Sialyltransferases and uses thereof

Abstract

Provided herein, inter alia, are methods, bacteria, nucleic acids, and polypeptides for producing sialylated oligosaccharides.

1 . A method for producing a sialylated oligosaccharide in a bacterium comprising providing a bacterium comprising an exogenous lactose-utilizing sialyltransferase enzyme, wherein the enzyme comprises an amino acid sequence that is
 (i) from 5% to 30% identical to the amino acid sequence of Pst6-224 (SEQ ID NO: 1) over a stretch of at least 250 amino acids; or   (ii) from 45% to 75% identical to the amino acid sequence of HAC1268 (SEQ ID NO: 8) over a stretch of at least 250 amino acids.   
     
     
         2 . The method of  claim 1 , wherein the enzyme comprises an amino acid sequence that is from 5% to 100% identical to the amino acid sequence of one or more of BstN (SEQ ID NO:10), BstC (SEQ ID NO: 2), Δ20BstC*2 (SEQ ID NO:27), Δ20BstC*3 (SEQ ID NO: 28), Δ20BstC*4 (SEQ ID NO: 29), Δ20BstC*5 (SEQ ID NO: 30), BstD (SEQ ID NO: 3), Δ20BstC* (SEQ ID NO: 15), Δ20BstC (SEQ ID NO: 18), BstE (SEQ ID NO: 4), BstE* (SEQ ID NO: 16), BstH (SEQ ID NO: 5), BstI (SEQ ID NO: 6), BstJ (SEQ ID NO: 7), or BstM (SEQ ID NO: 9). 
     
     
         3 . The method of  claim 1 , wherein the amino acid sequence of the enzyme
 i) is less than 100% identical to the amino acid sequence of BstN (SEQ ID NO:10), BstC (SEQ ID NO: 2), ABstC*2 (SEQ ID NO:27), Δ20BstC*3 (SEQ ID NO: 28), Δ20BstC*4 (SEQ ID NO: 29), Δ20BstC*5 (SEQ ID NO: 30), BstD (SEQ ID NO: 3), Δ20BstC (SEQ ID NO: 18), Δ20BstC* (SEQ ID NO: 15), BstE (SEQ ID NO: 4), BstE* (SEQ ID NO: 16), BstH (SEQ ID NO: 5), BstI (SEQ ID NO: 6), BstJ (SEQ ID NO: 7), or BstM (SEQ ID NO: 9);   ii) comprises no deletions or insertions compared to BstN (SEQ ID NO:10, BstC (SEQ ID NO: 2), ABstC*2 (SEQ ID NO:27), Δ20BstC*3 (SEQ ID NO: 28), Δ20BstC*4 (SEQ ID NO: 29), Δ20BstC*5 (SEQ ID NO: 30), BstD (SEQ ID NO: 3), Δ20BstC (SEQ ID NO: 18), Δ20BstC* (SEQ ID NO: 15), BstE (SEQ ID NO: 4), BstE* (SEQ ID NO: 16), BstH (SEQ ID NO: 5), BstI (SEQ ID NO: 6), BstJ (SEQ ID NO: 7), or BstM (SEQ ID NO: 9); and/or   iii) comprises one or more conservative amino acid substitutions to the amino acid sequence of BstN (SEQ ID N: 10), BstC (SEQ ID NO: 2), ΔBstC*2 (SEQ ID NO:27), Δ20BstC*3 (SEQ ID NO: 28), Δ20BstC*4 (SEQ ID NO: 29), Δ20BstC*5 (SEQ ID NO: 30), BstD (SEQ ID NO: 3), Δ20BstC (SEQ ID NO: 18), Δ20BstC* (SEQ ID NO: 15), BstE (SEQ ID NO: 4), BstE* (SEQ ID NO: 16), BstH (SEQ ID NO: 5), BstI (SEQ ID NO: 6), BstJ (SEQ ID NO: 7), or BstM (SEQ ID NO: 9).   
     
     
         4 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the sialyltransferase
 i) comprises an α(2,3) sialyltransferase or an α(2,6) sialyltransferase; and/or   ii) a mutation compared to a naturally occurring α(2,3) sialyltransferase.   
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 13 , wherein when the amino acid sequences of the enzyme and BstE* are aligned, then the enzyme comprises:
 i) a mutation;   ii) a non-conservative mutation; and/or   iii) a histidine or an alanine   at the position that aligns with position 13 and/or position 130 of the amino acid sequence of BstE* (SEQ ID NO: 16).   
     
     
         15 .- 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the C α  root-mean-square deviation (RMSD) between the backbone of the enzyme and a naturally occurring sialyltransferase is less than 3 Å. 
     
     
         23 .- 25 . (canceled) 
     
     
         26 . The method of  claim 1 , further comprising retrieving the sialylated oligosaccharide from the bacterium or from a culture supernatant of the bacterium. 
     
     
         27 . The method of  claim 1 , wherein the sialylated oligosaccharide comprises a sialyllactose. 
     
