequences; vectors.


REFERENCE
1 (bases 1 to 3585)


 AUTHORS
LaVallie,E.R., DiBlasio,E.A., Kovacic,S., Grant,K.L.,







Schendel,P.F.









and McCoy,J.M.


 TITLE
A thioredoxin gene fusion expression system that circumvents



inclusion body formation in the E. coli cytoplasm


 JOURNAL
Biotechnology (N.Y.) 11 (2), 187-193 (1993)


  PUBMED
7763371


REFERENCE
2 (bases 1 to 3585)


 AUTHORS
LaVallie,E.R.


 TITLE
Direct Submission


 JOURNAL
Submitted (03-NOV-1994) Edward R. LaVallie, Genetics Institute,







87









CambridgePark Drive, Cambridge, MA 02140, USA


FEATURES
   Location/Qualifiers


   Primer
   163..183



   /label=lacZR6


   CDS
   243..481



   /gene=“lgt”



   /note=“ECK2824:JW2796:b2828”



   /codon_start=1



   /transl_table=11



   /product=“phosphatidylglycerol-







prolipoproteindiacylglyceryl









   transferase”



   /protein_id=“BAE76897.1”



   /db_xref=“GI:85675644”



 /translation=“MTSSYLHFPEFDPVIFSIGPVALHWYGLMYLVGFIFAMWLATRRA



    NRPGSGWTKNEVENLLYAGFLGVFLGGRIGYVLFYNFPQFMADPLYLFRVWDSFHG







GLIGVIVVMIIFARRTKRSFFQVSDFIAPLIPFGLGAGRLGNFINGELWGRVDPNFPFA


MLFPGSRTEDILLLQTNPQWQSIFDTYGVLPRHPSQLYELLLEGVVLFIILNLYIRKPR


        PMGAVSGLFLIGYGAFRIIVEFFRQPDAQFTGAWVQYISMGQILSIPMIVAGVIMMVA


YRRSPQQHVS”








   Source
   complement(243..1365)



   /organism=“Escherichia coli W3110”



   /mol_type=“genomic DNA”



   /strain=“K-12”



   /sub_strain=“W3110”



   /db_xref=“taxon:316407”



   /note=“synonym: Escherichia coli str. K12 substr.







W3110”








   Source
   complement(242{circumflex over ( )}243)



   /organism=“Escherichia coli W3110”



   /mol_type=“genomic DNA”



   /strain=“K-12”



   /sub_strain=“W3110”



   /db_xref=“taxon:316407”



   /note=“synonym: Escherichia coli str. K12 substr.







W3110”








   Primer
   243..266



   /note=cagtcagtcaggcgccTCCTCAACCTGTATATTCGTAAAC



   /label=THYA-F


   Promoter
   359..364



   /label=“thyA -35”


   Promoter
   380..385



   /label=“thyA WEAK -10”


   Binding_site
   479..484



   /label=“thyA RBS”


   Gene
   488..1282



   /gene=“thyA”


   CDS
   488..1282



   /gene=“thyA”



   /note=“ECK2823:JW2795:b2827”



   /codon_start=1



   /transl_table=11



   /product=“thymidylate synthetase”



   /protein_id=“BAE76896.1”



   /db_xref=“GI:85675643”







        /translation=“MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRFNLQDGFPLV


          TTKRCHLRSIIHELLWFLQGDTNIAYLHENNVTIWDEWADENGDLGPVYGKQWRTP


         DGRHIDQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPCHAFFQFYVADGKLSL


          YQRSCDVFLGLPFNIASYALLVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQLSR


EPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPGIKAPVAI”








   Hairpin_loop
   1304..1310



   /label=Terminator


   Hairpin_loop
   1317..1323



   /label=Terminator


   Primer
   complement(1345..1365)



   /note=cagtcagtcaggcgccTTCGGGAAGGCGTCTCGAAGA



   /label=THYA-R


   Primer
   complement(1468..1489)



   /label=lacZF5


   Primer
   1508..1524



   /label=aspAseq


   Gene
   1536..1588



   /gene=“dsrB”


   Primer
   1536..1558



   /note=cagtcagtcaaagcttTCTTTAATGAAGCAGGGCATCAG



   /label=rcsA-R


   Hairpin_loop
   complement(1600..1610)



   /label=Hairpin


   Hairpin_loop
   complement(1615..1625)



   /label=Hairpin


   CDS
   complement(1632..2255)



   /gene=“rcsA”



   /note=“ECK1949:JW1935:b1951”



   /codon_start=1



   /transl_table=11



   /product=“DNA-binding transcriptional co-regulator



   withRcsB”



   /protein_id=“BAA15776.1”



   /db_xref=“GI:1736617”







        /translation=“MSTIIMDLCSYTRLGLTGYLLSRGVKKREINDIETVDDLAIACDS


QRPSVVFINEDCFIHDASNSQRIKLIINQHPNTLFIVFMAIANVHFDEYLLVRKNLLIS


        SKSIKPESLDDILGDILKKETTITSFLNMPTLSLSRTESSMLRMWMAGQGTIQISDQMN


IKAKTVSSHKGNIKRKIKTHNKQVIYHVVRLTDNVTNGIFVNMR”








   Promoter
   complement(2393..2398)



   /label=−10


   Promoter
   complement(2419..2424)



   /label=−35


   Primer
   complement(2473..2495)



   /note=cagtcagtcaaagcttCTACGAACATCTTCCAGGATACT



   /label=rcsA-F2


   Terminator
   complement(2502..2571)



   /note=“aspA transcription terminator”


   Primer
   complement(2553..2574)



   /note=cagtcagtcaCTCGAGGCTGCAGTAATCGTACAGGGTAG



   /label=PLvect2


   Primer_binding_
   2575..2644







/PCR_primers=cagtcagtcactcgagtTTAattcaaatcttcttcagaaatcaatt









   tttgttcAGCGTTATACTTTTGGGATTTTACCTC



   /label=“Primer 0011-futCMYC-4”


   CDS
   complement(2574{circumflex over ( )}2575)



   /note=“Identical to previously sequenced to



   BacteroidesfragilisSWALL:Q9F7604.3e-







123,coli(EMBL:AF461121)









   id”



   /transl_table=11



   /product=“putative LPS biosynthesis



   relatedalpha-1,2-fucosyltransferase”



   /gene=wcfW



   /locus_tag=BF1902



   /protein_id=CAH07600.1







/translation=MIVSSLRGGLGNQMFIYAMVKAMALRNNVPFAFNLTTDFANDEVYK


RKLLLSYFALDLPENKKLTFDFSYGNYYRRLSRNLGCHILHPSYRYICEERPPHFESRL


ISSKITNAFLEGYWQSEKYFLDYKQEIKEDFVIQKKLEYTSYLELEEIKLLDKNAIMIG


VRRYQESDVAPGGVLEDDYYKCAMDIMASKVTSPVFFCFSQDLEWVEKHLAGKYPVRLISKKEDDSGTIDDMFLM


MHFRNYIISNSSFYWWGAWLSKYDDKLVIAPGNFINKDSVPESWFKLNVR








POLYLINKER
complement(2575..2580)


   Protein
   complement(2578..2581)



   /label=“K.lactis alpha-factor leader”


   Gene
   complement(2582..3055)



   /locus_tag=“HP0093”



   /db_xref=“GeneID:900162”


   CDS
   complement(2582..3517)



   /label=“futC strain 26695 (fixed)”


   Source
   2582..3517



   /organism=“Helicobacter pylori 26695”



   /mol_type=“genomic DNA”



   /strain=“26695”



   /db_xref=“taxon:85962”


   MYC-tag
   complement(2585..2617)


   Primer
   2968..2990



   /note=GATAGTCAATACCAAGCTGACAG



   /label=3-6-R


   Primer
   2968..2990



   /label=“42 (3-6-R)”


   Gene
   complement(3052..3517)



   /locus_tag=“HP0094”



   /db_xref=“GeneID:899021”


   Primer
   complement(3495..3517)



   /note=GAAttcaagaaggagatataCATATGGCTTTTAAGGTGGTGCAAAT



   /label=pLfutC-F


   Source
   3517{circumflex over ( )}3518



   /organism=“Escherichia coli W3110”



   /mol_type=“genomic DNA”



   /strain=“K-12”



   /sub_strain=“W3110”



   /db_xref=“taxon:316407”



   /note=“synonym: Escherichia coli str. K12 substr.







W3110”








   RBS
   complement(3521..3535)



   /label=“T7 gene 10 RBS”


   Source
   complement(3541..3715)



   /note=“originates from LAMCG”



   /label=“lambda DNA”


   Primer
   3541..3567



   /note=cagtcagtcagaattcTAACAATTGATTGAATGTATGCAAATA



   /label=pLnut-R


   Region
   complement(3544)



   /label=“TR1 termination site”


   note
   3544



   /note=“original sequenced pEC2-BfT2-MYC plasmid is a



   mixture of C and A here. This plasmid is A”


   Misc._feature
   complement(3547..3553)



   /label=“TR1 rho-dep consensus box”


   Region
   complement(3566..3571)



   /label=“TR1 termination site”


   Misc._feature
   complement(3580..3599)



   /note=“rho utilization site B (rutB)”


   Misc._binding
   complement(3600..3616)



   /note=“N-utilization rightward; putative”



   /bound_moiety=“Nutr”


   Region
   complement(3601..3632)



   /label=nutR


   Misc._feature
   complement(3615..3632)



   /note=“rho utilization site A (rutA)”


   Variation
   complement(3636..3715)



   /note=“imm434 region”


   CDS
   complement(3640..3715)



   /codon_start=1



   /transl_table=11



   /product=“cro (antirepressor; also tof;66)”



   /protein_id=“AAA96582.1”



   /db_xref=“GI:215148”







/translation=“MEQRITLKDYAMRFGQTKTAKDLGVYQSAINKAIHAGRKIFLTIN









   ADGSVYAEEVKPFPSNKKTTA”


   Region
   complement(3729..3730)



   /label=“1/2 HaeIII site that Rosenberg used”


   CDS
   complement(3731..3907)



   /codon_start=1



   /transl_table=11



   /product=“N (early gene regulator;133)”



   /protein_id=“AAA96578.1”



   /db_xref=“GI:508997”







/translation=“MCQSRGVFVQDYNCHTPPKLTDRRIQMDAQTRRRERRAEKQAQWK


AANPLLVGVSAKPVNRPILSLNRKPKSRVESALNPIDLTVLAEYHKQIESNLQRIERKNQRTWYSKPGERGITCS


GRQKIKGKSIPLI”








   Source
   complement(3731..4023)



   /note=“originates from LAMCG rev”



   /label=“lambda DNA”


   Region
   complement(3731..3733)



   /label=“1/2 HpaI site that Rosenberg used”


   Variation
   3731..4184



   /note=“imm21 region”


   Misc._binding
   complement(3987..4003)



   /note=“N-utilization leftward.; putative”



   /bound_moiety=“Nutl”


   Region
   complement(3988..4018)



   /label=nutL


   Source
   complement(4024..4184)



   /note=“originates from LAMCG rev”



   /label=“lambda DNA”


   Primer
   4024..4044



   /note=cagtcagtcagaaTTCATGGTGGTCAGTGCGTCC



   /label=PLvect3


   Region
   complement(4024..4369)



   /note=“originates from EC1 pL region”



   /label=“EC1 pL region”


   mRNA
   complement(4051)



   /note=“mRNA-pl (alt.; via tl2a terminator)”



   /label=“pL1 mRNA start”


   Variation
   4052..4184



   /note=“imm434 region”


   Promoter
   complement(4058..4063)



   /label=“pL1 -10”


   operator
   complement(4060..4076)



   /note=“operator-11 (first base on comp strand)”



   /label=OL1


   Primer
   complement(4075..4092)



   /label=pLseq


   Promoter
   complement(4081..4086)



   /label=“pLl -35”


   operator
   complement(4084..4100)



   /note=“operator-l2 (first base on comp strand)”



   /label=OL2


   mRNA
   complement(4093)



   /label=“pL2 mRNA start”


   Promoter
   complement(4100..4105)



   /label=“pL2 -10”


   operator
   complement(4104..4120)



   /note=“operator-l3 (first base on comp strand)”



   /label=OL3


   Promoter
   complement(4122..4127)



   /label=“pL2 -35”


   Source
   complement(4185..6244)



   /label=“pUC18 DNA”


   Primer
   complement(4350..4369)



   /note=cagtcagtcaACATGTTCTTTCCTGCGTTA



   /label=pLnut-F


   Primer
   complement(4414..4430)



   /label=pLnutseq-F


   Replication_ ori
   complement(4425..5013)



   /label=“Replication origin”


   RNA_transcript
   complement(4425..4977)



   /label=RNAII


   Promoter
   4832..4836



   /label=“RNAI -35”


   Promoter
   4853..4858



   /label=“RNAI -10”


   RNA_transcript
   4867..4974



   /label=RNAI


   Promoter
   complement(4988..4992)



   /label=“RNAII -10”


   Promoter
   complement(5008..5013)



   /label=“RNAII -35”


   CDS
   complement(5184..6044)



   /label=beta-lactamase


   Restriction_sit
   5916..5927



   /label=“EcoK site”


   Signal_peptide
   complement(5976..6044)



   /label=beta-lactamase


   Promoter
   complement(6088..6093)



   /label=“beta-lactamase -10”


   Promoter
   complement(6109..6114)



   /label=“beta-lactamase -35”


 







