WO2020072617A1 - Use of substrate importers for the export of oligosaccharides - Google Patents

Use of substrate importers for the export of oligosaccharides

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Publication number
WO2020072617A1
WO2020072617A1 PCT/US2019/054258 US2019054258W WO2020072617A1 WO 2020072617 A1 WO2020072617 A1 WO 2020072617A1 US 2019054258 W US2019054258 W US 2019054258W WO 2020072617 A1 WO2020072617 A1 WO 2020072617A1
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WO
WIPO (PCT)
Prior art keywords
microorganism
cdt
hmo
seq
transporter
Prior art date
Application number
PCT/US2019/054258
Other languages
French (fr)
Inventor
James Cate
Kulika CHOMVONG
Oliver Kilian
Jingjing Liu
Jason Liu
Yong-Su Jin
Original Assignee
Zimitech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zimitech, Inc. filed Critical Zimitech, Inc.
Priority to CN201980075948.4A priority Critical patent/CN113056562A/en
Priority to CA3115210A priority patent/CA3115210A1/en
Priority to MX2021003702A priority patent/MX2021003702A/en
Priority to AU2019352624A priority patent/AU2019352624A1/en
Priority to KR1020217013154A priority patent/KR20210095128A/en
Priority to JP2021517938A priority patent/JP2022512574A/en
Priority to US17/282,636 priority patent/US20220064686A1/en
Priority to EP19869234.5A priority patent/EP3861123A4/en
Priority to BR112021006191-6A priority patent/BR112021006191A2/en
Publication of WO2020072617A1 publication Critical patent/WO2020072617A1/en

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Definitions

  • oligosaccharides have emerged as valuable components of food and dietary supplements. Their resistance to digestion and fermentation by colonic microbes has given oligosaccharides a nutritional edge. Apart from implications as dietary fibers, sweeteners, and humectants, they are hailed as prebiotics. Their beneficial effects extend from anti-oxidant, anti inflammatory, immunomodulatory, anti-hypertensive, and anti-allergic to anti-cancer, neuroprotective, and improvement of the skin barrier function and hydration. The rising popularity of bioactive oligosaccharides has accelerated the search for their generation from new, sustainable sources.
  • Oligosaccharides may be obtained from natural sources and may also be synthesized.
  • Various natural sources of oligosaccharides include milk, honey, sugarcane juice, rye, barley, wheat, soybean, lentils, mustard, fruits, and vegetables such as onion, asparagus, sugar beet, artichoke, chicory, leek, garlic, banana, yacon, tomato, and bamboo shoots.
  • oligosaccharide manufacturing methods include hydrolysis of polysaccharides, chemical, and enzymatic polymerization from disaccharide or monosaccharide substrates. Acid, alkali, and enzymatic hydrolysis of polysaccharides can generate oligosaccharides of desired structure and functional properties. In certain cases, enzymatic methods are preferred for oligosaccharide synthesis due to their high selectivity and yields, and environmental-friendly nature. In other cases, oligosaccharide-producing microbial strains may be engineered by introducing exogenous genes to enable oligosaccharide production.
  • Oligosaccharides produced in microorganisms will accumulate intracellularly if not actively transported out of the cell into the medium from where they can be further isolated. Accumulation within the cells in the absence of export processes requires isolation of the oligosaccharide from biomass and limits conversion of the substrate to fermentation product or oligosaccharide. The lack of export of fermentation products out of cells also increases costs of the fermentation processes since fermentation runs effectively have to be stopped once the cells accumulate significant amounts of oligosaccharide in order to recover the latter. In addition, recovery of oligosaccharide from cells require additional processes such as extraction or breakage of cells, or both, which might additionally increase costs and require significant purification steps to remove contaminating cell debris, or both.
  • substrate importers might act as exporters. For example, if oligosaccharides accumulate to high concentrations within cells, this along with the appropriate transporter may drive substrate flow out of the cell where the concentration is lower.
  • mutagenized versions of transporters might be impaired in regulation of transport processes in such a way that substrate export along a concentration gradient is facilitated.
  • modification of the same substrate transporter can lead to higher fermentation product or oligosaccharide export rates if expressed in an organism accumulating a suitable substrate within the cell.
  • transporters that can function as a substrate exporter, particularly for oligosaccharides.
  • Such transporters can also function as importers, and import oligosaccharides, such as an oligosaccharide different from that exported.
  • CDT-l (XP 963801.1) from the fungus Neurospora crassa is a substrate transporter from the major facilitator superfamily (MFS) that imports cellobiose into the cell.
  • MFS major facilitator superfamily
  • expression of a cellodextrin transporter in an engineered Saccharomyces cerevisiae strain capable of producing a lactose-based oligosaccharide, such as 2’-fucosyllactose (2’-FL) leads to an increase of 2’-FL released into the culture medium.
  • CDT-l acts as an exporter facilitating transport of oligosaccharides, such as 2’-FL, out of the cell.
  • CDT-2 is another substrate transporter from the fungus Neurospora crassa that can be used herein for exporting oligosaccharides, such as 2’-FL.
  • the present disclosure provides 2’-FL production strains expressing a CDT such as CDT-l, CDT-2 or a CDT mutant (i.e., having one or more alterations in a CDT amino acid sequence).
  • a CDT such as CDT-l, CDT-2 or a CDT mutant (i.e., having one or more alterations in a CDT amino acid sequence).
  • a microorganism comprises a heterologous cellodextrin transporter gene or a construct that enhances expression of the cellodextrin transporter, is provided.
  • the microorganisms described herein Compared to the parental microorganisms, the microorganisms described herein have an increased ability to produce oligosaccharide products of interest. Accordingly, methods of producing products of interest by culturing the microorganisms of the present disclosure in media containing the oligosaccharides and obtaining the products of interest from the media are provided.
  • a CDT mutant is CDT-l SY. These strains show increased export of oligosaccharides if compared to their parental strains not expressing CDT-l or a CDT-l analogue.
  • the present disclosure provides methods of producing oligosaccharides by culturing the microorganisms disclosed herein.
  • the microorganisms are bacteria or fungi, for example, filamentous fungi or yeasts.
  • the microorganisms are yeast, for example, Saccharomyces cerevisiae.
  • a method of producing an oligosaccharide comprising culturing a microorganism described herein in a culture medium and recovering the oligosaccharide is provided herein.
  • a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism described herein; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
  • a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism comprises a heterologous transporter and one or more heterologous HMO production gene(s); and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
  • a product suitable for animal consumption comprising the HMO produced by the microorganism described herein or according to the method described herein and at least one additional ingredient acceptable for animal consumption.
  • a product suitable for animal consumption comprising the
  • microorganism described herein and optionally at least one additional ingredient acceptable for animal consumption.
  • Fig. 1 shows a schematic of a cell expressing a CDT-l mutant and a lactose transporter.
  • the cell produces the oligosaccharide 2’-FL.
  • the cell is engineered to produce GDP-fucose.
  • the fucosyl residue in GDP-fucose is subsequently transferred onto lactose, thereby producing 2’-FL.
  • Lactose is imported by a transporter specific for lactose.
  • CDT-1SY facilitates export of oligosaccharides, such as 2’-FL, out of the cell.
  • the oligosaccharide can then be obtained from the growth medium.
  • Fig. 2 shows the level of 2’-FL in the supernatant in 2’-FL producing background strain either with, or without the transporter CDT-l mutant (such as CDT-l SY as specified in SEQ ID NO. 1).
  • the strain expressing CDT-l SY exhibits a -30% increase in product accumulation in the growth medium.
  • Fig. 3 shows lactose uptake activity and 2’-FL production by yeast strains expressing CDT-l M7 (CDT-l 209S 262Y) or Lacl2 as lactose transporter along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
  • Fig. 4 shows relative lactose uptake activity by yeast strains expressing different CDT-l mutants.
  • CDT-l CDT-l wild type
  • Ml CDT-l 91A
  • M2 CDT-l 213A
  • M3 CDT-l 256V
  • M4 CDT-l 335 A
  • M5 CDT-l 411 A
  • M6 CDT-l 209S 262W
  • M7 CDT-l 209S 262Y
  • M8 CDT-l 209S 262Y first 30 amino acid codons optimized.
  • Ctrl is control strain with no transporter expression.
  • Fig. 5 shows relative extracellular 2’-FL production by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
  • Ctrl is control strain without any lactose transporter expression.
  • Fig. 6 shows total 2’-FL production by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
  • Ctrl is control strain without any lactose transporter expression.
  • Fig. 7 shows extracellular 2’-FL ratio by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
  • Fig. 8 shows a schematic of production of fucosylated oligosaccharides within microbes. Shown is an example how the fucosylated oligosaccharide such as 2’-fucosyllacctose (2’-FL) is formed.
  • GDP-Mannose is dehydrated to GDP-4-dehydro-6-deoxy-D-mannose by a GDP- mannose dehydratase (GMD).
  • GDP-4-dehydro-6-deoxy-D-mannose is then reduced to GDP- Fucose by a GDP fucose synthase (GFS).
  • lactose had been imported into the cell by a specific lactose transporter and is then further fucosylated by a glycosyl transferase such as a fucosyl transferase (FT), e.g., alpha-l,2 fucosyltransferase to form 2’-FL.
  • FT fucosyl transferase
  • 2’-FL is then exported into the medium by an oligosaccharide transporter.
  • Fig. 9 shows 2’-FL production by introducing fucosyltransferase (FT) from different organisms to yeast strain with CDT-l M7, GMD and WcaG expression on plasmids.
  • Ctrl is control strain without FT expression.
  • Fig. 10 shows relative production of 2’-FL in yeast cells expressing plasmids with GMD, GFS and FT, relative to a base strain that contains a set of genomic GMD, GFS and FT genes.
  • the GFS gene carried on the expression plasmid was here selected from SEQ ID NOs: 20, 21,
  • Fig. 11 shows relative production of 2’-FL in yeast cells expressing plasmids with GMD, GFS and FT, relative to a base strain that contains a set of genomic GMD, GFS and FT genes.
  • the FT gene carried on the expression plasmid was selected from SEQ ID NOs: 38, 29, 30, 31, 32, and 40.
  • Fig. 12 shows relative production of 2’-FL in yeast cells expressing plasmids with (lst column) GMD, a FT and SEQ ID NO: 24 and (2nd column) plasmids with a FT and SEQ ID NO: 24 only, relative to a base strain that contains a set of genomic GMD, GFS and FT genes.
  • Fig. 13 shows production of 2’-FL by expression of plasmids in a control strain otherwise not capable of 2’-FL production (Ctrl).
  • Strains were transformed with plasmids expressing a GFS and a FT along with a plasmid carrying either SEQ ID NO: 17, 18, or 19, respectively.
  • the control strain carrying no plasmids does not produce any 2’-FL.
  • a microorganism comprises a heterologous cellodextrin transporter gene or a construct that enhances expression of the cellodextrin transporter, is provided.
  • the heterologous cellodextrin transporter is CDT-l .
  • the gene or construct that expresses CDT-l comprises a genetic modification that increases the oligosaccharide export activity of CDT-l relative to a corresponding wild-type gene or construct that expresses CDT-l .
  • the gene or construct that expresses CDT-l is MFS transporter gene (cdt-l) or a variant thereof.
  • the transporter comprises a PESPR motif.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%,
  • the CDT-l further comprises one or more mutations selected from the group consisting of 91A, 209S, 213A, 256V, 262Y, 335A, and 411 A of SEQ ID NO: 4.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 and a tyrosine at the position corresponding to residue 262 of SEQ ID No: 4.
  • the CDT-l has the sequence of SEQ ID NO: 1 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 of SEQ ID NO: 4.
  • the CDT-l has the sequence of SEQ ID NO: 2 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises a tyrosine at the position corresponding to residue 262 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 3 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 91 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 10 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 213 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 11 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises a valine at the position corresponding to residue 256 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 12 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 335 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 13 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 411 of SEQ ID NO: 4.
  • CDT-l has the sequence of SEQ ID NO: 14 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14.
  • the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 and a Tryptophan at the position corresponding to residue 262 of SEQ ID No: 4.
  • the CDT-l has the sequence of SEQ ID NO: 15 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15.
  • the CDT-l is encoded by a codon optimized nucleic acid.
  • the nucleic acid is optimized for yeast. In some embodiments, at least 5% of the nucleic acid is codon optimized. In some embodiments, at least 90 nucleotides of the nucleic acid are codon optimized. In some embodiments, the CDT-l is encoded by the nucleic acid of SEQ ID NO: 16.
  • the microorganism further comprising a genetic modification that increases the oligosaccharide export activity of CDT-l selected from: a) a promoter operably linked to the cdt-l gene b) extrachromosomal genetic material comprising cdt-l; c) one or more copies of cdt-l, wherein said copies are integrated into the genome of the microorganism; d) a modified cdt-l that encodes a constitutively active CDT-l compared to unmodified CDT-l; e) a modified cdt-l that encodes a CDT-l having increased oligosaccharide export activity compared to unmodified CDT-l ; f) extrachromosomal genetic material comprising a modified cdt-l that encodes a constitutively active CDT-l or a CDT-l having increased oligosaccharide export activity compared to the corresponding wild-type CDT-l ; or
  • the promoter operably linked to the cdt-l gene induces expression of cdt-l at a higher level than an endogenous promoter.
  • the promoter is specific for the microorganism in which it induces expression of cdt-l.
  • the heterologous cellodextrin transporter is CDT-2.
  • the CDT-2 has an amino acid sequence of SEQ ID NO: 9 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9.
  • the microorganism further comprising a gene or a construct that expresses a lactose permease.
  • the lactose permease is Lacl2.
  • the Lacl2 has an amino acid sequence of SEQ ID NO: 41 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 41.
  • the microorganism further comprising one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein.
  • the microorganism comprises the heterologous cellodextrin transporter CDT-l, or variant or mutation of CDT-l such as described herein and further comprising one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein.
  • the one or more HMO production protein is an enzyme capable of converting fucose and ATP to fucose- 1- phosphate, an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and/or a glucosyl transferase.
  • the one or more HMO production gene is a GDP-Mannose dehydratase gene or the one or more HMO production protein is a GDP-Mannose dehydratase protein.
  • the one or more HMO production gene is a GDP-L- fucose synthase gene or the one or more HMO production protein is a GDP-L-fucose synthase protein.
  • the one or more HMO production gene is a fucosyl transferase gene or the one or more HMO production protein is a fucosyl transferase protein.
  • the gene or construct that expresses GDP-Mannose dehydratase comprises a genetic modification that increases the oligosaccharide production activity of GDP-Mannose dehydratase relative to a corresponding wild-type gene or construct that expresses GDP- Mannose dehydratase.
  • the gene or construct that expresses GDP-Mannose dehydratase is GDP-Mannose dehydratase gene (gmd) or a variant thereof.
  • the GDP-Mannose dehydratase has an amino acid sequence of any one of SEQ ID NOs: 17-19, 42, and 61-63 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 17-19, 42, and 61-63.
  • the gene or construct that expresses GDP-L-fucose synthase comprises a genetic modification that increases the oligosaccharide production activity of GDP-L-fucose synthase relative to a corresponding wild-type gene or construct that expresses GDP-L-fucose synthase.
  • the gene or construct that expresses GDP-L-fucose synthase is GDP-L-fucose synthase gene (gfs) or a variant thereof.
  • the GDP-L-fucose synthase has an amino acid sequence of any one of SEQ ID NOs: 20-23 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 20-23.
  • the gene or construct that expresses GDP-L-fucose synthase is WcaG or a variant thereof.
  • the WcaG has an amino acid sequence of any one of SEQ ID NOs: 43-45 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 43-45.
  • the gene or construct that expresses GDP-L-fucose synthase is GMER or a variant thereof.
  • the GMER has an amino acid sequence of SEQ ID NO: 46 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 46.
  • the gene or construct that expresses fucosyl transferase comprises a genetic modification that increases the oligosaccharide production activity of fucosyl transferase relative to a corresponding wild-type gene or construct that expresses fucosyl transferase.
  • the gene or construct that expresses fucosyl transferase is fucosyl transferase gene (ft) or a variant thereof.
  • the fucosyl transferase is alpha 1 ,2-fucosyl transferase.
  • the fucosyl transferase has an amino acid sequence of any one of SEQ ID NOs: 26-40 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 26-40.
  • the gene or construct that expresses fucosyl transferase is wbgL or a variant thereof.
  • the wbgL has an amino acid sequence of SEQ ID NO: 47 or has at least 60%,
  • the gene or construct that expresses fucosyl transferase is futC or a variant thereof.
  • the futC has an amino acid sequence of SEQ ID NO: 48 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 48.
  • the gene or construct that expresses fucosyl transferase is wcfB or a variant thereof.
  • the wcfB has an amino acid sequence of SEQ ID NO: 49 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 49.
  • the gene or construct that expresses fucosyl transferase is wbgN or a variant thereof.
  • the wbgN has an amino acid sequence of SEQ ID NO: 50 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 50.
  • the gene or construct that expresses fucosyl transferase is wbwk or a variant thereof.
  • the wbwk has an amino acid sequence of any one of SEQ ID NO: 51 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 51.
  • the gene or construct that expresses fucosyl transferase is wbsJ or a variant thereof.
  • the wbsJ has an amino acid sequence of SEQ ID NO: 52 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 52.
  • the gene or construct that expresses fucosyl transferase is wbiQ or a variant thereof.
  • the wbiQ has an amino acid sequence of SEQ ID NO: 53 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 53.
  • the gene or construct that expresses fucosyl transferase is futB or a variant thereof.
  • the futB has an amino acid sequence of SEQ ID NO: 54 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 54.
  • the gene or construct that expresses fucosyl transferase is futL or a variant thereof.
  • the futL has an amino acid sequence of SEQ ID NO: 55 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 55.
  • the gene or construct that expresses fucosyl transferase is futF or a variant thereof.
  • the futF has an amino acid sequence of SEQ ID NO: 56 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 56.
  • the gene or construct that expresses fucosyl transferase is futG or a variant thereof.
  • the futG has an amino acid sequence of SEQ ID NO: 57 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 57.
  • the gene or construct that expresses fucosyl transferase is futN or a variant thereof.
  • the futN has an amino acid sequence of SEQ ID NO: 58 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 58.
  • the gene or construct that expresses fucosyl transferase is wcfw or a variant thereof.
  • the wcfw has an amino acid sequence of SEQ ID NO: 59 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 59.
  • the gene or construct that expresses fucosyl transferase is futA or a variant thereof.
  • the futA has an amino acid sequence of SEQ ID NO: 63 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 63.
  • the gene or construct that expresses fucosyl transferase is futD or a variant thereof.
  • the futD has an amino acid sequence of SEQ ID NO: 64 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 64.
  • the gene or construct that expresses fucosyl transferase is futE or a variant thereof.
  • the futE has an amino acid sequence of SEQ ID NO: 65 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 65.
  • the gene or construct that expresses fucosyl transferase is futH or a variant thereof.
  • the futH has an amino acid sequence of SEQ ID NO: 66 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 66.
  • the gene or construct that expresses fucosyl transferase is futJ or a variant thereof.
  • the futJ has an amino acid sequence of SEQ ID NO: 67 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 67.
  • the gene or construct that expresses fucosyl transferase is futK or a variant thereof.
  • the futK has an amino acid sequence of SEQ ID NO: 68 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 68.
  • the gene or construct that expresses fucosyl transferase is futM or a variant thereof.
  • the futM has an amino acid sequence of SEQ ID NO: 69 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 69.
  • the one or more HMO production gene an enzyme comprising two domains, wherein one domain has homology to GDP-Mannose dehydratase and the second domain has homology to fucosyl synthase.
  • the enzyme has an amino acid sequence of any one of SEQ ID NOs: 24-25 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 24-25.
  • the one or more HMO production gene is a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase and the one or more HMO production protein is a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase protein.
  • the bifunctional fucokinase/L-fucose-l-P-guanylyltransferase has an amino acid sequence of any one of SEQ ID NOs: 71-73 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 71-73.
  • the microorganism comprises one or more genetic modifications selected from: i) a genetic modification that increases the proton export activity of PMA1 in the microorganism compared to PMA1 activity in the parental microorganism, ii) a genetic modification that decreases the hexose sensing activity of SNF3 in the microorganism compared to SNF3 activity in the parental microorganism, iii) a genetic modification that decreases the hexose sensing activity of RGT2 in the microorganism compared to RGT2 activity in the parental microorganism, and iv) a genetic modification that decreases the hexose sensing activity of GPR1 in the microorganism compared to GPR1 activity in the parental microorganism.
  • the genetic modification that increases the proton export activity of PMA1 is a genetic modification to plasma membrane ATPase gene (pmal)
  • the genetic modification that decreases the hexose sensing activity of SNF3 is a genetic modification to sucrose non-fermenting gene (snf3)
  • the genetic modification that decreases the hexose sensing activity of RGT2 is a genetic modification that decreases the hexose sensing activity of RGT2 is a genetic
  • PMA1 has the sequence of SEQ ID NO: 5 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5
  • SNF3 has the sequence of SEQ ID NO: 6 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6
  • RGT2 has the sequence of SEQ ID NO: 7 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7
  • GPR1 has the sequence of SEQ ID NO: 8 or at least 60%, 65%, 70%, 75%, 80%,
  • the microorganism further comprises an exogenous nucleotide sequence encoding a chaperonin.
  • the chaperonin is gGroESL.
  • the microorganism is a eukaryotic organism
  • the fungus microorganism is a filamentous fungus or a yeast.
  • the microorganism is a Ascomycetes fungus.
  • the Ascomycetes fungus is selected from the group consisting of a Sacharomyces spp. , a Schizosaccharomyces spp. and a Pichia spp.
  • the microorganism is Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Neurospora sp, Torulaspora spp.,Torulaspora delbrueckii, Zygosaccharomyces s
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport an oligosaccharide selected from 2-fucosyllactose, 3- fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose, di-fucosyllactose, lacto-N- neotetraose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk oligosaccharide with a degree of polymerization of 3 out of the organism.
  • the human milk oligosaccharide is 2'-fucosyllactose, 3-fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, or 6’- sialyllactose.
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk oligosaccharide with a degree of polymerization of 4 out of the organism.
  • the human milk oligosaccharide is di-fucosyllactose, lacto-N-neotetraose, lacto-N-tetraose , sialyllacto-N-neotetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, disialyllacto-N-tetraose, fucosylsialyllacto-N- tetraose a, or fucosylsialyllacto-N-tetraose b.
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk
  • the human milk oligosaccharide is lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI.
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport 2’-fucosyllactose out of the organism.
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-tetraose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-neotetraose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport 3’-sialyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport 6’-sialyllactose out of the organism.
  • the microorganism has a higher capacity, compared to the parental microorganisms, to transport di-fucosyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-fucopentaose I out of the organism.
  • a microorganism for enhanced production of a human milk in another aspect, a microorganism for enhanced production of a human milk
  • HMO oligosaccharide
  • the microorganism is capable of producing and exporting the HMO.
  • the transporter is capable of exporting at least 20%, 30%, 40%, 50%, or 60% of the produced HMO.
  • the microorganism is capable of exporting at least 50% more of the HMO than a parental microorganism lacking the transporter.
  • the yeast comprises a transporter that has an amino sequence of SEQ ID NO:4 or a sequence with at least 80%, 85%, 90%, 95%, 98% or 99% homology thereto.
  • the transporter comprises a PESPR motif.
  • the transporter comprises a sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO:4.
  • the CDT-l is encoded by a codon optimized nucleic acid.
  • at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast.
  • the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411 A and any combination thereof.
  • the pathway gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L- fucose synthase, and an alpha- l,2-fucosyl transferase.
  • the microorganism comprises a second heterologous pathway gene.
  • the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose I (LNFP I).
  • the HMO is 2’- fucosyllactose.
  • the microorganism is an Ascomycetes fungus.
  • the Ascomycetes fungus is selected from the group consisting of a Sacharomyces spp., a Schizosaccharomyces spp. and a Pichia spp.
  • the Ascomycetes fungus is selected from the group consisting of Trichoderma, Kluyveromyces, Yarrowia, Aspergillus, and Neurospora.
  • one or both of the heterologous CDT-l transporter and the pathway gene are integrated into the yeast chromosome.
  • the microorganism comprises a set of pathway genes for production of the HMO.
  • the set comprises GDP-mannose 4,6-dehydratase (GMD), a GDP-L-fucose synthase (GFS), and a fucosyl transferase (FT).
  • GMD GDP-mannose 4,6-dehydratase
  • GFS GDP-L-fucose synthase
  • FT fucosyl transferase
  • the set comprises GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha-l,2- fucosyl transferase and wherein the HMO is 2’-FL.
  • the set comprises a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase.
  • the set comprises an enzyme capable of converting fucose and ATP to fucose-l -phosphate and an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and a glucosyl transferase.
  • the glucosyl transferase is an ⁇ -l,2-fucosyl transferase and wherein the HMO is 2’-FL.
  • the set of pathway genes comprises Gmd, WcaG and WbgL.
  • the GDP-mannose 4,6-dehydratase is selected from SEQ ID Nos. 17-19, 42, and 61-63 or a variant having at least 85% homology thereto.
  • the GDP-L-fucose synthase is selected from SEQ ID Nos. 20-23 or a variant having at least 85% homology thereto.
  • the alpha- l,2-fucosyl transferase is selected from SEQ ID Nos. 26-40 or a variant having at least 85% homology thereto.
  • a method of producing an oligosaccharide comprising culturing a microorganism described herein in a culture medium and recovering the oligosaccharide is provided herein.
  • a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism described herein; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
  • a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism comprises a heterologous transporter and one or more heterologous HMO production gene; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
  • the HMO is 2-fucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, 3’-sialyllactose, or 6’-sialyllactose di-fucosyllactose.
  • the method further comprising separating the culture medium from the microorganism.
  • the method further comprising isolating the HMO from the culture medium.
  • the heterologous transporter is CDT-l, CDT-2 or a variant thereof.
  • the HMO is 2’-FL.
  • the heterologous transporter gene is a CDT-l variant comprising an amino acid sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO: 1.
  • the CDT-l is encoded by a codon optimized nucleic acid.
  • the nucleic acid is optimized for yeast.
  • at least 5% of the nucleic acid is codon optimized.
  • at least 90 nucleotides of the nucleic acid are codon optimized.
  • the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411 A and any combination thereof.
  • the heterologous gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha- 1,2, fucosyl transferase.
  • the export of the HMO is increased as compared to a parental yeast strain that does not contain the heterologous transporter.
  • the heterologous transporter is capable of importing lactose and exporting the HMO.
  • the culture medium comprises lactose.
  • the ratio of the HMO in the culture medium to total HMO produced by the microorganism is at least about 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1 or greater than 4: 1.
  • the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose
  • a method of producing an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism expresses a heterologous transporter and one or more heterologous genes for the production of the HMO; and culturing microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture mediumis provided.
  • the method further comprises separating the culture medium from the microorganism.
  • the method further comprises isolating the HMO from the culture medium.
  • the heterologous transporter is CDT-l, CDT-2 or a variant thereof.
  • the HMO is 2’-FL.
  • the transporter is a CDT-l variant comprising an amino acid sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262,
  • the CDT-l is encoded by a codon optimized nucleic acid.
  • at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast.
  • the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411A and any combination thereof.
  • the heterologous gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha- l,2-fucosyl transferase.
  • the export of the HMO is increased as compared to a parental microorganism that does not contain the heterologous transporter.
  • the heterologous transporter is capable of importing lactose and exporting the HMO.
  • the culture medium comprises lactose.
  • the ratio of the HMO in the culture medium to total HMO produced by the microorganism is at least about 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1 or greater than 4: 1.
  • the HMO is selected from the group consisting of 2'- fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'- SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose
  • a product suitable for animal consumption comprising the HMO produced by the microorganism described herein or according to the method described herein and at least one additional ingredient acceptable for animal consumption.
  • a product suitable for animal consumption comprising the
  • microorganism described herein and optionally at least one additional ingredient acceptable for animal consumption.
  • the product is suitable for human consumption.
  • the product is an infant formula, an infant food, a nutritional supplement or a prebiotic product.
  • the product is suitable for mammalian consumption.
  • the product further comprising at least one additional human milk oligosaccharide.
  • the additional ingredient is selected from a protein, a lipid, a vitamin, a mineral or any combination thereof.
  • the product is suitable for use as an animal feed.
  • a product suitable for animal consumption comprising the
  • the product is suitable for human consumption.
  • the product is an infant formula, an infant food, a nutritional supplement or a prebiotic product.
  • the product is suitable for mammalian consumption.
  • the product further comprises at least one additional human milk oligosaccharide.
  • the additional consumable ingredient is selected from a protein, a lipid, a vitamin, a mineral or any combination thereof.
  • the product is suitable for use as an animal feed.
  • Ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Therefore, when ranges are stated for a value, any appropriate value within the range can be selected, and these values include the upper value and the lower value of the range. For example, a range of two to thirty represents the terminal values of two and thirty, as well as the intermediate values between two to thirty, and all intermediate ranges encompassed within two to thirty, such as two to five, two to eight, two to ten, etc.
  • genetic modification refers to altering the genomic DNA in a microorganism. Typically, a genetic modification alters the expression and/or activity of a protein encoded by the altered gene.