     
         28 . The method of  claim 1 , wherein the sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), 3′-sialyl-3-fucosyllactose (3′-S3FL), sialyllacto-N-tetraose a (SLNT a), sialyllacto-N-tetraose b (SLNT b), disialyllacto-N-tetraose (DSLNT), sialyllacto-N-fucopentaose II (SLNFP II), or sialyllacto-N-tetraose c (SLNT c). 
     
     
         29 . The method of  claim 1 , wherein the bacterium further comprises
 i) an exogenous or endogenous lactose-utilizing α(1,3) fucosyltransferase enzyme, an exogenous or endogenous lactose-utilizing α(1,4) fucosyltransferase enzyme, an exogenous or endogenous α(1,3) galactosyltransferase enzyme, an exogenous or endogenous α(1,4) galactosyltransferase enzyme, an exogenous or endogenous β-1,3-N-acetylglucosaminyltransferase, or any combination thereof;   ii) an exogenous or endogenous N-acetylneuraminate synthase, an exogenous or endogenous UDP-N-acetylglucosamine 2-epimerase, an exogenous or endogenous N-acetylneuraminate cytidylyltransferase, or any combination thereof;   iii) an exogenous N-acetylneuraminate synthase, UDP-N-acetylglucosamine 2-epimerase, and N-acetylneuraminate cytidylyltransferase from Campylobacter jejuni;   iv) a reduced level of β-galactosidase activity compared to a corresponding wild-type bacterium;   v) a deleted or inactivated endogenous β-galactosidase gene;   vi) a deleted or inactivated endogenous lacZ gene and/or a deleted or inactivated endogenous lacI gene;   vii) an endogenous β-galactosidase gene, wherein at least a portion of a promoter of the endogenous β-galactosidase gene has been deleted;   viii) an exogenous β-galactosidase enzyme with reduced enzymatic activity compared to an endogenous β-galactosidase enzyme in a corresponding wild-type bacterium;   ix) an exogenous β-galactosidase gene that is expressed at a lower level than to an endogenous β-galactosidase gene in a corresponding wild-type bacterium;   x) a lactose permease gene;   xi) a mutation in a thyA gene and/or a lacA gene;   xii) a laclq or lacPL8 promoter mutation; and/or   xiii) a nucleic acid construct comprising an isolated nucleic acid encoding the lactose-utilizing sialyltransferase enzyme.   
     
     
         30 .- 32 . (canceled) 
     
     
         33 . The method of  claim 29 , wherein the reduced level of β-galactosidase activity in iv)
 a) comprises reduced expression of a β-galactosidase gene or reduced β-galactosidase enzymatic activity; and/or 
 b) is less than 10% the level of the corresponding wild-type bacterium in the presence of lactose. 
 
     
     
         34 .- 39 . (canceled) 
     
     
         40 . The method of  claim 29 , wherein the bacterium
 i) comprises less than 50 units of β-galactosidase activity when cultured in the presence of lactose;   ii) does not express a β-galactoside transacetylase;   iii) accumulates intracellular lactose in the presence of exogenous lactose; and/or   iv) comprises the following genotype: PlacIq-lacY, Δ(lacI-lacZ), ΔlacA, ΔthyA::(0.8RBS lacZ+), ampC::(Ptrp M13g8 RBS-λcI+, CAT), ΔnanATE::scar.   
     
     
         41 .- 45 . (canceled) 
     
     
         46 . The method of  claim 1 , wherein the bacterium
 i) is an  Escherichia coli  ( E. coli ) bacterium;   ii) is a G1724 strain  E. coli  bacterium;   iii) is a member of the  Bacillus, Pantoea, Lactobacillus, Lactococcus, Streptococcus, Proprionibacterium, Enterococcus, Bifidobacterium, Sporolactobacillus, Micromomospora, Micrococcus, Rhodococcus, or Pseudomonas  genus; and/or   iv) is a  Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus thermophiles, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus,  and  Bacillus circulans, 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,  or  Pseudomonas aeruginosa  bacterium.   
     
     
         47 .- 52 . (canceled) 
     
     
         53 . The method of  claim 29 , wherein the nucleic acid in xii) is operably linked to a heterologous control sequence that directs the production of the enzyme in the bacterium. 
     
     
         54 . The method of  claim 53 , wherein the heterologous control sequence comprises a bacterial promoter, a bacterial operator, a bacterial ribosome binding site, a bacterial transcriptional terminator, or a plasmid selectable marker. 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 2 , wherein the enzyme comprises an amino acid sequence as set forth as SEQ ID NO: 15, 16, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. 
     
     
         57 . A nucleic acid encoding the lactose-utilizing sialyltransferase enzyme of  claim 58 . 
     
     
         58 . A lactose-utilizing sialyltransferase enzyme comprising amino acids in the sequence set forth as SEQ ID NO: 15, 16, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. 
     
     
         59 . An isolated bacterium comprising an exogenous lactose-utilizing sialyltransferase enzyme, wherein the enzyme comprises an amino acid sequence that is
 (i) from 5% to 30% identical to the amino acid sequence of Pst6-224 (SEQ ID NO: 1) over a stretch of at least 250 amino acids; or   (ii) from 45% to 75% identical to the amino acid sequence of HAC1268 (SEQ ID NO: 8) over a stretch of at least 250 amino acids.   
     
     
         60 . (canceled)