ORIGIN








1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA


61
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG


121
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC


181
ACCATATATG CGGTGTGAAA TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG


241
CCTCCTCAAC CTGTATATTC GTAAACCACG CCCAATGGGA GCTGTCTCAG GTTTGTTCCT


301
GATTGGTTAC GGCGCGTTTC GCATCATTGT TGAGTTTTTC CGCCAGCCCG ACGCGCAGTT


361
TACCGGTGCC TGGGTGCAGT ACATCAGCAT GGGGCAAATT CTTTCCATCC CGATGATTGT


421
CGCGGGTGTG ATCATGATGG TCTGGGCATA TCGTCGCAGC CCACAGCAAC ACGTTTCCTG


481
AGGAACCATG AAACAGTATT TAGAACTGAT GCAAAAAGTG CTCGACGAAG GCACACAGAA


541
AAACGACCGT ACCGGAACCG GAACGCTTTC CATTTTTGGT CATCAGATGC GTTTTAACCT


601
GCAAGATGGA TTCCCGCTGG TGACAACTAA ACGTTGCCAC CTGCGTTCCA TCATCCATGA


661
ACTGCTGTGG TTTCTGCAGG GCGACACTAA CATTGCTTAT CTACACGAAA ACAATGTCAC


721
CATCTGGGAC GAATGGGCCG ATGAAAACGG CGACCTCGGG CCAGTGTATG GTAAACAGTG


781
GCGCGCCTGG CCAACGCCAG ATGGTCGTCA TATTGACCAG ATCACTACGG TACTGAACCA


841
GCTGAAAAAC GACCCGGATT CGCGCCGCAT TATTGTTTCA GCGTGGAACG TAGGCGAACT


901
GGATAAAATG GCGCTGGCAC CGTGCCATGC ATTCTTCCAG TTCTATGTGG CAGACGGCAA


961
ACTCTCTTGC CAGCTTTATC AGCGCTCCTG TGACGTCTTC CTCGGCCTGC CGTTCAACAT


1021
TGCCAGCTAC GCGTTATTGG TGCATATGAT GGCGCAGCAG TGCGATCTGG AAGTGGGTGA


1081
TTTTGTCTGG ACCGGTGGCG ACACGCATCT GTACAGCAAC CATATGGATC AAACTCATCT


1141
GCAATTAAGC CGCGAACCGC GTCCGCTGCC GAAGTTGATT ATCAAACGTA AACCCGAATC


1201
CATCTTCGAC TACCGTTTCG AAGACTTTGA GATTGAAGGC TACGATCCGC ATCCGGGCAT


1261
TAAAGCGCCG GTGGCTATCT AATTACGAAA CATCCTGCCA GAGCCGACGC CAGTGTGCGT


1321
CGGTTTTTTT ACCCTCCGTT AAATTCTTCG AGACGCCTTC CCGAAGGCGC CATTCGCCAT


1381
TCAGGCTGCG CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC


1441
TGGCGAAAGG GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT


1501
CACGACGTTG TAAAACGACG GCCAGTGCCA AGCTTTCTTT AATGAAGCAG GGCATCAGGA


1561
CGGTATCTTT GTGGAGAAAG CAGAGTAATC TTATTCAGCC TGACTGGTGG GAAACCACCA


1621
GTCAGAATGT GTTAGCGCAT GTTGACAAAA ATACCATTAG TCACATTATC CGTCAGTCGG


1681
ACGACATGGT AGATAACCTG TTTATTATGC GTTTTGATCT TACGTTTAAT ATTACCTTTA


1741
TGCGATGAAA CGGTCTTGGC TTTGATATTC ATTTGGTCAG AGATTTGAAT GGTTCCCTGA


1801
CCTGCCATCC ACATTCGCAA CATACTCGAT TCGGTTCGGC TCAATGATAA CGTCGGCATA


1861
TTTAAAAACG AGGTTATCGT TGTCTCTTTT TTCAGAATAT CGCCAAGGAT ATCGTCGAGA


1921
GATTCCGGTT TAATCGATTT AGAACTGATC AATAAATTTT TTCTGACCAA TAGATATTCA


1981
TCAAAATGAA CATTGGCAAT TGCCATAAAA ACGATAAATA ACGTATTGGG ATGTTGATTA


2041
ATGATGAGCT TGATACGCTG ACTGTTAGAA GCATCGTGGA TGAAACAGTC CTCATTAATA


2101
AACACCACTG AAGGGCGCTG TGAATCACAA GCTATGGCAA GGTCATCAAC GGTTTCAATG


2161
TCGTTGATTT CTCTTTTTTT AACCCCTCTA CTCAACAGAT ACCCGGTTAA ACCTAGTCGG


2221
GTGTAACTAC ATAAATCCAT AATAATCGTT GACATGGCAT ACCCTCACTC AATGCGTAAC


2281
GATAATTCCC CTTACCTGAA TATTTCATCA TGACTAAACG GAACAACATG GGTCACCTAA


2341
TGCGCCACTC TCGCGATTTT TCAGGCGGAC TTACTATCCC GTAAAGTGTT GTATAATTTG


2401
CCTGGAATTG TCTTAAAGTA AAGTAAATGT TGCGATATGT GAGTGAGCTT AAAACAAATA


2461
TTTCGCTGCA GGAGTATCCT GGAAGATGTT CGTAGaagct tACTGCTCAC AAGAAAAAAG


2521
GCACGTCATC TGACGTGCCT TTTTTATTTG TACTACCCTG TACGATTACT GCAGCTCGAG


2581
TTTAattcaa atcttcttca gaaatcaatt tttgttcAGC GTTATACTTT TGGGATTTTA


2641
CCTCAAAATG GGATTCTATT TTCACCCACT CCTTACAAAG GATATTCTCA TGCCCAAAAA


2701
GCCAGTGTTT GGGGCCAATA ATGATTTTTT CTGGATTTTC TATCAAATAG GCCGCCCACC


2761
AGCTATAAGT GCTATTAGCG ATAATGCCAT GCTGACAAGA TTGCATGAGC AGCATGTCCC


2821
AATACGCCTC TTCTTCTTTA TCCCTAGTGG TCATGTCCAT AAAAGGGTAG CCAAGATCAA


2881
GATTTTGCGT GAATTCTAAG TCTTCGCAAA ACACAAAAAG CTCCATGTTT GGCACGCGCT


2941
TTGCCATATA CTCAAGCGCC TTTTTTTGAT AGTCAATACC AAGCTGACAG CCAATCCCCA


3001
CATAATCCCC TCTTCTTATA TGCACAAACA CGCTGTTTTT AGCGGCTAAA ATCAAAGAAA


3061
GCTTGCACTG ATATTCTTCC TCTTTTTTAT TATTATTCTT ATTATTTTCG GGtGGtGGtG


3121
GTAGAGTGAA GGTTTGCTTG ATTAAAGGGG ATATAGCATC AAAGTATCGT GGATCTTGGA


3181
AATAGCCAAA AAAATAAGTC AAGCGGCTTG GCTTTAGCAA TTTAGGCTCG TATTCAAAAA


3241
CGATTTCTTG ACTCACCCTA TCAAATCCCA TGCATTTGAG CGCGTCTCTT ACTAGCTTGG


3301
GGAGGTGTTG CATTTTAGCT ATAGCGATTT CTTTCGCGCT CGCATAGGGC AAATCAATAG


3361
GGAAAAGTTC TAATTGCATT TTCCTATCGC TCCAATCAAA AGAAGTGATA TCTAACAGCA


3421
CAGGCGTATT AGAGTGTTTT TGCAAACTTT TAGCGAAAGC GTATTGAAAC ATTTGATTCC


3481
CAAGCCCTCC GCAAATTTGC ACCACCTTAA AAGCCATATG tatatctcct tcttgaaTTC


3541
TAAaAATTGA TTGAATGTAT GCAAATAAAT GCATACACCA TAGGTGTGGT TTAATTTGAT


3601
GCCCTTTTTC AGGGCTGGAA TGTGTAAGAG CGGGGTTATT TATGCTGTTG TTTTTTTGTT


3661
ACTCGGGAAG GGCTTTACCT CTTCCGCATA AACGCTTCCA TCAGCGTTTA TAGTTAAAAA


3721
AATCTTTCGG AACTGGTTTT GCGCTTACCC CAACCAACAG GGGATTTGCT GCTTTCCATT


3781
GAGCCTGTTT CTCTGCGCGA CGTTCGCGGC GGCGTGTTTG TGCATCCATC TGGATTCTCC


3841
TGTCAGTTAG CTTTGGTGGT GTGTGGCAGT TGTAGTCCTG AACGAAAACC CCCCGCGATT


3901
GGCACATTGG CAGCTAATCC GGAATCGCAC TTACGGCCAA TGCTTCGTTT CGTATCACAC


3961
ACCCCAAAGC CTTCTGCTTT GAATGCTGCC CTTCTTCAGG GCTTAATTTT TAAGAGCGTC


4021
ACCTTCATGG TGGTCAGTGC GTCCTGCTGA TGTGCTCAGT ATCACCGCCA GTGGTATTTA


4081
TGTCAACACC GCCAGAGATA ATTTATCACC GCAGATGGTT ATCTGTATGT TTTTTATATG


4141
AATTTATTTT TTGCAGGGGG GCATTGTTTG GTAGGTGAGA GATCAATTCT GCATTAATGA


4201
ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC


4261
ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG


4321
GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC


4381
CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC


4441
CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA


4501
CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC


4561
CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT


4621
AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG


4681
CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC


4741
AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA


4801
GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT


4861
AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT


4921
GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG


4981
CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG


5041
TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA


5101
AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA


5161
TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG


5221
ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA


5281
CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG


5341
GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT


5401
GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT


5461
TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC


5521
TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA


5581
TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT


5641
AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC


5701
ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA


5761
TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA


5821
CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA


5881
AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT


5941
TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC


6001
GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA


6061
TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT


6121
TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC


6181
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT


6241
CGTC (SEQ ID NO: 1; plasmid G171)









The nucleic acid construct further comprises an α(1,2)fucosyltransferase gene comprising, e.g., at least 10% and less than 40% identity at the amino acid level to Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (futC). For example, the exogenous α(1,2)fucosyltransferase gene is selected from the group consisting of Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (futC), Vibrio cholera O22 wblA, Escherichia coli O126 wbgL, Helicobacter bilis ATCC 437879 futD, Helicobacter cinaedi CCUG 18818 alpha-1,2-fucosyltransferase (futE), Helicobacter mustelae 12198 (ATCC 43772) alpha-1,2-fucosyltransferase (futL), Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein (futN), Bacteroides ovatus ATCC 8483 futO, Escherichia coli O55:H7 (str. CB9615) fucosyltransferase (wbgN), Bacteroides fragilis (NCTC) 9343 alpha-1,2-fucosyltransferase (bft1), and Bacteroides fragilis (NCTC) 9343 fucosyl transferase (bft3/wcfB). The depiction of pG171 bears the alpha 1,2 FT gene futC to serve as an example.
Also within the invention is an isolated E. coli bacterium as described above and characterized as comprising a reduced level of β-galactosidase activity, a defective colonic acid synthesis pathway, a mutation in the lacA gene, a mutation in an ATP-dependant intracellular protease, and a mutation in a thyA gene. The invention also provides methods of identifying an α(1,2)fucosyltransferase gene capable of synthesizing 2′-fucosyllactose (2′-FL) in E. coli. The method of identifying non-FutC lactose-utilizing, α(1,2)fucosyltransferase enzyme comprises the following steps:
1) performing a computational search of sequence databases to define a broad group of simple sequence homologs of any known, lactose-utilizing α(1,2)fucosyltransferase;
2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list;
3) searching sequence databases, using a derived search profile based on the common sequence or structural motif from step (2) as query, and identifying a candidate sequences, wherein a sequence homology to a reference lactose-utilizing α(1,2)fucosyltransferase is 40% or less;
4) compiling a list of candidate organisms, said organisms being characterized as expressing α(1,2)fucosyl-glycans in a naturally-occurring state;
5) selecting candidate sequences that are derived from candidate organisms to generate a list of candidate lactose-utilizing enzymes;
6) expressing the candidate lactose-utilizing enzyme in a host organism; and
7) testing for lactose-utilizing α(1,2)fucosyltransferase activity, wherein detection of 2′-FL in said organism indicates that the candidate sequence comprises a non-FutC lactose-utilizing α(1,2)fucosyltransferase. For example, the sequence homology to a reference lactose-utilizing α(1,2)fucosyltransferase is 40% or less.
A purified fucosylated oligosaccharide produced by the methods described above is also within the invention. The purified oligosaccharide (2′-FL) obtained at the end of the process is a white/slightly off-white, crystalline, sweet powder. Unlike oligosaccharide production methods using FutC, the methods utilizing certain non-FutC enzymes (e.g. FutL) do not possess α(1,3)fucosyltransferase activity which leads to side reactions. The lack of a (1,3)fucosyltransferase activity associated with FutL contributes to its efficiency in producing 2′FL and is an advantage compared to FutC. FutL does not possess alpha 1,3 fucosyltransferase activity. For example, an engineered E. coli cell, E. coli culture supernatant, or E. coli cell lysate according to the invention comprises recombinant 2′-FL and does not substantially comprise α1,3 fucosylated lactose prior to purification of 2′-FL from the cell, culture supernatant, or lysate. Both FutN and WbgL appear to have alpha 1,3 fucosyltransferase activity (similar to FutC). However as a general matter, the fucosylated oligosaccharide produced by the methods contains a negligible amount of 3-FL in a 2′-FL-containing cell, cell lysate or culture, or supernatant, e.g., less than 1% of the level of 2′-FL or 0.5% of the level of 2′-FL.
A purified oligosaccharide, e.g., 2′-FL or LDFT, is one that is at least 90%, 95%, 98%, 99%, or 100% (w/w) of the desired oligosaccharide by weight. Purity is assessed by any known method, e.g., thin layer chromatography or other chromatographic techniques known in the art. The invention includes a method of purifying a fucosylated oligosaccharide produced by the genetically engineered bacterium described above, which method comprises separating the desired fucosylated oligosaccharide (e.g., 2′-FL) from contaminants in a bacterial cell lysate or bacterial cell culture supernatant of the bacterium.
The oligosaccharides are purified and used in a number of products for consumption by humans as well as animals, such as companion animals (dogs, cats) as well as livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry). For example, a pharmaceutical composition comprises purified 2′-FL and a pharmaceutically-acceptable excipient that is suitable for oral administration. Large quantities of 2′-FL are produced in bacterial hosts, e.g., an E. coli bacterium comprising an exogenous α(1,2)fucosyltransferase gene.
An E. coli bacterium comprising an enhanced cytoplasmic pool of lactose and GDP-fucose is useful in such production systems. Endogenous E. coli metabolic pathways and genes are manipulated in ways that result in the generation of increased cytoplasmic concentrations of lactose and/or GDP-fucose, as compared to levels found in wild type E. coli. For example, the bacteria contain at least 10%, 20%, 50%, or 2×, 5×, 10× or more of the levels compared to a corresponding wild type bacteria that lacks the genetic modifications described above.
A method of producing a pharmaceutical composition comprising a purified human milk oligosaccharide (HMOS) is carried out by culturing the bacterium described above, purifying the HMOS produced by the bacterium, and combining the HMOS with an excipient or carrier to yield a dietary supplement for oral administration. These compositions are useful in methods of preventing or treating enteric and/or respiratory diseases in infants and adults. Accordingly, the compositions are administered to a subject suffering from or at risk of developing such a disease.
The invention also provides methods for increasing the intracellular concentration of lactose in E. coli, for cells grown in the presence of lactose, by using manipulations of endogenous E. coli genes involved in lactose import, export, and catabolism. In particular, described herein are methods of increasing intracellular lactose levels in E. coli genetically engineered to produce a human milk oligosaccharide by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lad During construction of this deletion, the lacIq promoter is placed immediately upstream of (contiguous with) the lactose permease gene, lacY, i.e., the sequence of the lacIq promoter is directly upstream and adjacent to the start of the sequence encoding the lacY gene, such that the lacY gene is under transcriptional regulation by the lacIq promoter. The modified strain maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type chromosomal copy of the lacZ (encoding β-galactosidase) gene responsible for lactose catabolism. Thus, an intracellular lactose pool is created when the modified strain is cultured in the presence of exogenous lactose. Another method for increasing the intracellular concentration of lactose in E. coli involves deletion of the lacA gene. The lacA mutation prevents the formation of intracellular acetyl-lactose, which not only removes this molecule as a contaminant from subsequent purifications, but also eliminates E. coli's ability to export excess lactose from its cytoplasm (Danchin A. Cells need safety valves. Bioessays 2009, July; 31(7):769-73.), thus greatly facilitating purposeful manipulations of the E. coli intracellular lactose pool.
The invention also provides methods for increasing intracellular levels of GDP-fucose in Escherichia coli by manipulating the organism's endogenous colanic acid biosynthesis pathway. This increase is achieved through a number of genetic modifications of endogenous E. coli genes involved either directly in colanic acid precursor biosynthesis, or in overall control of the colanic acid synthetic regulon. In particular, described herein are methods of increasing intracellular GDP-fucose levels in E. coli genetically engineered to produce a human milk oligosaccharide by deletion of the weal gene, encoding the UDP-glucose lipid carrier transferase. In a wcaJ null background, GDP-fucose accumulates in the E. coli cytoplasm.
In one aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding an α(1,2)fucosyltransferase is 2′-FL (2′-fucosyllactose). Preferably the α(1,2)fucosyltransferase utilized is any α(1,2)fucosyltransferase capable of using lactose as the sugar acceptor substrate for 2′-FL synthesis. Preferably, the exogenous α(1,2)fucosyltransferase gene comprises at least 10% identity at the amino acid level and less than about 40% to Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (FutC).
The invention also provides compositions comprising E. coli genetically engineered to produce the human milk tetrasaccharide lactodifucotetraose (LDFT). The E. coli in this instance comprise an exogenous nucleic acid molecule encoding an α(1,2)fucosyltransferase that also possesses α(1,3)fucosyltransferase activity.
The invention provides a method of treating, preventing, or reducing the risk of infection in a subject comprising administering to said subject a composition comprising a purified recombinant human milk oligosaccharide, wherein the HMOS binds to a pathogen and wherein the subject is infected with or at risk of infection with the pathogen. In one aspect, the infection is caused by a Norwalk-like virus or Campylobacter jejuni. The subject is preferably a mammal in need of such treatment. The mammal is, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. In a preferred embodiment, the mammal is a human. For example, the compositions are formulated into animal feed (e.g., pellets, kibble, mash) or animal food supplements for companion animals, e.g., dogs or cats, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably, the purified HMOS is formulated into a powder (e.g., infant formula powder or adult nutritional supplement powder, each of which is mixed with a liquid such as water or juice prior to consumption) or in the form of tablets, capsules or pastes or is incorporated as a component in dairy products such as milk, cream, cheese, yogurt or kefir, or as a component in any beverage, or combined in a preparation containing live microbial cultures intended to serve as probiotics, or in prebiotic preparations to enhance the growth of beneficial microorganisms either in vitro or in vivo.
Polynucleotides, polypeptides, and oligosaccharides of the invention are purified and/or isolated. Purified defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or oligosaccharide, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. For example, purified HMOS compositions are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. For example, a “purified protein” refers to a protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Preferably, the protein constitutes at least 10, 20, 50, 70, 80, 90, 95, 99-100% by dry weight of the purified preparation.
Similarly, by “substantially pure” is meant an oligosaccharide that has been separated from the components that naturally accompany it. Typically, the oligosaccharide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
By “isolated nucleic acid” is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones.
A “heterologous promoter” is a promoter which is different from the promoter to which a gene or nucleic acid sequence is operably linked in nature.