  • a genetic modification encompasses a“variant”, which is a gene or protein sequence that deviates from a reference gene or protein, as further detailed below.
  • oligosaccharide refers to saccharide multimers of varying length and includes but is not limited to: sucrose (1 glucose monomer and 1 fructose monomer), lactose (1 glucose monomer and 1 galactose monomer), maltose (1 glucose monomer and 1 glucose monomer), isomaltose (2 glucose monomers), isomaltulose (1 glucose monomer and 1 fructose monomer), trehalose (2 glucose monomers), trehalulose (1 glucose monomer and 1 fructose monomer) cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), cellohexaose (6 glucose monomers), 2’- Fucosyllactose (2’-FL, 1 fucose monomer, 1 glucose monomer, and 1 galactose monomer), 3- Fucosyllactose (3’-FL, 1 fucose monomer, 1 glucose monomer, and
  • Monofucosyllacto-N-hexaose II (MFLNH II, 1 Fucose monomer, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers), Difucosyllacto-N-hexaose I
  • LNDFH I 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers
  • Difucosyllacto-N-hexaose II LNDFH II, 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers
  • Difucosyllacto- N-neohexaose LNnDFH, 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers
  • Difucosyl-para-lacto-N-Hexaose Difucosyl-para-lacto-N-Hexaose (DFpLNH, 2 N- acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers
  • FucosylSialyllacto-N-tetraose a (FLSTa, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), FucosylSialyllacto-N-tetraose b (FLSTb, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Fucosylsialyllacto-N-hexaose (FSLNH, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers), Fucosylsialyllacto-N-neohexaose
  • “human milk oligosaccharide”,“HMO”, and“human milk glycans” refer to oligosaccharides group that are be found in high concentrations in human breast milk.
  • the dominant oligosaccharide in 80% of all women is 2'-fucosyllactose.
  • HMOs include 3- fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose, di-fucosyllactose, lacto-N- neotetraose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose, monofucosyllacto-N-hexaose I, monofucosyllacto-N- hexaose II, difucosyllacto-N-
  • degree of polymerization is the number of monomeric units in a macromolecule or polymer or oligomer molecule.
  • microorganism refers to prokaryote or eukaryote microorganisms capable of oligosaccharides production or utilization with or without modifications.
  • enhanced utilization refers to an improvement in oligosaccharide production by a microorganism compared to a parental microorganism, specifically an increase in the oligosaccharides production rate, a decrease in the initial time before oligosaccharides production begins, an increase in the yield, defined as the ratio of product made to the starting material consumed, and/or a decrease in an overall time the microorganisms take to produce a given amount of an oligosaccharide.
  • parental microorganism refers to a microorganism that is manipulated to produce a genetically modified microorganism. For example, if a gene is mutated in a microorganism by one or more genetic modifications, the microorganism being modified is a parental microorganism of the microorganism carrying the one or more genetic modifications.
  • the term,“consumption rate” refers to an amount of oligosaccharides consumed by the microorganisms having a given cell density in a given culture volume in a given time period.
  • production rate refers to an amount of desired compounds produced by the microorganisms having a given cell density in a given culture volume in a given time period.
  • the term“gene” includes the coding region of the gene as well as the upstream and downstream regulatory regions.
  • the upstream regulatory region includes sequences comprising the promoter region of the gene.
  • the downstream regulatory region includes sequences comprising the terminator region. Other sequences may be present in the upstream and downstream regulatory regions.
  • a gene is represented herein in small caps and italicized format of the name of the gene, whereas, a protein is represented in all caps and non-italicized format of the name of the protein. For example, cdt-1 (italicized) represents a gene encoding the CDT-l protein, whereas CDT-l (non-italicized and all caps) represents CDT-l protein.
  • sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to a reference sequence refers to a comparison made between two sequences, preferably using the BLAST algorithm.
  • Algorithms for comparisons between two protein sequences that use protein structural information, such as sequence threading or 3D-1D profiles, are also known in the field.
  • A“variant” is a gene or protein sequence that deviates from a reference gene or protein.
  • the terms“isoform,”“isotype,” and“analog” also refer to“variant” forms of a gene or a protein.
  • the variant may have“conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a variant may have“nonconservative” changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both. Suitable amino acid residues that may be substituted, inserted, or deleted, and which are“conservative” or “nonconservative” may be determined by those of skill in the art, including by using computer programs well known in the art.
  • Exogenous nucleic acid refers to a nucleic acid, DNA, or RNA, which has been artificially introduced into a cell. Such exogenous nucleic acid may or may not be a copy of a sequence or fragments thereof which is naturally found in the cell into which it was introduced.
  • Endogenous nucleic acid refers to a nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is naturally present in a microorganism. An endogenous sequence is“native” to, i.e., indigenous to, the microorganism.
  • mutation refers to genetic modification to a gene including modifications to the open reading frame, upstream regulatory region, and/or downstream regulatory region.
  • a heterologous host cell for a nucleic acid sequence refers to a cell that does not naturally contain the nucleic acid sequence.
  • A“chimeric nucleic acid” comprises a first nucleotide sequence linked to a second nucleotide sequence, wherein the second nucleotide sequence is different from the sequence which is associated with the first nucleotide sequence in cells in which the first nucleotide sequence occurs naturally.
  • a constitutive promoter expresses an operably linked gene when RNA polymerase holoenzyme is available. Expression of a gene under the control of a constitutive promoter does not depend on the presence of an inducer.
  • An inducible promoter expresses an operably linked gene only in the presence of an inducer.
  • An inducer activates the transcription machinery that induces the expression of a gene operably linked to an inducible promoter.
  • microorganisms systems and methods for exporting oligosaccharides such as Human Milk Oligosaccharides (HMOs).
  • HMOs Human Milk Oligosaccharides
  • the present disclosure provides genetically engineered microorganisms capable of exporting oligosaccharides.
  • the microorganism described herein can export HMOs, such as 2’-fucosyllactose (2’- FL), such as into the growth medium where the microorganism resides.
  • the microorganism is genetically engineered to express a transporter that is capable of exporting oligosaccharides from the microorganism.
  • exemplary transporters include a cellodextrin transporter, which is CDT-l, CDT-2, or homologs and variants thereof.
  • the transporter CDT-l from the cellulolytic fungus Neurospora crassa (GenBank:
  • EAA34565.1 belongs to the major facilitator superfamily (MFS) class of transporters capable of transporting molecules comprising hexoses and related carbohydrates. This class of transporters is defined in PFAM under family PF00083 (see the World Wide Web at
  • CDT-l is capable of importing cellodextrins including cellobiose, cellotriose and cellotetraose, as well as lactose into Saccharomyces cerevisiae. However, it has not be shown or used previous to the disclosure herein as an exporter of engineered products in a microorganism. Surprisingly, another transporter FAC 12 from Kluyveromyces lactis is capable of importing lactose (like CDT-l), but as demonstrated herein, FAC12 does not function as an exporter for 2’- FF.
  • CDT-l is provided by the sequence of SEQ ID NO: 4, which is CDT-l from Neurospora crassa (Uniprot entry Q7SCU1). Homologues of CDT-l from microorganisms other than N crassa, particularly, from fungi, can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of CDT-l in the instant invention are represented by UmProt entries: A0A0B0E0J3, F8MZD6, G4U961, F7VQY4, Q7SCU1, A0A0J0XVF7, A0A0G2FA71, Q0CVN2, G4T6X5, A0A1Q5T2Z1, A0A0F7VA10,
  • A0A0S7E4Y9 A0A2T3AJM0, Q5B9G6, A0A2I1C7L5, A0A167H9D2, A0A2J6SE99, J3PJL4, A0A0C4EGH0, A0A135LD10, A0A0A2I302, A0A0G4NZP3, K9G9B1, K9G7S2,
  • CDT-2 is provided by the sequence of SEQ ID NO: 9.
  • cellodextrin transporter examples include Cellodextrin transporter cdt-g (ETniProt entry: R9ETSL5), Cellodextrin transporter cdt-d (ETniProt entry: R9ETTV3), Cellodextrin transporter cdt-c (ETniProt entry: R9ETR53), Cellodextrin transporter CdtG (ETniProt entry:
  • CDT-l Additional homologs of CDT-l are known in the art and such embodiments are within the purview of the invention.
  • the homologs of CDT-l have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1.
  • CDT-l is a substrate-proton symporter from the MFS family. It facilitates the import of beta-l,4-linked disaccharides such as lactose or cellobiose out of the growth medium into the cell.
  • CDT-l has been characterized as an importer of substrates such as cellobiose (such as used in the biofuel industry). For example, Ryan et al.
  • CDT-l variants of CDT-l, such as CDT-l N209S and CDT-1-F262Y have an improved capability to import the oligosaccharide cellobiose.
  • CDT-l nor any variants have been shown to be an exporter.
  • CDT-l has been characterized as lacking activity that would provide utility as an exporter (see e.g., Hollands K. et al., Metab Eng. 2019 Mar; 52: 232-242).
  • CDT-l -N209S/F262Y (or shortly: CDT-l SY): SEQ ID NO: 1
  • CDT-1-N209S (or shortly: CDT-ls): SEQ ID NO: 2
  • CDT-1-F262Y (or shortly: CDT-ly): SEQ ID NO: 3
  • a lactose permease a membrane protein
  • Lactose permease can be classified as a symporter, which uses the proton gradient towards the cell to transport b-galactosides such as lactose in the same direction into the cell.
  • the lactose importer is LAC12. Homologues of LAC12 can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of LAC12 in the instant invention are represented by UniProt entries: Q9FLB5, B9FJH4, P07921, A0A1 J6J8V9, A0A251TUB0, A0A0A9W3I8, D0E8H2, W0THP1, A0A1 S9RK01,
  • A0A151V9Y9 A0A1C1CDD3, W0TAG2, A0A151W5N5, A0A151WE7, A0A151WBL8, A0A151V6X4, A0A151W4U2, A0A1C7LPV6, W0T7D8, W0T8B1, A0A1C1CKJ6,
  • lactose permease are encoded by LacY gene (UniProt entry: P02920, P22733, P47234, P18817, P59832), LacE (UniProt entry: P11162, P24400, P23531, Q4L869, Q5HE15, P50976, Q931G6, Q8CNF7, Q5HM40, Q99S77, Q7A092, Q6GEN9, Q6G7C4, A0A0H3BYW2), LacS gene (UniProt entry: P23936, Q48624, Q7WTB2), LacP (UniProt entry: 033814).
  • Lactose permease can be expressed in a microorganism and provide lactose uptake. In some aspects, lactose can then be used by the microorganism as a substrate for the production of other oligosaccharides such as HMOs.
  • a lactose permease such as Lac 12 when expressed in a microorganism does not act as an exporter with respect to oligosaccharides such as HMOs.
  • Lacl2 does not export 2’-FL when Lacl2 is expressed in a yeast such as Sacharomyces cerevisae.
  • a cellobiose transporter acting as an importer within Neurospora crassa can act as an exporter when expressed in a microorganism such as when expressed in Saccharomyces cerevisiae strains producing an HMO.
  • the HMO exported by such transporter is a non-branched HMO comprised of a lactose core with modifications to the galactose ring.
  • the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N- tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto- difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I).
  • the HMO is 2’-FL.
  • the transporter for export of HMOs is a CDT-l, a CDT-2 or homolog thereof.
  • the transporter for export of HMOs is a variant, such as a mutant CDT-l, where one or more amino acids are altered as compared to a CDT-l amino acid sequence.
  • a mutant CDT-l for exporting HMOs comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 98%, 99% or greater than 99% homology with SEQ ID NO: 1.
  • the mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions 91, 209, 213, 256, 262, 335, and 411 of SEQ ID NO: 1.
  • the mutant CDT-l can comprise SEQ ID NO: 1 having one or more ammo acid substitutions selected from G91A, N209S, F213A, L256V, F262Y, F262W, F335A, S411A.
  • the mutant CDT-l is CDT-l N209S F262Y (SEQ ID NO: 1), CDT-l G91A (SEQ ID NO: 10), CDT-l F213A (SEQ ID NO: 11), CDT-l L256V (SEQ ID NO: 12), CDT-l F335A (SEQ ID NO: 13), CDT-l S411A (SEQ ID NO: 14), or CDT-l N209S F262W (SEQ ID NO: 15).
  • the CDT transporter such as a CDT-l or mutant CDT-l when expressed in a microorganism exports HMO such as 2’-FL.
  • HMO such as 2’-FL.
  • CDT-l N209S/F262Y (encoding CDT-l N209S/F262Y) was expressed within a background strain (microorganism) producing 2’-FL and 2’-FL accumulation in the growth medium during a fermentation experiment was compared to the same strain without the cdt-l-sy gene. Unexpectedly, the expression of CDT-l N209S/F262Y significantly increases the accumulation of 2’-FL within the growth medium indicating that CDT-l SY can act as an efficient substrate exporter.
  • Lactose permease mutant (CDT-l G91A) [Neurospora crassa] SEQ ID NO: 10
  • Lactose permease mutant (CDT-l L256V) ⁇ Neurospora crassa] SEQ ID NO: 12
  • a variant of CDT-l and related transporters for use as an HMO exporter can include one or more mutations of amino acids predicted to be near the sugar substrate binding pocket (e.g., N209S in CDT-l) or near the highly-conserved PESPR motif in the sugar porter family PF00083 (e.g., F262Y in CDT-l).
  • Exemplary mutations include amino acids in CDT-l predicted to be in the substrate binding pocket such as G336, Q337, N341, and G471.
  • modifications of a microorganism expressing a transporter such as CDT-l or a CDT-l mutant can be engineered to increase the activity of the transporter.
  • Non limiting examples of genetic modifications to cdt-1 that can increase the activity of CDT-l as a substrate exporter in the microorganisms compared to CDT-l substrate import activity in the parental microorganisms include one or more of: a) replacement of an endogenous promoter with an exogenous promoter operably linked to the endogenous cdt-1; b) expression of a cdt-1 via an extrachromosomal genetic material; c) integration of one or more copies of cdt-1 into the genome of the microorganism; d) a modification to the endogenous cdt-1 to produce a modified CDT-l that encodes a transporter protein that has an increased activity as a substrate exporter; e) introduction into the microorganism on extrachromosomal genetic material comprising a cdt-1 or a variant of cdt-1 (mutant cdt-l) such as encoding CDT-l N209S F262Y or one or more of the
  • an expression of cdt-l or its variants is varied by utilizing different promoters or changes immediately adjacent to the introduced cdt-l gene.
  • the deletion of a URA3 cassette adjacent to an introduced cdt-l sy expression cassette leads to a further improvement of HMO export, such as 2’-FL export.
  • the endogenous promoter is replaced with an exogenous promoter that induces the expression of cdt-l at a higher level than the endogenous promoter.
  • the exogenous promoter is specific for the microorganism in which the exogenous promoter replaces the endogenous promoter.
  • a yeast specific exogenous promoter can be used if the microorganism being modified is a yeast.
  • the exogenous promoter can be a constitutive promoter or inducible promoter.
  • Non-limiting examples of constitutive yeast specific promoters include: pCYCl, pADHl, p STE5, pADHl, pCYClOO minimal, p CYC70 minimal, p CYC43 minimal, p CYC28 minimal, pCYC16, pPGKl, pCYC, p GPD or p TDH3. Additional examples of constitutive promoters from yeast and examples of constitutive promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
  • Non-limiting examples of inducible yeast specific promoters include: pGALl, pMFAl, pMFA2, p STE3, p URA3, pFIGl, pEN02, pDLD, pJENl, pmCYC, and p STE2. Additional examples of inducible promoters from yeast and examples of inducible promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
  • the microorganisms comprise a modification to the wildtype cdt- 1 to produce a modified cdt-1 that encodes a transporter with an increased capability to export 2’-FL from the cell.
  • modification of the wildtype cdt-1 produces a modified cdt-1 that encodes a CDT-l with increased export rates of 2’-FL.
  • wildtype cdt-1 is mutated around the conserved PEPSR motif which is conserved in hexose transporters.
  • cdt-1 is modified leading to the production of a protein CDT-1-F262Y.
  • the mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions 91, 209, 213, 256, 262, 262, 335, and 411 of SEQ ID NO: 1.
  • the mutant CDT- 1 can comprise SEQ ID NO: 1 having one or more amino acid substitutions selected from G91A, N209S, F213A, L256V, F262Y, F262W, F335A, S411A.
  • the mutant CDT-l is CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, or CDT-l N209S F262W.
  • the mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket and/or the PESPR motif such as positions G336, Q337, N341, and G471.
  • wild-type cdt-1 is mutated around the amino acid residues within CDT-l which are interacting with the oligosaccharide substrate.
  • cdt-1 is modified leading to the production of a protein CDT-1-N209S.
  • cdt-1 is modified leading to the production of a protein CDT-1-N209S F262Y.
  • cdt-1 is modified leading to the production of a protein CDT-l G91 A.
  • cdt-1 is modified leading to the production of a protein CDT-l F213A.
  • cdt-1 is modified leading to the production of a protein CDT-l L256V. .
  • cdt-1 is modified leading to the production of a protein CDT-l F335A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l S411 A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l N209S F262W.
  • a microorganism preferably, a fungus such as a yeast, more preferably, a Saccharomyces spp., and even more preferably, S. cerevisiae is provided, the microorganism comprising the genetic modifications or the combinations of genetic
  • HMOs are generally comprised of monosaccharides linked together, and typically with a lactose molecule at one end.
  • the monomer might be a monosaccharide.
  • the monomer might be glucose, galactose, N-acetylglucosamine, fucose, and/or N-acetylneuraminic acid.
  • production can include i) the biosynthesis of GDP-fucose and ii) the transfer of the fucosyl domain of GDP-fucose onto an acceptor oligosaccharide.
  • the acceptor oligosaccharide is the disaccharide lactose.
  • GDP-fucose is synthesized from GDP-Mannose by two successive reactions: First, GDP Mannose is dehydrated by a GDP-Mannose dehydratase (GMD) to produce GDP-4-dehydro-6- deoxy-D-mannose. Second, GDP-4-dehydro-6-deoxy-D-mannose is further reduced to GDP-L- fucose by a GDP-L-fucose synthase (GFS).
  • GDP-fucose can then be transferred to the disaccharide lactose by a fucosyl transferase (FT), forming a fucosylated oligosaccharide.
  • the FT is an alpha 1 ,2-fucosyl transferase.
  • the fucosylated oligosaccharide is 2’-FL or 3’-FL.
  • microorganisms that exhibit increased utilization of oligosaccharides are provided.
  • the microorganism further comprises one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein.
  • “HMO production gene” expresses“HMO production protein”.
  • “HMO production protein” is an enzyme that participates in a pathway for HMO production.
  • Exemplary enzymes that participate in pathways for HMO production are enzymes capable of converting fucose and ATP to fucose- 1- phosphate, an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and/or a glucosyl transferase.
  • Examples of HMO production protein are a GDP-Mannose dehydratase (GMD), a GDP-L-fucose synthase (GFS), and a fucosyl transferase (FT).
  • the microorganisms comprise one or more genetic modifications that: i) increase the activity of a GDP-Mannose dehydratase (GMD), and/or ii) increase the activity of a GDP-L-fucose synthase (GFS), and/or iii) increase the activity of glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1,2 -fucosyl transferase.
  • GMD GDP-Mannose dehydratase
  • GFS GDP-L-fucose synthase
  • FT fucosyl transferase
  • these genetic modifications that result in i), ii), and iii) are produced by introduction of a GDP-Mannose dehydratase gene (GMD), GDP-L-fucose synthase gene (GFS), and a glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1,2 -fucosyl transferase gene, respectively.
  • GMD GDP-Mannose dehydratase gene
  • GFS GDP-L-fucose synthase gene
  • FT fucosyl transferase
  • the microorganism comprises a heterologous GDP- Mannose dehydratase gene or a construct that enhances expression of the GDP-Mannose dehydratase.
  • the microorganism comprises a heterologous GDP-L- fucose synthase gene or a construct that enhances expression of the GDP-L-fucose synthase.
  • the microorganism comprises a heterologous glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1 ,2-fucosyl transferase gene or a construct that enhances expression of the glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1 ,2-fucosyl transferase.
  • FT fucosyl transferase
  • microorganisms comprising one or more genetic modifications selected from:
  • GMD GDP-Mannose dehydratase gene
  • GFS GDP-L-fucose synthase gene
  • FT transferase
  • alpha 1,2 -fucosyl transferase gene or its analogues.
  • HMOs such as 2’-FL can be produced in a microorganism.
  • a microorganism is genetically engineered by incorporating one or more nucleic acids that encode for an enzyme for one or more steps in the production of an HMO.
  • an HMO pathway is supplied entirely by such genetic engineering.
  • an HMO pathway is comprised of one or more endogenous activities from the host microorganism, and others through genetic engineering.
  • the host microorganism synthesizes an HMO using endogenous activities.
  • the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I).
  • the HMO is a fucosyllactose, such as 2’-FL.
  • fucosyllactose, such as 2’-FL is synthesized in a host microorganism through a de novo pathway.
  • the pathway can comprise GMD (GDP-mannose dehydratase), GFS (GDP-fucose synthase), and FT (fucosyltransferase), where GMD supplies an enzymatic activity to convert GDP-Mannose to GDP-4-keto-6-deoxymanose.
  • GFS for example, WcaG, converts GDP-4- keto-6-deoxymanose to GDP-fucose and FT converts GDP-fucose to 2’-FL.
  • the FT is an alpha l,2-fucosyl transferase.
  • GMD GDP-Mannose dehydratase
  • SEQ ID NOs: 17-19 are GDP-Mannose dehydratases from Idsiularia Solaris, Cladosiphon okamuranus, and Cladosiphon okamuranus, respectively.
  • Homologues of GMD from microorganisms other than Fistularia solans and Cladosiphon okamuranus, in particular, from other heteronochphytes and from fungi, can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of GMD in the instant invention are represented by UniProt entries: P93031, 060547, Q18801, Q51366, Q93VR3, P0AC88, Q9VMW9, 045583, A3C4S4, Q9SNY3, Q8K0C9, Q8K3X3, Q9JRN5, Q56872,
  • homologs of GMD are known in the art and such embodiments are within the purview of the invention.
  • the homologs of GMD have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 17-19 and 42.
  • GDP-mannose 4,6-dehydratase catalyzes the conversion of GDP- mannose to GDP-4-keto-6-deoxymannose, the first step in the synthesis of GDP-fucose from GDP-mannose, using NAD+ as a cofactor.
  • This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds.
  • the systematic name of this enzyme class is GDP-mannose 4,6-hydro-lyase (GDP-4-dehydro-6-deoxy-D-mannose-forming).
  • guanosine 5'-diphosphate-D-mannose oxidoreductase guanosine diphosphomannose oxidoreductase
  • guanosine diphosphomannose 4,6-dehydratase GDP-D-mannose dehydratase
  • GDP-D-mannose 4,6-dehydratase Gmd
  • GDP-mannose 4,6- hydro-lyase This enzyme participates in fructose and mannose metabolism. It employs one cofactor, NAD+.
  • GMD and/or GFS are derived from E. coli, Helicobacter pylori, Arabidopsis thaliana, and/or Mortierella alpina (Ren et al., Biochem Biophys Res Commun. 2010 Jan 22;39l(4): 1663-9; Hollands K. et al., Metab Eng. 2019 Mar; 52: 232-242).
  • GMD is encoded by one of the sequences listed in Table 1 or a variant thereof.
  • GMD from Arabidopsis thaliana SEQ ID NO: 61 1 MASENNGSRS DSESITAPKA DSTWEPRKI ALITGITGQD GSYLTEFLLG
  • GMD from Mortierella alpine SEQ ID NO: 62
  • GFS GDP-fucose synthase
  • SEQ ID NOs: 20-23 are GDP-L-fucose synthases from Cladosiphon okamuranus, Phaeodaciylum tricomutum, Saccharina japonica, and Mucor circinelloides f circinelloides 1006PhL, respectively.
  • Non-limiting examples of the homologs of GFSs in the instant invention are represented by ETniProt entries: Q13630, P32055, 049213, P23591, Q9W1X8, Q9LMU0, G5EER4, Q8K3X2, P33217, Q5RBE5, F0F7M8, Q67WR2, P55353, Q67WR5, D9RW33, F2KZP1, G1WDT9, D7NG24, C9MLN8, Q9S5F8, X6PWX2, H1HNE5, D1QPT8, G6AG96, 10TA81, G1VAH6, A0A0K1NMZ0, U2KFA0, F0H551, A0A2K9HDD8, A0A095YQN3, D3I452, A0A096ARU1, A0A095ZVW3, A0A096ACH9, A0A1B1IBP6, Q55C77, A0
  • homologs of GFS’s are known in the art and such embodiments are within the purview of the invention.
  • the homologs of GFS’s have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 20-23.
  • a GDP-L-fucose synthase (EC 1.1.1.271) is an enzyme that catalyzes the chemical reaction GDP-4-dehydro-6-deoxy-D-mannose + NADPH + H + ⁇ — > GDP-L-fucose + NADP + .
  • the three substrates of this enzyme are GDP-4-dehydro-6-deoxy-D-mannose, NADPH, and H + , whereas its two products are GDP-L-fucose and NADP + .
  • This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of a donor with NAD + or NADP + as acceptor.
  • GFS is encoded by one of the sequences listed in Table 2 or a variant thereof.
  • GMER (WcaG) from Arabidopsis thaliana SEQ ID NO: 44
  • GMD and GFS activities are supplied by a single enzyme, such as one of those listed in Table 3 or a variant thereof.
  • FTs fucosyl transferases
  • alpha- 1,2 -fucosyl transferase alpha 1,2 -fucosyl transferases
  • Dictyostelium discoideum AX4 Homo sapiens, Pisum sativa, Rhizobium marinum,
  • Herbaspirillum rubrisubalbicans Citrobacter freundii, Lactobacillus helveticus, Neocallimastix californiae, Gracilariopsis chorda, Lactobacillus gasseri, Octopus bimaculoides, and
  • Herbaspirillum rubrisubalbicans Citrobacter freundii, Lactobacillus helveticus, Neocallimastix californiae, Gracilariopsis chorda, Lactobacillus gasseri, Octopus bimaculoides, and
  • Chryseobacterium scophthalmum particularly, from fungi, can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of FTs in the instant invention are represented by FTniProt entries: 030511, P51993, Ql 1128, G5EFP5, G5EE06, P56434, Q11130, Q11131, P56433, Q8HYJ7, Q8HYJ6, Q17WZ9, Q9ZFI3, D0ISI2, D0ITD1, Q9ZKD7, C7BXF2, E6NNI5, E6NPH4, B6JFN9, C7BZU7, E6NJ21, E6NI06, E6NRI2,
  • Analogues of FTs can be used in the microorganisms and methods described herein.
  • FT is selected from a-l,2-fucosyltransferases (FTs) from
  • H. pylori 26695 FetC
  • Bacteroides fragilis WcfB
  • E. coli such as WbgF, WbgN, and WbwK, for example, wbwK from E. coli086, wbsJ from E. coli 0128, wbgF from E. coli 0126, wbiQ from E. coli 0127
  • futB from H. pylori, futL from H. mustelae, futF from H. bibs, , futG from C. jejuni, futN from B. vulgatus ATCC 8482, and wcfB and wcfW from B. fragilis).
  • FT is encoded by one of the sequences listed in Table 4 or a variant thereof.
  • the nucleic acids encoding an enzyme sequence include a targeting sequence, such as for localization to a specific cellular organelle.
  • such sequence is removed from the nucleic acid prior to providing it as a heterologous sequence through genetic engineering into a microorganism.
  • the targeting sequence of SEQ ID Nos. 27, 28, 33, 38, 39 or 40 can be removed before the encoded FT is genetic engineered for expression in a microorganism.
  • homologs of FTs are known in the art and such embodiments are envisioned for use with the engineered microorganisms and methods here.
  • the homologs of FTs have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to SEQ ID NOs: 26-40.
  • an HMO such as 2’-FF
  • a microorganism can utilize lactose and fucose substrates to synthesize 2’-FF using an enzyme to convert fucose and ATP to fucose-l- phosphate and an enzyme to convert the fucose- 1 -phosphate and GTP to GDP-fucose, which then can be converted by a fucosyl transferase (FT) to 2’-FF.
  • FT fucosyl transferase
  • a bifunctional fucokinase/F-fucose-l-P-guanylyltransferase (FKP) enzyme such as fkp from Bacteroides fragilis performs the two enzymatic steps from fucose to GDP-fucose and then a FT coverts the GDP-fucose to 2’-FF.
  • the kfp is from B. fragilis 9343, B. thetaiotaomircon or B. ovatus .
  • the FT may be fuel 2 from Heliobacter pylori or any of the FTs described herein.
  • lactose is supplied exogenously to the microorganism and a transporter such as Fac 12, CDT-l, CDT-2 or a variants or homolog thereof imports the lactose intracellularly for conversion to the HMO.
  • a transporter such as Fac 12, CDT-l, CDT-2 or a variants or homolog thereof imports the lactose intracellularly for conversion to the HMO.
  • one or more modification are made to a microorganism (such as by genetic engineering) and/or to one or more nucleic acids encoding an enzyme for a step in making an HMO.