The term “overexpress” or “overexpression” refers to a situation in which more factor is expressed by a genetically-altered cell than would be, under the same conditions, by a wild type cell. Similarly, if an unaltered cell does not express a factor that it is genetically altered to produce, the term “express” (as distinguished from “overexpress”) is used indicating the wild type cell did not express the factor at all prior to genetic manipulation.
The terms “treating” and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. The terms “preventing” and “prevention” refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.
By the terms “effective amount” and “therapeutically effective amount” of a formulation or formulation component is meant a nontoxic but sufficient amount of the formulation or component to provide the desired effect.
The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
The host organism used to express the non-FutC lactose-accepting fucosyltransferase gene product is typically the enterobacterium Escherichia coli K12 (E. coli). E. coli K-12 is not considered a human or animal pathogen nor is it toxicogenic. E. coli K-12 is a standard production strain of bacteria and is noted for its safety due to its poor ability to colonize the colon and establish infections (see, e.g., epa.gov/oppt/biotech/pubs/fra/fra004.htm). However, a variety of bacterial species may be used in the oligosaccharide biosynthesis methods, e.g., Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration showing the synthetic pathway of the fucosyl oligosaccharides of human milk. Se and Le indicate synthesis by fucosyltransferases of the secretor and Lewis genes, respectively. The abbreviated biochemical name [with alternate biochemical structure in brackets] is given (histo-blood group antigen analog in parentheses).
FIG. 2 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer 2′-fucosyllactose (2′-FL) synthesis in Escherichia coli (E. coli).
FIG. 3 is a series of photographs showing thin layer chromatography analysis of 2′-FL produced in E. coli strains by candidate α(1,2)fucosyltransferases. FIG. 3A shows significant production of 2′-FL by WbgL. FIG. 3B shows significant production of 2′-FL by FutL. FIG. 3C shows significant production of 2′-FL by FutN.
FIG. 4 is a chart and a photograph of thin layer chromatography analysis showing that fucosidase digestion confirms synthesis of bona fide 2′-FL by WbgL. Oligosaccharides produced by an E. coli strain expressing wbgL were isolated and subjected to overnight digestion with different fucosidases. Reaction products were analyzed by TLC. The production of fucose and lactose by treatment with α(1,2)fucosidase is illustrated in lane 2.
FIG. 5 is a diagram of plasmid pG171.
FIG. 6 is a diagram of a PlacIq lacY+ chromosomal construct.
FIG. 7 is a diagram of the chromosomal deletion of wcaJ.
FIG. 8 is a diagram of the kan, lacZ+ insertion into the ion locus.
FIG. 9 is a diagram of plasmid pG204.
FIG. 10 is a diagram of plasmid pG216.
FIG. 11 is a diagram of plasmid pG217.



DETAILED DESCRIPTION OF THE INVENTION
While some studies suggest that human milk glycans could be used as antimicrobial anti-adhesion agents, the difficulty and expense of producing adequate quantities of these agents of a quality suitable for human consumption has limited their full-scale testing and perceived utility. What has been needed is a suitable method for producing the appropriate glycans in sufficient quantities at reasonable cost. Prior to the invention described herein, there were attempts to use several distinct synthetic approaches for glycan synthesis. Some chemical approaches can synthesize oligosaccharides (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003)), but reactants for these methods are expensive and potentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Enzymes expressed from engineered organisms (Albermann, C., Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16, 205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J. Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficient synthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999)); Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but the high cost of the reactants, especially the sugar nucleotides, limits their utility for low-cost, large-scale production. Microbes have been genetically engineered to express the glycosyltransferases needed to synthesize oligosaccharides from the bacteria's innate pool of nucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)). However, prior to the invention described herein, there was a growing need to identify and characterize additional glycosyltransferases that are useful for the synthesis of HMOS in metabolically engineered bacterial hosts.
Not all α(1,2)fucosyltransferases can utilize lactose as an acceptor sugar. A desired enzyme utilizes GDP-fucose as a donor, and lactose is the acceptor for that donor. A method of identifying novel α(1,2)fucosyltransferase enzymes capable of utilizing lactose as an acceptor was carried out using the following steps:: 1) performing a computational search of sequence databases to define a broad group of simple sequence homologs of any known, lactose-utilizing α(1,2)fucosyltransferase; 2) using the list of homologs from step 1 to derive a search profile containing common sequence and/or structural motifs shared by the members of the broad group, e.g. by using computer programs such as MEME (Multiple Em for Motif Elicitation available at http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) or PSI-BLAST (Position-Specific Iterated BLAST available at ncbi.nlm.nih.gov/blast with additional information at cnx.org/content/m11040/latest/); 3) searching sequence databases (e.g., using computer programs such as PSI-BLAST, or MAST (Motif Alignment Search Tool available at http://meme.sdsc.edu/meme/cgi-bin/mast.cgi); using this derived search profile as query, and identifying “candidate sequences” whose simple sequence homology to the original lactose-accepting α(1,2)fucosyltransferase is 40% or less; 4) scanning the scientific literature and developing a list of “candidate organisms” known to express α(1,2)fucosyl-glycans; 5) selecting only those “candidate sequences” that are derived from “candidate organisms” to generate a list of “candidate lactose-utilizing enzymes”; and 6) expressing each “candidate lactose-utilizing enzyme” and testing for lactose-utilizing α(1,2)fucosyltransferase activity.
The MEME suite of sequence analysis tools (meme.sdsc.edu/meme/cgi-bin/meme.cgi) can also be used as an alternative to PSI-BLAST. Sequence motifs are discovered using the program “MEME”. These motifs can then be used to search sequence databases using the program “MAST”. The BLAST and PHI-BLAST search algorithms are other well known alternatives.
To test for lactose-utilizing activity, the production of 2′-FL is evaluated in a host organism that expresses the candidate enzyme and which contains both cytoplasmic GDP-fucose and lactose pools. The production of 2′-FL indicates that the candidate enzyme-encoding sequence functions as a lactose-utilizing α(1,2)fucosyltransferase.
To find enzymes with similarity to FutC, entire amino acid of FutC was used as a query in PSI-BLAST. The results of the lactose-utilizing α(1,2)fucosyltransferase identification method of this invention are surprising, because the % identity of several of the lactose-utilizing α(1,2)fucosyltransferases identified are less than 40% of the reference FutC sequence. Another most surprising aspect of the study is that 8 of the 10 candidates tested were able to utilize lactose as an acceptor, 3 of which did so at levels very close to the “gold-standard” enzyme FutC. This was a higher “hit rate” was anticipated. While 6 out of 10 of the candidate enzymes are found in bacteria that incorporate α(1,2)fucose into their LPS structure, the oligosaccharides to which the fucose is attached are very different than the lactose each candidate enzyme is being asked to utilize in the query. Moreover, it was surprising that both WblA and WbgN could utilize lactose as an acceptor, because both of these enzymes are found in bacteria that do not incorporate fucose into their LPS structure. Rather, they utilize a related sugar called colitose.
Human Milk Glycans
Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides (Kunz, C., Rudloff, S., Baier, W., Klein, N., and Strobel, S. (2000). Annu Rev Nutr 20, 699-722; Bode, L. (2006). J Nutr 136, 2127-130). More than 130 different complex oligosaccharides have been identified in human milk, and their structural diversity and abundance is unique to humans. Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome (Marcobal, A., Barboza, M., Froehlich, J. W., Block, D. E., et al. J Agric Food Chem 58, 5334-5340 (2010)), in the prevention of disease (Newburg, D. S., Ruiz-Palacios, G. M. & Morrow, A. L. Annu Rev Nutr 25, 37-58 (2005)), and in immune function (Newburg, D. S. & Walker, W. A. Pediatr Res 61, 2-8 (2007)). Despite millions of years of exposure to human milk oligosaccharides (HMOS), pathogens have yet to develop ways to circumvent the ability of HMOS to prevent adhesion to target cells and to inhibit infection. The ability to utilize HMOS as pathogen adherence inhibitors promises to address the current crisis of burgeoning antibiotic resistance. Human milk oligosaccharides produced by biosynthesis represent the lead compounds of a novel class of therapeutics against some of the most intractable scourges of society.
One alternative strategy for efficient, industrial-scale synthesis of HMOS is the metabolic engineering of bacteria. This approach involves the construction of microbial strains overexpressing heterologous glycosyltransferases, membrane transporters for the import of precursor sugars into the bacterial cytosol, and possessing enhanced pools of regenerating nucleotide sugars for use as biosynthetic precursors (Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Ruffing, A., and Chen, R. R. (2006). Microb Cell Fact 5, 25). A key aspect of this approach is the heterologous glycosyltransferase selected for overexpression in the microbial host. The choice of glycosyltransferase can significantly affect the final yield of the desired synthesized oligosaccharide, given that enzymes can vary greatly in terms of kinetics, substrate specificity, affinity for donor and acceptor molecules, stability and solubility. A few glycosyltransferases derived from different bacterial species have been identified and characterized in terms of their ability to catalyze the biosynthesis of HMOS in E. coli host strains (Dumon, C., Bosso, C., Utille, J. P., Heyraud, A., and Samain, E. (2006). Chembiochem 7, 359-365; Dumon, C., Samain, E., and Priem, B. (2004). Biotechnol Prog 20, 412-19; Li, M., Liu, X. W., Shao, J., Shen, J., Jia, Q., Yi, W., Song, J. K., Woodward, R., Chow, C. S., and Wang, P. G. (2008). Biochemistry 47, 378-387). The identification of additional glycosyltransferases with faster kinetics, greater affinity for nucleotide sugar donors and/or acceptor molecules, or greater stability within the bacterial host significantly improves the yields of therapeutically useful HMOS. Prior to the invention described herein, chemical syntheses of HMOS were possible, but were limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). The invention overcomes the shortcomings of these previous attempts by providing new strategies to inexpensively manufacture large quantities of human milk oligosaccharides (HMOS) for use as dietary supplements. Advantages include efficient expression of the enzyme, improved stability and/or solubility of the gene product (2′-FL) and reduced toxicity to the host organism. For example, α(1,2)fucosyltransferases derived from E. coli strains (e.g. WbgL) are more stable and are expressed at higher levels within E. coli production hosts strains compared to FutC. In another example, highly active fucosyltransferase (futN) is derived from a commensal microbe (Bacteroides) rather than a pathogen. Since many engineered production strains use fucosyltransferase genes obtained from pathogens, safety and/or increased consumer acceptance are added advantages of this sequence/enzyme.
As described in detail below, E. coli (or other bacteria) is engineered to produce 2′-FL in commercially viable levels. For example, yields are >5 grams/liter in a bacterial fermentation process.
Role of Human Milk Glycans in Infectious Disease
Human milk glycans, which comprise both unbound oligosaccharides and their glycoconjugates, play a significant role in the protection and development of the infant gastrointestinal (GI) tract. Neutral fucosylated oligosaccharides, including 2′-fucosyllactose (2′-FL), protect infants against several important pathogens. Milk oligosaccharides found in various mammals differ greatly, and the composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Approximately 200 distinct human milk oligosaccharides have been identified and combinations of simple epitopes are responsible for this diversity (Newburg D. S., 1999 Curr_Med Chem, 6:117-127; Ninonuevo M. et al., 2006 J Agric Food Chem, 54:7471-74801).
Human milk oligosaccharides are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). Human milk oligosaccharides are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J. et al., 1999 Anal Biochem, 273:261-277; Martin-Sola et al., 2003 J Dairy Sci, 86:52-59; Parkkinen J. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001 J Chromatogr A, 921:315-321).
Approximately 70-80% of oligosaccharides in human milk are fucosylated, and their synthetic pathways are believed to proceed as shown in FIG. 1 (Type I and Type II pathways begin with different precursor molecules). A smaller proportion of the oligosaccharides are sialylated or both fucosylated and sialylated, but their synthetic pathways are not fully defined. Understanding of the acidic (sialylated) oligosaccharides is limited in part by the ability to measure these compounds. Sensitive and reproducible methods for the analysis of both neutral and acidic oligosaccharides have been designed. Human milk oligosaccharides as a class survive transit through the intestine of infants very efficiently, being essentially indigestible (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L. K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)).
Human Milk Glycans Inhibit Binding of Enteropathogens to their Receptors
Human milk glycans have structural homology to cell receptors for enteropathogens and function as receptor decoys. For example, pathogenic strains of Campylobacter bind specifically to glycans containing H-2, i.e., 2′-fucosyl-N-acetyllactosamine or 2′-fucosyllactose (2′FL); Campylobacter binding and infectivity are inhibited by 2′-FL and other glycans containing this H-2 epitope. Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by human milk oligosaccharides containing 2-linked fucose moieties. Several major strains of human caliciviruses, especially the noroviruses, also bind to 2-linked fucosylated glycans, and this binding is inhibited by human milk 2-linked fucosylated glycans. Consumption of human milk that has high levels of these 2-linked fucosyloligosaccharides was associated with lower risk of norovirus, Campylobacter, ST of E. coli-associated diarrhea, and moderate-to-severe diarrhea of all causes in a Mexican cohort of breastfeeding children (Newburg D. S. et al., 2004 Glycobiology, 14:253-263; Newburg D. S. et al., 1998 Lancet, 351:1160-1164). Several pathogens utilize sialylated glycans as their host receptors, such as influenza (Couceiro, J. N., Paulson, J. C. & Baum, L. G. Virus Res 29, 155-165 (1993)), parainfluenza (Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81, 8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P., Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317 (1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori (Mandavi, J., Sonden, B., Hurtig, M., Olfat, F. O., et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J., Ruvoen-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).
Engineering of E. coli to Produce Human Milk Oligosaccharide 2′-FL
Described herein is a gene screening approach, which was used to identify new α(1,2)fucosyltransferases (α(1,2) FTs) for the synthesis of fucosyl-linked oligosaccharides in metabolically engineered E. coli. Of particular interest are α(1,2) FTs that are capable of the synthesis of the HMOS 2′-fucosyllactose (2′-FL). 2′-FL is the most abundant fucosylated oligosaccharide present in human milk, and this oligosaccharide provides protection to newborn infants against infectious diarrhea caused by bacterial pathogens such as Campylobacter jejuni (Ruiz-Palacios, G. M., et al. (2003). J Biol Chem 278, 14112-120; Morrow, A. L. et al. (2004). J Pediatr 145, 297-303; Newburg, D. S. et al. (2004). Glycobiology 14, 253-263).
The synthetic pathway of the fucosyl oligosaccharides of human milk is illustrated in FIG. 1. Structurally, 2′-FL consists of a fucose molecule α1,2 linked to the galactose portion of lactose (Fucα1-2Galβ1-4Glc). An α(1,2) FT from H. pylori strain 26695 termed FutC has been utilized to catalyze the synthesis of 2′-FL in metabolically engineered E. coli (Drouillard, S. et al. (2006). Angew Chem Int Ed Engl 45, 1778-780). Therefore, the amino acid sequence of FutC was used as a query in the search algorithm PSI-BLAST (Position Specific Iterated Basic Local Alignment Search Tool) to identify candidate novel α(1,2) FTs for the production of 2′-FL in E. coli. Using PSI-BLAST, a list of closely related protein sequences is created based on the query sequence. The algorithm then generates a profile sequence, which summarizes significant motifs present in these sequences. This profile is then used as a new query to identify a larger group of candidate sequences, and the process is iterated to generate an even larger group of candidates.
The FutC amino acid sequence was used as a query for 2 iterations of the PSI-BLAST search algorithm. This search yielded a group of 277 candidates with similarity to FutC, some of which were more closely related (shared amino acid identity greater than 25%) as well as a group that was more distantly related to FutC (shared amino acid identity less than 25%). Of the more closely related group, the predicted α(1,2) FTs from bacterial species that incorporate fucose into the O-antigen of their lipopolysaccharide (LPS) or into the polysaccharide subunits that compose the cell surface capsule were analyzed. α(1,2) FTs from these types of organisms are more likely to utilize fucose as a substrate, given the presence of fucose in their surface carbohydrate structures. α(1,2) FTs from known enteric bacterial species, either commensals or pathogens were also analyzed. Such organisms sometimes display carbohydrate structures on their cell-surface that contain fucose and mimic various 2′-fucosyl containing Lewis antigen structures found in higher organisms (Appelmelk, B. J. et al. (1998). Infect Immun 66, 70-76; Coyne, M. J. et al. (2005). Science 307, 1778-781). Candidate α(1,2) FTs from these types of organisms are more likely to utilize fucose as a substrate and also to catalyze the linkage of fucose to useful acceptor oligosaccharides.
Ten α(1,2) FTs with greater than 25% homology at the amino acid level to FutC identified from the screen were analyzed (Table 1).







TABLE 1







Summary of candidate α (1,2) fucosyltransferases tested for their ability to


promote 2′-FL in engineered E. coli strains. The activity of each candidate


was compared to FutC and described semi-quantitatively using the “+”


symbol in the last column, where FutC is assessed the highest activity


with 4 “+” symbols.












Gene
Accession

% Identity
Fucose in LPS
2′-FL


Name
No. (NCBI)
Organism
w/FutC
or capsule?
Synthesis





futC
NP_206893
H. pylori 26695
—
Yes
++++



NP_206894


wblA
BAA33632
V. cholerae
28%
No
+




O22


wbgL
ADN43847
E. Coli O126
25%
Yes
+++


futD
ZP_04580654
H. bilis ATCC
39%
Yes
+




437879


futE
ZP_07805473
H. cinaedi
44%
Unknown
−




CCUG 18818


futL
YP_003517185
H. mustelae
70%
Yes
+++




ATCC 43772


futN
YP_001300461
B. vulgatus
27%
Unknown
++




ATCC 8482


futO
ZP_02065239
B. ovatus ATCC
27%
Unknown
−




8483


wbgN
YP_003500093
E coli O55:H7
28%
No
+


bft1
CAH09369
B. fragilis 9343
34%
Yes
−


bft3/wcfB
CAH06753
B. fragilis 9343
28%
Yes
+









The amino acid sequence of Helicobacter pylori 26695 alpha-(1,2)fucosyltransferase (FutC) is set forth below (SEQ ID NO: 2; GenBank Accession Number NP—206893 and NP—206894 (GI:15644723 and 15644724), incorporated herein by reference).









1
mafkvvqicg glgnqmfqya fakslqkhln tpvllditsf dwsnrkmqle lfpidlpyas



 


61
akeiaiakmq hlpklvrdtl kcmgfdrvsq eivfeyepgl lkpsrltyfy gyfqdpryfd


 


121
aisplikqtf tlpppengnn kkkeeeyhrk lalilaakns vfvhvrrgdy vgigcqlgid


 


181
yqkkaleyia krvpnmelfv fcedlkftqn ldlgypfmdm ttrdkeeeay wdmllmqsck


 


241
hgiianstys wwaaylinnp ekiiigpkhw lfghenilck ewvkieshfe vkskkyna






The amino acid sequence of Vibrio cholera O22 WblA is set forth below (SEQ ID NO: 3; GenBank Accession Number BAA33632 (GI:3721682), incorporated herein by reference).









1
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61
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121
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181
vdyikekiea pvffvfsndv awckdnfnfi dspvfiedtq teiddlmlmc qcqhnivans


 


241
sfswwaawln snvdkiviap ktwmaenpkg ykwvpdswre i






The amino acid sequence of Escherichia coli O126 WbgL is set forth below (SEQ ID NO: 4; GenBank Accession Number ADN43847 (GI:307340785), incorporated herein by reference).









1
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61
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121
ffhkhildlk effipknvse qanllaakil esqsslsihi rrgdyiknkt atlthgvcsl


 


181
eyykkalnki rdlamirdvf ifsddifwck enietllskk yniyysedls qeedlwlmsl


 


241
anhhiianss fswwgaylgt sasqiviypt pwyditpknt yipivnhwin vdkhssc






The amino acid sequence of Helicobacter bilis ATCC 437879 FutD is set forth below (SEQ ID NO: 5; GenBank Accession Number ZP—04580654 (GI: 237750174), incorporated herein by reference).









1
mgdykivelt cglgnqmfqy afakalqkhl qvpvlldktw ydtqdnstqf sldifnvdle



 


61
yatntqieka karvsklpgl lrkmfglkkh niaysqsfdf hdeyllpndf tyfsgffqna


 


121
kylkgleqel ksifyydsnn fsnfgkqrle lilqaknsif ihirrgdyck igwelgmdyy


 


181
kraiqyimdr veepkffifg atdmsfteqf qknlglnenn sanlsektit qdnqhedmfl


 


241
mcyckhaila nssysfwsay lnndannivi aptpwlldnd niicddwiki ssk






The amino acid sequence of Helicobacter cinaedi CCUG 18818 alpha-1,2-fucosyltransferase (FutE) is set forth below (SEQ ID NO: 6; GenBank Accession Number ZP—07805473 (GI:313143280), incorporated herein by reference).









1
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61
afpyginmsf vysndvyall kddfrlkipl rydnamlkkq iqntdksvfl hirrgdylqs


 


121
eglyvvlgvt yyqkaleilk skitnphifv fsndmcwcke ylmryvdfsg ctidfiegnt


 


181
egnaveemel mrscqhaiia nstfswwaay lienpdkivi mpkeylndss rflpkqflal


 


241
knwflvdhiw gsvelan






The amino acid sequence of Helicobacter mustelae 12198 (ATCC 43772) alpha-1,2-fucosyltransferase (FutL) is set forth below (SEQ ID NO: 7; GenBank Accession Number YP—003517185 (GI:291277413), incorporated herein by reference).









1
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61
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121
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181
iaymakrvqn lelflfcedl efvqnldlgy pfvdmttrdg aahwdmmlmq sckhgiitns


 


241
tyswwaayli knpekiiigp shwiygneni lckdwvkies qfetks






One α(1,2)fucosyltransferase identified through the screen that possessed comparable enzymatic activity relative to FutC was termed FutL. FutL was found to direct the synthesis of 2′-FL at  ̃75% the level of FutC in the metabolically engineered E. coli production strain (Table 1 and FIG. 3). In addition, the data indicated that FutL is significantly less efficient at promoting the synthesis of LDFT, a byproduct that was observed with other α(1,2)FTs Therefore, FutL offers advantages over the others, e.g., the ability to robustly produce 2′-FL without the concern of concurrently producing other undesirable contaminating oligosaccharides. FutL is derived from Helicobacter mustelae and is 70% identical to FutC at the amino acid level.
The amino acid sequence of Bacteroides vulgatus ATCC 8482 glycosyl transferase family protein (FutN) is set forth below (SEQ ID NO: 8; GenBank Accession Number YP—001300461 (GI:150005717), incorporated herein by reference).









1
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61
inqplkkvie flffkkiyer kqapnslraf ekkyfwplly fkgfyqserf fadikdevre


 


121
sftfdknkan srslnmleil dkdenayslh irrgdylqpk hwattgsvcq lpyyqnaiae


 


181
msrrvaspsy yifsddiawv kenlplqnav yidwntdeds wqdmmlmshc khhiicnstf


 


241
swwgawlnpn mdktvivpsr wfqhseapdi yptgwikvpv s






The amino acid sequence of Bacteroides ovatus ATCC 8483 FutO is set forth below (SEQ ID NO: 9; GenBank Accession Number ZP—02065239 (GI:160884236), incorporated herein by reference).









1
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61
aalsqlarva ypffnykswq lmrhflplrk smasgttqip fdysevtrnd nvyydgywqn


 


121
eknflsirdk vikaftfpef rdeknkalsd klksvktasc hirrgdylkd piygvcnsdy


 


181
ytraitelnq svnpdmycif sddigwcken fkfligdkev vfvdwnkgqe sfydmqlmsl


 


241
chyniianss fswwgawlnn nddkvvvape rwmnktlend picdnwkrik ve






The amino acid sequence of Escherichia coli O55:H7 (str. CB9615) fucosyltransferase (WbgN) is set forth below (SEQ ID NO: 10; GenBank Accession Number YP—003500093 (GI:291283275), incorporated herein by reference).









1
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61
vmskkemcif pnfivraink fpklslcskr feseqyskki ngsmkgsvef igfwqneryf


 


121
lehkeklrei ftpininlda kelsdvirct nsysvhirrg dyvsnvealk ihglcteryy


 


181
idsirylker fnnlvffvfs ddiewckkyk neifsrsddv kfiegntqev dmwlmsnaky


 


241
hiianssfsw wgawlknYdl gitiaptpwf ereelnsfdp cpekwvriek






The amino acid sequence of Bacteroides fragilis (NCTC) 9343 alpha-1,2-fucosyltransferase (Bft1) is set forth below (SEQ ID NO: 11, GenBank Accession Number CAH09369 (GI:60494568), incorporated herein by reference).