  • modification can include, but are not limited to: a) replacement of an endogenous promoter with an exogenous promoter operably linked to the endogenous enzyme, such as gmd, gfs,fkp, and/or ft: b) expression of GMD, GFS, FKP and/or FT via an endogenous promoter with an exogenous promoter operably linked to the endogenous enzyme, such as gmd, gfs,fkp, and/or ft: b) expression of GMD, GFS, FKP and/or FT via an endogenous promoter with an exogenous promoter operably linked to the endogenous enzyme, such as gmd, gfs,fkp, and/or ft: b) expression of GMD, GFS, FK
  • extrachromosomal genetic material c) integration of one or more copies of gmd, gfs,fkp, and/or ft into the genome of the microorganism; or d) a modification to the endogenous gmd , gfs,fkp and/or ft to produce a modified gmd , gfs,fkp, and/or ft that encodes a protein that has an increased activity or any combination of modifications a) to d) described in this paragraph.
  • an expression of GMD, GFS, and/or FT is varied by utilizing different promoters or changes immediately adjacent to the introduced gmd, gfs,fkp and/or ft genes.
  • the deletion of a URA3 cassette adjacent to an introduced gmd, gfs, fkp, and/or ft expression cassette leads to a further improvement of 2’-FL production.
  • the endogenous promoter is replaced with an exogenous promoter that induces the expression at a higher level than the endogenous promoter.
  • the exogenous promoter is specific for the microorganism in which the exogenous promoter replaces the endogenous promoter.
  • a yeast specific exogenous promoter can be used if the microorganism being modified is a yeast.
  • the exogenous promoter can be a constitutive promoter or inducible promoter.
  • Non-limiting examples of constitutive yeast specific promoters include: pCYCl, pADHl, p STE5, pADHl, pCYClOO minimal, p CYC70 minimal, p CYC43 minimal, p CYC28 minimal, pCYC16, pPGKl, pCTC, p GPD or pTDH3. Additional examples of constitutive promoters from yeast and examples of constitutive promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
  • Non-limiting examples of inducible yeast specific promoters include: pGALl, pMFAl, pMFA2, p STE3, p URA3, pFIGl, pEN02, pDLD, pJENl, pmCYC, and rL7 ' //2. Additional examples of inducible promoters from yeast and examples of inducible promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention. Microorganisms used to produce the genetically modified microorganisms described herein may be selected from Saccharomyces spp., such as S. cerevisiae, S. pastorianus, S.
  • I.orientalis Kloeckera spp. such as K. apiculata; Aureobasidium spp. such as A. pullulans; Torulaspora spp., Torulaspora delbrueckii, Zygosaccharomyces spp., Zygosaccharomyces bailii, Brettanomyces spp., Brettannomyces intermedius, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera spp., Dekkera bruxellensis, Dekkera anomala, Metschmkowia spp., Issatchenkia spp., Issatchenkia orientalis, Issatchenkia terricola, Kloeckera spp., Kloeckera apiculate,
  • Aureobasidium spp. Aureobasidium pullulans, Rhodotorula spp., Rhodotorula glutinis, Rhodotorula cladiensis, Rhodosporidiumspp., Rhodosporidum toruloides, Cryptococcus spp., Cryptococcus neoformans, Cryptococcus albidus, Yarrowia spp, Yarrowia lipolytica, Kuraishia spp, Kuraishia capsulata, Kuraishia molischiana, Komagataella spp., Komagataella phaffii, Komagataella pastoris, Hanseniaspora spp., Hanseniaspora guilliermondii, Hanseniaspora uvarum, Hasegawaea spp., Hasegawaea japonica, Ascoidea spp., Ascoidea asiatica,
  • Cephaloascus spp. Cephaloascus fragrans, Lipomyces spp., Lipomyces starkeyi, Kawasakia spp., Kawasakia arxii, Zygozyma spp, Zygozyma oligophaga, Metschmkowia spp.,
  • Coccidiodes spp. Coccidiodes immitis, Neurospora discreta, Neurospora africana, Aspergillus spp., Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus, Mucor spp., Mucor circinelloides, Mucor racemosus, Rhizopus spp., Rhizopus oryzae, Rhizopus stolonifera, Umbelopsis spp., Umbelapsis isabelline,
  • Mortierella spp Mortierella alpine, Alternariaspp., Altemaria alternate, Botrytis spp., Botrytis cinereal, Fusarium spp., Fusarium graminarium, Geotrichum spp., Geotrichum candidum, Penicillium spp., Penicillum chrysogenum, Chaetomium spp., Chaetomium thermophila, Magnaporthe spp., Magnaporthe grisea, Emericella spp., Emericella discophora, Trichoderma spp., Trichodema reesei, Talaromyces spp., Talaromyces emersonii, Sordaria spp., or Sordaria macrospora.
  • a microorganism preferably, a fungus, such as a yeast, more preferably, a Saccharomyces spp., and even more preferably, S. cerevisiae is provided as the microorganism host.
  • Yeast such as Saccharomyces spp. can be genetically engineered as described herein or using a multitude of available tools.
  • Ascomycetes fungi can also serve as suitable hosts. Many ascomycetes are useful industrial hosts for fermentation production. Exemplary genera include Trichoderma,
  • Exemplary species include Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma reesei, Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis, Kluyveromyces marxianus , Neurospora crassa, Hansenula polymorpha, Yarrowia lipolytica, and Saccharomyces boulardii.
  • Cloning tools are widely known to those skilled in the art. See e.g., Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei, Robert H. Bischof, Microbial Cell Factories Volume 15, Article number: 106 (2016)), Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant
  • Kluyveromyces marxianus yeast strain Yumiko Nambu-Nishida, , Scientific Reportsvolume 7, Article number: 8993 (2017); Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host, Paul Cernak, mBio Sep 2018, 9 (5) e0l4l0-l 8; DOI:
  • the production and/or export of an HMO can be enhanced through genetic modification of an HMO-producing microorganism.
  • an HMO-producing microorganism can be modified by one or more of the following:
  • PMA1 is a genetic modification to plasma membrane ATPase gene (pmal ), ii) the genetic modification that decreases the activity of SNF3 is a genetic modification to sucrose non fermenting gene ( snf3 ), iii) the genetic modification that decreases the activity of RGT2 is a genetic modification to glucose transport gene ( rgt2 ), and iv) the genetic modification that decreases the activity of GPR1 is a genetic modification to G protein-coupled receptor 1 gene ( , gprl ).
  • Examples of PMA1, SNF3, RGT2, and GPR1 are described in International Patent Application No. PCT/US2018/040351, the contents of which are incorporated herein by reference.
  • PMA1 is provided by the sequence of SEQ ID NO: 5, which is PMA1 from Saccharomyces cerevisiae.
  • Homologs of PMA1 from microorganisms other than A cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure.
  • Non-limiting examples of the homologs of PMA1 useful in the instant disclosure are represented by Umprot entries: A0A1U8I9G6, A0A1U8H4C1, A0A093V076, A0A1U8FCY1, Q08435, A0A1U7Y482, A0A1U8GLU7, P22180, A0A1U8G6C0, A0A1U8IAV5,
  • homologs of PMA1 are known in the art and such embodiments are within the purview of the present disclosure.
  • the homologs of PMA1 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5.
  • SNF3 is provided by the sequence of SEQ ID NO: 6, which is SNF3 from S. cerevisiae. Homologs of SNF3 from microorganisms other than S. cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure.
  • Non limiting examples of the homologs of SNF3 useful in the instant disclosure are represented by Umprot entries: W0TFH8, Q6FNU3, A0A0W0CEX1, G2WBX2, A6ZXD8, J6EGX9, P10870, C7GV56, B3LH76, A0A0L8RL87, A0A0K3C9L0, M7WSX8, A0A1U8HEQ5, G5EBN9, A8X3G5, A3LZS0, G3AQ67, A0A1E4RGT4, A0A1B2J9B3, F2QP27, E3MDL0,
  • the Uniprot entries listed herein are incorporated by reference in their entireties.
  • homologs of SNF3 are known in the art and such embodiments are within the purview of the present disclosure.
  • the homologs of SNF3 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6.
  • RGT2 is provided by the sequence of SEQ ID NO: 7, which is RGT2 from S. cerevisiae. Homologs of RGT2 from organisms other than S. cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure.
  • Non limiting examples of the homologs of RGT2 are represented by Uniprot entries: A0A0FHMAJ7, N4TG48, A0A1Q8RPY1, N4U7I0, A0A1L7SSQ2, A0A1L7VB15, A0A0C4E497,
  • A0A0G2E6D5 A0A1J9R914, A0A0F4GQX7, A0A1 S9RLB9, A3M0N3, J9PF54,
  • A0A178DQW4 A0A167V6F7, A0A166WR60, A0A162KLT6, A0A1L7X3D1,
  • homologs of RGT2 are known in the art and such embodiments are within the purview of the present disclosure.
  • the homologs of RGT2 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7.
  • GPR1 is provided by the sequence of SEQ ID NO: 8, which is GPR1 from S. cerevisiae. Homologs of GPR1 from microorganisms other than S. cerevisiae, particularly, from yeasts, can be used in the microorganisms and methods of the present disclosure.
  • Non limiting examples of the homologs of GPR1 are represented by Uniprot entries: A0A1S3ALF0, A0A0Q3MD25, A0A146RBQ8, A0A0P5SHA9, A2ARI4, Q9BXB1, Q9Z2H4, F1MLX5, U3DQD9, 12CVT9, 10FI44, K7D663, K7ASZ6, A0A1U7Q769, U3ESI5, T1E5B8,
  • A0A1A8P7N2 A0A1A8HF38, E7FE13, A0A1 S3FZF3, A0A0P7WFQ9, H2KQN3,
  • A0A1 S3FZK9 A0A1U7TUH0, A0A1U8BX93, A0A091DKN5, A0A146W919, A0A147B2K7, A0A146XNF4, A0A091DTX9, A0A0Q3UQB0, A0A146WH37, E9QDD1, Q58Y75,
  • homologs of GPR1 are known in the art and such embodiments are within the purview of the present disclosure.
  • the homologs of GPR1 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8.
  • the present disclosure provides microorganisms comprising one or more genetic modifications that provide for import and/or enhanced uptake of one or more substrates that can be used by the microorganism to make an HMO.
  • a microorganism comprising one or more genetic modifications that provide for import and/or enhanced uptake of one or more substrates that can be used by the microorganism to make an HMO.
  • microorganism can include:
  • analogues which increases the uptake of lactose and/or other substrate into the microorganism
  • a transporter which can both import a substrate, such as lactose and export a produced HMO, such as the wild type cellodextrin transporter gene ceil- 1 or a variant of the cellodextrin transporter gene ceil- 1 such as those described herein (for example, CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CD
  • Lactose transporter (Lacl2) [Kluyveromyces lactis] SEQ ID NO:4l
  • the microorganisms described herein are capable of producing HMOs such as 2’-FL. In some embodiments, the microorganisms are capable of converting lactose into 2’-FL. In particular embodiments, the microorganisms described herein have higher capacity, compared to the parental microorganisms, of converting lactose into 2’-FL. In specific embodiments, the conversion of lactose into 2’-FL occurs in the cytosol of the microorganisms.
  • the disclosure provides methods of producing 2’-FL by culturing the microorganisms described herein in culture media containing lactose under appropriate conditions for an appropriate period of time and recovering 2’-FL from the culture media.
  • the microorganisms belong to Saccharomyces spp. In even more preferred embodiments, the microorganisms are S. cerevisiae.
  • the media contains about 10 g/L yeast extract, 20 g/L peptone, and about 40 g/L oligosaccharide, particularly, lactose or sucrose.
  • the microorganisms, particularly, yeast are grown at 30 °C.
  • the present disclosure provides methods for producing
  • an HMO is separated from the cells (microorganism) that produce the HMO.
  • an HMO can be further isolated from other constituents of the culture media (fermentation broth) in which the HMO-producing cells are grown.
  • an HMO is recovered from the fermentation broth (also referred to a culture medium).
  • fermentation broth also referred to a culture medium.
  • Many methods are available for separation of cells and/or cell debris and other broth constituents from the produced HMO.
  • cell/debris separation can be achieved through centrifugation and/or filtration.
  • the filtration can be microfiltration or ultrafiltration or a combination thereof.
  • Ion exchange chromatography can be cation or anion exchange chromatography, and can be performed in normal mode or as simulated moving bed (SMB) chromatography.
  • SMB simulated moving bed
  • Other types of chromatography may be used to separate based upon size (size exclusion chromatography) or affinity towards a specific target molecule (affinity chromatography).
  • size exclusion chromatography size exclusion chromatography
  • affinity chromatography affinity chromatography
  • Absorption techniques such as adsorption using activated charcoal, can also be used as a separation step and in particular is useful for removal of color bodies or separation of oligosaccharides from monomers.
  • An HMO product can also be pasteurized, filtered, or otherwise sterilized for food quality purposes.
  • a product suitable for animal consumption includes one or more HMO produced by the microorganisms or methods herein.
  • the product can include one or more additional consumable ingredients, such as a protein, a lipid, a vitamin, a mineral or any combination thereof.
  • the product can be suitable for mammalian consumption, human consumption or consumption as an animal feed or supplement for livestock and companion animals.
  • the product is suitable for mammalian consumption, such as for human consumption and is an infant formula, an infant food, a nutritional supplement or a prebiotic product.
  • Products can have 1 , 2, 3 or more than 3 HMOs, and one or more of the HMOs can be produced by the microorganisms or by the methods described herein.
  • the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I), or any combinations thereof.
  • an engineered microorganism for production of an HMO comprises one or more of the following genetic modifications
  • a genetic modification producing a transporter for export of an HMO for example CDT-l or a variant of CDT-l such as one of CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411 A, CDT-l N209S F262W, one or more ammo acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket and/or the PESPR motif such as positions G336, Q337, N341, and G471 ;
  • CDT-l N209S F262Y CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, CDT-l N209S F262W;
  • a genetic modification of any of the embodiments (a)-(f) and the CDT-l can have one or more amino acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket the PESPR motif such as positions G336, Q337, N341, and
  • HMO is a non-branched HMO comprised of a lactose core, such as 2'-fucosyllactose (2'-FL), 3'- fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I)
  • a lactose core such as 2'-fucosyllactose (2'-FL), 3'- fucosyllactose (3'-FL), 3’
  • microorganism any of a)-l) wherein the microorganism is a Ascomycetes fungus, including but not limited to, a Sacharomyces spp., a Schizosaccharomyces spp., a Pichia spp., Trichoderma,
  • Kluyveromyces Kluyveromyces, Yarrowia, Aspergillus, and Neurospora.
  • Example 1 Improved 2’-FL production in Saccharomyces cerevisiae expressing GMD, GFS, and/or FT
  • GMD t GFS t
  • FT_t FT-t-t expression vectors conferring well known activities for the enzymes GMD, GFS and FT (named GMD t, GFS t and FT_t) were generated for expression in the yeast Saccharomyces cerevisiae. Under selection pressure, these expression vectors are believed to occur in multiple tens of copies per cell and thus expression of a plasmid born gene is likely higher than from a single genomic locus if comparable promoters are used.
  • Constructs expressing heterologous GMD, GFS or FT genes were then co-transformed with plasmids containing all but the genes for which enzymatic activity was to be tested.
  • the acceptor strain was a genetically modified Saccharomyces cerevisiae strain producing low titers of 2’-FL if grown on lactose.
  • the strain also expresses Lad 2 from Kluyveromyces lactis for improved import of lactose and an engineered oligosaccharide transporter for improved export of 2’-FL as indicated in Fig. 8.
  • the base strain was auxotrophic for the synthesis of Leucine, Histidine and Uracil while plasmids carried individual gene cassettes restoring auxotrophy for the respective compounds, respectively.
  • Putative GFSs were tested by transforming an expression construct comprising the putative GFS gene together with expression constructs containing GMD t and FT_t. After transformation, cells were selected on respective media omitting the compound for which transformed plasmids conferred auxotrophy for.
  • Colonies forming after the transformation were grown in drop out medium (omitting the compound the transformed plasmids conferred auxotrophy for) overnight at 30°C and 250 rpm shaking. Cells were then washed and then transferred into YP4D0.4L medium, which is YPD medium with 0.4 g/L lactose and 4 g/L Glucose, and grown for 6 days under identical conditions. Supernatants were analyzed by HPLC analysis.
  • Fig. 9 shows 2’-FL production by introducing a heterologous fucosyltransferase (FT) from different organisms to a yeast strain which also expresses CDT-l M7, GMD and WcaG from plasmids.
  • Ctrl is control strain without FT expression.
  • Fig. 10 shows 2’-FL formation compared to the base strain, which was capable of producing lower amounts of 2’-FL with integrated 2’-FL pathway consist of GMD, WcaG and WbgL.
  • putative FTs were tested by preparing expression constructs containing GMD t and GFS t.
  • An additional plasmid carrying each one of the Fucose transferase genes from SEQ ID NO: 38, 29, 30, 31, 32, and 40 was included in each of these transformations.
  • Cells were transformed with expression plasmids GMD t, GFS t and expression plasmids carrying each one of the FTs genes from SEQ ID NO: 38, 29, 30, 31, 32, and 40 and then selected, grown an analyzed as indicated above.
  • Fig. 11 shows that strains expressing various FTs accumulate more 2’-FL compared to the base strain.
  • SEQ ID NO: 24 The activity of an enzyme represented by SEQ ID NO: 24 was tested.
  • This enzyme consists of 2 modules, one that has homology to GDP-Mannose-Dehydratases and one that shares homology with GDP fucose synthases.
  • An enzyme comprising both, GMD and GFS, activities would hence be able to produce GDP fucose from GDP Mannose, NADPFEFE and GTP.
  • plasmids expressing i) a GMD, a FT and SEQ ID NO: 24 and ii) a FT and SEQ ID NO: 24 only.
  • Cells were transformed, selected and grown as described above. Compared to the base strain, both combinations yielded higher 2’-FL production when compared to the base strain without expression of additional plasmids.
  • the addition of plasmids expressing SEQ ID NO: 24 in absence of an additional plasmids expressing a fucose synthase significantly increases 2’-FL production compared to the base strain.
  • Expression of a plasmid carrying a GMD gene in addition to plasmids carrying a FT and SEQ ID NO: 24 further 2’-FL production.
  • Fig. 12 shows relative production of 2’-FL in yeast cells expressing plasmids with (lst column) GMD, a FT and SEQ ID NO: 24 and (2nd column) plasmids with a FT and SEQ ID NO: 24 only, relative to a base strain that contains a set of genomic GMD, GFS and FT genes. Fermentation and metabolite analysis
  • 2’-FL concentration as determined using an ICS-3000 Ion Chromatography System (Dionex, Sunnyvale, CA, EISA) equipped with CarboPac PA20 column. The column was eluted with KOH gradient at a flow rate of 0.4 mL/min, 30 °C.
  • Example 2 2’-FL production in Saccharomyces cerevisiae, which lacks 2’-FL biosynthesis, by expressing GMD, GFS, and/or FT
  • a base strain only carrying Lacl2 for improved lactose import and an engineered membrane transporter for improved 2’-FL export as indicated in Fig. 8 was prepared. However, while this strain lacks any genes for 2’-FL biosynthesis it also had not been improved for 2’-FL biosynthesis.
  • This base strain was transformed with plasmids expressing the GMDs encoded by SEQ ID NOs i) 17, ii) 18, and iii) 19. 2’-FL was produced in all these strains indicating that GMDs encoded by SEQ ID NOs: 17, 18, and 19, respectively all confer GMD activity if expressed in yeast cells.
  • Fig. 13 shows production of 2’-FL by expression of plasmids in a control strain otherwise not capable of 2’-FL production (Ctrl). Strains were transformed with plasmids expressing a GFS and a FT along with a plasmid carrying either SEQ ID NO: 17, 18, or 19, respectively. The control strain carrying no plasmids does not produce any 2’-FL.
  • Example 3 Increase in 2’-FL production in Saccharomyces cerevisiae expressing CDT-1 N209S/F262Y
  • S. cerevisiae was grown and maintained on YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 30 °C. All genes were expressed chromosomally.
  • the cdt-lsy gene (encoding CDT-l N209S/F262Y) was expressed within a background strain producing 2’-FL and 2’-FL accumulation in the growth medium was during a fermentation experiment was compared to the 2’-FL accumulation produced from the same strain without the cdt-l-sy gene.
  • the 2’-FL producing utilizing strain contains GDP-mannose-4, 6-dehydratase ( gmdl ), GDP-L-fucose synthase (wcaG), lactose permease ⁇ LAC 12) and two fucosyltransferases ( FucT2 , wbgL ).
  • YPDL medium (10 g/L yeast extract, 20 g/L peptone, 30 g/L glucose 2 g/L lactose) at 30 °C.

Abstract

Disclosed herein are genetically modified microorganisms and related methods for the enhanced export of oligosaccharides. The microorganisms described herein express major facility superfamily proteins such as CDT-1 which allows for the export of oligosaccharides. Variants of CDT-1 exhibit higher activity regarding oligosaccharide export. Means to export oligosaccharides into the growth medium are provided herein.

Description

USE OF SUBSTRATE IMPORTERS FOR THE EXPORT OF OLIGOSACCHARIDES
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/740,049, filed October 2, 2018, and U.S. Provisional Application No. 62/801,755, filed February 6, 2019. The contents of each of these applications are hereby incorporated by reference in their entirety.
BACKGROUND
Functional oligosaccharides have emerged as valuable components of food and dietary supplements. Their resistance to digestion and fermentation by colonic microbes has given oligosaccharides a nutritional edge. Apart from implications as dietary fibers, sweeteners, and humectants, they are hailed as prebiotics. Their beneficial effects extend from anti-oxidant, anti inflammatory, immunomodulatory, anti-hypertensive, and anti-allergic to anti-cancer, neuroprotective, and improvement of the skin barrier function and hydration. The rising popularity of bioactive oligosaccharides has accelerated the search for their generation from new, sustainable sources.
Oligosaccharides may be obtained from natural sources and may also be synthesized. Various natural sources of oligosaccharides include milk, honey, sugarcane juice, rye, barley, wheat, soybean, lentils, mustard, fruits, and vegetables such as onion, asparagus, sugar beet, artichoke, chicory, leek, garlic, banana, yacon, tomato, and bamboo shoots. Common
oligosaccharide manufacturing methods include hydrolysis of polysaccharides, chemical, and enzymatic polymerization from disaccharide or monosaccharide substrates. Acid, alkali, and enzymatic hydrolysis of polysaccharides can generate oligosaccharides of desired structure and functional properties. In certain cases, enzymatic methods are preferred for oligosaccharide synthesis due to their high selectivity and yields, and environmental-friendly nature. In other cases, oligosaccharide-producing microbial strains may be engineered by introducing exogenous genes to enable oligosaccharide production.
SUMMARY OF THE INVENTION
Oligosaccharides produced in microorganisms will accumulate intracellularly if not actively transported out of the cell into the medium from where they can be further isolated. Accumulation within the cells in the absence of export processes requires isolation of the oligosaccharide from biomass and limits conversion of the substrate to fermentation product or oligosaccharide. The lack of export of fermentation products out of cells also increases costs of the fermentation processes since fermentation runs effectively have to be stopped once the cells accumulate significant amounts of oligosaccharide in order to recover the latter. In addition, recovery of oligosaccharide from cells require additional processes such as extraction or breakage of cells, or both, which might additionally increase costs and require significant purification steps to remove contaminating cell debris, or both.
Exporter proteins for oligosaccharides are not readily available since organisms typically evolved mechanisms to import, not export, substrates for consumption, sensing or both. The identification of functional substrate transporters allowing for oligosaccharide export which is functional in eukaryotic cells is thus paramount for the production of oligosaccharides in yeasts and other eukaryotic production hosts.
It has been discovered that substrate importers might act as exporters. For example, if oligosaccharides accumulate to high concentrations within cells, this along with the appropriate transporter may drive substrate flow out of the cell where the concentration is lower.
Additionally, mutagenized versions of transporters might be impaired in regulation of transport processes in such a way that substrate export along a concentration gradient is facilitated.
Additionally, modification of the same substrate transporter can lead to higher fermentation product or oligosaccharide export rates if expressed in an organism accumulating a suitable substrate within the cell.
Accordingly, provided herein are transporters that can function as a substrate exporter, particularly for oligosaccharides. Such transporters can also function as importers, and import oligosaccharides, such as an oligosaccharide different from that exported.
CDT-l (XP 963801.1) from the fungus Neurospora crassa is a substrate transporter from the major facilitator superfamily (MFS) that imports cellobiose into the cell. Unexpectedly, expression of a cellodextrin transporter in an engineered Saccharomyces cerevisiae strain capable of producing a lactose-based oligosaccharide, such as 2’-fucosyllactose (2’-FL), leads to an increase of 2’-FL released into the culture medium. In such circumstances, CDT-l acts as an exporter facilitating transport of oligosaccharides, such as 2’-FL, out of the cell. Moreover, mutated versions of CDT-l can act as 2’-FL exporters and in some cases, such mutations further increase 2’-FL export out of the cell, if compared to the non-mutated version of this transporter. CDT-2 is another substrate transporter from the fungus Neurospora crassa that can be used herein for exporting oligosaccharides, such as 2’-FL.
In certain aspects, the present disclosure provides 2’-FL production strains expressing a CDT such as CDT-l, CDT-2 or a CDT mutant (i.e., having one or more alterations in a CDT amino acid sequence).
In one aspect a microorganism comprises a heterologous cellodextrin transporter gene or a construct that enhances expression of the cellodextrin transporter, is provided.
Compared to the parental microorganisms, the microorganisms described herein have an increased ability to produce oligosaccharide products of interest. Accordingly, methods of producing products of interest by culturing the microorganisms of the present disclosure in media containing the oligosaccharides and obtaining the products of interest from the media are provided.
In some embodiments, a CDT mutant is CDT-l SY. These strains show increased export of oligosaccharides if compared to their parental strains not expressing CDT-l or a CDT-l analogue.
In certain aspects, the present disclosure provides methods of producing oligosaccharides by culturing the microorganisms disclosed herein. In some embodiments, the microorganisms are bacteria or fungi, for example, filamentous fungi or yeasts. In some embodiments, the microorganisms are yeast, for example, Saccharomyces cerevisiae.
In one aspect a method of producing an oligosaccharide comprising culturing a microorganism described herein in a culture medium and recovering the oligosaccharide is provided herein. In another aspect, a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism described herein; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided. In another aspect, a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism comprises a heterologous transporter and one or more heterologous HMO production gene(s); and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided. In another aspect, a product suitable for animal consumption comprising the HMO produced by the microorganism described herein or according to the method described herein and at least one additional ingredient acceptable for animal consumption.
In another aspect, a product suitable for animal consumption comprising the
microorganism described herein and optionally at least one additional ingredient acceptable for animal consumption.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic of a cell expressing a CDT-l mutant and a lactose transporter. In this example the cell produces the oligosaccharide 2’-FL. The cell is engineered to produce GDP-fucose. The fucosyl residue in GDP-fucose is subsequently transferred onto lactose, thereby producing 2’-FL. Lactose is imported by a transporter specific for lactose. CDT-1SY facilitates export of oligosaccharides, such as 2’-FL, out of the cell. The oligosaccharide can then be obtained from the growth medium.
Fig. 2 shows the level of 2’-FL in the supernatant in 2’-FL producing background strain either with, or without the transporter CDT-l mutant (such as CDT-l SY as specified in SEQ ID NO. 1). The strain expressing CDT-l SY exhibits a -30% increase in product accumulation in the growth medium.
Fig. 3 shows lactose uptake activity and 2’-FL production by yeast strains expressing CDT-l M7 (CDT-l 209S 262Y) or Lacl2 as lactose transporter along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
Fig. 4 shows relative lactose uptake activity by yeast strains expressing different CDT-l mutants. CDT-l (CDT-l wild type), Ml (CDT-l 91A), M2 (CDT-l 213A), M3 (CDT-l 256V), M4 (CDT-l 335 A), M5 (CDT-l 411 A), M6 (CDT-l 209S 262W), M7 (CDT-l 209S 262Y), M8 (CDT-l 209S 262Y first 30 amino acid codons optimized). Ctrl is control strain with no transporter expression.
Fig. 5 shows relative extracellular 2’-FL production by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL. Ctrl is control strain without any lactose transporter expression. Fig. 6 shows total 2’-FL production by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL. Ctrl is control strain without any lactose transporter expression.
Fig. 7 shows extracellular 2’-FL ratio by yeast strains expressing different CDT-l mutants along with plasmid based 2’-FL pathway expression consist of GMD, WcaG, and WbgL.
Fig. 8 shows a schematic of production of fucosylated oligosaccharides within microbes. Shown is an example how the fucosylated oligosaccharide such as 2’-fucosyllacctose (2’-FL) is formed. GDP-Mannose is dehydrated to GDP-4-dehydro-6-deoxy-D-mannose by a GDP- mannose dehydratase (GMD). GDP-4-dehydro-6-deoxy-D-mannose is then reduced to GDP- Fucose by a GDP fucose synthase (GFS). In this example, lactose had been imported into the cell by a specific lactose transporter and is then further fucosylated by a glycosyl transferase such as a fucosyl transferase (FT), e.g., alpha-l,2 fucosyltransferase to form 2’-FL. 2’-FL is then exported into the medium by an oligosaccharide transporter.
Fig. 9 shows 2’-FL production by introducing fucosyltransferase (FT) from different organisms to yeast strain with CDT-l M7, GMD and WcaG expression on plasmids. Ctrl is control strain without FT expression.
Fig. 10 shows relative production of 2’-FL in yeast cells expressing plasmids with GMD, GFS and FT, relative to a base strain that contains a set of genomic GMD, GFS and FT genes. The GFS gene carried on the expression plasmid was here selected from SEQ ID NOs: 20, 21,
22, and 23.
Fig. 11 shows relative production of 2’-FL in yeast cells expressing plasmids with GMD, GFS and FT, relative to a base strain that contains a set of genomic GMD, GFS and FT genes. The FT gene carried on the expression plasmid was selected from SEQ ID NOs: 38, 29, 30, 31, 32, and 40.
Fig. 12 shows relative production of 2’-FL in yeast cells expressing plasmids with (lst column) GMD, a FT and SEQ ID NO: 24 and (2nd column) plasmids with a FT and SEQ ID NO: 24 only, relative to a base strain that contains a set of genomic GMD, GFS and FT genes.
Fig. 13 shows production of 2’-FL by expression of plasmids in a control strain otherwise not capable of 2’-FL production (Ctrl). Strains were transformed with plasmids expressing a GFS and a FT along with a plasmid carrying either SEQ ID NO: 17, 18, or 19, respectively. The control strain carrying no plasmids does not produce any 2’-FL.
DETAILED DESCRIPTION
In one aspect, a microorganism comprises a heterologous cellodextrin transporter gene or a construct that enhances expression of the cellodextrin transporter, is provided.
Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the heterologous cellodextrin transporter is CDT-l . In some embodiments, the gene or construct that expresses CDT-l comprises a genetic modification that increases the oligosaccharide export activity of CDT-l relative to a corresponding wild-type gene or construct that expresses CDT-l . In some embodiments, the gene or construct that expresses CDT-l is MFS transporter gene (cdt-l) or a variant thereof. In some embodiments, the transporter comprises a PESPR motif. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, one or more amino acid is replaced at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO: 4. In some embodiments, the CDT-l further comprises one or more mutations selected from the group consisting of 91A, 209S, 213A, 256V, 262Y, 335A, and 411 A of SEQ ID NO: 4. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 and a tyrosine at the position corresponding to residue 262 of SEQ ID No: 4. In some embodiments, the CDT-l has the sequence of SEQ ID NO: 1 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 of SEQ ID NO: 4. In some embodiments, the CDT-l has the sequence of SEQ ID NO: 2 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises a tyrosine at the position corresponding to residue 262 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 3 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 91 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 10 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 213 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 11 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11. In some
embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises a valine at the position corresponding to residue 256 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 12 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 335 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 13 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the amino acid sequence comprises an alanine at the position corresponding to residue 411 of SEQ ID NO: 4. In some embodiments, CDT-l has the sequence of SEQ ID NO: 14 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the CDT-l has an amino acid sequence of SEQ ID NO: 4 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, and wherein the CDT-l amino acid sequence comprises a serine at the position corresponding to residue 209 and a Tryptophan at the position corresponding to residue 262 of SEQ ID No: 4. In some embodiments, the CDT-l has the sequence of SEQ ID NO: 15 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the CDT-l is encoded by a codon optimized nucleic acid. In some embodiments, the nucleic acid is optimized for yeast. In some embodiments, at least 5% of the nucleic acid is codon optimized. In some embodiments, at least 90 nucleotides of the nucleic acid are codon optimized. In some embodiments, the CDT-l is encoded by the nucleic acid of SEQ ID NO: 16. In some embodiments, the microorganism further comprising a genetic modification that increases the oligosaccharide export activity of CDT-l selected from: a) a promoter operably linked to the cdt-l gene b) extrachromosomal genetic material comprising cdt-l; c) one or more copies of cdt-l, wherein said copies are integrated into the genome of the microorganism; d) a modified cdt-l that encodes a constitutively active CDT-l compared to unmodified CDT-l; e) a modified cdt-l that encodes a CDT-l having increased oligosaccharide export activity compared to unmodified CDT-l ; f) extrachromosomal genetic material comprising a modified cdt-l that encodes a constitutively active CDT-l or a CDT-l having increased oligosaccharide export activity compared to the corresponding wild-type CDT-l ; or g) one or more copies of cdt-l or a modified cdt-l that encode a constitutively active CDT-l or a CDT-l having increased oligosaccharide export activity compared to the corresponding wild- type CDT-l, wherein said copies are integrated into the genome of the microorganism. In some embodiments, the promoter operably linked to the cdt-l gene induces expression of cdt-l at a higher level than an endogenous promoter. In some embodiments, the promoter is specific for the microorganism in which it induces expression of cdt-l. In some embodiments, the heterologous cellodextrin transporter is CDT-2. In some embodiments, the CDT-2 has an amino acid sequence of SEQ ID NO: 9 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the microorganism further comprising a gene or a construct that expresses a lactose permease. In some embodiments, the lactose permease is Lacl2. In some embodiments, the Lacl2 has an amino acid sequence of SEQ ID NO: 41 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 41. In some embodiments, the microorganism further comprising one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein. In some embodiments, the microorganism comprises the heterologous cellodextrin transporter CDT-l, or variant or mutation of CDT-l such as described herein and further comprising one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein. In some embodiments, the one or more HMO production protein is an enzyme capable of converting fucose and ATP to fucose- 1- phosphate, an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and/or a glucosyl transferase. In some embodiments, the one or more HMO production gene is a GDP-Mannose dehydratase gene or the one or more HMO production protein is a GDP-Mannose dehydratase protein. In some embodiments, the one or more HMO production gene is a GDP-L- fucose synthase gene or the one or more HMO production protein is a GDP-L-fucose synthase protein. In some embodiments, the one or more HMO production gene is a fucosyl transferase gene or the one or more HMO production protein is a fucosyl transferase protein. In some embodiments, the gene or construct that expresses GDP-Mannose dehydratase comprises a genetic modification that increases the oligosaccharide production activity of GDP-Mannose dehydratase relative to a corresponding wild-type gene or construct that expresses GDP- Mannose dehydratase. In some embodiments, the gene or construct that expresses GDP-Mannose dehydratase is GDP-Mannose dehydratase gene (gmd) or a variant thereof. In some
embodiments, the GDP-Mannose dehydratase has an amino acid sequence of any one of SEQ ID NOs: 17-19, 42, and 61-63 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 17-19, 42, and 61-63. In some embodiments, the gene or construct that expresses GDP-L-fucose synthase comprises a genetic modification that increases the oligosaccharide production activity of GDP-L-fucose synthase relative to a corresponding wild-type gene or construct that expresses GDP-L-fucose synthase. In some embodiments, the gene or construct that expresses GDP-L-fucose synthase is GDP-L-fucose synthase gene (gfs) or a variant thereof. In some embodiments, the GDP-L-fucose synthase has an amino acid sequence of any one of SEQ ID NOs: 20-23 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 20-23. In some embodiments, the gene or construct that expresses GDP-L-fucose synthase is WcaG or a variant thereof. In some embodiments, the WcaG has an amino acid sequence of any one of SEQ ID NOs: 43-45 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 43-45. In some embodiments, the gene or construct that expresses GDP-L-fucose synthase is GMER or a variant thereof. In some embodiments, the GMER has an amino acid sequence of SEQ ID NO: 46 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 46. In some embodiments, In some embodiments, the gene or construct that expresses fucosyl transferase comprises a genetic modification that increases the oligosaccharide production activity of fucosyl transferase relative to a corresponding wild-type gene or construct that expresses fucosyl transferase. In some embodiments, the gene or construct that expresses fucosyl transferase is fucosyl transferase gene (ft) or a variant thereof. In some embodiments, the fucosyl transferase is alpha 1 ,2-fucosyl transferase. In some embodiments, the fucosyl transferase has an amino acid sequence of any one of SEQ ID NOs: 26-40 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 26-40. In some embodiments, the gene or construct that expresses fucosyl transferase is wbgL or a variant thereof. In some embodiments, the wbgL has an amino acid sequence of SEQ ID NO: 47 or has at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 47. In some embodiments, the gene or construct that expresses fucosyl transferase is futC or a variant thereof. In some embodiments, the futC has an amino acid sequence of SEQ ID NO: 48 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 48. In some embodiments, the gene or construct that expresses fucosyl transferase is wcfB or a variant thereof. In some embodiments, the wcfB has an amino acid sequence of SEQ ID NO: 49 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 49. In some embodiments, the gene or construct that expresses fucosyl transferase is wbgN or a variant thereof. In some embodiments, the wbgN has an amino acid sequence of SEQ ID NO: 50 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 50. In some embodiments, the gene or construct that expresses fucosyl transferase is wbwk or a variant thereof. In some embodiments, the wbwk has an amino acid sequence of any one of SEQ ID NO: 51 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 51. In some embodiments, the gene or construct that expresses fucosyl transferase is wbsJ or a variant thereof. In some embodiments, the wbsJ has an amino acid sequence of SEQ ID NO: 52 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 52. In some embodiments, the gene or construct that expresses fucosyl transferase is wbiQ or a variant thereof. In some embodiments, the wbiQ has an amino acid sequence of SEQ ID NO: 53 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 53. In some embodiments, the gene or construct that expresses fucosyl transferase is futB or a variant thereof. In some embodiments, the futB has an amino acid sequence of SEQ ID NO: 54 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 54. In some embodiments, the gene or construct that expresses fucosyl transferase is futL or a variant thereof. In some embodiments, the futL has an amino acid sequence of SEQ ID NO: 55 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 55. In some embodiments, the gene or construct that expresses fucosyl transferase is futF or a variant thereof. In some embodiments, the futF has an amino acid sequence of SEQ ID NO: 56 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 56. In some embodiments, the gene or construct that expresses fucosyl transferase is futG or a variant thereof. In some embodiments, the futG has an amino acid sequence of SEQ ID NO: 57 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 57. In some embodiments, the gene or construct that expresses fucosyl transferase is futN or a variant thereof. In some embodiments, the futN has an amino acid sequence of SEQ ID NO: 58 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 58. In some embodiments, the gene or construct that expresses fucosyl transferase is wcfw or a variant thereof. In some embodiments, the wcfw has an amino acid sequence of SEQ ID NO: 59 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 59. In some embodiments, the gene or construct that expresses fucosyl transferase is futA or a variant thereof. In some embodiments, the futA has an amino acid sequence of SEQ ID NO: 63 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 63. In some embodiments, the gene or construct that expresses fucosyl transferase is futD or a variant thereof. In some embodiments, the futD has an amino acid sequence of SEQ ID NO: 64 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 64. In some embodiments, the gene or construct that expresses fucosyl transferase is futE or a variant thereof. In some embodiments, the futE has an amino acid sequence of SEQ ID NO: 65 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 65. In some embodiments, the gene or construct that expresses fucosyl transferase is futH or a variant thereof. In some embodiments, the futH has an amino acid sequence of SEQ ID NO: 66 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 66. In some embodiments, the gene or construct that expresses fucosyl transferase is futJ or a variant thereof. In some embodiments, the futJ has an amino acid sequence of SEQ ID NO: 67 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 67. In some embodiments, the gene or construct that expresses fucosyl transferase is futK or a variant thereof. In some embodiments, the futK has an amino acid sequence of SEQ ID NO: 68 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 68. In some embodiments, the gene or construct that expresses fucosyl transferase is futM or a variant thereof. In some embodiments, the futM has an amino acid sequence of SEQ ID NO: 69 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity of SEQ ID NO: 69. In some embodiments, the one or more HMO production gene an enzyme comprising two domains, wherein one domain has homology to GDP-Mannose dehydratase and the second domain has homology to fucosyl synthase. In some embodiments, the enzyme has an amino acid sequence of any one of SEQ ID NOs: 24-25 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO: 24-25. In some embodiments, the one or more HMO production gene is a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase and the one or more HMO production protein is a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase protein. In some embodiments, the bifunctional fucokinase/L-fucose-l-P-guanylyltransferase has an amino acid sequence of any one of SEQ ID NOs: 71-73 or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 71-73. In some embodiments, the microorganism comprises one or more genetic modifications selected from: i) a genetic modification that increases the proton export activity of PMA1 in the microorganism compared to PMA1 activity in the parental microorganism, ii) a genetic modification that decreases the hexose sensing activity of SNF3 in the microorganism compared to SNF3 activity in the parental microorganism, iii) a genetic modification that decreases the hexose sensing activity of RGT2 in the microorganism compared to RGT2 activity in the parental microorganism, and iv) a genetic modification that decreases the hexose sensing activity of GPR1 in the microorganism compared to GPR1 activity in the parental microorganism. In some embodiments, i) the genetic modification that increases the proton export activity of PMA1 is a genetic modification to plasma membrane ATPase gene (pmal), ii) the genetic modification that decreases the hexose sensing activity of SNF3 is a genetic modification to sucrose non-fermenting gene (snf3), iii) the genetic modification that decreases the hexose sensing activity of RGT2 is a genetic
modification to restores glucose transport gene (rgt2), and iv) the genetic modification that decreases the hexose sensing activity of GPR1 is a genetic modification to G protein-coupled receptor 1 gene (gprl). In some embodiments, i) PMA1 has the sequence of SEQ ID NO: 5 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5, n) SNF3 has the sequence of SEQ ID NO: 6 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6, iii) RGT2 has the sequence of SEQ ID NO: 7 or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7, iv) GPR1 has the sequence of SEQ ID NO: 8 or at least 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the microorganism further comprises an exogenous nucleotide sequence encoding a chaperonin. In some embodiments, the chaperonin is gGroESL. In some embodiments, the microorganism is a eukaryotic organism In some embodiments, the fungus microorganism is a filamentous fungus or a yeast. In some embodiments, the microorganism is a Ascomycetes fungus. In some
embodiments, the Ascomycetes fungus is selected from the group consisting of a Sacharomyces spp. , a Schizosaccharomyces spp. and a Pichia spp. In some embodiments, the microorganism is Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Neurospora sp, Torulaspora spp.,Torulaspora delbrueckii, Zygosaccharomyces spp., Zygosaccharomyces bailii, Brettanomyces spp., Brettannomyces intermedius,Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces
naardenensis, Brettanomyces nanus, Dekkera spp.,Dekkera bruxellensis, Dekkera anomala, Metschmkowia spp., Issatchenkia spp., Issatchenkia orientalis, Issatchenkia terricola, Kloeckera spp. ,Kloeckera apiculate, Aureobasidium spp., Aureobasidium pullulans, Rhodotorula spp., Rhodotorula glutinis, Rhodotorula cladiensis, Rhodosporidiumspp., Rhodosporidum toruloides, Cryptococcus spp., Cryptococcus neoformans, Cryptococcus albidus, Yarrowia spp, Yarrowia lipolytica, Kuraishia spp, Kuraishia capsulata, Kuraishia molischiana, Komagataella spp., Komagataella phaffii, Komagataella pastoris, Hanseniaspora spp., Hanseniaspora guilliermondii, Hanseniaspora uvarum, Hasegawaea spp., Hasegawaea japonica, Ascoidea spp., Ascoidea asiatica, Cephaloascus spp., Cephaloascus fragrans, Lipomyces spp., Lipomyces starkeyi, Kawasakia spp., Kawasakia arxii, Zygozyma spp, Zygozyma oligophaga,
Metschnikowia spp., Metschnikowia pulcherrima, Coccidiodes spp., Coccidiodes immitis, Neurospora discreta, Neurospora africana, Aspergillus spp., Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus, Mucor spp., Mucor circinelloides, Mucor racemosus, Rhizopus spp., Rhizopus oryzae, Rhizopus stolonifera, Umbelopsis spp., Umbelapsis isabelline, Mortierella spp, Mortierella alpine, Alternariaspp., Altemaria alternate, Botrytis spp., Botrytis cinereal, Fusarium spp., Fusarium graminarium, Geotrichum spp., Geotrichum candidum, Penicillium spp., Penicillum chrysogenum, Chaetomium spp., Chaetomium thermophila, Magnaporthe spp., Magnaporihe grisea, Emericella spp., Emericella discophora, Trichoderma spp., Trichodema reesei, Talaromyces spp., Talaromyces emersonii, Sordaria spp., or Sordaria macrospora. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport an oligosaccharide out of the
microorganism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport an oligosaccharide selected from 2-fucosyllactose, 3- fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose, di-fucosyllactose, lacto-N- neotetraose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose, monofucosyllacto-N-hexaose I, monofucosyllacto-N- hexaose II, difucosyllacto-N-hexaose I, difucosyllacto-N-hexaose II, difucosyllacto-N- neohexaose, difucosyl-para-lacto-N-neohexaose, difucosyl-para-lacto-N-hexaose,
trifucosyllacto-N-hexaose, sialyllacto-N-neotetraose a, sialyllacto-N-tetraose b, sialyllacto-N- tetraose c, disialyllacto-N-tetraose, fucosylsialyllacto-N-tetraose a, fucosylsialyllacto-N-tetraose b, fucosylsialyllacto-N-hexaose, fucosylsialyllacto-N-neohexaose I, or fucosyldisialyllacto-N- hexaose II out of the microorganism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk oligosaccharide with a degree of polymerization of 3 out of the organism. In some embodiments, the human milk oligosaccharide is 2'-fucosyllactose, 3-fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, or 6’- sialyllactose. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk oligosaccharide with a degree of polymerization of 4 out of the organism. In some embodiments, the human milk oligosaccharide is di-fucosyllactose, lacto-N-neotetraose, lacto-N-tetraose , sialyllacto-N-neotetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, disialyllacto-N-tetraose, fucosylsialyllacto-N- tetraose a, or fucosylsialyllacto-N-tetraose b. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport a human milk
oligosaccharide with a degree of polymerization of 5 out of the organism. In some embodiments, the human milk oligosaccharide is lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport 2’-fucosyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-tetraose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-neotetraose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport 3’-sialyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport 6’-sialyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport di-fucosyllactose out of the organism. In some embodiments, the microorganism has a higher capacity, compared to the parental microorganisms, to transport lacto-N-fucopentaose I out of the organism.
In another aspect, a microorganism for enhanced production of a human milk
oligosaccharide (HMO) comprising a heterologous CDT-l transporter or a variant thereof and at least one heterologous pathway gene for production of the HMO, is provided.
As described above, certain embodiments are applicable to any microorganism described herein. For example, in some embodiments, the microorganism is capable of producing and exporting the HMO. In some embodiments, the transporter is capable of exporting at least 20%, 30%, 40%, 50%, or 60% of the produced HMO. In some embodiments, the microorganism is capable of exporting at least 50% more of the HMO than a parental microorganism lacking the transporter. In some embodiments, the yeast comprises a transporter that has an amino sequence of SEQ ID NO:4 or a sequence with at least 80%, 85%, 90%, 95%, 98% or 99% homology thereto. In some embodiments, the transporter comprises a PESPR motif. In some embodiments, the transporter comprises a sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO:4. In some embodiments, the CDT-l is encoded by a codon optimized nucleic acid. In some embodiments, at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast. In some embodiments, the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411 A and any combination thereof. In some
embodiments, the pathway gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L- fucose synthase, and an alpha- l,2-fucosyl transferase. In some embodiments, the microorganism comprises a second heterologous pathway gene. In some embodiments, the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose I (LNFP I). In some embodiments, the HMO is 2’- fucosyllactose. In some embodiments, the microorganism is an Ascomycetes fungus. In some embodiments, the Ascomycetes fungus is selected from the group consisting of a Sacharomyces spp., a Schizosaccharomyces spp. and a Pichia spp. In some embodiments, the Ascomycetes fungus is selected from the group consisting of Trichoderma, Kluyveromyces, Yarrowia, Aspergillus, and Neurospora. In some embodiments, one or both of the heterologous CDT-l transporter and the pathway gene are integrated into the yeast chromosome. In some
embodiments, one or both of the heterologous CDT-l transporter and the pathway gene are episomal. In some embodiments, the microorganism comprises a set of pathway genes for production of the HMO. In some embodiments, the set comprises GDP-mannose 4,6-dehydratase (GMD), a GDP-L-fucose synthase (GFS), and a fucosyl transferase (FT). In some embodiments, the set comprises GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha-l,2- fucosyl transferase and wherein the HMO is 2’-FL. In some embodiments, the set comprises a bifunctional fucokinase/L-fucose-l-P-guanylyltransferase. In some embodiments, the set comprises an enzyme capable of converting fucose and ATP to fucose-l -phosphate and an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and a glucosyl transferase. In some embodiments, the glucosyl transferase is an□-l,2-fucosyl transferase and wherein the HMO is 2’-FL. In some embodiments, the set of pathway genes comprises Gmd, WcaG and WbgL. In some embodiments, the GDP-mannose 4,6-dehydratase is selected from SEQ ID Nos. 17-19, 42, and 61-63 or a variant having at least 85% homology thereto. In some embodiments, the GDP-L-fucose synthase is selected from SEQ ID Nos. 20-23 or a variant having at least 85% homology thereto. In some embodiments ,the alpha- l,2-fucosyl transferase is selected from SEQ ID Nos. 26-40 or a variant having at least 85% homology thereto.
In another aspect, a method of producing an oligosaccharide comprising culturing a microorganism described herein in a culture medium and recovering the oligosaccharide is provided herein.
In another aspect, a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism described herein; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
In another aspect, a method of isolating an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism comprises a heterologous transporter and one or more heterologous HMO production gene; and culturing the microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture medium is provided.
As described above, certain embodiments are applicable to any method described herein. For example, in some embodiments, the HMO is 2-fucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, 3’-sialyllactose, or 6’-sialyllactose di-fucosyllactose. In some embodiments, the method further comprising separating the culture medium from the microorganism. In some embodiments, the method further comprising isolating the HMO from the culture medium. In some embodiments, the heterologous transporter is CDT-l, CDT-2 or a variant thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the heterologous transporter gene is a CDT-l variant comprising an amino acid sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO: 1. In some embodiments, the CDT-l is encoded by a codon optimized nucleic acid. In some embodiments, the nucleic acid is optimized for yeast. In some embodiments, at least 5% of the nucleic acid is codon optimized. In some embodiments, at least 90 nucleotides of the nucleic acid are codon optimized. In some embodiments, the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411 A and any combination thereof. In some embodiments, the heterologous gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha- 1,2, fucosyl transferase. In some embodiments, the export of the HMO is increased as compared to a parental yeast strain that does not contain the heterologous transporter. In some embodiments, the heterologous transporter is capable of importing lactose and exporting the HMO. In some embodiments, the culture medium comprises lactose. In some embodiments, the ratio of the HMO in the culture medium to total HMO produced by the microorganism is at least about 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1 or greater than 4: 1. In some embodiments, the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose I (LNFP I).
In another aspect, a method of producing an HMO comprising: providing a culture medium with at least one carbon source; providing a microorganism capable of producing and exporting an HMO, wherein the microorganism expresses a heterologous transporter and one or more heterologous genes for the production of the HMO; and culturing microorganism in the culture medium; wherein a substantial portion of the HMO is exported into the culture mediumis provided.
As described above, certain embodiments are applicable to any method described herein. For example, in some embodiments, the method further comprises separating the culture medium from the microorganism. In some embodiments, the method further comprises isolating the HMO from the culture medium. In some embodiments, the heterologous transporter is CDT-l, CDT-2 or a variant thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the transporter is a CDT-l variant comprising an amino acid sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262,
335, 411 of SEQ ID NO:4. In some embodiments, the CDT-l is encoded by a codon optimized nucleic acid. In some embodiments, at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast. In some embodiments, the transporter comprises an amino acid replacement selected from the group consisting of 91 A, 209S, 213A, 256V, 262Y, 262W, 335A, 411A and any combination thereof. In some embodiments, the heterologous gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an alpha- l,2-fucosyl transferase. In some embodiments, the export of the HMO is increased as compared to a parental microorganism that does not contain the heterologous transporter. In some embodiments, the heterologous transporter is capable of importing lactose and exporting the HMO. In some embodiments, the culture medium comprises lactose. In some embodiments, the ratio of the HMO in the culture medium to total HMO produced by the microorganism is at least about 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1 or greater than 4: 1. In some embodiments, the HMO is selected from the group consisting of 2'- fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'- SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose I (LNFP I). In some embodiments, the microorganism is according to any one of claims 1-29.
In another aspect, a product suitable for animal consumption comprising the HMO produced by the microorganism described herein or according to the method described herein and at least one additional ingredient acceptable for animal consumption.
In another aspect, a product suitable for animal consumption comprising the
microorganism described herein and optionally at least one additional ingredient acceptable for animal consumption.
As described above, certain embodiments are applicable to any product described herein. For example, in some embodiments, the product is suitable for human consumption. In some embodiments, the product is an infant formula, an infant food, a nutritional supplement or a prebiotic product. In some embodiments, the product is suitable for mammalian consumption. In some embodiments, the product further comprising at least one additional human milk oligosaccharide. In some embodiments, the additional ingredient is selected from a protein, a lipid, a vitamin, a mineral or any combination thereof. In some embodiments, the product is suitable for use as an animal feed.
In another aspect, a product suitable for animal consumption comprising the
microorganism according described herein, the HMO produced by the microorganism described herein or according to the method described herein and at least one additional consumable ingredient. As described above, certain embodiments are applicable to any product described herein. For example, in some embodiments, the product is suitable for human consumption. In some embodiments, the product is an infant formula, an infant food, a nutritional supplement or a prebiotic product. In some embodiments, the product is suitable for mammalian consumption. In some embodiments, the product further comprises at least one additional human milk oligosaccharide. In some embodiments, the additional consumable ingredient is selected from a protein, a lipid, a vitamin, a mineral or any combination thereof. In some embodiments, the product is suitable for use as an animal feed.
Definitions
For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
As used herein, the singular forms“a,”“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the values measured or determined, i.e., the limitations of the measurement system. Where the terms “about” or“approximately” are used in the context of compositions containing amounts of ingredients or conditions such as temperature, these values include the stated value with a variation of 0-10% around the value (X ± 10%).
The terms“including,”“includes,”“having,”“has,”“with,” or variants thereof are inclusive in a manner similar to the term“comprising.” The term“consisting” and the grammatical variations of consist encompass embodiments with only the listed elements and excluding any other elements. The phrases“consisting essentially of’ or“consists essentially of’ encompass embodiments containing the specified materials or steps and those including materials and steps that do not materially affect the basic and novel characteristic(s) of the embodiments.
Ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Therefore, when ranges are stated for a value, any appropriate value within the range can be selected, and these values include the upper value and the lower value of the range. For example, a range of two to thirty represents the terminal values of two and thirty, as well as the intermediate values between two to thirty, and all intermediate ranges encompassed within two to thirty, such as two to five, two to eight, two to ten, etc.
The term“genetic modification” as used herein refers to altering the genomic DNA in a microorganism. Typically, a genetic modification alters the expression and/or activity of a protein encoded by the altered gene. A genetic modification encompasses a“variant”, which is a gene or protein sequence that deviates from a reference gene or protein, as further detailed below.
The term“oligosaccharide” refers to saccharide multimers of varying length and includes but is not limited to: sucrose (1 glucose monomer and 1 fructose monomer), lactose (1 glucose monomer and 1 galactose monomer), maltose (1 glucose monomer and 1 glucose monomer), isomaltose (2 glucose monomers), isomaltulose (1 glucose monomer and 1 fructose monomer), trehalose (2 glucose monomers), trehalulose (1 glucose monomer and 1 fructose monomer) cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), cellohexaose (6 glucose monomers), 2’- Fucosyllactose (2’-FL, 1 fucose monomer, 1 glucose monomer, and 1 galactose monomer), 3- Fucosyllactose (3’-FL, 1 fucose monomer, 1 glucose monomer, and 1 galactose monomer), 6’- Fucosyllactose (6’-FL, 1 fucose monomer, 1 glucose monomer, and 1 galactose monomer), 3’- Sialyllactose (3’-SL, 1 N-Acetylneuraminic acid monomer, 1 glucose monomer, and 1 galactose monomer), 6’- Sialyllacotse (6’-SL, 1 N-Acetylneuraminic acid monomer, 1 glucose monomer, and 1 galactose monomer), Di-fucosyllactose (DF-L, 2 fucose monomers, 1 glucose monomer, and 1 galactose monomer), Lacto-N- triose (LNT II, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 1 galactose monomer), Lacto-N-neotetraose (LNnT , 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N-tetraose (LNT, 1 N- acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N- fucopentaose I (LNFP I, 1 fucose monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N-fucopentaose II (LNFP II, 1 fucose monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N fucopentaose III (LNFP III, 1 fucose monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N-fucopentaose IV (LNFP IV, 1 fucose monomer,
1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N- Fucopentaose V (LNFP V, 1 fucose monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N-fucopentaose VI (LNFP VI, 1 fucose monomer,
1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Lacto-N- hexaose (LNH, 2 N-acetylglucosamine monomers, 1 glucose monomer, and 3 galactose monomers), Lacto-N-neohexaose (LNnH, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers), Monofucosyllacto-N-hexaose I (MFLNH I, 1 Fucose monomer, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers),
Monofucosyllacto-N-hexaose II (MFLNH II, 1 Fucose monomer, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers), Difucosyllacto-N-hexaose I
(LNDFH I, 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers), Difucosyllacto-N-hexaose II (LNDFH II, 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers), Difucosyllacto- N-neohexaose (LNnDFH, 2 N-acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers), Difucosyl-para-lacto-N-Hexaose (DFpLNH, 2 N- acetylglucosamine monomers, 1 glucose monomer, 2 fucose monomers and 3 galactose monomers), Difucosyl-para-lacto-N neohexaose (DFpLNnH, 2 N-acetylglucosamine monomers,
1 glucose monomer, 2 fucose monomers and 3 galactose monomers), Trifucosyllacto-N-hexaose (TFLNH, 2 N-acetylglucosamine monomers, 1 glucose monomer, 3 fucose monomers and 3 galactose monomers), Sialyllacto-N-neotetraose c (LSTc, 1 N-acetylneuraminic acid monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Sialyllacto-N- tetraose a (LSTa, 1 N-acetylneuraminic acid monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Sialyllacto-N-tetraose b (LSTb, 1 N- acetylneuraminic acid monomer, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Disialyllacto-N-tetraose (DSLNT, 2 N-acetylneuraminic acid monomers,
1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers),
FucosylSialyllacto-N-tetraose a (FLSTa, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), FucosylSialyllacto-N-tetraose b (FLSTb, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 1 N-acetylglucosamine monomer, 1 glucose monomer, and 2 galactose monomers), Fucosylsialyllacto-N-hexaose (FSLNH, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers), Fucosylsialyllacto-N-neohexaose I (FSLNnH I, 1 fucose monomer, 1 N-acetylneuraminic acid monomers, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers) and Fucosyldisialyllacto-N-hexaose II( FDSLNH II, 1 fucose monomer, 2 N-acetylneuraminic acid monomers, 2 N-acetylglucosamine monomer, 1 glucose monomer, and 3 galactose monomers).