1
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61
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121
sykyftniea airgqfhfsk vlsdknefik kqmqnsnsys lhvrlgdyvn npaysnicts


 


181
ayynkainii qskvsepkff vfsddtvwck dhlkipnchi idwnnkeesy wdmclmtyck


 


241
hniianssfs wwgawlntnp eriviapgkw inddrvqvsd iipsdwicv






The amino acid sequence of Bacteroides fragilis (NCTC) 9343 fucosyl transferase (Bft3/WcfB) is set forth below (SEQ ID NO: 12; GenBank Accession Number CAH06753 (GI:60491992), incorporated herein by reference).









1
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61
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121
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181
kryyfientt plldlyiqsl cthniisnss fswwgawlne nsnkiviapq mwfgisvklg


 


241
vsdllpvswv rlpnnytlgr ycfalykvve dyllnilrli wkrkknm






Homology Comparison Matrix of Fucosyltransferases Examined in this Study:











Homology comparison matrix of fucosyltransferases examined in this study:




















FutC
WbsJ
WbgL
WbIA
WbgN
Bft1
Bft3
FutD
FutE
FutL
FutN
FutO






















FutC
—
30%
28%
28%
28%
34%
28%
39%
44%
70%
27%
27%


WbsJ
30%
—
33%
33%
36%
36%
40%
30%
35%
30%
33%
36%


WbgL
28%
33%
—
33%
37%
32%
39%
31%
32%
25%
32%
33%


WbIA
28%
33%
33%
—
36%
37%
33%
31%
38%
29%
31%
35%


WbgN
28%
36%
37%
36%
—
32%
37%
30%
38%
30%
32%
35%


Bft1
34%
36%
32%
37%
32%
—
30%
32%
37%
33%
35%
38%


Bft3
28%
40%
39%
33%
37%
30%
—
30%
33%
29%
34%
35%


FutD
39%
30%
31%
31%
30%
32%
30%
—
34%
40%
28%
31%


FutE
44%
35%
32%
38%
38%
37%
33%
34%
—
33%
33%
36%


FutL
70%
30%
25%
29%
30%
33%
29%
40%
33%
—
30%
28%


FutN
27%
33%
32%
31%
32%
35%
34%
28%
34%
30%
—
37%


FutO
27%
36%
33%
35%
35%
38%
35%
31%
36%
28%
37%
—









All of these proteins are found in bacteria that interact with the gastrointestinal system of higher organisms. In addition, 6 of the 10 selected incorporate fucose into their cell surface glycans. Such genes were predicted to have the strongest activity in terms of fucosyl-oligosaccharide synthesis. In this group of 10 candidates, 2 enzymes found in bacterial strains that do not incorporate fucose into cell surface glycans (WblA and WbgN) were also included. It was predicted that these candidates would have little or no fucosyl-oligosaccharide synthesis activity, and therefore might serve as a useful negative control to validate the screening approach.
Candidate α(1,2) FTs were cloned by standard molecular biological techniques into an expression plasmid. This plasmid utilizes the strong leftwards promoter of bacteriophage λ, (termed PL) to direct expression of the candidate genes (Sanger, F. et al. (1982). J Mol Biol 162, 729-773). The promoter is controllable, e.g., a trp-cI construct is stably integrated the into the E. coli host's genome (at the ampC locus), and control is implemented by adding tryptophan to the growth media. Gradual induction of protein expression is accomplished using a temperature sensitive cI repressor. Another similar control strategy (temperature independent expression system) has been described (Mieschendahl et al., 1986, Bio/Technology 4:802-808). The plasmid also carries the E. coli rcsA gene to up-regulate GDP-fucose synthesis, a critical precursor for the synthesis of fucosyl-linked oligosaccharides. In addition, the plasmid carries a β-lactamase (bla) gene for maintaining the plasmid in host strains by ampicillin selection (for convenience in the laboratory) and a native thyA (thymidylate synthase) gene as an alternative means of selection in thyA− hosts. Alternative selectable markers include the proBA genes to complement proline auxotrophy (Stein et al., (1984), J Bacteriol 158:2, 696-700 (1984) or purC to complement adenine auxotrophy (Parker, J., (1984), J Bacteriol 157:3, 712-7). To act as plasmid selectable markers each of these genes are first inactivated in the host cell chromosome, then wild type copies of the genes are provided on the plasmid. Alternatively a drug resistance gene may be used on the plasmid, e.g. beta-lactamase (this gene is already on the expression plasmid described above, thereby permitting selection with ampicillin). Ampicilline selection is well known in the art and described in standard manuals such as Maniatis et al., (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring, N.Y.
The expression constructs were transformed into a host strain useful for the production of 2′-FL. Biosynthesis of 2′-FL requires the generation of an enhanced cellular pool of both lactose and GDP-fucose (FIG. 2). The wild-type Eschericia coli K12 prototrophic strain W3110 was selected as the parent background to test the ability of the candidates to catalyze 2′-FL production (Bachmann, B. J. (1972). Bacteriol Rev 36, 525-557). The particular W3110 derivative employed was one that previously had been modified by the introduction (at the ampC locus) of a tryptophan-inducible PtrpB cI+ repressor cassette, generating an E. coli strain known as GI724 (LaVallie, E. R. et al. (2000). Methods Enzymol 326, 322-340). Other features of GI724 include lacIq and lacPL8 promoter mutations. E. coli strain GI724 affords economical production of recombinant proteins from the phage λ PL promoter following induction with low levels of exogenous tryptophan (LaVallie, E. R. et al. (1993). Biotechnology (N Y) 11, 187-193; Mieschendahl, et al. (1986). Bio/Technology 4, 802-08). Additional genetic alterations were made to this strain to promote the biosynthesis of 2′-FL. This was achieved in strain GI724 through several manipulations of the chromosome using λ Red recombineering (Court, D. L. et al. (2002). Annu Rev Genet. 36, 361-388) and generalized P1 phage transduction.
First, the ability of the E. coli host strain to accumulate intracellular lactose was engineered by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lad). During construction of this deletion, the lacIq promoter was placed immediately upstream of the lactose permease gene, lacY. The modified strain maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type copy of the lacZ (β-galactosidase) gene responsible for lactose catabolism. Therefore, an intracellular lactose pool is created when the modified strain is cultured in the presence of exogenous lactose. A schematic of the PlacIq lacY+ chromosomal construct is shown in FIG. 6.
Genomic DNA sequence of the PlacIq lacY+ chromosomal construct is set forth below (SEQ ID NO: 13):







CACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCG


 


GAAGAGAGTCAAGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTC


 


TAGAGAATAGGAACTTCGGAATAGGAACTTCGGAATAGGAACTAAGGAG


 


GATATTCATATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTT


 


TATTCTTTTTCTTTTACTTTTTTATCATGGGAGCCTACTTCCCGTTTTT


 


CCCGATTTGGCTACATGACATCAACCATATCAGCAAAAGTGATACGGGT


 


ATTATTTTTGCCGCTATTTCTCTGTTCTCGCTATTATTCCAACCGCTGT


 


TTGGTCTGCTTTCTGACAAACTCGGGCTGCGCAAATACCTGCTGTGGAT


 


TATTACCGGCATGTTAGTGATGTTTGCGCCGTTCTTTATTTTTATCTTC


 


GGGCCACTGTTACAATACAACATTTTAGTAGGATCGATTGTTGGTGGTA


 


TTTATCTAGGCTTTTGTTTTAACGCCGGTGCGCCAGCAGTAGAGGCATT


 


TATTGAGAAAGTCAGCCGTCGCAGTAATTTCGAATTTGGTCGCGCGCGG


 


ATGTTTGGCTGTGTTGGCTGGGCGCTGTGTGCCTCGATTGTCGGCATCA


 


TGTTCACCATCAATAATCAGTTTGTTTTCTGGCTGGGCTCTGGCTGTGC


 


ACTCATCCTCGCCGTTTTACTCTTTTTCGCCAAAACGGATGCGCCCTCT


 


TCTGCCACGGTTGCCAATGCGGTAGGTGCCAACCATTCGGCATTTAGCC


 


TTAAGCTGGCACTGGAACTGTTCAGACAGCCAAAACTGTGGTTTTTGTC


 


ACTGTATGTTATTGGCGTTTCCTGCACCTACGATGTTTTTGACCAACAG


 


TTTGCTAATTTCTTTACTTCGTTCTTTGCTACCGGTGAACAGGGTACGC


 


GGGTATTTGGCTACGTAACGACAATGGGCGAATTACTTAACGCCTCGAT


 


TATGTTCTTTGCGCCACTGATCATTAATCGCATCGGTGGGAAAAACGCC


 


CTGCTGCTGGCTGGCACTATTATGTCTGTACGTATTATTGGCTCATCGT


 


TCGCCACCTCAGCGCTGGAAGTGGTTATTCTGAAAACGCTGCATATGTT


 


TGAAGTACCGTTCCTGCTGGTGGGCTGCTTTAAATATATTACCAGCCAG


 


TTTGAAGTGCGTTTTTCAGCGACGATTTATCTGGTCTGTTTCTGCTTCT


 


TTAAGCAACTGGCGATGATTTTTATGTCTGTACTGGCGGGCAATATGTA


 


TGAAAGCATCGGTTTCCAGGGCGCTTATCTGGTGCTGGGTCTGGTGGCG


 


CTGGGCTTCACCTTAATTTCCGTGTTCACGCTTAGCGGCCCCGGCCCGC


 


TTTCCCTGCTGCGTCGTCAGGTGAATGAAGTCGCTTAAGCAATCAATGT


 


CGGATGCGGCGCGAGCGCCTTATCCGACCAACATATCATAACGGAGTGA


 


TCGCATTGTAAATTATAAAAATTGCCTGATACGCTGCGCTTATCAGGCC


 


TACAAGTTCAGCGATCTACATTAGCCGCATCCGGCATGAACAAAGCGCA


 


GGAACAAGCGTCGCA






Second, the ability of the host E. coli strain to synthesize colanic acid, an extracellular capsular polysaccharide, was eliminated by the deletion of the wcaJ gene, encoding the UDP-glucose lipid carrier transferase (Stevenson, G. et al. (1996). J Bacteriol 178, 4885-893). In a wcaJ null background GDP-fucose accumulates in the E. coli cytoplasm (Dumon, C. et al. (2001). Glycoconj J 18, 465-474). A schematic of the chromosomal deletion of wcaJ is shown in FIG. 7.
The sequence of the chromosomal region bearing the ΔwcaJ::FRT mutation is set forth below (SEQ ID NO: 14):







GTTCGGTTATATCAATGTCAAAAACCTCACGCCGCTCAAGCTGGTGATC


 


AACTCCGGGAACGGCGCAGCGGGTCCGGTGGTGGACGCCATTGAAGCCC


 


GCTTTAAAGCCCTCGGCGCGCCCGTGGAATTAATCAAAGTGCACAACAC


 


GCCGGACGGCAATTTCCCCAACGGTATTCCTAACCCACTACTGCCGGAA


 


TGCCGCGACGACACCCGCAATGCGGTCATCAAACACGGCGCGGATATGG


 


GCATTGCTTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTGACGAAAA


 


AGGGCAGTTTATTGAGGGCTACTACATTGTCGGCCTGTTGGCAGAAGCA


 


TTCCTCGAAAAAAATCCCGGCGCGAAGATCATCCACGATCCACGTCTCT


 


CCTGGAACACCGTTGATGTGGTGACTGCCGCAGGTGGCACGCCGGTAAT


 


GTCGAAAACCGGACACGCCTTTATTAAAGAACGTATGCGCAAGGAAGAC


 


GCCATCTATGGTGGCGAAATGAGCGCCCACCATTACTTCCGTGATTTCG


 


CTTACTGCGACAGCGGCATGATCCCGTGGCTGCTGGTCGCCGAACTGGT


 


GTGCCTGAAAGATAAAACGCTGGGCGAACTGGTACGCGACCGGATGGCG


 


GCGTTTCCGGCAAGCGGTGAGATCAACAGCAAACTGGCGCAACCCGTTG


 


AGGCGATTAACCGCGTGGAACAGCATTTTAGCCGTGAGGCGCTGGCGGT


 


GGATCGCACCGATGGCATCAGCATGACCTTTGCCGACTGGCGCTTTAAC


 


CTGCGCACCTCCAATACCGAACCGGTGGTGCGCCTGAATGTGGAATCGC


 


GCGGTGATGTGCCGCTGATGGAAGCGCGAACGCGAACTCTGCTGACGTT


 


GCTGAACGAGTAATGTCGGATCTTCCCTTACCCCACTGCGGGTAAGGGG


 


CTAATAACAGGAACAACGATGATTCCGGGGATCCGTCGACCTGCAGTTC


 


GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAGCC


 


TACAGTTAACAAAGCGGCATATTGATATGAGCTTACGTGAAAAAACCAT


 


CAGCGGCGCGAAGTGGTCGGCGATTGCCACGGTGATCATCATCGGCCTC


 


GGGCTGGTGCAGATGACCGTGCTGGCGCGGATTATCGACAACCACCAGT


 


TCGGCCTGCTTACCGTGTCGCTGGTGATTATCGCGCTGGCAGATACGCT


 


TTCTGACTTCGGTATCGCTAACTCGATTATTCAGCGAAAAGAAATCAGT


 


CACCTTGAACTCACCACGTTGTACTGGCTGAACGTCGGGCTGGGGATCG


 


TGGTGTGCGTGGCGGTGTTTTTGTTGAGTGATCTCATCGGCGACGTGCT


 


GAATAACCCGGACCTGGCACCGTTGATTAAAACATTATCGCTGGCGTTT


 


GTGGTAATCCCCCACGGGCAACAGTTCCGCGCGTTGATGCAAAAAGAGC


 


TGGAGTTCAACAAAATCGGCATGATCGAAACCAGCGCGGTGCTGGCGGG


 


CTTCACTTGTACGGTGGTTAGCGCCCATTTCTGGCCGCTGGCGATGACC


 


GCGATCCTCGGTTATCTGGTCAATAGTGCGGTGAGAACGCTGCTGTTTG


 


GCTACTTTGGCCGCAAAATTTATCGCCCCGGTCTGCATTTCTCGCTGGC


 


GTCGGTGGCACCGAACTTACGCTTTGGTGCCTGGCTGACGGCGGACAGC


 


ATCATCAACTATCTCAATACCAACCTTTCAACGCTCGTGCTGGCGCGTA


 