The terms“human milk oligosaccharide”,“HMO”, and“human milk glycans” refer to oligosaccharides group that are be found in high concentrations in human breast milk. The dominant oligosaccharide in 80% of all women is 2'-fucosyllactose. Other HMOs include 3- fucosyllactose, 6’-fucosyllactose, 3’-sialyllactose, 6’-sialyllactose, di-fucosyllactose, lacto-N- neotetraose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose, monofucosyllacto-N-hexaose I, monofucosyllacto-N- hexaose II, difucosyllacto-N-hexaose I, difucosyllacto-N-hexaose II, difucosyllacto-N- neohexaose, difucosyl-para-lacto-N-neohexaose, difucosyl-para-lacto-N-hexaose,
trifucosyllacto-N-hexaose, sialyllacto-N-neotetraose a, sialyllacto-N-tetraose b, sialyllacto-N- tetraose c, disialyllacto-N-tetraose, fucosylsialyllacto-N-tetraose a, fucosylsialyllacto-N-tetraose b, fucosylsialyllacto-N-hexaose, fucosylsialyllacto-N-neohexaose I, fucosyldisialyllacto-N- hexaose II.
The term“degree of polymerization”, or DP, is the number of monomeric units in a macromolecule or polymer or oligomer molecule.
The term“microorganism” refers to prokaryote or eukaryote microorganisms capable of oligosaccharides production or utilization with or without modifications.
The term,“enhanced utilization” refers to an improvement in oligosaccharide production by a microorganism compared to a parental microorganism, specifically an increase in the oligosaccharides production rate, a decrease in the initial time before oligosaccharides production begins, an increase in the yield, defined as the ratio of product made to the starting material consumed, and/or a decrease in an overall time the microorganisms take to produce a given amount of an oligosaccharide.
The term“parental microorganism” refers to a microorganism that is manipulated to produce a genetically modified microorganism. For example, if a gene is mutated in a microorganism by one or more genetic modifications, the microorganism being modified is a parental microorganism of the microorganism carrying the one or more genetic modifications. The term,“consumption rate” refers to an amount of oligosaccharides consumed by the microorganisms having a given cell density in a given culture volume in a given time period.
The term,“production rate” refers to an amount of desired compounds produced by the microorganisms having a given cell density in a given culture volume in a given time period.
The term“gene” includes the coding region of the gene as well as the upstream and downstream regulatory regions. The upstream regulatory region includes sequences comprising the promoter region of the gene. The downstream regulatory region includes sequences comprising the terminator region. Other sequences may be present in the upstream and downstream regulatory regions. A gene is represented herein in small caps and italicized format of the name of the gene, whereas, a protein is represented in all caps and non-italicized format of the name of the protein. For example, cdt-1 (italicized) represents a gene encoding the CDT-l protein, whereas CDT-l (non-italicized and all caps) represents CDT-l protein.
The sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to a reference sequence refers to a comparison made between two sequences, preferably using the BLAST algorithm. Algorithms for comparisons between two protein sequences that use protein structural information, such as sequence threading or 3D-1D profiles, are also known in the field.
A“variant” is a gene or protein sequence that deviates from a reference gene or protein. The terms“isoform,”“isotype,” and“analog” also refer to“variant” forms of a gene or a protein. The variant may have“conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. A variant may have“nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Suitable amino acid residues that may be substituted, inserted, or deleted, and which are“conservative” or “nonconservative” may be determined by those of skill in the art, including by using computer programs well known in the art.
“Exogenous nucleic acid” refers to a nucleic acid, DNA, or RNA, which has been artificially introduced into a cell. Such exogenous nucleic acid may or may not be a copy of a sequence or fragments thereof which is naturally found in the cell into which it was introduced. “Endogenous nucleic acid” refers to a nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is naturally present in a microorganism. An endogenous sequence is“native” to, i.e., indigenous to, the microorganism.
The term“mutation” refers to genetic modification to a gene including modifications to the open reading frame, upstream regulatory region, and/or downstream regulatory region.
A heterologous host cell for a nucleic acid sequence refers to a cell that does not naturally contain the nucleic acid sequence.
A“chimeric nucleic acid” comprises a first nucleotide sequence linked to a second nucleotide sequence, wherein the second nucleotide sequence is different from the sequence which is associated with the first nucleotide sequence in cells in which the first nucleotide sequence occurs naturally.
A constitutive promoter expresses an operably linked gene when RNA polymerase holoenzyme is available. Expression of a gene under the control of a constitutive promoter does not depend on the presence of an inducer.
An inducible promoter expresses an operably linked gene only in the presence of an inducer. An inducer activates the transcription machinery that induces the expression of a gene operably linked to an inducible promoter.
Microorganisms, systems and methods for exporting Human Milk Oligosaccharides
I. Transporters
Provided herein are microorganisms, systems and methods for exporting oligosaccharides such as Human Milk Oligosaccharides (HMOs). In certain aspects, the present disclosure provides genetically engineered microorganisms capable of exporting oligosaccharides. For example, the microorganism described herein can export HMOs, such as 2’-fucosyllactose (2’- FL), such as into the growth medium where the microorganism resides.
In some embodiments, the microorganism is genetically engineered to express a transporter that is capable of exporting oligosaccharides from the microorganism. Exemplary transporters include a cellodextrin transporter, which is CDT-l, CDT-2, or homologs and variants thereof.
The transporter CDT-l from the cellulolytic fungus Neurospora crassa (GenBank:
EAA34565.1) belongs to the major facilitator superfamily (MFS) class of transporters capable of transporting molecules comprising hexoses and related carbohydrates. This class of transporters is defined in PFAM under family PF00083 (see the World Wide Web at
pfam. xfam. org/family/PF 00083).
CDT-l is capable of importing cellodextrins including cellobiose, cellotriose and cellotetraose, as well as lactose into Saccharomyces cerevisiae. However, it has not be shown or used previous to the disclosure herein as an exporter of engineered products in a microorganism. Surprisingly, another transporter FAC 12 from Kluyveromyces lactis is capable of importing lactose (like CDT-l), but as demonstrated herein, FAC12 does not function as an exporter for 2’- FF.
An example of CDT-l is provided by the sequence of SEQ ID NO: 4, which is CDT-l from Neurospora crassa (Uniprot entry Q7SCU1). Homologues of CDT-l from microorganisms other than N crassa, particularly, from fungi, can be used in the microorganisms and methods described herein. Non-limiting examples of the homologs of CDT-l in the instant invention are represented by UmProt entries: A0A0B0E0J3, F8MZD6, G4U961, F7VQY4, Q7SCU1, A0A0J0XVF7, A0A0G2FA71, Q0CVN2, G4T6X5, A0A1Q5T2Z1, A0A0F7VA10,
A0A1 S9RFP6, A0A0U1LZX5, A0A0C2J3L3, U7PNA2, A0A0F2M9E7, A0A2I1D8G2, A0A2J5HR99, A0A2I2EZ95, A0A0C2IUQ7, U7PNU1, A0A1L7XY52, A0A2J6PQH9, A0A165JU51, A0A167P382, A0A1W2TJP3, A0A175VST0, A1CN94, S3DBB4, L7IWM4, G4NAG6, L7HX81, G4NAG7, A0A1Y2BF25, G0SC27, A0A0F7SHM7, A0A2P5HRQ8, A0A194VWR4, A0A194UTG8, B8M4C1, A0A2J6RYZ2, S8AIR7, R9UR53, Q4WR71, B0XPA9, A0A0J5PH40, A0A0K8LME8, A0A1Y2V0X9, A0A0F8VMB5, A1D134.
A0A0S7E4Y9, A0A2T3AJM0, Q5B9G6, A0A2I1C7L5, A0A167H9D2, A0A2J6SE99, J3PJL4, A0A0C4EGH0, A0A135LD10, A0A0A2I302, A0A0G4NZP3, K9G9B1, K9G7S2,
A0A161ZL14, A0A0A2KJ45, A0A136JJM0, and A0A090D3T9.
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKFAQF
WGRFVFGFG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPFI
241 FQAFTCFIVM SSVFFFPESP RFFF AN GRD A EAV AFFVKYH GNGDPNSKFV FFETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH SGRWRMAQVF MISIFGQFSG NGFGYFNTVI FKNIGVTSTS 361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA (SEQ ID NO: 4)
Another example of cellodextrin transporter is CDT-2 from Neurospora crassa (UniProt entry: A0A2P5IEX1). CDT-2 is provided by the sequence of SEQ ID NO: 9.
1 MGIFNKKP V A QAVDLNQIQE EAPQFERVDW KKDPGLRKLY FYAFILCIAS ATTGYDGMFF
61 NSVQNFETWI KYFGDPRGSE LGLLGALYQI GSIGSIPFVP LLTDNFGRKT
PIIIGCVIMI
121 VGAVLQATAK NLDTFMGGRT MLGFGNSLAQ IASPMLLTEL AHPQHRARLT TIYNCLWNVG
181 ALVVSWLAFG TNYINNDWSW RIPALLQAFP SIIQLLGIWW VPESPRFLIA KDKHDEALHI
241 LAKYHANGDP NHPTVQFEFR EIKETIRLEM ESTKNSSYLD FFKSRGNRYR LAILLSLGFF
301 SQWSGNAIIS NYSSKLYETA GVTDSTAKLG LSAGQTGLAL IVSVTMALLV DKLGRRLAFL
361 ASTGGMCGTF VTWTLTAGLY GEHRLKGADK AMIFFIWVFG IFYSLAWSGL LVGYAIEILP
421 YRLRGKGLMV MNMS VQC ALT LNTYANPVAF DYFGPDHSWK LYLIYTCWIA AEFVFVFFMY
481 VETKGPTLEE LAKVIDGDEA DVAHIDIHQV EKEVEIHEHE GKSVA (SEQ ID NO:
9)
Other examples of cellodextrin transporter are Cellodextrin transporter cdt-g (ETniProt entry: R9ETSL5), Cellodextrin transporter cdt-d (ETniProt entry: R9ETTV3), Cellodextrin transporter cdt-c (ETniProt entry: R9ETR53), Cellodextrin transporter CdtG (ETniProt entry:
S8A015), Putative Cellodextrin transporter CdtD (ETniProt entry: A0A0ET5GS76), Cellodextrin transporter CdtC (ETniProt entry: S8AIR7), Cellodextrin transporter CdtD (ETniProt entry:
S8AVE0), and Putative Cellodextrin transporter cdt-c (ETniProt entry: A0A0F7VA10).
The ETniProt entries listed herein are incorporated by reference in their entireties.
Additional homologs of CDT-l are known in the art and such embodiments are within the purview of the invention. For example, the homologs of CDT-l have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1. CDT-l is a substrate-proton symporter from the MFS family. It facilitates the import of beta-l,4-linked disaccharides such as lactose or cellobiose out of the growth medium into the cell. Prior to the discoveries described herein, CDT-l has been characterized as an importer of substrates such as cellobiose (such as used in the biofuel industry). For example, Ryan et al. (2014) have shown that variants of CDT-l, such as CDT-l N209S and CDT-1-F262Y have an improved capability to import the oligosaccharide cellobiose. A variant with both mutations CDT-1-N209S/F262Y (or shortly: CDT-1SY) exhibited a further improved uptake of cellobiose. Mapping of the mutations on related MFS transporters revealed that the position N209 of the wildtype CDT-l is predicted to interact with the oligosaccharide molecule inside the channel. However, neither CDT-l nor any variants have been shown to be an exporter. To the contrary, outside of the discoveries herein, CDT-l has been characterized as lacking activity that would provide utility as an exporter (see e.g., Hollands K. et al., Metab Eng. 2019 Mar; 52: 232-242).
CDT-l -N209S/F262Y (or shortly: CDT-l SY): SEQ ID NO: 1
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF
WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYSC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RYLFANGRDA EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET
481 HRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA
CDT-1-N209S (or shortly: CDT-ls): SEQ ID NO: 2
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF 121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYSC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RFLFANGRDA EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA
CDT-1-F262Y (or shortly: CDT-ly): SEQ ID NO: 3
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RYLFANGRDA EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNY IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA
A lactose permease, a membrane protein, is a member of the major facilitator superfamily. Lactose permease can be classified as a symporter, which uses the proton gradient towards the cell to transport b-galactosides such as lactose in the same direction into the cell. In some embodiments, the lactose importer is LAC12. Homologues of LAC12 can be used in the microorganisms and methods described herein. Non-limiting examples of the homologs of LAC12 in the instant invention are represented by UniProt entries: Q9FLB5, B9FJH4, P07921, A0A1 J6J8V9, A0A251TUB0, A0A0A9W3I8, D0E8H2, W0THP1, A0A1 S9RK01,
A0A151V9Y9, A0A1C1CDD3, W0TAG2, A0A151W5N5, A0A151WE7, A0A151WBL8, A0A151V6X4, A0A151W4U2, A0A1C7LPV6, W0T7D8, W0T8B1, A0A1C1CKJ6,
A0A1C1CH50, A0A1C1D058, A0A1C1C6W6, A0A1C1CIT2, A0A1C1CFR6, A0A2N6NU09, A0A1C1C6I1, A0A1C7LTH2, A0A2N6N8U0, A0A2N6NP59, A0A0F8AZD4, Q8X109,
A0A1 J6IEJ6, A0A034W1B8, A0A1C7LRQ8, A0A1C1CWY2, A0A1C1CTI7, A0A1C1CQ74, A0A1C7M6U6, A0A1C7LT95, A0A2N6NIJ0, A0A2C5X4W3, A0A1C7M1E6, A0A2H8TQZ2, A0A2N6NWY5, A0A1T4IZL8, A0A1T4IZJ1, A0A1T4IZJ3, A0A1T4IZM1, A0A1T4IZL0, A0A1T4IZJ8, A0A0A9YFY8, W8BTJ3, A0A1C7LK22, A0A0C9QF59, and A0A0A9WYQ6.
Other examples of lactose permease are encoded by LacY gene (UniProt entry: P02920, P22733, P47234, P18817, P59832), LacE (UniProt entry: P11162, P24400, P23531, Q4L869, Q5HE15, P50976, Q931G6, Q8CNF7, Q5HM40, Q99S77, Q7A092, Q6GEN9, Q6G7C4, A0A0H3BYW2), LacS gene (UniProt entry: P23936, Q48624, Q7WTB2), LacP (UniProt entry: 033814).
The Uniprot entries listed herein are incorporated by reference in their entireties.
Lactose permease can be expressed in a microorganism and provide lactose uptake. In some aspects, lactose can then be used by the microorganism as a substrate for the production of other oligosaccharides such as HMOs. However, unlike a CDT transporter, a lactose permease, such as Lac 12, when expressed in a microorganism does not act as an exporter with respect to oligosaccharides such as HMOs. For example, Lacl2 does not export 2’-FL when Lacl2 is expressed in a yeast such as Sacharomyces cerevisae.
As described herein, a cellobiose transporter acting as an importer within Neurospora crassa can act as an exporter when expressed in a microorganism such as when expressed in Saccharomyces cerevisiae strains producing an HMO. In some embodiments, the HMO exported by such transporter is a non-branched HMO comprised of a lactose core with modifications to the galactose ring. In some embodiments, the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N- tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto- difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I). In some embodiments, the HMO is 2’-FL. In some embodiments, the transporter for export of HMOs is a CDT-l, a CDT-2 or homolog thereof. In some embodiments, the transporter for export of HMOs is a variant, such as a mutant CDT-l, where one or more amino acids are altered as compared to a CDT-l amino acid sequence. In some embodiments, a mutant CDT-l for exporting HMOs comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 98%, 99% or greater than 99% homology with SEQ ID NO: 1. The mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions 91, 209, 213, 256, 262, 335, and 411 of SEQ ID NO: 1. The mutant CDT-l can comprise SEQ ID NO: 1 having one or more ammo acid substitutions selected from G91A, N209S, F213A, L256V, F262Y, F262W, F335A, S411A. In some embodiments, the mutant CDT-l is CDT-l N209S F262Y (SEQ ID NO: 1), CDT-l G91A (SEQ ID NO: 10), CDT-l F213A (SEQ ID NO: 11), CDT-l L256V (SEQ ID NO: 12), CDT-l F335A (SEQ ID NO: 13), CDT-l S411A (SEQ ID NO: 14), or CDT-l N209S F262W (SEQ ID NO: 15). The CDT transporter, such as a CDT-l or mutant CDT-l when expressed in a microorganism exports HMO such as 2’-FL. For example, cdt-lsy gene
(encoding CDT-l N209S/F262Y) was expressed within a background strain (microorganism) producing 2’-FL and 2’-FL accumulation in the growth medium during a fermentation experiment was compared to the same strain without the cdt-l-sy gene. Unexpectedly, the expression of CDT-l N209S/F262Y significantly increases the accumulation of 2’-FL within the growth medium indicating that CDT-l SY can act as an efficient substrate exporter.
Lactose permease mutant (CDT-l G91A) [Neurospora crassa] SEQ ID NO: 10
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYFAIFVA FCCACANGYD ASLMTGIIAM DKFQNQFHTG DTGPKVSVTF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF
WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RFLF AN GRD A EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET 481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA Lactose permease mutant (CDT-l F213A) [Neurospora crassa] SEQ ID NO: 11
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWAGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RFLF AN GRD A EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA
Lactose permease mutant (CDT-l L256V) \Neurospora crassa] SEQ ID NO: 12
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFVPESP RFLF AN GRD A EAVAFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIFGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNY IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA Factose permease mutant (CDT-l F335A) \Neurospora crassa] SEQ ID NO: 13
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKLAQF WGRFVLGLG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPLI
241 LQAFTCLIVM SSVFFLPESP RFLF AN GRD A EAV AFLVKYH GNGDPNSKLV LLETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VL MISIAGQFSG NGLGYFNTVI FKNIGVTSTS
361 QQLAYNILNS VISAIGALTA V SMTDRMPRR AVLIIGTFMC AAALATNSGL SATLDKQTQR
421 GTQINLNQGM NEQDAKDNAY LHVDSNYAKG ALAAYFLFNV IFSFTYTPLQ GVIPTEALET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIALH NIGYKYIFVF V GWDLIETV A WYFFGVESQG
541 RTLEQLEWVY DQPNPVKASL KVEKVWQAD GHVSEAIVA lactose permease mutant (CDT-l S411 A) \Neurospora crassa] SEQ ID NO: 14
1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKFAQF WGRFVFGFG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYNC GWFGGSIPAA CITYGCYFIK SNWSWRIPFI
241 FQAFTCFIVM SSVFFFPESP RFFF AN GRD A EAVAFFVKYH GNGDPNSKFV PPETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VF MISIFGQFSG NGFGYFNTVI FKNIGVTSTS
361 QQFAYNIFNS VISAIGALTA V SMTDRMPRR AVFIIGTFMC AAAFATNSGF AATFDKQTQR
421 GTQINFNQGM NEQDAKDNAY FHVDSNYAKG AFAAYFFFNY IFSFTYTPFQ GVIPTEAFET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIAFH NIGYKYIFVF V GWDFIETV A WYFFGVESQG
541 RTFEQFEWVY DQPNPVKASF KVEKVWQAD GHVSEAIVA lactose permease mutant (CDT-l N209S F262W) \Neurospora crassa] SEQ ID NO: 15 1 MSSHGSHDGA STEKHLATHD IAPTHDAIKI VPKGHGQTAT KPGAQEKEVR NAALFAAIKE
61 SNIKPWSKES IHLYF AIFVA FCCACANGYD GSLMTGIIAM DKFQNQFHTG DTGPKVSVIF
121 SLYTVGAMVG APFAAILSDR F GRKKGMFIG GIFIIVGSII VASSSKFAQF WGRFVFGFG
181 IAIMTVAAPA YSIEIAPPHW RGRCTGFYSC GWFGGSIPAA CITYGCYFIK SNWSWRIPFI
241 FQAFTCFIVM SSVFFFPESP RWFFANGRDA EAV AFFVKYH GNGDPNSKFV FFETEEMRD G
301 IRTDGVDKVW WDYRPLFMTH S GRWRMAQ VF MISIFGQFSG NGFGYFNTVI FKNIGVTSTS
361 QQFAYNIFNS VISAIGALTA V SMTDRMPRR AVFIIGTFMC AAAFATNSGF SATFDKQTQR
421 GTQINFNQGM NEQDAKDNAY FHVDSNYAKG AFAAYFFFNY IFSFTYTPFQ GVIPTEAFET
481 TIRGKGLALS GFIVNAMGFI NQFAGPIAFH NIGYKYIFVF V GWDFIETV A WYFFGVESQG
541 RTFEQFEWVY DQPNPVKASF KVEKVWQAD GHVSEAIVA lactose permease mutant (CDT-l 209S 262Y first 30 amino acid codons optimized by yeast) [Neurospora crassa] SEQ ID NO: 16
1 ATGTCCTCTC ATGGTTCTCA TGATGGTGCT TCTACTGAAA AACATTTGGC CACTCATGAT
61 ATTGCTCCAA CTCATGATGC TATCAAGATC GTGCCCAAGG GCCATGGCCA GACAGCCACA
121 AAGCCCGGCG CCCAAGAGAA GGAGGT CCGC AACGCCGCCC TATTTGCGGC CATCAAGGAG
181 TCCAATATCA AGCCCTGGAG CAAGGAGTCC ATCCACCTCT ATTTCGCCAT CTTCGTCGCC
241 TTTTGTTGTG CATGCGCCAA CGGTTACGAT GGTTCACTCA TGACCGGAAT CATCGCTATG
301 GACAAGTTCC AGAACCAATT CCACACTGGT GACACTGGTC CTAAAGTCTC TGTCATCTTT
361 TCTCTCTATA CCGTGGGTGC CATGGTTGGA GCTCCCTTCG CTGCTATCCT CTCTGATCGT
421 TTTGGCCGTA AGAAGGGCAT GTTCATCGGT GGTATCTTTA TCATTGTCGG CTCCATTATT
481 GTTGCTAGCT CCTCCAAGCT CGCTCAGTTT GTCGTTGGCC GCTTCGTTCT TGGCCTCGGT
541 ATCGCCATCA TGACCGTTGC TGCCCCGGCC TACTCCATCG AAATCGCCCC TCCTCACTGG
601 CGCGGCCGCT GCACTGGCTT CTACAgCTGC GGTTGGTTCG GAGGTT CGAT TCCTGCCGCC 661 TGCATCACCT ATGGCTGCTA CTTCATTAAG AGCAACTGGT CATGGCGTAT CCCCTTGATC
721 CTTCAGGCTT TCACGTGCCT TATCGTCATG TCCTCCGTCT TCTTCCTCCC AGAATCCCCT
781 CGCTaCCTAT TTGCCAACGG CCGCGACGCT GAGGCTGTTG CCTTTCTTGT CAAGTATCAC
841 GGCAACGGCG ATCCCAATTC CAAGCTGGTG TTGCTCGAGA CTGAGGAGAT GAGGGACGGT
901 ATCAGGACCG ACGGTGTCGA CAAGGTCTGG TGGGATTACC GCCCGCTCTT CATGACCCAC
961 AGCGGCCGCT GGCGCATGGC CCAGGTGCTC ATGATCTCCA TCTTTGGCCA GTTCTCCGGC
1021 AACGGTCTCG GTTACTTCAA TACCGTCATC TTCAAGAACA TTGGTGTCAC CAGCACCTCC
1081 CAACAGCTCG CCTACAACAT CCTCAACTCC GTCATCTCCG CTATCGGTGC CTTGACCGCC
1141 GTCTCCATGA CTGATCGTAT GCCCCGCCGC GCGGTGCTCA TTATCGGTAC CTTCATGTGC
1201 GCCGCTGCTC TTGCCACCAA CTCGGGTCTT TCGGCTACTC TCGACAAGCA GACTCAAAGA
1261 GGCACGCAAA TCAACCTGAA CCAGGGTATG AACGAGCAGG
ATGCCAAGGA CAACGCCTAC
1321 CTCCACGTCG ACAGCAACTA CGCCAAGGGT GCCCTGGCCG CTTACTTCCT CTTCAACGTC
1381 ATCTTCTCCT TCACCTACAC TCCCCTCCAG GGTGTTATTC CCACCGAGGC TCTCGAGACC
1441 ACCATCCGTG GCAAGGGTCT TGCCCTTTCC GGCTTCATTG TCAACGCCAT GGGCTTCATC
1501 AACCAGTTCG CTGGCCCCAT CGCTCTCCAC AACATTGGCT ACAAGTACAT CTTTGTCTTT
1561 GTCGGCTGGG ATCTTATCGA GACCGTCGCT TGGTACTTCT TTGGTGTCGA ATCCCAAGGC
1621 CGTACCCTCG AGCAGCTCGA ATGGGTCTAC GACCAGCCCA ACCCCGTCAA GGCCTCCCTA
1681 AAAGT CGAAA AGGTCGTCGT CCAGGCCGAC GGCCATGTGT CCGAAGCTAT CGTTGCTTAA
In some embodiments, a variant of CDT-l and related transporters for use as an HMO exporter can include one or more mutations of amino acids predicted to be near the sugar substrate binding pocket (e.g., N209S in CDT-l) or near the highly-conserved PESPR motif in the sugar porter family PF00083 (e.g., F262Y in CDT-l). Exemplary mutations include amino acids in CDT-l predicted to be in the substrate binding pocket such as G336, Q337, N341, and G471. In some embodiments, modifications of a microorganism expressing a transporter such as CDT-l or a CDT-l mutant can be engineered to increase the activity of the transporter. Non limiting examples of genetic modifications to cdt-1 that can increase the activity of CDT-l as a substrate exporter in the microorganisms compared to CDT-l substrate import activity in the parental microorganisms include one or more of: a) replacement of an endogenous promoter with an exogenous promoter operably linked to the endogenous cdt-1; b) expression of a cdt-1 via an extrachromosomal genetic material; c) integration of one or more copies of cdt-1 into the genome of the microorganism; d) a modification to the endogenous cdt-1 to produce a modified CDT-l that encodes a transporter protein that has an increased activity as a substrate exporter; e) introduction into the microorganism on extrachromosomal genetic material comprising a cdt-1 or a variant of cdt-1 (mutant cdt-l) such as encoding CDT-l N209S F262Y or one or more of the variants described herein (e.g., CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411 A, or CDT-l N209S F262W) ; f) integration into the genome of the microorganism of one or more copies of cdt-l or a variant of cdt-l encoding a transporter such as CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, or CDT-l N209S F262W; (g) introduction through extrachromosomal genetic material or through integration of a variant of cdt-l encoding CDT-l with one or more mutations of amino acids predicted to be near the sugar substrate binding pocket and/or the PESPR motif such as positions G336, Q337, N341, and G471; and/or (h) codon optimization of part of or all of cdt-l or a variant of cdt-l.
Any combinations of the modifications (a) to (h) described in this paragraph are also envisioned. In some embodiments, an expression of cdt-l or its variants is varied by utilizing different promoters or changes immediately adjacent to the introduced cdt-l gene. For example, in certain embodiments the deletion of a URA3 cassette adjacent to an introduced cdt-l sy expression cassette leads to a further improvement of HMO export, such as 2’-FL export.