TTCTCGGCGCGGGCGTGGCAGGGGGATACAACCTGGCGTACAACGTGGC


 


CGTTGTGCCACCGATGAAGCTGAACCCAATCATCACCCGCGTGTTGTTT


 


CCGGCATTCGCCAAAATTCAGGACGATACCGAAAAGCTGCGTGTTAACT


 


TCTACAAGCTGCTGTCGGTAGTGGGGATTATCAACTTTCCGGCGCTGCT


 


CGGGCTAATGGTGGTGTCGAATAACTTTGTACCGCTGGTCTTTGGTGAG


 


AAGTGGAACAGCATTATTCCGGTGCTGCAATTGCTGTGTGTGGTGGGTC


 


TGCTGCGCTCCG






Third, the magnitude of the cytoplasmic GDP-fucose pool was enhanced by the introduction of a null mutation into the lon gene. Lon is an ATP-dependant intracellular protease that is responsible for degrading RcsA, which is a positive transcriptional regulator of colanic acid biosynthesis in E. coli (Gottesman, S. & Stout, V. Mol Microbiol 5, 1599-1606 (1991)). In a lon null background, RcsA is stabilized, RcsA levels increase, the genes responsible for GDP-fucose synthesis in E. coli are up-regulated, and intracellular GDP-fucose concentrations are enhanced. The lon gene was almost entirely deleted and replaced by an inserted functional, wild-type, but promoter-less E. coli lacZ+ gene (Δlon::(kan, lacZ+). λ Red recombineering was used to perform the construction. A schematic of the kan, lacZ+ insertion into the ion locus is shown in FIG. 8.
Genomic DNA sequence surrounding the lacZ+ insertion into the lon region in the E. coli strain is set forth below (SEQ ID NO: 15):







GTGGATGGAAGAGGTGGAAAAAGTGGTTATGGAGGAGTGGGTAATTGAT


 


GGTGAAAGGAAAGGGTTGGTGATTTATGGGAAGGGGGAAGGGGAAGAGG


 


GATGTGGTGAATAATTAAGGATTGGGATAGAATTAGTTAAGGAAAAAGG


 


GGGGATTTTATGTGGGGTTTAATTTTTGGTGTATTGTGGGGGTTGAATG


 


TGGGGGAAAGATGGGGATATAGTGAGGTAGATGTTAATAGATGGGGTGA


 


AGGAGAGTGGTGTGATGTGATTAGGTGGGGGAAATTAAAGTAAGAGAGA


 


GGTGTATGATTGGGGGGATGGGTGGAGGTGGAGTTGGAAGTTGGTATTG


 


TGTAGAAAGTATAGGAAGTTGAGAGGGGTTTTGAAGGTGAGGGTGGGGG


 


AAGGAGTGAGGGGGGAAGGGGTGGTAAAGGAAGGGGAAGAGGTAGAAAG


 


GGAGTGGGGAGAAAGGGTGGTGAGGGGGGATGAATGTGAGGTAGTGGGG


 


TATGTGGAGAAGGGAAAAGGGAAGGGGAAAGAGAAAGGAGGTAGGTTGG


 


AGTGGGGTTAGATGGGGATAGGTAGAGTGGGGGGTTTTATGGAGAGGAA


 


GGGAAGGGGAATTGGGAGGTGGGGGGGGGTGTGGTAAGGTTGGGAAGGG


 


GTGGAAAGTAAAGTGGATGGGTTTGTTGGGGGGAAGGATGTGATGGGGG


 


AGGGGATGAAGATGTGATGAAGAGAGAGGATGAGGATGGTTTGGGATGA


 


TTGAAGAAGATGGATTGGAGGGAGGTTGTGGGGGGGGTTGGGTGGAGAG


 


GGTATTGGGGTATGAGTGGGGAGAAGAGAGAATGGGGTGGTGTGATGGG


 


GGGGTGTTGGGGGTGTGAGGGGAGGGGGGGGGGGTTGTTTTTGTGAAGA


 


GGGAGGTGTGGGGTGGGGTGAATGAAGTGGAGGAGGAGGGAGGGGGGGT


 


ATGGTGGGTGGGGAGGAGGGGGGTTGGTTGGGGAGGTGTGGTGGAGGTT


 


GTGAGTGAAGGGGGAAGGGAGTGGGTGGTATTGGGGGAAGTGGGGGGGG


 


AGGATGTGGTGTGATGTGAGGTTGGTGGTGGGGAGAAAGTATGGATGAT


 


GGGTGATGGAATGGGGGGGGTGGATAGGGTTGATGGGGGTAGGTGGGGA


 


TTGGAGGAGGAAGGGAAAGATGGGATGGAGGGAGGAGGTAGTGGGATGG


 


AAGGGGGTGTTGTGGATGAGGATGATGTGGAGGAAGAGGATGAGGGGGT


 


GGGGGGAGGGGAAGTGTTGGGGAGGGTGAAGGGGGGATGGGGGAGGGGG


 


AGGATGTGGTGGTGAGGGATGGGGATGGGTGGTTGGGGAATATGATGGT


 


GGAAAATGGGGGGTTTTGTGGATTGATGGAGTGTGGGGGGGTGGGTGTG


 


GGGGAGGGGTATGAGGAGATAGGGTTGGGTAGGGGTGATATTGGTGAAG


 


AGGTTGGGGGGGAATGGGGTGAGGGGTTGGTGGTGGTTTAGGGTATGGG


 


GGGTGGGGATTGGGAGGGGATGGGGTTGTATGGGGTTGTTGAGGAGTTG


 


TTGTAATAAGGGGATGTTGAAGTTGGTATTGGGAAGTTGGTATTGTGTA


 


GAAAGTATAGGAAGTTGGAAGGAGGTGGAGGGTAGATAAAGGGGGGGGT


 


TATTTTTGAGAGGAGAGGAAGTGGTAATGGTAGGGAGGGGGGGTGAGGT


 


GGAATTGGGGGGATAGTGAGGGGGTGGAGGAGTGGTGGGGAGGAATGGG


 


GATATGGAAAGGGTGGATATTGAGGGATGTGGGTTGTTGGGGGTGGAGG


 


AGATGGGGATGGGTGGTTTGGATGAGTTGGTGTTGAGTGTAGGGGGTGA


 


TGTTGAAGTGGAAGTGGGGGGGGGAGTGGTGTGGGGGATAATTGAATTG


 


GGGGGTGGGGGAGGGGAGAGGGTTTTGGGTGGGGAAGAGGTAGGGGGTA


 


TAGATGTTGAGAATGGGAGATGGGAGGGGTGAAAAGAGGGGGGAGTAAG


 


GGGGTGGGGATAGTTTTGTTGGGGGGGTAATGGGAGGGAGTTTAGGGGG


 


TGTGGTAGGTGGGGGAGGTGGGAGTTGAGGGGAATGGGGGGGGGATGGG


 


GTGTATGGGTGGGGAGTTGAAGATGAAGGGTAATGGGGATTTGAGGAGT


 


AGGATGAATGGGGTAGGTTTTGGGGGTGATAAATAAGGTTTTGGGGTGA


 


TGGTGGGAGGGGTGAGGGGTGGTAATGAGGAGGGGATGAGGAAGTGTAT


 


GTGGGGTGGAGTGGAAGAAGGGTGGTTGGGGGTGGTAATGGGGGGGGGG


 


GTTGGAGGGTTGGAGGGAGGGGTTAGGGTGAATGGGGGTGGGTTGAGTT


 


AGGGGAATGTGGTTATGGAGGGGTGGAGGGGTGAAGTGATGGGGGAGGG


 


GGGTGAGGAGTTGTTTTTTATGGGGAATGGAGATGTGTGAAAGAAAGGG


 


TGAGTGGGGGTTAAATTGGGAAGGGTTATTAGGGAGGTGGATGGAAAAA


 


TGGATTTGGGTGGTGGTGAGATGGGGGATGGGGTGGGAGGGGGGGGGGA


 


GGGTGAGAGTGAGGTTTTGGGGGAGAGGGGAGTGGTGGGAGGGGGTGAT


 


GTGGGGGGGTTGTGAGGATGGGGTGGGGTTGGGTTGGAGTAGGGGTAGT


 


GTGAGGGAGAGTTGGGGGGGGGTGTGGGGGTGGGGTAGTTGAGGGAGTT


 


GAATGAAGTGTTTAGGTTGTGGAGGGAGATGGAGAGGGAGTTGAGGGGT


 


TGGGAGGGGGTTAGGATGGAGGGGGAGGATGGAGTGGAGGAGGTGGTTA


 


TGGGTATGAGGGAAGAGGTATTGGGTGGTGAGTTGGATGGTTTGGGGGG


 


ATAAAGGGAAGTGGAAAAAGTGGTGGTGGTGTTTTGGTTGGGTGAGGGG


 


TGGATGGGGGGTGGGGTGGGGAAAGAGGAGAGGGTTGATAGAGAAGTGG


 


GGATGGTTGGGGGTATGGGGAAAATGAGGGGGGTAAGGGGAGGAGGGGT


 


TGGGGTTTTGATGATATTTAATGAGGGAGTGATGGAGGGAGTGGGAGAG


 


GAAGGGGGGGTGTAAAGGGGGATAGTGAGGAAAGGGGTGGGAGTATTTA


 


GGGAAAGGGGGAAGAGTGTTAGGGATGGGGTGGGGGTATTGGGAAAGGA


 


TGAGGGGGGGGGTGTGTGGAGGTAGGGAAAGGGATTTTTTGATGGAGGA


 


TTTGGGGAGAGGGGGGAAGGGGTGGTGTTGATGGAGGGGGGGGTAGATG


 


GGGGAAATAATATGGGTGGGGGTGGTGTGGGGTGGGGGGGGTTGATAGT


 


GGAGGGGGGGGGAAGGATGGAGAGATTTGATGGAGGGATAGAGGGGGTG


 


GTGATTAGGGGGGTGGGGTGATTGATTGGGGAGGGAGGAGATGATGAGA


 


GTGGGGTGATTAGGATGGGGGTGGAGGATTGGGGTTAGGGGTTGGGTGA


 


TGGGGGGTAGGGAGGGGGGATGATGGGTGAGAGGATTGATTGGGAGGAT


 


GGGGTGGGTTTGAATATTGGGTTGATGGAGGAGATAGAGGGGGTAGGGG


 


TGGGAGAGGGTGTAGGAGAGGGGATGGTTGGGATAATGGGAAGAGGGGA


 


GGGGGTTAAAGTTGTTGTGGTTGATGAGGAGGATATGGTGGAGGATGGT


 


GTGGTGATGGATGAGGTGAGGATGGAGAGGATGATGGTGGTGAGGGTTA


 


AGGGGTGGAATGAGGAAGGGGTTGGGGTTGAGGAGGAGGAGAGGATTTT


 


GAATGGGGAGGTGGGGGAAAGGGAGATGGGAGGGTTGTGGTTGAATGAG


 


GGTGGGGTGGGGGGTGTGGAGTTGAAGGAGGGGAGGATAGAGATTGGGG


 


ATTTGGGGGGTGGAGAGTTTGGGGTTTTGGAGGTTGAGAGGTAGTGTGA


 


GGGGATGGGGATAAGGAGGAGGGTGATGGATAATTTGAGGGGGGAAAGG


 


GGGGGTGGGGGTGGGGAGGTGGGTTTGAGGGTGGGATAAAGAAAGTGTT


 


AGGGGTAGGTAGTGAGGGAAGTGGGGGGAGATGTGAAGTTGAGGGTGGA


 


GTAGAGGGGGGGTGAAATGATGATTAAAGGGAGTGGGAAGATGGAAATG


 


GGTGATTTGTGTAGTGGGTTTATGGAGGAAGGAGAGGTGAGGGAAAATG


 


GGGGTGATGGGGGAGATATGGTGATGTTGGAGATAAGTGGGGTGAGTGG


 


AGGGGAGGAGGATGAGGGGGAGGGGGTTTTGTGGGGGGGGTAAAAATGG


 


GGTGAGGTGAAATTGAGAGGGGAAAGGAGTGTGGTGGGGGTAAGGGAGG


 


GAGGGGGGGTTGGAGGAGAGATGAAAGGGGGAGTTAAGGGGATGAAAAA


 


TAATTGGGGTGTGGGGTTGGTGTAGGGAGGTTTGATGAAGATTAAATGT


 


GAGGGAGTAAGAAGGGGTGGGATTGTGGGTGGGAAGAAAGGGGGGATTG


 


AGGGTAATGGGATAGGTGAGGTTGGTGTAGATGGGGGGATGGTAAGGGT


 


GGATGTGGGAGTTTGAGGGGAGGAGGAGAGTATGGGGGTGAGGAAGATG


 


GGAGGGAGGGAGGTTTGGGGGAGGGGTTGTGGTGGGGGAAAGGAGGGAA


 


AGGGGGATTGGGGATTGAGGGTGGGGAAGTGTTGGGAAGGGGGATGGGT


 


GGGGGGGTGTTGGGTATTAGGGGAGGTGGGGAAAGGGGGATGTGGTGGA


 


AGGGGATTAAGTTGGGTAAGGGGAGGGTTTTGGGAGTGAGGAGGTTGTA


 


AAAGGAGGGGGAGTGAATGGGTAATGATGGTGATAGTAGGTTTGGTGAG


 


GTTGTGAGTGGAAAATAGTGAGGTGGGGGAAAATGGAGTAATAAAAAGA


 


GGGGTGGGAGGGTAATTGGGGGTTGGGAGGGTTTTTTTGTGTGGGTAAG


 


TTAGATGGGGGATGGGGGTTGGGGTTATTAAGGGGTGTTGTAAGGGGAT


 


GGGTGGGGTGATATAAGTGGTGGGGGTTGGTAGGTTGAAGGATTGAAGT


 


GGGATATAAATTATAAAGAGGAAGAGAAGAGTGAATAAATGTGAATTGA


 


TGGAGAAGATTGGTGGAGGGGGTGATATGTGTAAAGGTGGGGGTGGGGG


 


TGGGTTAGATGGTATTATTGGTTGGGTAAGTGAATGTGTGAAAGAAGG






Fourth, a thyA (thymidylate synthase) mutation was introduced into the strain by P1 transduction. In the absence of exogenous thymidine, thyA strains are unable to make DNA and die. The defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort, M., Maley, G. F., and Maley, F. (1983). Proc Natl Acad Sci USA 80, 1858-861). This complementation was used here as a means of plasmid maintenance.
An additional modification that is useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of 2′-FL) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the E. coli cytoplasm. High intracellular osmolarity (e.g., caused by a high intracellular lactose pool) can inhibit bacterial growth, and E. coli has evolved a mechanism for protecting itself from high intra cellular osmlarity caused by lactose by “tagging” excess intracellular lactose with an acetyl group using LacA, and then actively expelling the acetyl-lactose from the cell (Danchin, A. Bioessays 31, 769-773 (2009)). Production of acetyl-lactose in E. coli engineered to produce 2′-FL or other human milk oligosaccharides is therefore undesirable: it reduces overall yield. Moreover, acetyl-lactose is a side product that complicates oligosaccharide purification schemes. The incorporation of a lacA mutation resolves these problems. Sub-optimal production of fucosylated oligosaccharides occurs in strains lacking either or both of the mutations in the colanic acid pathway and the lon protease. Diversion of lactose into a side product (acetyl-lactose) occurs in strains that don't contain the lacA mutation. A schematic of the lacA deletion and corresponding genomic sequence is provided above (SEQ ID NO: 13).
The strain used to test the different α(1,2) FT candidates incorporates all the above genetic modifications and has the following genotype:
ΔampC::PtrpBcI, Δ(lacI-lacZ)::FRT, PlacIqlacY+, ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ+), ΔlacA
The E. coli strains harboring the different α(1,2) FT candidate expression plasmids were analyzed. Strains were grown in selective media (lacking thymidine) to early exponential phase. Lactose was then added to a final concentration of 1%, and tryptophan (200 μM) was added to induce expression of each candidate α(1,2) FT from the PL promoter. At the end of the induction period ( ̃20 h) equivalent OD 600 units of each strain were harvested. Lysates were prepared and analyzed for the presence of 2′-FL by thin layer chromatography (TLC). As shown in FIG. 3A, a control strain producing FutC-Myc was efficient in the biosynthesis of 2′-FL and also produced a smaller amount of the tetrasaccharide lactodifucotetraose (LDFT). The previously characterized α(1,2) FT WbsJ from E. coli O128:B12 was also capable of catalyzing 2′-FL synthesis, although only at  ̃30% the level produced by FutC-Myc (FIG. 3A, lanes 5 and 6). WblA (derived from V. cholerae O22) was able to promote 2′-FL synthesis, although at a significantly lower level compared to FutC (FIG. 3A, lanes 7 and 8). This result was not unexpected, as V. cholerae O22 does not incorporate fucose into cell surface glycans (Cox, A. D. et al. (1997). Carbohydr Res 304, 191-208). The strain producing WbgL (derived from E. coli strain O126) from plasmid pG204 synthesized a significant amount of 2′-FL, approximately  ̃75% of the amount produced by FutC-Myc (FIG. 3A, lanes 9 and 10). WbgL was also capable of synthesizing LDFT. The strain producing FutL (derived from H. mustelae ATCC 43772) from plasmid pG216 was capable of directing the synthesis of robust amounts of 2′-FL, comparable to the levels obtained utilizing FutC-Myc and WbgL (FIG. 3B, lanes 7 and 8). Furthermore, a strain producing FutN (derived from B. vulgatus ATCC 8482) from plasmid pG217 also produced significant amounts of 2′-FL, approximately  ̃50% the amount produced by FutC-Myc (FIG. 3C, lanes 5 and 6). FutN is derived from the commensal bacterium B. vulgatus, and therefore may not be subject to the same concerns associated with utilization of an α(1,2) FT obtained from a pathogenic bacterium for the production of a food additive.
A map of plasmid pG204 is shown in FIG. 9. The sequence of plasmid pG204 is set forth below (SEQ ID NO: 16):







AATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACCATAGG


 


TGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGG


 


GGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCT


 


TCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGA


 


ACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATT


 


GAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCAT


 


CTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCC


 


TGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCG


 


CACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCCTTCTG


 


CTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTT


 


CATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGG


 


TATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATC


 


TGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGT


 


AGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGA


 


GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC


 


TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC


 


GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG


 


TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC


 


TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG


 


ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG


 


GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC


 


CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT


 


TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC


 


TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCG


 


CCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT


 


ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT


 


GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA


 


CTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT


 


CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT


 


AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAG


 


GATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG


 


GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG


 


ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT


 


AAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG


 


TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC


 


TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTG


 


GCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGA


 


TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT


 


CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG


 


CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT


 


TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC


 


AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT


 


GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA


 


GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT


 


CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT


 


CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG


 


CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAA


 


GTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCT


 


TACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG


 


ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA


 


GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT


 


GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGG


 


TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA


 


CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT


 


AAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC


 


GAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA


 


CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCG


 


GGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCG


 


GGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCAC


 


CATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCG


 


CATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGA


 


GCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCATCATTG


 


TTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGTGCA


 


GTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCGGGT


 


GTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTT


 


CCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGA


 


CGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCCATT


 


TTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGGTGA


 


CAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTGGTT


 


TCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCACC


 


ATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATG


 


GTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCA


 


GATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGCCGC


 


ATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGCTGG


 


CACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACTCTC


 


TTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTC


 


AACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCG


 


ATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCTGTA


 


CAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCGCGT


 


CCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCGACT


 


ACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT


 


TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACG


 


CCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCT


 


TCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC


 


GATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATG


 


TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGA


 


CGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGGGCA


 


TCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCCTGA


 


CTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAAAAT


 


ACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACCTGT


 


TTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATGAAA


 


CGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCCCTG


 


ACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAATGAT


 


AACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCAGAA


 


TATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGAACT


 


GATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACATTG


 


GCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAATGA


 


TGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTCCTC


 


ATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCAAGG


 


TCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTCTAC


 


TCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATCCAT


 


AATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAATTCC


 


CCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCACCT


 


AATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAAAGT


 


GTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGCGAT


 


ATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGGAAG


 


ATGTTCGTgAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCTGA


 


CGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAGCT


 


AACACGAGCTATGTTTATCCACGTTTATCCAGTGATTGACTATGGGGAT


 


ATAAGTATTTTTTGGAGTTATATCGTACCAAGGAGTAGGATAAATAACA


 


ATCTGTGACGCTGATGTACCTAAATAAGCCCCCCACCAACTAAAACTAC


 


TATTCGCTATAATATGATGGTTAGCTAAGCTCATTAACCATAAATCTTC


 


TTCTTGTGATAAATCTTCTGAATAATATATATTATATTTTTTACTGAGT


 


AATGTTTCGATATTTTCTTTACACCAAAAAATATCATCACTGAAAATAA


 


ACACGTCACGTATCATTGCCAAATCGCGTATTTTATTTAAAGCTTTTTT


 


GTAATACTCTAACGAACAAACGCCATGAGTTAAAGTAGCTGTTTTGTTT


 


TTTATATAATCTCCTCTTCTTATATGAATAGAAAGTGATGATTGAGATT


 


CAAGAATTTTTGCTGCAAGTAAATTTGCTTGTTCAGACACATTCTTTGG


 


AATAAAAAATTCTTTTAGATCTAATATATGTTTATGGAAAAAGTGCTCA


 


GATTGCCAATACCCTATATATTTTTTGGATTTCCATTTTTGCGCTATAT


 


ATTCAAAATCATAACCATAGGCATGAAATTCATTGCAAAAACCTAAAAA


 


AAGAAAGATTTCAGGATATAATCTTGACCCACGAACCAAAAATTTATAA


 


ATATTATTAATTTTTGGTGTGTAATACTGTAAATATTCCTCTGGAATTT


 


GTAGATTGTTTAGCCTGTAACCACCATGATCATCATTTTCAGCATAATG


 


ACTTATATCAAAATATAATGGTGTCCCATTAATTTTGGAAAGCGCATAC


 


CCAAATGAGAACTGAAAAAGTTGATTTCCAAGTCCGCCTTGTAATCTTA


 


TAATAGACATTATATCTCCTTCTTG






A map of plasmid pG216 is shown in FIG. 10. The sequence of plasmid pG216 is set forth below (SEQ ID NO: 17):








TCTAGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACC


 


ATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAG


 


AGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTA


 


CCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTT


 


TCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTT


 


CCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCA


 


TCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGT


 


AGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGG


 


AATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCC


 


TTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTC


 


ACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCC


 


AGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGG


 


TTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGT


 


TTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGG


 


GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGA


 


CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA


 


AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA


 


ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC


 


GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA


 


AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT


 


ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC


 


CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG


 


GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG


 


TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG


 


CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC


 


GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA


 


GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG


 


CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT


 


ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG


 


CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA


 


AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT


 


CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA


 


AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC


 


AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA


 


ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG


 


TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC


 


ATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT


 


CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA


 


GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG


 


GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT


 


GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT


 


CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT


 


GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA


 


AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA


 


ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA


 


GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC


 


TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT


 


TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAG


 


GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC


 


AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA


 


AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA


 


ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT


 


CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA


 


ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA


 


CGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT


 


ATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACC


 


TCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGA


 


TGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGG


 


TGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG


 


TGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA


 


TACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAA


 


TGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCAT


 


CATTGTTGAGTMTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGGGT


 


GCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCGCG


 


GGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACG


 


TTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCT


 


CGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTTCC


 


ATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCTGG


 


TGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTGTG


 


GTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTC


 


ACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGT


 


ATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGA


 


CCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGCGC


 


CGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGCGC


 


TGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAACT


 


CTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCG


 


TTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGT


 


GCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCATCT


 


GTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAACCG


 


CGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTTCG


 


ACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGG


 


CATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCG


 


ACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACG


 


CCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAG


 


GGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG


 


ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCA


 


CGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCAGG


 


GCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAGCC


 


TGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACAAA


 


AATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAACC


 


TGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGATG


 


AAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTTCC


 


CTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCAAT


 


GATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTTCA


 


GAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTAGA


 


ACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAACA


 


TTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATTAA


 


TGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAGTC


 


CTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGGCA


 


AGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCCTC


 


TACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAATC


 


CATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATAAT


 


TCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGTCA


 


CCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGTAA


 


AGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTTGC


 


GATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCTGG


 


AAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCATCT


 


GACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCGAG


 


TTAGGATTTCGTTTCGAATTGGGATTCGATTTTAACCCAGTCTTTGCAC


 


AGGATGTTTTCGTTACCGTAAATCCAGTGGGACGGACCAATGATAATTT


 


TTTCCGGATTTTTGATCAGGTAGGCTGCCCACCAGGAGTAAGTGCTGTT


 


AGTGATGATACCGTGTTTGCAAGACTGCATCAGCATCATGTCCCAGTGG


 


GCTGCACCATCACGCGTCGTCATGTCAACAAACGGGTAACCCAGATCCA


 


GGTTCTGTACGAATTCCAGATCCTCGCAGAACAGGAACAGTTCCAGATT


 


TTGAACACGTTTTGCCATATACGCAATGGCGCGCAGCTGGTAGGAGATG


 


TCCAGCTGCCAGCCCAGGCGCATGTAATCGCCACGGCGGATGTGAACGA


 


ACACAGAGTTTTTCGCAGCCAGGATCTGGGACAGTTTACGAGAGTACTG


 


TTCCGCGTGTTCGGTCGGGTGAGGCAGGGTGAAAGTTTGTTTGATCAGA


 


GGGGAGATATCTTCGAAATAGCGCGGGTCCTGAAAGTAGCCATGGAAAT


 


ACGCAATGCGGCTCGGTTCAAACAGTTCCGGCATGTACTCGAATACAAT


 


TTCTTTGCTAACGCGGCCCAGACCCATACGACGCAGTGCACCACGCACC


 


AGACGCGGCAGGTTCTGCATGTGTGCCGCGGCGATCTGCTGGGCGGACG


 


CACACTGCAGGTCGATCGGGAACAGGTGCAGGCCCAGTTCACGGTTACC


 


GTAATCGAACCAAGTGGTATCCAGCAGTACCGGAATGTTCAGGTGAGTC


 


TGCAGAGATTTAGCGAATGCGTACTGGAACATCTGGTTACCCAGGCCGC


 


CGTGCACCTGAACGATTTTGAAATCCATTATATCTCCTTCTTG






A map of plasmid pG217 is shown in FIG. 11. The sequence of plasmid pG217 is set forth below (SEQ ID NO: 18):







TCTAGAATTCTAAAAATTGATTGAATGTATGCAAATAAATGCATACACC


 


ATAGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAG


 


AGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTA


 


CCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTT


 


TCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTT


 


CCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCA


 


TCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGT


 


AGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGG


 


AATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCC


 


TTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTC


 


ACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCC


 


AGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGG


 


TTATCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGT


 


TTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACGCGCGG


 


GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGA


 


CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA


 


AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA


 


ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC


 


GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA


 


AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT


 


ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC


 


CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG


 


GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG


 


TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG


 


CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC


 


GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA


 


GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG


 


CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT


 


ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG


 


CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA


 


AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT


 


CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA


 


AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC


 


AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA


 


ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG


 


TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC


 


ATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCT


 


CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA


 


GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG


 


GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT


 


GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT


 


CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT


 


GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA


 


AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA


 


ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA


 


GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC


 


TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT


 


TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAG


 


GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC


 


AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA


 


AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA


 


ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT


 


CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA


 


ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA


 


CGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT


 


ATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACC


 


TCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGA


 


TGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGG


 


TGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG


 


TGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA


 


TACCGCATCAGGCGCCTCCTCAACCTGTATATTCGTAAACCACGCCCAA


 


TGGGAGCTGTCTCAGGTTTGTTCCTGATTGGTTACGGCGCGTTTCGCAT


 


CATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTTTACCGGTGCCTGG


 


GTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGTCG


 


CGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACA


 


CGTTTCCTGAGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTG


 


CTCGACGAAGGCACACAGAAAAACGACCGTACCGGAACCGGAACGCTTT


 


CCATTTTTGGTCATCAGATGCGTTTTAACCTGCAAGATGGATTCCCGCT


 


GGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGAACTGCTG


 


TGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATG


 


TCACCATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGT


 


GTATGGTAAACAGTGGCGCGCCTGGCCAACGCCAGATGGTCGTCATATT


 


GACCAGATCACTACGGTACTGAACCAGCTGAAAAACGACCCGGATTCGC


 


GCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACTGGATAAAATGGC


 


GCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAAA


 


CTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGC


 


CGTTCAACATTGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCA


 


GTGCGATCTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGACACGCAT


 


CTGTACAGCAACCATATGGATCAAACTCATCTGCAATTAAGCCGCGAAC


 


CGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATCCATCTT


 


CGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCG


 


GGCATTAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGC


 


CGACGCCAGTGTGCGTCGGTTTTTTTACCCTCCGTTAAATTCTTCGAGA


 


CGCCTTCCCGAAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA


 


AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG


 


GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT


 


CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTTCTTTAATGAAGCA


 


GGGCATCAGGACGGTATCTTTGTGGAGAAAGCAGAGTAATCTTATTCAG


 


CCTGACTGGTGGGAAACCACCAGTCAGAATGTGTTAGCGCATGTTGACA


 


AAAATACCATTAGTCACATTATCCGTCAGTCGGACGACATGGTAGATAA


 


CCTGTTTATTATGCGTTTTGATCTTACGTTTAATATTACCTTTATGCGA


 


TGAAACGGTCTTGGCTTTGATATTCATTTGGTCAGAGATTTGAATGGTT


 


CCCTGACCTGCCATCCACATTCGCAACATACTCGATTCGGTTCGGCTCA


 


ATGATAACGTCGGCATATTTAAAAACGAGGTTATCGTTGTCTCTTTTTT


 


CAGAATATCGCCAAGGATATCGTCGAGAGATTCCGGTTTAATCGATTTA


 


GAACTGATCAATAAATTTTTTCTGACCAATAGATATTCATCAAAATGAA


 


CATTGGCAATTGCCATAAAAACGATAAATAACGTATTGGGATGTTGATT


 


AATGATGAGCTTGATACGCTGACTGTTAGAAGCATCGTGGATGAAACAG


 


TCCTCATTAATAAACACCACTGAAGGGCGCTGTGAATCACAAGCTATGG


 


CAAGGTCATCAACGGTTTCAATGTCGTTGATTTCTCTTTTTTTAACCCC


 


TCTACTCAACAGATACCCGGTTAAACCTAGTCGGGTGTAACTACATAAA


 


TCCATAATAATCGTTGACATGGCATACCCTCACTCAATGCGTAACGATA


 


ATTCCCCTTACCTGAATATTTCATCATGACTAAACGGAACAACATGGGT


 


CACCTAATGCGCCACTCTCGCGATTTTTCAGGCGGACTTACTATCCCGT


 


AAAGTGTTGTATAATTTGCCTGGAATTGTCTTAAAGTAAAGTAAATGTT


 


GCGATATGTGAGTGAGCTTAAAACAAATATTTCGCTGCAGGAGTATCCT


 


GGAAGATGTTCGTAGAAGCTTACTGCTCACAAGAAAAAAGGCACGTCAT


 


CTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGCTCG


 


AGTTAGGATACCGGCACTTTGATCCAACCAGTCGGGTAGATATCCGGTG


 


CTTCGGAGTGCTGGAACCAACGGCTCGGCACAATAACAGTCTTATCCAT


 


ATTAGGGTTCAGCCAGGCACCCCACCAAGAAAACGTGCTGTTACAAATG


 


ATGTGATGTTTGCAATGAGACATCAGCATCATATCCTGCCAGGAGTCTT


 


CATCAGTGTTCCAGTCAATATAAACCGCATTCTGCAGTGGCAGATTTTC


 


TTTAACCCACGCGATATCGTCGGAGAAGATATAGTAAGATGGGCTAGCA


 


ACACGACGGGACATTTCCGCGATAGCATTCTGGTAATACGGCAGCTGGC


 


ACACGGAACCGGTAGTAGCCCAGTGTTTCGGCTGCAGATAGTCACCACG


 


ACGAATGTGCAGGGAAACCGCGTTTTCATCTTTGTCCAGGATTTCCAGC


 


ATGTTCAGGCTGCGGGAATTTGCTTTGTTCTTATCAAAGGTGAAGGATT


 


CACGCACTTCGTCTTTGATATCAGCGAAGAAACGCTCGCTCTGATAGAA


 


ACCTTTAAAGTACAGCAGCGGCCAGAAATACTTCTTCTCGAACGCACGC


 


AGAGAGTTCGGCGCCTGCTTGCGTTCGTAGATTTTTTTAAAAAACAGGA


 


ATTCGATAACTTTTTTCAGCGGTTGGTTGATGCAGAATTCGGTGTGCGG


 


CAGGTTGAACACGCGGTGCATTTCGTAACCGTAATGGACTTTGTAATGC


 


ATCATGTCGCTCAGGTCGATACGGACCTTCGGGTAATACTTTTTCATAC


 


GCAGATAGAAAGCATAGATAAACATCTGGTTGCCCAGACCGCCAGTCAC


 


TTTGATCAGACGCATTATATCTCCTTCTTG






Fucosylated oligosaccharides produced by metabolically engineered E. coli cells are purified from culture broth post-fermentation. An exemplary procedure comprises five steps. (1) Clarification: Fermentation broth is harvested and cells removed by sedimentation in a preparative centrifuge at 6000×g for 30 min. Each bioreactor run yields about 5-7 L of partially clarified supernatant. (2) Product capture on coarse carbon: A column packed with coarse carbon (Calgon 12×40 TR) of  ̃1000 ml volume (dimension 5 cm diameter×60 cm length) is equilibrated with 1 column volume (CV) of water and loaded with clarified culture supernatant at a flow rate of 40 ml/min. This column has a total capacity of about 120 g of sugar. Following loading and sugar capture, the column is washed with 1.5 CV of water, then eluted with 2.5 CV of 50% ethanol or 25% isopropanol (lower concentrations of ethanol at this step (25-30%) may be sufficient for product elution.) This solvent elution step releases about 95% of the total bound sugars on the column and a small portion of the color bodies. In this first step capture of the maximal amount of sugar is the primary objective. Resolution of contaminants is not an objective. (3) Evaporation: A volume of 2.5 L of ethanol or isopropanol eluate from the capture column is rotary-evaporated at 56 C.° and a sugar syrup in water is generated. Alternative methods that could be used for this step include lyophilization or spray-drying. (4) Flash chromatography on fine carbon and ion exchange media: A column (GE Healthcare HiScale50/40, 5×40 cm, max pressure 20 bar) connected to a Biotage Isolera One FLASH Chromatography System is packed with 750 ml of a Darco Activated Carbon G60 (100-mesh): Celite 535 (coarse) 1:1 mixture (both column packings were obtained from Sigma). The column is equilibrated with 5 CV of water and loaded with sugar from step 3 (10-50 g, depending on the ratio of 2′-FL to contaminating lactose), using either a celite loading cartridge or direct injection. The column is connected to an evaporative light scattering (ELSD) detector to detect peaks of eluting sugars during the chromatography. A four-step gradient of isopropanol, ethanol or methanol is run in order to separate 2′-FL from monosaccharides (if present), lactose and color bodies. Fractions corresponding to sugar peaks are collected automatically in 120-ml bottles, pooled and directed to step 5. In certain purification runs from longer-than-normal fermentations, passage of the 2′-FL-containing fraction through anion-exchange and cation exchange columns can remove excess protein/DNA/caramel body contaminants. Resins tested successfully for this purpose are Dowex 22
The identity of the major oligosaccharide synthesized by WbgL was tested and confirmed to be bona fide 2′-FL. Oligosaccharides synthesized in the WbgL strain were immobilized on a carbon column, eluted and resuspended in distilled water. This material was subjected to overnight digestion with fucosidases of different specificities, and the reactions were analyzed by TLC. As shown in FIG. 4, the untreated material consisted primarily of an oligosaccharide with the same mobility as the 2′-FL standard (lane 1). Treatment with α1,2 fucosidase yielded both lactose and fucose, while the presumptive 2′-FL spot was significantly diminished in staining intensity (lane 2). Treatment of the oligosaccharides with an α1,3-4 fucosidase had no effect. These results demonstrate that WbgL is capable of the biosynthesis of bona fide 2′-FL in metabolically engineered E. coli. 
The gene screening approach was successfully utilized to identify new α(1,2) FTs for the efficient biosynthesis of 2′-FL in metabolically engineered E. coli host strains. The results of the screen are summarized in Table 1. Specifically, WbgL and FutL both direct the synthesis of 2′-FL at approximately 75% the levels attained by the previously characterized α(1,2) FT FutC. In addition, WbgL also was capable of synthesizing LDFT, which is another therapeutically useful HMO. Furthermore, FutN from the commensal enteric bacterium B. vulgatus was identified as another α(1,2) FT useful for the synthesis of fucosylated oligosaccharides. The approach described herein is useful in the analysis of additional candidate α(1,2) FTs and identifies additional enzymes that are useful for the large-scale production of HMOS.
Production Host Strains
E. coli K-12 is a well-studied bacterium which has been the subject of extensive research in microbial physiology and genetics and commercially exploited for a variety of industrial uses. The natural habitat of the parent species, E. coli, is the large bowel of mammals. E. coli K-12 has a history of safe use, and its derivatives are used in a large number of industrial applications, including the production of chemicals and drugs for human administration and consumption. E. coli K-12 was originally isolated from a convalescent diphtheria patient in 1922. Because it lacks virulence characteristics, grows readily on common laboratory media, and has been used extensively for microbial physiology and genetics research, it has become the standard bacteriological strain used in microbiological research, teaching, and production of products for industry and medicine. E. coli K-12 is now considered an enfeebled organism as a result of being maintained in the laboratory environment for over 70 years. As a result, K-12 strains are unable to colonize the intestines of humans and other animals under normal conditions. Additional information on this well known strain is available at http://epa.gov/oppt/biotech/pubs/fra/fra004.htm. In addition to E. coli K12, other bacterial strains are used as production host strains, e.g., a variety of bacterial species may be used in the oligosaccharide biosynthesis methods, e.g., Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).
Suitable host strains are amenable to genetic manipulation, e.g., they maintain expression constructs, accumulate precursors of the desired end product, e.g., they maintain pools of lactose and GDP-fucose, and accumulate endproduct, e.g., 2′-FL. Such strains grow well on defined minimal media that contains simple salts and generally a single carbon source. The strains engineered as described above to produce the desired fucosylated oligosaccharide(s) are grown in a minimal media. An exemplary minimal medium used in a bioreactor, minimal “FERM” medium, is detailed below.
Ferm (10 liters): Minimal medium comprising:
40 g (NH4)2HPO4 
100 g KH2PO4 
10 g MgSO4.7H2O
40 g NaOH
Trace elements:
1.3 g NTA (nitrilotriacetic acid)
0.5 g FeSO4.7H2O
0.09 g MnCl2.4H2O
0.09 g ZnSO4.7H2O
0.01 g CoCl2.6H2O
0.01 g CuCl2.2H2O
0.02 g H3BO3 
0.01 g Na2MoO4.2H2O (pH 6.8)
Water to 10 liters
DF204 antifoam (0.1 ml/L)
150 g glycerol (initial batch growth), followed by fed batch mode with a 90% glycerol-1% MgSO4-1× trace elements feed, at various rates for various times.
A suitable production host strain is one that is not the same bacterial strain as the source bacterial strain from which the fucosyltransferase-encoding nucleic acid sequence was identified. For example, the fucosyltransferase-encoding nucleic acid sequence FutL was identified in Helicobacter mustelae and a suitable host strain is a bacteria other than Helicobacter mustelae, e.g., FutL is produced in production host strain E. coli K12 or any of the other strains described above.
Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and a fucosylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The fucosylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.
Other Embodiments
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method for producing a fucosylated oligosaccharide in a bacterium comprising providing the bacterium comprising an exogenous lactose-utilizing α(1,2) fucosyltransferase enzyme, wherein an amino acid sequence of said enzyme comprises SEQ ID NO:8 and wherein said bacterium produces 2′fucosyllactose (2′-FL).

2. The method of claim 1, further comprising retrieving the fucosylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.

3. The method of claim 1, wherein said bacterium comprises a nucleic acid construct comprising an isolated nucleic acid encoding said α(1,2) fucosyltransferase enzyme.

4. The method of claim 3, wherein said nucleic acid encoding said α(1,2) fucosyltransferase enzyme is operably linked to one or more heterologous control sequences that direct the production of the α(1,2) fucosyltransferase enzyme in the bacterium.

5. The method of claim 4, wherein said heterologous control sequence comprises a bacterial promoter and operator, a bacterial ribosome binding site, a bacterial transcriptional terminator, or a plasmid selectable marker.

6. The method of claim 1, wherein said fucosylated oligosaccharide comprises 2′-fucosyllactose (2′-FL) or lactodifucotetraose (LDFT), Lacto-N-fucopentaose I (LNF I), or lacto-N-difucohexaose I (LDFH I).

7. The method of claim 1, wherein said bacterium further comprises a reduced level of β-galactosidase activity, a defective colonic acid synthesis pathway, a mutation in an ATP dependent intracellular protease, a mutation in a thyA gene, or a combination thereof.

8. The method of claim 1, wherein said method further comprises culturing said bacterium in the presence of tryptophan and in the absence of thymidine.

9. The method of claim 1, wherein an endogenous lacZ gene and an endogenous lacI gene of said bacterium are deleted.

10. The method of claim 1, wherein said bacterium comprises a lacIq gene promoter immediately upstream of a lacY gene.

11. The method of claim 1, wherein an endogenous wcaJ gene of said bacterium is deleted.

12. The method of claim 7, wherein said mutation in said ATP-dependent intracellular protease is a null mutation in a lon gene.

13. The method of claim 1, wherein said bacterium accumulates intracellular lactose in the presence of exogenous lactose.

14. The method of claim 1, wherein said bacterium accumulates intracellular GDP-fucose.

15. The method of claim 1, wherein said bacterium is a member of Streptococcus, Proprionibacterium, Enterococcus, Bifidobacterium, Sporolactobacillus, Micromomospora, Micrococcus, Rhodococcus, or Pseudomonas genus.

16. The method of claim 1, wherein said bacterium is E. coli.

17. The method of claim 1, wherein said bacterium is selected from the group consisting of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus thermophiles, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Escherichia coli K12, Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, Xanthomonas campestris Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactococcus lactis, Streptococcus thermophiles, Proprionibacterium freudenreichii, Enterococcus faecium, Enterococcus thermophiles, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Pseudomonas fluorescens and Pseudomonas aeruginosa.

18. A method for producing a fucosylated oligosaccharide in a bacterium comprising providing the bacterium comprising an exogenous lactose-utilizing α(1,2) fucosyltransferase enzyme, wherein an amino acid sequence of said enzyme comprises SEQ ID NO:8 and wherein said fucosylated oligosaccharide comprises a fucose group linked to a galactose group through an alpha(1,2) linkage.