In some embodiments the endogenous promoter is replaced with an exogenous promoter that induces the expression of cdt-l at a higher level than the endogenous promoter. In certain embodiments, the exogenous promoter is specific for the microorganism in which the exogenous promoter replaces the endogenous promoter. For example, a yeast specific exogenous promoter can be used if the microorganism being modified is a yeast. The exogenous promoter can be a constitutive promoter or inducible promoter. Non-limiting examples of constitutive yeast specific promoters include: pCYCl, pADHl, p STE5, pADHl, pCYClOO minimal, p CYC70 minimal, p CYC43 minimal, p CYC28 minimal, pCYC16, pPGKl, pCYC, p GPD or p TDH3. Additional examples of constitutive promoters from yeast and examples of constitutive promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
Non-limiting examples of inducible yeast specific promoters include: pGALl, pMFAl, pMFA2, p STE3, p URA3, pFIGl, pEN02, pDLD, pJENl, pmCYC, and p STE2. Additional examples of inducible promoters from yeast and examples of inducible promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
In certain embodiments, the microorganisms comprise a modification to the wildtype cdt- 1 to produce a modified cdt-1 that encodes a transporter with an increased capability to export 2’-FL from the cell.
Accordingly, in certain embodiments, modification of the wildtype cdt-1 produces a modified cdt-1 that encodes a CDT-l with increased export rates of 2’-FL. In certain
embodiments wildtype cdt-1 is mutated around the conserved PEPSR motif which is conserved in hexose transporters. In certain embodiments cdt-1 is modified leading to the production of a protein CDT-1-F262Y. The mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions 91, 209, 213, 256, 262, 262, 335, and 411 of SEQ ID NO: 1. The mutant CDT- 1 can comprise SEQ ID NO: 1 having one or more amino acid substitutions selected from G91A, N209S, F213A, L256V, F262Y, F262W, F335A, S411A. In some embodiments, the mutant CDT-l is CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, or CDT-l N209S F262W. The mutant CDT-l can have one or more amino acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket and/or the PESPR motif such as positions G336, Q337, N341, and G471.
In certain embodiments wild-type cdt-1 is mutated around the amino acid residues within CDT-l which are interacting with the oligosaccharide substrate. In certain embodiments cdt-1 is modified leading to the production of a protein CDT-1-N209S. In yet other embodiments cdt-1 is modified leading to the production of a protein CDT-1-N209S F262Y. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l G91 A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l F213A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l L256V. . In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l F335A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l S411 A. In some certain embodiments cdt-1 is modified leading to the production of a protein CDT-l N209S F262W.
In specific embodiments, a microorganism, preferably, a fungus such as a yeast, more preferably, a Saccharomyces spp., and even more preferably, S. cerevisiae is provided, the microorganism comprising the genetic modifications or the combinations of genetic
modifications listed below:
1) A genetic modification producing a CDT-l conferring the cell with oligosaccharide-, and in particular, HMO-export activity, such as 2’-FL-export activity.
2) A genetic modification producing a CDT-l with mutated amino acid residues increasing export activity of CDT-l for oligosaccharides, HMO-export activity, such as and in particular 2’-FL.
II. Production of HMOs in Microorganisms
HMOs are generally comprised of monosaccharides linked together, and typically with a lactose molecule at one end. Generally, the production of HMOs in microbes requires the presence of a starting monomer and one or more heterologous enzymes introduced into the microorganism. The monomer might be a monosaccharide. The monomer might be glucose, galactose, N-acetylglucosamine, fucose, and/or N-acetylneuraminic acid. For example, for the production of fucosylated HMOs, production can include i) the biosynthesis of GDP-fucose and ii) the transfer of the fucosyl domain of GDP-fucose onto an acceptor oligosaccharide. For the production of fucosylated oligosaccharide such as 2’-fucosyllactose (2’-FL) or 3-Fucosyllactose (3’-FL), the acceptor oligosaccharide is the disaccharide lactose.
GDP-fucose is synthesized from GDP-Mannose by two successive reactions: First, GDP Mannose is dehydrated by a GDP-Mannose dehydratase (GMD) to produce GDP-4-dehydro-6- deoxy-D-mannose. Second, GDP-4-dehydro-6-deoxy-D-mannose is further reduced to GDP-L- fucose by a GDP-L-fucose synthase (GFS). In some embodiments, GDP-fucose can then be transferred to the disaccharide lactose by a fucosyl transferase (FT), forming a fucosylated oligosaccharide. In some embodiments, the FT is an alpha 1 ,2-fucosyl transferase. In some embodiments, the fucosylated oligosaccharide is 2’-FL or 3’-FL.
Microorganisms that exhibit increased utilization of oligosaccharides are provided. In some embodiments, the microorganism further comprises one or more heterologous HMO production gene or a construct that enhances the expression of one or more HMO production protein. As described herein“HMO production gene” expresses“HMO production protein”. As described herein,“HMO production protein” is an enzyme that participates in a pathway for HMO production. Exemplary enzymes that participate in pathways for HMO production, such as for a fucosylated HMO, are enzymes capable of converting fucose and ATP to fucose- 1- phosphate, an enzyme capable of converting the fucose- 1 -phosphate and GTP to GDP-fucose, and/or a glucosyl transferase. Examples of HMO production protein are a GDP-Mannose dehydratase (GMD), a GDP-L-fucose synthase (GFS), and a fucosyl transferase (FT).
In certain embodiments, the microorganisms comprise one or more genetic modifications that: i) increase the activity of a GDP-Mannose dehydratase (GMD), and/or ii) increase the activity of a GDP-L-fucose synthase (GFS), and/or iii) increase the activity of glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1,2 -fucosyl transferase. In certain embodiments, these genetic modifications that result in i), ii), and iii) are produced by introduction of a GDP-Mannose dehydratase gene (GMD), GDP-L-fucose synthase gene (GFS), and a glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1,2 -fucosyl transferase gene, respectively. In some embodiments, the microorganism comprises a heterologous GDP- Mannose dehydratase gene or a construct that enhances expression of the GDP-Mannose dehydratase. In some embodiments, the microorganism comprises a heterologous GDP-L- fucose synthase gene or a construct that enhances expression of the GDP-L-fucose synthase. In some embodiments, the microorganism comprises a heterologous glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1 ,2-fucosyl transferase gene or a construct that enhances expression of the glycosyl transferase such as fucosyl transferase (FT), e.g., alpha 1 ,2-fucosyl transferase.
In certain embodiments, the present disclosure provides microorganisms comprising one or more genetic modifications selected from:
i) a genetic modification that introduces a GDP-Mannose dehydratase gene (GMD), or its analogues, ii) a genetic modification that introduces a GDP-L-fucose synthase gene ( GFS ), or its analogues, and
iii) a genetic modification that introduces a glycosyl transferase such as fucosyl
transferase (FT), e.g., alpha 1,2 -fucosyl transferase gene, or its analogues.
HMOs, such as 2’-FL can be produced in a microorganism. In some embodiments, a microorganism is genetically engineered by incorporating one or more nucleic acids that encode for an enzyme for one or more steps in the production of an HMO. In some embodiments, an HMO pathway is supplied entirely by such genetic engineering. In some embodiments, an HMO pathway is comprised of one or more endogenous activities from the host microorganism, and others through genetic engineering. In yet other embodiments, the host microorganism synthesizes an HMO using endogenous activities.
In some embodiments, the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I).
In some cases, the HMO is a fucosyllactose, such as 2’-FL. In some embodiments, fucosyllactose, such as 2’-FL is synthesized in a host microorganism through a de novo pathway. For example, the pathway can comprise GMD (GDP-mannose dehydratase), GFS (GDP-fucose synthase), and FT (fucosyltransferase), where GMD supplies an enzymatic activity to convert GDP-Mannose to GDP-4-keto-6-deoxymanose. A GFS, for example, WcaG, converts GDP-4- keto-6-deoxymanose to GDP-fucose and FT converts GDP-fucose to 2’-FL. In some
embodiments, the FT is an alpha l,2-fucosyl transferase.
An example of GDP-Mannose dehydratase (GMD) is provided by the sequence of SEQ ID NOs: 17-19, which are GDP-Mannose dehydratases from Idsiularia Solaris, Cladosiphon okamuranus, and Cladosiphon okamuranus, respectively. Homologues of GMD from microorganisms other than Fistularia solans and Cladosiphon okamuranus, in particular, from other heterokontophytes and from fungi, can be used in the microorganisms and methods described herein. Non-limiting examples of the homologs of GMD in the instant invention are represented by UniProt entries: P93031, 060547, Q18801, Q51366, Q93VR3, P0AC88, Q9VMW9, 045583, A3C4S4, Q9SNY3, Q8K0C9, Q8K3X3, Q9JRN5, Q56872,
A0A1B4XBH2, P55354, 085713, Q06952, Q1ZXF7, Q56598, P0AC90, P0AC91, P0AC89, B9UJ29, A8Y0L5, 067175, P71790, A0A1H3VGZ0, A0A078KV89, Q7UVN9, Q7NMK1, Q89TZ1, A0A132P8J4, P72586, Q2R1V8, A0A0G1U600, A2Z7B3, D4ZMX8, K9QEY2, L0A7V1, C3SCZ0, B5W8Q3, K1XEL2, A0A0G1FQB5, H1WIZ0, and Q63JM9.
The UniProt entries listed herein are incorporated by reference in their entireties.
Additional homologs of GMD are known in the art and such embodiments are within the purview of the invention. For example, the homologs of GMD have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 17-19 and 42.
GDP-mannose 4,6-dehydratase (GMD; EC 4.2.1.47) catalyzes the conversion of GDP- mannose to GDP-4-keto-6-deoxymannose, the first step in the synthesis of GDP-fucose from GDP-mannose, using NAD+ as a cofactor. This enzyme belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds. The systematic name of this enzyme class is GDP-mannose 4,6-hydro-lyase (GDP-4-dehydro-6-deoxy-D-mannose-forming). Other names in common use include guanosine 5'-diphosphate-D-mannose oxidoreductase, guanosine diphosphomannose oxidoreductase, guanosine diphosphomannose 4,6-dehydratase, GDP-D-mannose dehydratase, GDP-D-mannose 4,6-dehydratase, Gmd, and GDP-mannose 4,6- hydro-lyase. This enzyme participates in fructose and mannose metabolism. It employs one cofactor, NAD+.
In some embodiments, GMD and/or GFS are derived from E. coli, Helicobacter pylori, Arabidopsis thaliana, and/or Mortierella alpina (Ren et al., Biochem Biophys Res Commun. 2010 Jan 22;39l(4): 1663-9; Hollands K. et al., Metab Eng. 2019 Mar; 52: 232-242). In some embodiments, GMD is encoded by one of the sequences listed in Table 1 or a variant thereof.
Many of the proteins involved in GDP-fucose synthesis presented here have been identified in heterokontophytes, a group of algae which includes diatoms and kelps and which had been shown to contain large amounts of fucose in their cell walls. In addition, fusion proteins which appear to consist of a GMD and a GFS protein domain were identified.
Table 1 GMD activity:
Figure imgf000042_0001
Figure imgf000043_0001
SEQ ID NO: 17
1 MSSERKCALI TGITGQDGSY LTELLLEKGY EVHGIVRRSS CFNTGRIDHL YKDR HETGVK
61 LFLHYGDLCD ATNLISIISN VKPTEVYNLG AMSHVKV SFD MPEYTADCDG VGV LRMLDAI
121 RAAGMEKTVK FYQASTSELY GKVQEVPQSE TTPFYPRSPY AVAKQYAFWI LV NYREAYGM
181 HLTNGILFNH ESPRRGRTFV TRKITCGVAA IHHGKQKTLF LGNLDAKRDW GH ARDYVEGM
241 WRMLQQETSD DYVLATGETH TVREFVEKAF AWNTTVQWQ GEKGTVDE V G VDAADPSRIL
301 VRIDPRYFRP TEVDLLLGNP AKAKEKLGW S SSTPFDALVK EMVEADLAIL RGE MADADNT
361 FD
SEQ ID NO: 18
1 MAEPETKKTK VDEGTVKKAI ITGITGQDGS YLAEFLLEKG YEVHGIIRRS SSFNT QRIDH
61 IYRDRHESAV RLKLHY GDLT DSTNLMHIIY EVQPDEIYNL GAMSHVKV SF EMS EYTAEAD
121 GV GVLRLLNA IRSAGLEKKT RLYQASTSEL YGKVQEIPQK ETTPFYPRSP YGV AKQFGYW
181 MLINYREAYG MHLTNGILFN HESPRRGPTF VTRKITRAVA RIHRGKQKCI YLG NLDAKRD
241 WGHAKDYIKG MWLMV QRDEP SDYVLSTGEC HSVKEFVQEA FAYVGIDITW V GEGVEEYGH
301 VKGDPENYLV RVDPRYFRPT EVELLLGDCT KAKKELGWVP EITFKELVKD M MQADIANYD
361 AGNDHT
SEQ ID NO: 19
1 MQKTALITGI TGQDGAYLAE LLLEKGYTVH GIKRRSSSFN TGRIDHLYQD PH DRDVKLHL
61 HYGDMTDSTN LIRIMQETQP DEVYNLA AQ S HVQVSFETPE YTGNADALGT LR LLEAIRLL
121 GLSEKTRFYQ ASTSELYGKV QEVPQSETTP FYPRSPYAAA KLYAYWIWN YR EAYGMHAS
181 NGILFNHESP IRGETFVTRK ITRAAAAIKL GLQDKLYLGN LDAERDWGHA KDY VRGMWLM
241 LQQDKADDYV LATGEKHSVR EFVEQAFAEL EINIRWEGRG LDEQGFDTKT EK AWAVDPR
301 YFRPTEVDLL LGSPKKARKA LGWAPTTPFR DMIKQMVRSD LNSVSEDSKK GS QASWIKTG
SEQ ID NO: 42
1 MSKVALITGV TGQDGSYLAE FLLEKGYEVH GIKRRASSFN
TERVDHIYQD PHTCNPKFHL
61 HYGDLSDTSN LTRILREVQP DEVYNLGAMS HVAVSFESPE YTADVDAMGT LRLLEAIRFL
121 GLEKKTRF Y Q ASTSELYGLV QEIPQKETTP FYPRSPYAVA KLYAYWITVN YRESYGMY AC
181 NGILFNHESP RRGETFVTRK ITRAIANIAQ GLESCLYLGN MDSLRDWGHA KDYVKMQWMM
241 LQQEQPEDFV IATGVQYSVR QFVEMAAAQL GIKLRFEGTG VEEKGVWSV TGHDAPGVKP
301 GDVIIAVDPR YFRPAEVETL LGDPTKAHEK LGWKPEITLR EMV SEMV AND LEAAKKHSLL
361 KSHGYDVAIA LES
GMD from Helicobacter pylori SEQ ID NO: 60
1 MKEKIALITG VTGQDGSYLA EYLLNLGYEV HGLKRRSSSI NTSRIDHLYE DLHSDHKRRF
61 FLHYGDMTDS SNLIHLIATT KPTEIYNLAA QSHVKVSFET PEYTANADGI GTLRILEAMR
121 ILGLEKKTRF YQASTSELYG EVLETPQNEN TPFNPRSPYA VAKMYAFYIT KNYREAYNLF
181 AVNGILFNHE SRVRGETFVT RKITRAASAI AYNLTDCLYL GNLDAKRDWG HAKDYVKMMH
241 LMLQAPIPQD YVIATGKTTS VRDFVKMSFE FIGINLEFQN TGIKEIGLIK
SVDEKRANAL
301 KLNLSHLKKG QIWRIDERY FRPTEVDLLL GDPTKAEKEL DWVREYDLKE LVKDMLEYDL
361 KECQKNLYLQ DGGYILRNFY E
GMD from Arabidopsis thaliana : SEQ ID NO: 61 1 MASENNGSRS DSESITAPKA DSTWEPRKI ALITGITGQD GSYLTEFLLG
KGYEVHGLIR
61 RSSNFNTQRI NHIYIDPHNV NKALMKLHYA DLTDASSLRR WIDVIKPDEV YNLAAQSHVA
121 VSFEIPDYTA DWATGALRL LEAVRSHTID S GRTVKYY Q A GSSEMFGSTP PPQSETTPFH
181 PRSPYAASKC AAHWYTVNYR EAYGLFACNG ILFNHESPRR GENFVTRKIT RALGRIKV GL
241 QTKLFLGNLQ ASRDWGFAGD YVEAMWLMLQ QEKPDDYWA TEEGHTVEEF LDVSFGYLGL
301 NWKDYVEIDQ RYFRPAEVDN LQGDASKAKE VLGWKPQ V GF EKLVKMMVDE DLELAKREKV
361 LVDAGYMDAK QQP
GMD from Mortierella alpine SEQ ID NO: 62
1 MSSPIEWNM SPADYRNRKV ALITGITGQD GSYLAELLIE KGYQVHGIIR RSSSFNTGRI
61 EHLYKDAHEN PKMRLHHGDL TDSTCLVHII SKVLPTEIYN LGAQSHVKVS FDMSEYTADV
121 D AV GTLRLLD AIRTCGLSHL VRFYQASTSE LYGKVAEIPQ SETTPFYPRS PYGVAKMYAY
181 WITINYREAY DMYACNGILF NHESPRRGRT FVTRKITCAV ASIHLGKQEC LYLGNLDAKR
241 DWGHARDYVE GMWRMLQQET AEDFVLATGE MHTVREFVEK SFKAIGSTIR WEGSAEEEVG
301 LDEKGVIRVR VDPAYYRPTE VELLLGNPAK ANEKLGWKRQ VEFDALVEEM VKSDLIGVAA
361 GDVFN
An example of a GFS (GDP-fucose synthase) is provided by the sequence of SEQ ID NOs: 20-23, which are GDP-L-fucose synthases from Cladosiphon okamuranus, Phaeodaciylum tricomutum, Saccharina japonica, and Mucor circinelloides f circinelloides 1006PhL, respectively. Homologues of GFSs from microorganisms other than Cladosiphon okamuranus, Phaeodactylum tricomutum, Saccharina japonica, and Mucor circinelloides f. circinelloides 1006PhL, in particularly from heterokontophytes and from fungi, can be used in the
microorganisms and methods described herein. Non-limiting examples of the homologs of GFSs in the instant invention are represented by ETniProt entries: Q13630, P32055, 049213, P23591, Q9W1X8, Q9LMU0, G5EER4, Q8K3X2, P33217, Q5RBE5, F0F7M8, Q67WR2, P55353, Q67WR5, D9RW33, F2KZP1, G1WDT9, D7NG24, C9MLN8, Q9S5F8, X6PWX2, H1HNE5, D1QPT8, G6AG96, 10TA81, G1VAH6, A0A0K1NMZ0, U2KFA0, F0H551, A0A2K9HDD8, A0A095YQN3, D3I452, A0A096ARU1, A0A095ZVW3, A0A096ACH9, A0A1B1IBP6, Q55C77, A0A1F0MVW9, A0A1F0P341, A0A1T4MGU5, W4UTD5, A0A0G0Z978, Q5V3C6, A0A2U0U1K6, A0A2T4T802, and A0A2T4TH79.
The UniProt entries listed herein are incorporated by reference in their entireties.
Additional homologs of GFS’s are known in the art and such embodiments are within the purview of the invention. For example, the homologs of GFS’s have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 20-23.
A GDP-L-fucose synthase (EC 1.1.1.271) is an enzyme that catalyzes the chemical reaction GDP-4-dehydro-6-deoxy-D-mannose + NADPH + H+ <— > GDP-L-fucose + NADP+. Thus, the three substrates of this enzyme are GDP-4-dehydro-6-deoxy-D-mannose, NADPH, and H+, whereas its two products are GDP-L-fucose and NADP+. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of a donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is GDP-L-fucose:NADP+ 4- oxidoreductase (3,5-epimerizing). This enzyme is also called GDP-4-keto-6-deoxy-D-mannose- 3,5-epimerase-4-reductase. This enzyme participates in fructose and mannose metabolism.
In some embodiments, GFS is encoded by one of the sequences listed in Table 2 or a variant thereof.
Table 2 GFS activity:
Figure imgf000046_0001
SEQ ID NO: 20
1 MEATKAIFEK YKPTHVIHLA ARVGGLFSNL KYKVEFFREN ILINDNVMEC CRIY KVAKLV
61 SCLSTCIFPD KTTYPIDETM VHNGPPHTSN EGYAYAKRMI DVLNRCYKDE YGC NFTSVIP
121 TNIYGKGDNF SIDNGHVLPG LIHKCYKAKQ AGEDLHVW GT GSPLRQFIYN VD LGALMVWT 181 MRNYHEVDPI ILSVGEEDEV SIADAAKMIA SAMDFEGNYV FDTDKSDGQF KK TACNDLLK
241 KKNPDFKFTS MQDGLKAACD WFCENFETAR K
SEQ ID NO: 21
1 MVTGGSGLVG AAIREYVEGT GALENESWIY LN SKEGDLRN RADTEKIFAK YQP THVIHLA
61 AKVGGLFANM AQKVEFFREN ILINDNIMEC SRIYKVEKLV SFLSTCIFPD KTTY PIDETM
121 LHDGPPHPSN EGYAYAKRLI DTMNRAYAEE YGCNFTSIIP TNIYGPHDNF SIQN GHVTPG
181 LIHKCYLAKK DNTPFTIWGS GTPLRQFVYS RDLAELTVWV MREYHDPTPI TLS VDEEEEV
241 SIKDVALAVA KAMQFDGQIV FDTSKADGQF KKTACNKKLR SLKADYEFTS MP DGIQQSVD
301 WFVANYDSCR K
SEQ ID NO: 22
1 MAETSGTDAA PKKWMVTGG TGLVGCGIKE FVESDAEAKE KEEYIFLSSK DGD IRNMEET
61 KLIFEKYKPT HVIHLAARVG GLFSNLKYKV EFFRENILIN DNVMECCRIY KVEK LVSCLS
121 TCIFPDKTTY PIDETMVHNG PPHVSNEGYA YAKRMIDVLN RCYKEEYGCN FTS VIPTNIY
181 GKGDNFSIDN GHVLPGLIHK CYKAKQAGED LHVWGTGSPL RQFIYNYDLG AL MIWTMRNY
241 HEVDPIILSV GEEDEVSIAD AAKMIASAMD FEGNYVFDTD KSDGQFKKTA CN DLLKQKNP
301 DFKFTPMKEG LKQACEWFCE NYETARK
SEQ ID NO: 23
1 MATESVILVT GGSGLVGEAV KWVIENDKSE RY GKKENEKW VFLSSKDGDL RKEQDVKAIF
61 EKYKPTHVIH LAAMVGGLFK NMKYKLDFLR ENMLMNDNIL WQSKEYNVKK WSCLSTCIF
121 PDKTTYPIDE TMVHN GPPHE SNFGYAHGKR MIDVYNHAYH EQFGCHFTSV IP TNIFGPHD
181 NYDLEGSHVL PGLTHKCYLA KKNNTPFVVW GSGKPLRQFI YSRDLAKLFI WT LREYEEID 241 PIILSVGEKD EVSIKDVADS IVKAMDFQGE YSFDSTKADG QYKKTASNEK LMK YIPDFEF
301 TPFDVAIKES VEWFVENYDT LRK
GDP-L-fucose synthetase (WcaG) [. Escherichia coli] SEQ ID NO: 43
1 MSKQRVFIAG HRGMVGSAIR RQLEQRGDVE LVLRTRDELN LLDSRAVHDF FASERIDQVY
61 LAAAKVGGIV ANNTYPADFI YQNMMIESNI IHAAHQNDVN KLLFLGSSCI YPKLAKQPMA
121 ESELLQGTLE PTNEPYAIAK IAGIKLCESY NRQYGRDYRS VMPTNLY GPH DNFHPSNSHV
181 IPALLRRFHE ATAQNAPDW VWGSGTPMRE FLHVDDMAAA SIHVMELAHE VWLENTQPML
241 SHINVGTGVD CTIRELAQTI AKWGYKGRV VFDASKPDGT PRKLLDVTRL HQLGWYHEIS
301 LEAGLASTYQ WFLEN QDRFR G
GMER (WcaG) from Arabidopsis thaliana SEQ ID NO: 44
1 MAETIGSEVS SMSDKSAKIF V AGHRGL V GS AIVRKLQEQG FTNLVLKTHA ELDLTRQADV
61 ESFFSQEKPV YVILAAAKVG GIHANNTYPA DFIGVNLQIQ TNYIHSAYEH GVKKLLFLGS
121 SCIYPKFAPQ PIPESALLTA SLEPTNEWYA IAKIAGIKTC QAYRIQHGWD AISGMPTNLY
181 GPNDNFHPEN SHVLPALMRR FHEAKVNGAE EVWWGTGSP LREFLHVDDL ADACVFLLDR
241 YSGLEHVNIG SGQEVTIREL AELVKEWGF EGKLGWDCTK PDGTPRKLMD SSKLASLGWT
301 PKVSLRDGLS QTYDWYLKNY CNR
GMER (WcaG) from Helicobacter pylori SEQ ID NO: 45
1 MNEIILITGA YGMV GQNTAL YFKKNKPDVT LLTPKKSELC LLDKDNYQAY LKEYKPTGII
61 HCAGRVGGIV ANMNDLSTYM VENLLMGLYL FSSALDSGVK KAINLASSCA YPKFAPNPLK
121 ESDLLNGSLE PTNEGYALAK LSVMKYCEYV SAEKGVFYKT LVPCNLYGEF DKFEEKIAHM
181 IPGLIARMHT AKLKNEKEF A MWGDGTARRE YLNAKDLARF ISLAYENIAS IPSVMNYGSG
241 VDYSIEEYYE KVAQVLDYKG VFVKDLSKPV GMQQKLMDIS KQRALKWELE IPLEQGIKEA
301 YEYYLKLLEV GMER from Mortierella alpine SEQ ID NO: 46
1 MSPSKSVIMV TGGSGLVGKA IDWWENDSK Y GKREGEEWV FLTSKDGNLI DPAQTKAIFE
61 KYRPTHVIHL AAKVGGLFGN MAANLDYFRD NLLINDNVLH NAKEF GVKKV VSCLSTCIFP
121 DKTSYPIDET MVHQGPPHDS NYGYSHSKRM VDVMNRAYNQ QYGCNFTSVI PTNVF GPHDN
181 FHLVN SHVIP GLIHKCYLAQ QNNTPFIMAG TGRPLRQFIY SRDLARLFIW TLREYQEITP
241 LILSVPEEEE VSIKQVGDSI VKAMGYTGDY RFDTTKADGQ YKKT ASNKKL MSLNPDFQFT
301 PFDVALSETV EWFKENYDTI RK
In some embodiments, GMD and GFS activities are supplied by a single enzyme, such as one of those listed in Table 3 or a variant thereof.
Table 3 genes for GFS and GMD activity
Figure imgf000049_0001
SEQ ID NO: 24
1 MKKALITGIT GQDGSYLAEL LLEKGYEVHG IIRRASTFNT RDHYEDPfflN GKKF LHYGDL
61 ADGV QMVKLL YNLQPDEIYH LGAQSHVRVS FDVPEYTGDV TGLGTLRLEA IR EVGLNNKC
121 RFYQASSSEM FGMVQEVPQT EKTPYPRSPG CAKVYAYWLT VNYRESYNLH A TNGILFNHE
181 SPRRGETFVT RKITRAATRI KMGLQDKLYL GNLDAKRDWG YAKEYVEAMW L MLQQDSGDD
241 YVMATNETHS VKEVQETFAQ LDMDWEAFVE YDKRYERPTE VDLIGDPSKA K KQLDWEPKV
301 RFKDLVKIMV EADLEIARKE AAFKAATEQS FRLMNKDAKI YVAGHRGMV G S AWRALEEN
361 GFQSIITRTH AELDLTDQSE VRAFFQSNNI QYAVIAAAKV GGIHANNSYP AEFI YENLAI
421 AQNTIHEAYA SGVRLLFLGS TCYPKFAKQP IQEASLLTDA LEPTNEAYAI ARIA GLKLCQ 481 FYRQYGVLYH S AMPTNLY GR GDNYHPEN SH VMPALIRRIH E AKE V GAPE V V VWGTGKPLR
541 EFLHSEDAAS GIVHLLNIEN PPDWVNLGSG REISIGDLAQ MISSIIGYDG VLKFD TSKPD
601 GTPRKVTDIQ LISDTGWSPQ ISLEEGVASA YQEFLFELKQ GTVRF
SEQ ID NO: 25
1 MQAEFLLEKG YEVHGVKRRA SLFNTQRVDH LYEDPHDSDT RLKLHY GDLT DT SNLTRLLR
61 DIEPDEVYNL GAQSHVAVSF E APE YT AD VD ATGTLRLLEA IRFLGLEEKT RFYQ ASTSEL
121 YGKVQEIPQS ETTPFHPRSP YAVAKMYAYW ITVNYRESYG MYACNGILFN HE SPRRGETF
181 VTRKITRGLS NIAMGLEPCL YMGNIDALRD WGHAKDYVRM QWMMLQQDEP EDFVIATGVQ
241 YSVREFIRWT ARELGMELEF SGTGTDEIAR VASITGDRVK ALKVGDWMR IDP RYFRPAE
301 VETLLGNPAK AKAKLGWVPE ITAQEMCKRI WVAGHRGMV G GAWRRLERE DCEVICAARD
361 WDLTRQQEV QDWMAETRPD AIIMAAAKVG GILANDTRPV DFLLQNLQIE TN IVEAAHQV
421 DVERFLFLGS SCIYPKMAPQ PIPEDSLLTG PLEPTNEWYA IAKIAGIKLM QAYR KQYGRD
481 WISAMPTNLY GPGDNYDLAS SHVLPALLRK FHEAKVAGAK HVELWGSGTP L REFMHCDDL
541 ADALVFLLQR YSGHDHVNVG SGSEVSIREL AETIAQWGY EAEIVFDSSK PDG TPRKLMD
601 SARLHDMGWN NARSLLDGLR DTYARGTWF KSVADEIRTV DVADYSILPV G WQWLETDG
661 AGDSYNIASR LDFAPNPDIA VISALRPLSN LTPIQRVFHL GGGNQHILLM RMISS QPEDV
721 HNIPHLGWYM RTGVRVIVIS AALSSGGLFA IGWILQSSGH AY GRVLIGGA VFL FPMILAE
781 AVMNLARARG SFFMALLPRD IIWRTLVIAI ALGLLLALPT GWSGLQLMLI CAG SLMICLL
841 VQIRLAWGLY AGHIPPQTAP DWPNWRAQSL WLWISSLAGN ISGNLAVLII SMT LSLEAAG
901 VFFAALRLSM VLALPLNALN IAVAPRFSHL HARQDYNALQ TYGLRMTQVI AL PTLAALAL
961 IVAYGDQALS WFDSEITGGW GALCLLAIGY TLRTCAGASG VMMLMTGHER K AVRIFFQTE
1021 GLSLLVLPLA AHFYGIEGAA ACLALGV AAS SVLSNLHLRR SFRVDPGLHS VLL APRSDQG
1081 IF
Examples of fucosyl transferases (FTs), e.g., alpha- 1,2 -fucosyl transferase are provided by the sequences of SEQ ID NOs: 26-40, which are alpha 1,2 -fucosyl transferases from
Dictyostelium discoideum AX4, Homo sapiens, Pisum sativa, Rhizobium marinum,
Herbaspirillum rubrisubalbicans, Citrobacter freundii, Lactobacillus helveticus, Neocallimastix californiae, Gracilariopsis chorda, Lactobacillus gasseri, Octopus bimaculoides, and
Chryseobacterium scophthalmum, respectively. Homologues of FTs from microorganisms other than Dictyostelium discoideum AX4, Homo sapiens, Pisum sativa, Rhizobium marinum,
Herbaspirillum rubrisubalbicans, Citrobacter freundii, Lactobacillus helveticus, Neocallimastix californiae, Gracilariopsis chorda, Lactobacillus gasseri, Octopus bimaculoides, and
Chryseobacterium scophthalmum, particularly, from fungi, can be used in the microorganisms and methods described herein. Non-limiting examples of the homologs of FTs in the instant invention are represented by FTniProt entries: 030511, P51993, Ql 1128, G5EFP5, G5EE06, P56434, Q11130, Q11131, P56433, Q8HYJ7, Q8HYJ6, Q17WZ9, Q9ZFI3, D0ISI2, D0ITD1, Q9ZKD7, C7BXF2, E6NNI5, E6NPH4, B6JFN9, C7BZU7, E6NJ21, E6NI06, E6NRI2,
E6NSJ6, E6NEQ5, E6NDP7, J0NAV4, and Q9F8S4. Analogues of FTs can be used in the microorganisms and methods described herein.
In some embodiments, FT is selected from a-l,2-fucosyltransferases (FTs) from
Helicobacter pylori 26695 (FutC), Bacteroides fragilis (WcfB), or E. coli (such as WbgF, WbgN, and WbwK, for example, wbwK from E. coli086, wbsJ from E. coli 0128, wbgF from E. coli 0126, wbiQ from E. coli 0127), or futB from H. pylori, futL from H. mustelae, futF from H. bibs, , futG from C. jejuni, futN from B. vulgatus ATCC 8482, and wcfB and wcfW from B. fragilis).
In some embodiments, FT is encoded by one of the sequences listed in Table 4 or a variant thereof.
Table 4 genes for FT activity.
Figure imgf000051_0001
Figure imgf000052_0001
SEQ ID NO: 26
1 MNDSPIISW LPFLIKDNDD KSLNYQGINN LIISIDSIIE QTFKEWELIL VDDGSNN
EIL
61 EQLLSKRYST DNRIKFIINK ENKGIVKSLN DAILNHCSPT SKYLARMDSD DISHP TRLQS
121 QLKYLQSNET IDILGCPIKM FNNNKLIEIL NNNNNNNNIN NNVKELINII NNEES FKFIQ
181 HPDKDILMWS MFFNCCIVHP SVIFKRSIFT IEHCYEENNQ FPFIEDYLFW LKSLI MKGLN
241 ISNIQSSTPL LYLRKHNNSI SFKNIEKQKD STANASCYYL NILFKRFNID SEIIQNS SLS
301 MKEIIQFFQL SPSSLSKINN ISIELFEFAF KYLELIEKSC TKQQPNYSNS IKDAANE KMG
361 ELVSLCLSNY PNNQKSSLLW EKWLSRNPTS QLLSLLSNLN VKSSTTIINN NINN NNNNNN
421 NNNNNNNNNN NNNNNNNNNN SILNFISGIN SNKINTPKSN NNKFKEN GIR IICF SKDRAF
481 QLKEYLRTFF K YLKNDDN GN DKFEIIVDVL FTYSNEKFKN SYQLVIESFP QVNF IKEENF
541 TDQLINLVQK TNKLEYVMFS VDDILYYNEF NLKEYCLSLN SEPLALGFYM KL NKNITYCH
601 TCNQDITIPL NSNTISRTEN NFKYLKWNRN DNDCKKDWNY PWDLCSTIYR CN DID SUNG
661 IVKYYGIRNG INHPNRFEFN GNRPIIQKQI YQNKPYCLCL SDHYSPMSVV TINR VQDVYD
721 NPIYDQTLSL DDLDQLLYSN KSLNDEKYKE NSLSLNFKSV fflGELFIS
SEQ ID NO: 27
1 MLVVQMPFSF PMAHFILFVF TVSTIFHVQQ RLAKIQAMWE LPVQIPVLAS TSKA LGPSQL
61 RGMWTINAIG RLGNQMGEYA TLYALAKMNG RPAFIPAQMH STLAPIFRIT LPV LHSATAS
121 RIPWQNYHLN DWMEEEYRHI PGEYVRFTGY PCSWTFYHHL RQEILQEFTL HD HVREEAQK
181 FLRGLQVNGS RPGTFVGVHV RRGDYVHVMP K V WKGVY ADR RYLQQALDWF RARYSSLIFV
241 VTSNGMAWCR ENIDTSHGDV VFAGDGIEGS PAKDFALLTQ CNHTIMTIGT FGI WAAYLTG
301 GDHYLANYT LPDSPFLKIF KPEAAFLPEW TGIAADLSPL LKH
SEQ ID NO: 28
1 MNMLIKRVIA IKNPRGDDNN NNKLSDLETL TDKCTTCPLT LMRVMAFFVV S FMLFSVLFS
61 LSWLRDPPS DAAISSTTTL FQLNQGLGSD DFDSVELLND KLLGGLLADG FDEK SCLSRY
121 QSAIFGKGLS GKPSSYLISR LRKYEARHKQ CGPYTESYNK TVKELGSGQF SESV DCKYVV
181 WISFSGLGNR ILTLVSAFLY ALLTDRVLLV DPGVDMTDLF CEPFPDASWF VPP DFPLNSH
241 LNNFNQESNQ CHGKILKTKS ITNSTVPSFV YLHLAHDYDD HDKLFFCDEE QLF LQNYPLL
301 IMKTDNYFIP SLFLMPSFEQ ELNDLFPKKE KVFHFLGRYL LHPTNNVWGL VVR YYDAYLA
361 KVDERIGIQI RVFDTDPGPF QHVLDQVLAC TLKESILPDV NREQNINSSS GTPKS KAVLI
421 TSLSSGYFEK VRDMYWEFPT ETGEVVGIYQ PSHEGYQQTQ KQFHNQKAWA E MYLLSLTDV
481 LVTSSWSTFG YVAQGLGGLK PWILYKPENR TAPNPPCQRA MSMEPCFHAP PF YDCKAKRG
541 TDTGALVPHV RHCEDMS W GL KLVDN
SEQ ID NO: 29
1 MITVKLLGGL GNQMFQFATG RAVARRLGSE LLLDISSFEH YDLRRFELED WAI NARVATA
61 SELARAGWP SPPRMLTRIS RLLGLAFPAT TFRESSFAYD PGILQVTDPV YLDG YFQSER
121 YFSDVAGHLR EEFVLRQPTD AKNKAMEALI RDAGPLAVSL HIRRGDYVAN AQ TAKYHGV C
181 SLDYYSAAVD fflAEQVGGGH YFVFSDDLAW VRENLKITQP MTLVDVNGPD K GAWDMALMT
241 ACRHHIIANS SFSWWGAWLN PRPDKIIVAP KRWF AGASHD TTDLVPASWI RL
SEQ ID NO: 30
1 MIVSRLIGGL GNQMFQYAVG RALAEHMHTP LLLDVSGFVH YDLRRYELDG FNI KAKPASE
61 EELARLGVKA GVKPSMYERA MRKLGIRREP SILREASFTY DARIETVEAP LYLD GYWQSQ
121 RYFAAIRPQL LQEFSLKDSW GSANDALAEQ IGLAGDGAVS LHVRRGDYVN NA QTAQYHGV
181 CSLDYYRQAV AYIVARVAAP HFFVFSDDHA WVSANLDTGC PTTFVQTNSP DQ GIFDMALM
241 KTCRHHIIAN SSFSWWGAWL NANDEKIWA PQRWFNEASK DTSDLIPAGW VR L
SEQ ID NO: 31
1 MQVNRVYVRP MGGLGNQLFQ YAVAYGVARK HSAQLIIDTR FFDNYELHGG FR LYNLNISV
61 SEMTNADLKK FPEWKCKLLS KFPQVTRFFN EYIYDKVGDL NEIKSNDAML LG YWQNETNF
121 HQYKNELVTI FKPKIISEND NKKAESILAT NSWIfflRRG DYINNPIAYK HHGVC SLNYY
181 KQAINEMKKN TKNIFFYIFS DDIEWCRENI TPLFSEYDSF SFVRGETQEV DMWL MSCGKY
241 HIIANSSFSW WGAFLSTNPD QIVIAPTPWF DITQKYTGDP SLPQWIKIDK Y
SEQ ID NO: 32
1 MLYMRLKGRL GNQLFIYAFA RELVYKYNQQ VLLYDRKDEK DSMWYSHLDN Y PLNTNVHFT
61 SNRRDMKIGN FKSKLRFIYD RVAIRHLPPR ERYNFQIRNL KKNEKNSLFL LMDG YAPLPK
121 KINDGTFFDG YFQSPKYFNN IREELIKELN PVHTYSEEEK KFINKIKNTE SVCVT IRLGD
181 YINNSTHQVC SKEFYLNAMD KLKKIYPDCT FFIFSDEVDK AQQIFDFKYP VIYD SGKMQD
241 YV SLHVMSMC KHFIISNSSF SWWAQYLSTN PQKIVIAPDK WYAQDVPCDI YE DNWVLMKG
301 K SEQ ID NO: 33
1 MKILIFSVSF SFFYLLHLFF ILYYIISKAS KEIRIVKLCG GLGNQMFQYA YGKSLE HKLQ
61 EKVLFDVSWY KYLNKKKNEK LTKREYGLGI FNLKISFPTK KQLKKCNNKT FEK KSYIYDE
121 ELLQNKGSSY YVGYFQNEKY FKDIKDNIKK IYTFPKIHDT DKFNQQWINK IKN VKNSVFI
181 HIRRADYIYL DGWVLSMDYY KKAIEYIKKN VENPTFFIFC YQCKDYVEEQ FKL DDTIQFI
241 GETNSINNEN WKDMVLMKEC KYAIIANSSF SWWAAWLGRA NEEGIVIAPS PFI KNNDEII
301 CDNWIKINSN NSS
SEQ ID NO: 34
1 MGLRERLHSV WFLWFVAFSI IAVGFLSRSV RTPSVPQQLK STVLVTLSGR LGNQ LFQVAA
61 SEFITARIKP QKVEFERNNY SAETDFSQGV FRDFKHVN S V SEACRGFRRN YYS HKRMSCS
121 HVRRNQFKGE CFIVEGFFQC PHFANAGSSF VRSFFESSFI ASKAEETYRS YAAV SPASPV
181 VAIHIRRGDY TKRFNRNFLE PFPMKYYIRA TKFMPKNAIY LVFSDDTAWC KSN EPEEFRK
241 IPHSRFIFVK ETDASISEAE MSFADHFIIA NSTFSWWAAF FRRFEKKIVV SPKNW FGDRV
301 TEKNKIYPRK WIRY
SEQ ID NO: 35
1 MLYVEMDGRC GNQLFHYAVA RYIQLAIGNK EKLCLNFNKI FEKKDENN GW ID YLKDFKTV
61 PYSYYSKSGT ILKNESNFIQ KIAIGLKAIQ IKSLTKKSRQ EQADKAEVGQ RTLNK LGVYW
121 VREGVNQIYP YKNNKILVSG ICESNFIYEI QEQLQKELIP VTPVSSLNKS LLEKID NCNS
181 VCISVRRGDF FNNKNAKKYG VCSPEYYIRA KKYFDKKRLE NTVYFCFSDD IE WCKENLKF
241 TDKNVIFVSQ EMPVYETLRL MSRCKHFILS NSTFSWWGQF LSEYKDKIVV SPA RWNNDGY
301 DTNLIDKNWI LIDA SEQ ID NO: 36
1 MLLPCWLYHC YCFYHDVAAV GVFHS AFCVK LLIFFIVFFL GVIIFHYLDI LGVIY TINYL
61 VHRQQDDTKV LCPKFVGGLG NQMFQYASLY GIAKSKNMTL LIDAECELNQ LF SISAVTLP
121 HVACWFLKTR TDYRPCAFNK DTMNFSADQN YQMQGYLQSW QYFHRAEPAL RQIFKFKAAI
181 REKAESILKQ AIEVHQKQVR NQ ALTFIAIH IRRGDITKDN FKTYGYNTAS LDYI RRAMQY
241 FSERYHRILF LV CTNDMEWA KRYLHKKNYY FVENQPREVD MALMAS CNHT I MTVGSFGWW
301 SAWLANGEW YYRYPASRGS KLRKAFSKEM TDYYYPKWKP ML
SEQ ID NO: 37
1 MVAVELIGGL GNQMFQYATA RALSLHRDDS LLLDSRLFDN YKLHS YCLNH FNI GAAWKN
61 DLSLKTPGFS KRWDKFFQK IDAFTFQNKI FNTYQEKNFF FDDSFFRNSK KNIY FKGYFQ
121 SEKYFAKYED QFRKDFEIVT PEKKETTDEE KIIEAENSVS FHIRRGDYIS NPAAN AVHGT
181 CDFNYYHRAI EIIKEKIEHP IFFIFSDDID WAKENEKEEN TTYFVDFNDA STNYE DLKLM
241 SACKNNITAN SSFSWWGAWL NANKSKIVIA PSKWFNTDVL NSQDIIPESW MKI
SEQ ID NO 38
1 MLAKJQAMWE LPVQIPVLAS TSKALGPSQL RGMWΉNAIG RLGNQMGEYA TL YALAKMNG
61 RPAFIPAQMH STLAPIFRIT LPVLHSATAS RIPWQNYHLN DWMEEEYRHI PGEY VRFTGY
121 PCSWTFYHHL RQEILQEFTL HDHVREEAQK FLRGLQVNGS RPGTFVGVHV RR GDYVHVMP
181 KVWKGVY ADR RYLQQALDWF RARYSSLIFV VTSNGMAWCR ENIDTSHGDV VFAGDGIEGS
241 PAKDFALLTQ CNHTIMTIGT FGIWAAYLTG GDTIYLANYT LPDSPFLKIF KPEA AFLPEW
301 TGIAADLSPL LKH
SEQ ID NO: 39
1 MGLGSDDFDS VELLNDKLLG GLLADGFDEK SCLSRYQSAI FGKGLSGKPS SYLI SRLRKY
61 EARHKQCGPY TESYNKTVKE LGSGQFSESV DCKYWWISF SGLGNRILTL VSA FLYALLT
121 DRVLLVDPGV DMTDLFCEPF PDASWFVPPD FPLNSHLNNF NQESNQCHGK IL KTKSITNS
181 TVPSFVYLHL AHDYDDHDKL FFCDEEQLFL QNVPLLIMKT DNYFIPSLFL MPSF EQELND
241 LFPKKEKVFH FLGRYLLHPT NNVWGLVVRY YDAYLAKVDE RIGIQIRVFD TD PGPFQHVL
301 DQVLACTLKE SILPDVNREQ NINSSSGTPK SKAVLITSLS S GYFEKVRDM YWEF PTETGE
361 WGIYQPSHE GYQQTQKQFH NQKAWAEMYL LSLTDVLVTS SWSTFGYVAQ G LGGLKPWIL
421 YKPENRT APN PPCQRAMSME PCFHAPPFYD CKAKRGTDTG ALVPHVRHCE D MSWGLKLVD
481 N
SEQ ID NO: 40
1 MKLCGGLGNQ MFQYAYGKSL EHKLQEKVLF D V S WYKYLNK KKNEKLTKRE YGLGIFNLKI
61 SFPTKKQLKK CNNKTFEKKS YIYDEELLQN KGSSYYVGYF QNEKYFKDIK DNI KKIYTFP
121 KIHDTDKFNQ QWINKIKNVK NSVFIHIRRA DYIYLDGWVL SMDYYKKAIE YIK KNVENPT
181 FFIFCYQCKD YVEEQFKLDD TIQFIGETNS INNENWKDMV LMKECKYAII ANSS FSWWAA
241 WLGRANEEGI VIAPSPFIKN NDEIICDNWI KINSNNSS alpha- 1 ,2-fucosyltransferase (WbgL) [ Escherichia coli] SEQ ID NO: 47
1 MRSIIRLQGG LGNQLFQFSF GYALSKINGT PLYFDISHYA ENDDHGGYRL NNLQIPEEYL
61 QYYTPKINNI YKFLVRGSRL YPEIFLFLGF CNEFHAYGYD FEYIAQKWKS KKYIGYWQ SE
121 HFFHKHILDL KEFFIPKNYS EQANLLAAKI LESQSSLSIH IRRGDYIKNK
TATLTHGV CS
181 LEYYKKALNK IRDLAMIRDV FIFSDDIFWC KENIETLLSK KYNIYYSEDL SQEEDLWLMS
241 LANHHIIANS SFSWWGAYLG TSASQIVIYP TPWYDITPKN TYIPIVNHWI NVDKHSSC futC_Hp26695 from H. pylori SEQ ID NO: 48 1 MAFKWQICG 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 WDMLLMQ S CK
241 HGIIANSTYS WWAAYLINNP EKIIIGPKHW LFGHENILCK EWVKIESHFE VKSKKYNA
Putative fucosyl transferase from Bacteroides fragilis SEQ ID NO: 49
1 MLYVILRGRL GNNLFQIATA ASLTQNFIFC TVNKDQERQV LLYKDSFFKN IKVMKGVPDG
61 IPYYKEPFHE FSRIPYEEGK DLIIDGYFQS EKYFKRSWL DLYRITDELR KKIWNICGNI
121 LEKGETVSIH VRRGDYLKLP HALPFCGKSY YKNAIQYIGE DKIFIICSDD IDWCKKNFIG
181 KRYYFIENTT PLLDLYIQSL CTHNIISNSS FSWWGAWLNE NSNKIVIAPQ MWFGISVKLG
241 VSDLLPVSWV RLPNNYTLGR YCFALYKVVE DYLLNILRLI WKRKKNM wbgN from A. coli SEQ ID NO: 50
1 MSIWARLAG GLGNQMFQYA KGYAESVERN SSLKLDLRGY KNYTLHGGFR LDKLNIDNTF
61 VMSKKEMCIF PNFIVRAINK FPKLSLCSKR FESEQYSKKI NGSMKGSVEF IGFWQNERYF
121 LEHKEKLREI FTPININLDA KELSDVIRCT NSVSVHIRRG DYVSNVEALK IHGLCTERYY
181 IDSIRYLKER FNNLVFFVFS DDIEWCKKYK NEIFSRSDDV KFIEGNTQEV DMWLMSNAKY
241 HIIANSSFSW WGAWLKNYDL GITIAPTPWF EREELNSFDP CPEKWVRIEK wbwK from E. coli SEQ ID NO: 51
1 MYSCLSGGLG NQMFQYAAAY ILQRKLKQRS LVLDDSYFLD CSNRDTRRRF ELNQFNICYD
61 RLTTSKEKKE ISIIRHVNRY RLPLFVTNSI F GVLLKKN YL PEAKFYEFLN NCKLQVKNGY
121 CLFSYFQDAT LIDSHRDMIL PLFQINEDLL HLCNDLHIYK KVICENANTT SLHIRRGDYI 181 TNPHASKFHG VLPMDYYEKA IRYIEDVQGE QVIIVFSDDV KWAENTF AN Q PNYYWNN SE
241 CEYSAIDMFL MSKCKNNIIA NSTYSWWGAW LNTFEDKIVV SPRKWF AGNN KSKLTMDSWI
301 NL
wbsJ from E. coli SEQ ID NO: 52
1 MEVKIIGGLG NQMFQYATAF AIAKRTHQNL TVDISDAVKY KTHPLRLVEL SCSSEFVKKA
61 WPFEKYLFSE KIPHFMKKGM FRKHYVEKSL EYDPDIDTKS INKKIVGYFQ TEKYFKEFRH
121 ELIKEFQPKT KFNSYQNELL NLIKENDTCS LHIRRGDYVS SKIANETHGT CSEKYFERAI
181 DYLMNKGVIN KKTLLFIFSD DIKWCRENIF FNNQICFVQG DAYHVELDML LMSKCKNNII
241 SNSSFSWWAA WLNENKNKTV IAPSKWFKKD IKHDIIPESW VKL wbiQ from . coli SEQ ID NO: 53
1 MVMMYCCLSG GLGNQMFQYA AAYILKQHFP DTIFVFDDSY YFNQPQKi RHLELDQFKI
61 IFDRFSSKDE KVKINRLRKH KKIPLLNSFL QFTAIKLCNK YSLNDASYYN PESIKNIDVA
121 CLFSFYQDSK LLNEHRDLIL PLFEIRDDLR VLCHNLQIYS LITDSKNITS IHVRRGDYVN
181 NKHAAKFHGT LSMDYYISAM EYIESECGSQ TFIIFTDDVI WAKEKFSKYS NCLVADADEN
241 KFSVIDMYFM SFCNNNIIAN STYSWWGAWL NRSEDKFVIA PKQWYISGNE CSLKNENWIA
301 M futB from H. Pylori SEQ ID NO: 54
1 MVFQPLLDAF IESASIEKMV SKSPPPPLKI AVANWWGDEE IKEFKKSVLY FILSQRYAIT
61 LHQNPNESSD LVFSNPLGAA RKILSYQNTK RVFYTGENES PNFNLFDYAI GFDELDFNDR
121 YLRMPLYYAH LHYEAELVND TTAPYKLKDN SLYALKKPSH HFKENHPNLC AWNDESDLL
181 KRGFASFVAS NANAPMRNAF YDALNSIEPV TGGGSVRNTL GYKV GNKSEF LSQYKFNLCF
241 ENSQGYGYVT EKILDAYFSH ΉPIYWGSPS VAKDFNPKSF VNVHDFNNFD EAIDYIKYLH
301 THPN A YLDML YENPLNTLDG KAYFYQDLSF KKIFDFFKTI LENDTIYHNN PFIFYRDLHE 361 PLISIDDLRV NYDDLRVNYD DLRVNYDDLR VNYDDLRVNY DDLRVNYDDL RVNYDDLRVN
421 YDDLRVNYDD LRVNYDRLLQ NASPLLELSQ NTTFKIYRKA YQKSLPLLRT IRRWVKK futL from if. mustelae SEQ ID NO: 55
1 MDFKIVQVHG GLGNQMFQYA FAKSLQTHLN IPVFFDTTWF DYGNREFGFH FFPIDFQCAS
61 AQQIAAAHMQ NLPRLVRGAL RRMGFGRV SK EIVFEYMPEF FEPSRIAYFH GYFQDPRYFE
121 DISPLIKQTF TLPHPTEHAE QYSRKLSQIL AAKNSVFVHI RRGDYMRLGW QLDISYQLRA
181 I A YMAKRV QN LELFLFCEDL EF V QNLDLGY PFVDMTTRDG AAHWDMMLMQ SCKHGIITNS
241 TYSWWAAYLI KNPEKIIIGP SHWIYGNENI LCKDWVKIES QFETKS futF from H. bilis SEQ ID NO: 56
1 MEDNLIIVRV DGGIASQIGF VALGKAFEEK GYQVKYDLSW FETSGKGFYN TINGYDRIYD
61 LTFDMPKAFP QLEMKIASED EVKRYNKLYF IDDEKVITHK PPLYV GGYLG RHYDIYFARH
121 FATYFSPKEI EQKDAPFYIL LQEILNTQSC GIfflRRGDLS QNHIVYGEPT SLTYFERVIQ
181 LVAQMNSKSV FYLFSDDVAW VREfflAPLLK DKQFKICDIN TPEQGYLDLY LLSRCKVIVA
241 SHGSLGA Y AK ILAPHNPLLI APRVRNVFFE MENVMLVNWG AKLQITQPCN NYITPPPHCQ
301 NLTLRYRLFL YLYNRLRSKL LRKGVIQ futG from H. jejuni SEQ ID NO: 57
1 MLESNFVIIR VDGGIVSQLY FFAIGKLFEK KGYKVKYDIT WFEEEGLGFY NINKGYDKTY
61 NINWDIPKIF PNISIEIASK SEIDQYKKFR VDSELVLEYQ PPLYWGYNS KCDIVEICRE
121 IREFFNPLEL LSDNKIKFLA NEIKRNRSCG VHVRRGDLSQ EHWYGKPTS VDYFFKCINI
181 VRSMYSDAKF YFF SDDNKWV KDNIAPHIEN LDYFICDINT PEKGYLDLYF LSLCKIIIGS
241 HGSMGLGAKL LSQEETLFIT PKYNYMLF SM SNIMMINFEP KNMEPFNPKI KKIKYKILIK
301 IYYYIRQILL RKFLIKGSD futN from B. vulgatus SEQ ID NO: 58
1 MRLIKVTGGL GNQMFIYAFY LRMKKYYPKV RIDLSDMMHY KVHYGYEMHR VFNLPHTEFC 61 INQPLKKVTE FLFFKKIYER KQAPNSLRAF EKKYFWPLLY FKGFYQSERF FADIKDEVRE
121 SFTFDKNKAN SRSLNMLEIL DKDENAVSLH IRRGDYLQPK HWATTGSVCQ LPYYQNAIAE
181 MSRRVASPSY YIFSDDIAWV KENLPLQNAV YIDWNTDEDS W QDMMLMSHC KHHIICNSTF
241 SWW GAWLNPN MDKTVIVPSR WFQHSEAPDI YPTGWIKVPV S wcfW from B. fragilis SEQ ID NO: 59
1 MIVSSLRGGL GNQMFIYAMV KAMALRNNYP FAFNLTTDFA NDEVYKRKFF FSYFAFDFPE
61 NKKFTFDFSY GNYYRRFSRN LGCHILHPSY RYICEERPPH FESRFISSKI TN AFFEGYW Q
121 SEKYFFDYKQ EIKEDFVIQK KFEYTSYFEF EEIKFFDKNA IMIGVRRYQE SD V APGGVFE
181 DDYYKCAMDI MASKVTSPVF FCFSQDFEWV EKHFAGKYPV RFISKKEDDS GTIDDMFFMM
241 HFRNYIISNS SFYWWGAWLS KYDDKLVIAP GNFINKDSVP ESWFKLNYR futA SEQ ID NO: 63
1 MGFQPFFDAF IESASIEKMA SKSPPPPFKI AVANWWGDEE IKEFKKSVFY FIFSQRYAIT
61 FHQNPNEFSD FVFSNPFGAA RKIFSYQNTK RVFYTGENES PNFNLFDYAI GFDEFDFNDR
121 YLRMPLYYAH FHYKAEFVND TTAPYKFKDN SFYALKKPSH HFKENHPNLC AWNDESDFF
181 KRGFASFVAS NANAPMRNAF YDAFNSIEPV TGGGSVRNTL GYKV GNKSEF FSQYKFNFCF
241 ENSQGYGYVT EKIFDAYFSH ΉPIYWGSPS VAKDFNPKSF VNYHDFNNFD EAIDYIKYFH
301 THPNAYLDML YENPENTEDG KAYFYQDFSF KKILDFFKTI LENDTIYHKF STSFMWEYDF
361 HKPFVSIDDF RVNYDDFRVN YDRFFQNASP FFEFSQNTTF KIYRKAYQKS EPEERAVRKL
421 VKKFGF futD SEQ ID NO: 64
1 MDKQILNMRV LDWWTEDNEQ NFYDNIFIRL LQRKYEWYS DTPDFVLCGP FGYKHLEYRG
61 VRIFCTGENV RPDFNLVDYA ISFDYAVFGD RHLRTPLMFL CDDYVEDMQK VLNSRAHLIK
121 SKIKFCSFIA SNNYMTEMRD SFFEALCTYK KVDSGGKWKN NIGVYVDDKI EWLKSYKFNI
181 CFENDSSPGY LTEKLFDAFM GGCVPIYWGD TSLRCKVDNE CGNLIETQEI GYHLNLEQTK 241 KEVDFVYNGG GYGMFDTRIP NIPAYLFDYK INPKAFINAH DFPTFKELID EIKRIDNDEQ
301 AFKDMLNEPV FLNNFNPKEF YSQKTFHFLD YIVSQGPVCA KRIGRGSRLQ RKENIMRMFP
361 YDTDSVLIPN FMSYCVKHKK IIDRVRRVCG FPRDIMRΉR GK futE SEQ ID NO: 65
1 MQKQQVKMRV LDWWNKDCEE NFYNNFFIQI LQKKYDWYS DKPDFILYGP CGYEHLKYDC
61 VRIFYTAENI RPDYNIADYS IDYDYIKFGD RHLRLPYMFW VFCDEMRQKE MDNRISLLDK
121 KEKFCGFMVS NNALTDKRDM FFEALNKYKR VDSGGRWKNN IGGNYDDKIE WLKSYKFNLC
181 FENSSYPGYL TEKLFDAFLA GCVPIYWGDT SLRVHKNTCA DSKNSENINN RGGGGNDTFD
241 MRIPNISHSL IDYEINPKAF INAHNFPTFK DLIDEIKRID NDSYAFESIL
REPIFLNNFS
301 PYEFYTEQIS AFLDHIIMQG ANDARRCGDG YWLRTHLEFR RISAKYWNLP SDFLHYCFKY
361 RKIIQGVRDI SEYPRNFMRF LRRK FutH SEQ ID NO: 66
1 MAQNLQTPQD SKTKKRIYFC DGAVKGKIPA ILSRHYDIEI TPHNPDYVFY SVMGNEHINY
61 DCIRIFSTGE NYRADFNF CD YAIGFDYMQF EDRYLRYPFY LHYKEAMEKA RNKHLHITPQ
121 TLENKKRFCT FVVSNGKADS IRSQFFDKLM QYKHIDSGGK YKNNIGAP V A DKLAFLSEGK
181 FNIAFENSSA NGYTTEKLIE AFAAGTIPLY WGDESVSLPL DSSGGGVNPK SFVRLNDFAS
241 FEEAIAYIEF LDTHNDAYLA ILREETFLDS NHEAIFDKKL ESFLLfflFNQ PLEKAYRRGF
301 GQWRCNIEKR YKKYQRIRSL TNTCVNIIKN PIRRIKKLFK FutJ SEQ ID NO: 67
1 MKDDLVILHP DGGIASQIAF VALGLAFEQK GAKVKYDLSW F AEGAKGF WN PSNGYDKVYD
61 ITWDISKAFP ALHIEIANEE EIERYKSKYL IDNDRVIDYA PPLYCYGYKG RIFHYLYAPF
121 FAQSFAPKEA QDSHTPFAAL LQEIESSPSP CGVfflRRGDL SQPfflVYGNP TSNEYFAKSI
181 ELMCLLHPQS SFYLFSDDLA FVKEQIVPLL KGKTYRICDV NNPSQGYLDL YLLSRCRNII
241 GSQGSMGEFA KVLSPHNPLL ITPRYRNIFK EVENYMCVNW GESVQHPPLV CSAPPPLVSQ
301 LKRNAPLNSR LYKEKDNASA FutK SEQ ID NO: 68
1 MNQGCTKTHK PTKKVYF CDG A VKGKI V ALL EQHYELILTN KDPDYIFYSC MGFEHLNYNK
61 VRIFATGENL RADFNF CD Y A IGYDYIHFED RYLRYPLYLH CESDMQKAMN KHLHITPETL
121 QNKSRFCTFV VSNGKADEIR TQFFDFLSQY NRVDSGGRYK NNIGNPVVDK Y AFLKEGKFN
181 IAFENSSTNG YITEKLIQAF AAHΉPIYWG DERISLPLDK MGGGINPKSF
INMHKYESYK
241 EVLETIYFLD THDEAYLSML SEPVFLDKNH QKIFDEKLEN FLLfflFNQPL EKAYRRGFGQ
301 WRCNIEKRYK KAQKARQIVN NFANIFQIPL RTLKKYLLSI YLSATSKSFV FFTKERTSK
FutM SEQ ID NO: 69
1 MCDCLSIILL VKMKKIYLKF VDFWDGFDTI SNFIVDALSI QYEWLSNEP
DYLFYSCFGT
61 SHLEYDCIKI MFIGENIVPD FNVCDYAIGF NYIDFGDRYL RLPLYAIYDG
FSNLQNKKID
121 VNKALDRKFC SIWSNNKWA DPIRETFFKL LSSYKKVDSG GRAWNNIGGP VDNKLDFISQ
181 YKFNIAFENS RVLGYTTEKI MEPMQVNSIP VYWGNPLVGK DFNVDSFVNA HDFDSLERLV
241 EYIIELDSSK DKYLEMLEKP WLLDKTYLDW KQLLLNFINN IMMKSYKDAK YLVNY GHAGK
301 YRNEQRFWGR CERKFKLQRI IEYYSQLFDR K
In some embodiments, the nucleic acids encoding an enzyme sequence include a targeting sequence, such as for localization to a specific cellular organelle. In some
embodiments, such sequence is removed from the nucleic acid prior to providing it as a heterologous sequence through genetic engineering into a microorganism. For example, the targeting sequence of SEQ ID Nos. 27, 28, 33, 38, 39 or 40 can be removed before the encoded FT is genetic engineered for expression in a microorganism.
Other FTs that can be used for HMO production in a microorganism, include, but are not limited to, UmProt entries 030511, P51993, Q11128, G5EFP5, G5EE06, P56434, Q11130, Q11131, P56433, Q8HYJ7, Q8HYJ6, Q17WZ9, Q9ZLI3, D0ISI2, D0ITD1, Q9ZKD7, C7BXF2, E6NNI5, E6NPH4, B6JLN9, C7BZU7, E6NJ21, E6NI06, E6NRI2, E6NSJ6, E6NEQ5, E6NDP7, J0NAV4, and Q9L8S4. Analogues and homologs of FTs also can be used in the microorganisms and methods described herein.
The UniProt entries listed herein are incorporated by reference in their entireties.
Additional homologs of FTs are known in the art and such embodiments are envisioned for use with the engineered microorganisms and methods here. For example, the homologs of FTs have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to SEQ ID NOs: 26-40.
In some embodiments, an HMO such as 2’-FF, can be synthesized using so-called salvage pathway enzymes. For example, for 2’-FF, a microorganism can utilize lactose and fucose substrates to synthesize 2’-FF using an enzyme to convert fucose and ATP to fucose-l- phosphate and an enzyme to convert the fucose- 1 -phosphate and GTP to GDP-fucose, which then can be converted by a fucosyl transferase (FT) to 2’-FF. In some embodiments, a bifunctional fucokinase/F-fucose-l-P-guanylyltransferase (FKP) enzyme such as fkp from Bacteroides fragilis performs the two enzymatic steps from fucose to GDP-fucose and then a FT coverts the GDP-fucose to 2’-FF. In some embodiments, the kfp is from B. fragilis 9343, B. thetaiotaomircon or B. ovatus . For example, the FT may be fuel 2 from Heliobacter pylori or any of the FTs described herein. In some embodiments, lactose is supplied exogenously to the microorganism and a transporter such as Fac 12, CDT-l, CDT-2 or a variants or homolog thereof imports the lactose intracellularly for conversion to the HMO.
Bifunctional fucokinase/F-fucose-l-P-guanylyltransferase (FKP) [ Bacteroides fragilis ] SEQ ID NO: 70
1 MQKFFSFPPN FVQSFHEFER VNRTDWFCTS DP V GKKFGS G GGTSWFFEEC YNEYSDGATF
61 GEWFEKEKRI FFHAGGQSRR FPGYAPSGKI FTPVPVFRWE RGQHLGQNLL SFQFPFYEKI
121 MSFAPDKFHT FIASGDVYIR SEKPFQSIPE ADWCYGFWV DPSFATHHGV FASDRKHPEQ
181 FDFMFQKPSF AEFESFSKTH FFFMDIGIWF FSDRAVEIFI KRSHKESSEE FKYYDFYSDF
241 GLALGTHPRI EDEEVNTFSV AIFPFPGGEF YHYGTSKEFI SSTFSVQNKV YDQRRIMHRK 301 VKPNPAMFVQ NAWRIPLCA ENADLWIENS fflGPKWKIAS RHIITGVPEN DWSLAVPAGV
361 CVDWPMGDK GFVARPYGLD D VFKGDLRD S KTTLTGIPFG EWMSKRGLSY TDLKGRTDDL
421 QAASVFPMVN SVEELGLVLR WMLSEPELEE GKNIWLRSER FSADEISAGA NLKRLYAQRE
481 EFRKGNWKAL AVNHEKSVFY QLDLADAAED FVRLGLDMPE LLPGDALQMS RIHNRMLRAR
541 ILKLDGKDYR PEEQAAFDLL RDGLLDGISN RKSTPKLDVY SDQIVWGRSP VRIDMAGGWT
601 DTPPYSLYSG GNYVNLAIEL NGQPPLQVYV KPCKDFHIVL RSIDMGAMEI VSTFDELQDY
661 KKIGSPFSIP KAALSLAGFA PAFSAVSYAS LEEQLKDFGA GIEVTLLAAI PAGSGLGTSS
721 ILASTVLGAI NDFCGLAWDK NEICQRTLVL EQLLTTGGGW QDQYGGVLQG VKLLQTEAGF
781 AQ SPLVRWLP DHLFTHPEYK DCHLLYYTGI TRTAKGILAE IVSSMFLNSS LHLNLLSEMK
841 AHALDMNEAI QRGSFVEFGR LV GKTWEQNK ALDSGTNPPA VEAIIDLIKD YTLGYKLPGA
901 GGGGYLYMVA KDPQAAVRIR KILTENAPNP RARFVEMTLS DKGFQVSRS
Bifunctional fucokinase/L-fucose-l-P-guanylyltransferase (FKP) [Bacteroides thetaiotaomicron ] SEQ ID NO: 71
1 MPEPICCFLL CRHSAIAGIQ SCYKPINTDS TMQKLLSLPP NLIDSFHQLE EVNHTDWFCT
61 SDPVGSKLGS GGGTTWLLQA CHQAFAPEET FSKWIGNEKK ILLHAGGQSR RLPGYAPSGK
121 ILTPIPVFSW ERGQKLGQNL LSLQLPLYER IMKQAPKGLN TLIASGDVYI RSEKPLQDIP
181 EVDVVCYGLW VNPSLATHHG VFVSDRKKPE VLDFMLQKPS LEELEGLSKT HLFLMDIGIW 241 ILSDRAVEVL MKRSLKEGTN DISYYDLYSD Y GLALGEHPQ TTDDEVNKLS VAILPLPGGE
301 FYHFGTSREL ISSTLAIQDK VRDQRRIMHR KVKPNPAIFI QNSFTQVKLS AENANLWIEN
361 SHV GEGWKLG SRQIITGVPE NHWNINLPDG VCIDIVPMGD AAFVARPYGL DDVFKGDLSN
421 DSTTYLGNSF TQWMKEREIG LEDIKGRTDD LQAAPVFPVT TSIEELGILI RWMT AEPQLK
481 EGKELWLRAE KLSADEISAQ ANLERLYAQR S AFRRDNWKG LSANYEKSVF YQLDLQDAAN
541 EFVRLNLDVP AVLKEDAAPM VRIHNRMLRA RILKLQGNEG CKGEEQAAFQ LLRDGLLEAV
601 AGKKNYPKLN VYSDQIVWGR SPVRIDVAGG WTDTPPYSLY SGGSWNLAI ELNGQPPLQV
661 YVKPCHEFHI VLRSIDMGAV EVIRSYEELQ DYKKVGSPFS IPKAALTLAG FAPLFAAESH
721 ASLEEHLKAF GSGLEITLLA AIPAGSGLGT SSILASTVLG AINDFCGLAW DRNDICNYTL
781 VLEQLLTTGG GWQDQYGGVF PGVKLLQSES GFEQHPLVRW LPDQLFVQPE YRDCHLLYYT
841 GITRTAKGIL AEIVSSMFLN SGKHLSLLAE MKAHAMDMSE AILRGNFETF GNLVGKSWIQ
901 NQALDSGTNP PAVAAIIEQI KDYTLGYKLP GAGGGGYLYM VAKDPQAAGC IRRILTEQAP
961 NPRARFVEMT LSDKGLQVSR S
Bifunctional fucokinase/L-fucose-l-P-guanylyltransferase (FKP) \Bacteroides ovatus] SEQ ID NO: 72
1 MQKLLSLPPN LIHCFHELEE VNHTDWFCTS DPIGSKLGSG GGTTWLLQAC HQAFAPQESF 61 SNWIGHEKRI LLHAGGQSRR LPSYGPSGKI LTPIPIFSWE RGQKLGQNLL SLQLPLYERI
121 MNQAPAGLNT LIASGDVYIR SEKPLQDIPN ADWCYGLWV NPSLATHHGV FVSDRKKPEV
181 LDFMLQKPSL EELEGLSKTH LFLMDIGIWI LSDRAIEVLM KRSLKEGTKD ITYYDLYSDY
241 GLTLGEHPKT KDEEINQLSV AILPLPGGEF YHYGTSHELI SSTLAIQDKV RDQRRIMHRK
301 VKPNPAIFIQ NSITQVSLSA DNANLWIEN S Q V GKEWKLGS RQIITGVPEN QWSINLPDGV
361 CIDIIPIGEN EFVARPYGLD DVFKGALDKI TTTYLNYPFT RWMEDRGITW EDIKGRTDDL
421 QSASIFPKVA SVEDLGILVR WMTSEPQLEE GKKLWLKAEK VS ADEISASA NLKRLYEQRN
481 AFRKENWKGL AANYEKSVFY QLDLLDAANE FVRFNLDMPD VLKEDAAPML RIHNRMLRAR
541 IMKLREDKDC AKEEQAAFQL LRDGLLGVMS ERKSHPILNV YSDQIVWGRS PVRIDVAGGW
601 TDTPPYSLYS GGSVVNLAIE LNGQPPLQVY VKPCKEYHIT LRSIDMGAME VIRNYEELQD
661 YKKVGSPFSI PKAALTLAGF APAFSTESYP SLAKQLEDFG SGIEITLLAA IPAGSGLGTS
721 SILASTVLGA INDFCGLAWD KNDICSYTLV LEQLLTTGGG WQDQYGGVFS GIKLLQSEAG
781 FEQNPLVRWL PDQFFVHPDY RDCHLLYYTG ITRTAKSILA EIVSSMFLNS GPHLSLLAEM
841 KAHAMDMSEA ILRSNFESFG RLVGKTWIQN QALDCGTNPP A V A ATTEKTK DYTLGYKLPG
901 AGGGGYLYMV AKDPQAAGQI RRILTEQAPN PRARFVEMTL SDKGLQVSRS In some embodiments, one or more modification are made to a microorganism (such as by genetic engineering) and/or to one or more nucleic acids encoding an enzyme for a step in making an HMO. Such modification can include, but are not limited to: a) replacement of an endogenous promoter with an exogenous promoter operably linked to the endogenous enzyme, such as gmd, gfs,fkp, and/or ft: b) expression of GMD, GFS, FKP and/or FT via an
extrachromosomal genetic material; c) integration of one or more copies of gmd, gfs,fkp, and/or ft into the genome of the microorganism; or d) a modification to the endogenous gmd , gfs,fkp and/or ft to produce a modified gmd , gfs,fkp, and/or ft that encodes a protein that has an increased activity or any combination of modifications a) to d) described in this paragraph.
In some embodiments, an expression of GMD, GFS, and/or FT is varied by utilizing different promoters or changes immediately adjacent to the introduced gmd, gfs,fkp and/or ft genes. For example, in certain embodiments the deletion of a URA3 cassette adjacent to an introduced gmd, gfs, fkp, and/or ft expression cassette leads to a further improvement of 2’-FL production.
In some embodiments the endogenous promoter is replaced with an exogenous promoter that induces the expression at a higher level than the endogenous promoter. In certain embodiments, the exogenous promoter is specific for the microorganism in which the exogenous promoter replaces the endogenous promoter. For example, a yeast specific exogenous promoter can be used if the microorganism being modified is a yeast. The exogenous promoter can be a constitutive promoter or inducible promoter.
Non-limiting examples of constitutive yeast specific promoters include: pCYCl, pADHl, p STE5, pADHl, pCYClOO minimal, p CYC70 minimal, p CYC43 minimal, p CYC28 minimal, pCYC16, pPGKl, pCTC, p GPD or pTDH3. Additional examples of constitutive promoters from yeast and examples of constitutive promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention.
Non-limiting examples of inducible yeast specific promoters include: pGALl, pMFAl, pMFA2, p STE3, p URA3, pFIGl, pEN02, pDLD, pJENl, pmCYC, and rL7'//2. Additional examples of inducible promoters from yeast and examples of inducible promoters from microorganisms other than yeast are known to a skilled artisan and such embodiments are within the purview of the invention. Microorganisms used to produce the genetically modified microorganisms described herein may be selected from Saccharomyces spp., such as S. cerevisiae, S. pastorianus, S.
beticus, S. fermentati, S. paradoxus, S. uvarum and S. bayanus; Schizosaccharomyces spp., such as S. pombe, S. japonicus, S. octosporus and S. cryophilus; Torulaspora spp. such as T.
delbrueckii; Kluyveromyces spp. such as K. marxianus; Pichia spp. such as P. stipitis, P. pastoris or P. angusta, Zygosaccharomyces spp. such as Z bailii; Brettanomyces spp. such as B. inter medius, B. bruxellensis, B. anomalus, B. custersianus, B. naardenensis, B. nanus; Dekkera spp., such as /). bruxellensis and I) anomala; Metschmkowia spp.; Issatchenkia spp. such as
I.orientalis, Kloeckera spp. such as K. apiculata; Aureobasidium spp. such as A. pullulans; Torulaspora spp., Torulaspora delbrueckii, Zygosaccharomyces spp., Zygosaccharomyces bailii, Brettanomyces spp., Brettannomyces intermedius, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera spp., Dekkera bruxellensis, Dekkera anomala, Metschmkowia spp., Issatchenkia spp., Issatchenkia orientalis, Issatchenkia terricola, Kloeckera spp., Kloeckera apiculate,
Aureobasidium spp., Aureobasidium pullulans, Rhodotorula spp., Rhodotorula glutinis, Rhodotorula cladiensis, Rhodosporidiumspp., Rhodosporidum toruloides, Cryptococcus spp., Cryptococcus neoformans, Cryptococcus albidus, Yarrowia spp, Yarrowia lipolytica, Kuraishia spp, Kuraishia capsulata, Kuraishia molischiana, Komagataella spp., Komagataella phaffii, Komagataella pastoris, Hanseniaspora spp., Hanseniaspora guilliermondii, Hanseniaspora uvarum, Hasegawaea spp., Hasegawaea japonica, Ascoidea spp., Ascoidea asiatica,
Cephaloascus spp., Cephaloascus fragrans, Lipomyces spp., Lipomyces starkeyi, Kawasakia spp., Kawasakia arxii, Zygozyma spp, Zygozyma oligophaga, Metschmkowia spp.,
Metschmkowia pulcherrima, Coccidiodes spp., Coccidiodes immitis, Neurospora discreta, Neurospora africana, Aspergillus spp., Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus, Mucor spp., Mucor circinelloides, Mucor racemosus, Rhizopus spp., Rhizopus oryzae, Rhizopus stolonifera, Umbelopsis spp., Umbelapsis isabelline,
Mortierella spp, Mortierella alpine, Alternariaspp., Altemaria alternate, Botrytis spp., Botrytis cinereal, Fusarium spp., Fusarium graminarium, Geotrichum spp., Geotrichum candidum, Penicillium spp., Penicillum chrysogenum, Chaetomium spp., Chaetomium thermophila, Magnaporthe spp., Magnaporthe grisea, Emericella spp., Emericella discophora, Trichoderma spp., Trichodema reesei, Talaromyces spp., Talaromyces emersonii, Sordaria spp., or Sordaria macrospora.
In specific embodiments, a microorganism, preferably, a fungus, such as a yeast, more preferably, a Saccharomyces spp., and even more preferably, S. cerevisiae is provided as the microorganism host. Yeast such as Saccharomyces spp. can be genetically engineered as described herein or using a multitude of available tools.
Other Ascomycetes fungi can also serve as suitable hosts. Many ascomycetes are useful industrial hosts for fermentation production. Exemplary genera include Trichoderma,
Kluyveromyces, Yarrowia, Aspergillus, Schizosaccharomyces, Neurospora, Pichia ( Hansenula ) and Saccharomyces. Exemplary species include Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma reesei, Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis, Kluyveromyces marxianus , Neurospora crassa, Hansenula polymorpha, Yarrowia lipolytica, and Saccharomyces boulardii.
Cloning tools are widely known to those skilled in the art. See e.g., Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei, Robert H. Bischof, Microbial Cell Factories Volume 15, Article number: 106 (2016)), Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant
Kluyveromyces marxianus yeast strain, Yumiko Nambu-Nishida, , Scientific Reportsvolume 7, Article number: 8993 (2017); Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host, Paul Cernak, mBio Sep 2018, 9 (5) e0l4l0-l 8; DOI:
l0. H28/mBio.0l4l0-l 8; How a fungus shapes biotechnology: 100 years of Aspergillus niger research, Timothy C. Cairns, Fungal Biology and Biotechnology Volume 5, Article number: 13 (2018), GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris, Roland Prielhofer, BMC Systems Biology Volume 11, Article number: 123 (2017)), Aiko Ozaki,“Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose,” Metabolic Engineering Communications Volume 5, December 2017, Pages 60-67, World J Microbiol Biotechnol. 2019; 35(1): 10.“ Yarrowia lipolytica : a beneficious yeast in biotechnology as a rare opportunistic fungal pathogen: a minireview,” Bartlomiej Zieniuk (2014)“Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast
Saccharomyces boulardii PLOS ONE 9(11)).;“Metabolic Engineering of Probiotic Saccharomyces boulardii ,” Liu J-J, Kong II, 2016. Metabolic engineering of probiotic
Saccharomyces boulardii. Appl Environ Microbiol 82:2280 -2287; David Havlik,
“Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product”, Microb Cell Fact. 2017; 16: 128.; Ho, C.C. (April 1986). "Identity and characteristics of Neurospora intermedia responsible for oncom fermentation in Indonesia" . Food Microbiology. 3 (2): 115-132.
III. Enhancement of Production and Export of HMOs
In some embodiments, the production and/or export of an HMO can be enhanced through genetic modification of an HMO-producing microorganism. For example, an HMO-producing microorganism can be modified by one or more of the following:
i) a genetic modification that increases the activity of PMA1 in the microorganism compared to PMA1 activity in the parental microorganism,
ii) a genetic modification that decreases the activity of SNF3 in the microorganism compared to SNF3 activity in the parental microorganism,
iii) a genetic modification that decreases the activity of RGT2 in the microorganism compared to RGT2 activity in the parental microorganism, and
iv) a genetic modification that decreases the activity of GPR1 in the microorganism compared to GPR1 activity in the parental microorganism.
In particular embodiments, i) the genetic modification that increases the activity of
PMA1 is a genetic modification to plasma membrane ATPase gene (pmal ), ii) the genetic modification that decreases the activity of SNF3 is a genetic modification to sucrose non fermenting gene ( snf3 ), iii) the genetic modification that decreases the activity of RGT2 is a genetic modification to glucose transport gene ( rgt2 ), and iv) the genetic modification that decreases the activity of GPR1 is a genetic modification to G protein-coupled receptor 1 gene (, gprl ). Examples of PMA1, SNF3, RGT2, and GPR1 are described in International Patent Application No. PCT/US2018/040351, the contents of which are incorporated herein by reference.
An example of PMA1 is provided by the sequence of SEQ ID NO: 5, which is PMA1 from Saccharomyces cerevisiae. Homologs of PMA1 from microorganisms other than A cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure. Non-limiting examples of the homologs of PMA1 useful in the instant disclosure are represented by Umprot entries: A0A1U8I9G6, A0A1U8H4C1, A0A093V076, A0A1U8FCY1, Q08435, A0A1U7Y482, A0A1U8GLU7, P22180, A0A1U8G6C0, A0A1U8IAV5,
A0A1U8FQ89, P09627, A0A199VNH3, P05030, P28877, A0A1U8I3U0, Q0EXL8,
A0A1U8I3V7, P49380, Q07421, A0A1D8PJ01, P54211, P37367, P07038, Q0Q5F2, G8BGS3, A0A167F957, M5ENE2, A0A1B8GQT5, 074242, Q9GV97, Q6VAU4, A0A177AKN9, A0A1J6KB29, A0A2H9ZYJ6, A0A251UIM1, A0A251USM2, D2DVW3, M5BX73, Q6FXU5, A3LP36, G3ARI4, 9NSP9, A0A167C712, G2WE85, F2QNM0, A6ZUY5, C7GK65,
A0A142GRJ4, W0T7K4, B3LDT4, A0A0H5BY16, A0A1B2J5T9, E7DB83, Q9UR20,
F4NA03, Q96TH7, F4NA02, 12G7P2, C4PGL3, F4NA00, F4N9Z6, Q7Z8B7, F4N9Z9, A0A1L4AAP4, 094195, A0A1D1YKT6, A0A0U1YLR0, A0A0F8DBR8, A0A1C7N6N1, A0A2N6P2L5, A0A2C5WY03, 014437, T1VYW7, T1VY71, A1KAB0, C0QE12, K0NAG7, A0A0H3J1I1, A0A1Q9D817, A0A068MZP7, D1JED6, A0A2K8WRE9, A0A1A8YFD7, A0A1A8YG89, 12G7P8, D9PN36, D1JI19, B6IUJ9, B1XP54, H8W7G4, H6SL18, G8LCW3, L8AJP6, Q5ZFR6, A0A1D7QSR3, A0A1Q2TYG8, F4N054, A0A1Q9CTB2, A0A1Q9EJY5, A0A1D1XEE3, A0A0F7GAE0, D2DVW4, A0A0A9YX23, A0A1Q9ELW6. The Umprot entries listed herein are incorporated by reference in their entireties.
Additional homologs of PMA1 are known in the art and such embodiments are within the purview of the present disclosure. For example, the homologs of PMA1 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5.
SEQ ID 5:
1 mtdtssssss ssassvsahq ptqekpakty ddaasessdd ddidalieel qsnhgvdded
61 sdndgpvaag earpvpeeyl qtdpsyglts devlkrrkky glnqmadeke slvvkfvmff
121 vgpiqfvmea aailaaglsd wvdfgvicgl lmlnagvgfv qefqagsivd elkktlanta
181 vvirdgqlve ipanewpgd ilqledgtvi ptdgrivted cflqidqsai tgeslavdkh
241 ygdqtfssst vkrgegfmw tatgdntfvg raaalvnkaa ggqghftevl ngigiillvl
301 viatlllvwt acfyrtngiv rilrytlgit iigvpvglpa wtttmavga aylakkqaiv
361 qklsaiesla gveilcsdkt gtltknklsl hepytvegvs pddlmltacl aasrkkkgld
421 aidkaflksl kqypkakdal tkykvlefhp fdpvskkvta vvespegeri vcvkgaplfv
481 lktveedhpi pedvhenyen kvaelasrgf ralgvarkrg eghweilgvm pcmdpprddt
541 aqtvsearhl glrvkmltgd avgiaketcr qlglgtniyn aerlglgggg dmpgseladf
601 venadgfaev fpqhkyrwe ilqnrgylva mtgdgvndap slkkadtgia vegatdaars
661 aadivflapg lsaiidalkt srqifhrmys ywyrialsl hleiflglwi aildnsldid
721 livfiaifad vatlaiaydn apyspkpvkw nlprlwgmsi ilgivlaigs witlttmflp 781 kggiiqnfga mngimflqis ltenwlifit raagpfwssi pswqlagavf avdiiatmft
841 lfgwwsenwt divtvvrvwi wsigifcvlg gfyyemstse afdrlmngkp mkekkstrsv
901 edfmaamqrv stqheket
An example of SNF3 is provided by the sequence of SEQ ID NO: 6, which is SNF3 from S. cerevisiae. Homologs of SNF3 from microorganisms other than S. cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure. Non limiting examples of the homologs of SNF3 useful in the instant disclosure are represented by Umprot entries: W0TFH8, Q6FNU3, A0A0W0CEX1, G2WBX2, A6ZXD8, J6EGX9, P10870, C7GV56, B3LH76, A0A0L8RL87, A0A0K3C9L0, M7WSX8, A0A1U8HEQ5, G5EBN9, A8X3G5, A3LZS0, G3AQ67, A0A1E4RGT4, A0A1B2J9B3, F2QP27, E3MDL0,
A0A2C5X045, G0NWE1, A0A0H5S3Z1, A0A2G5VCG9, A0A167ER19, A0A167DDU9, A0A167CY60, A0A167CEW8, A0A167ER43, A0A167F8X4, A0A1B8GC68, A0A177A9B0, E3EIS7, E3E8B6, A0A0A9Z0Q2. The Uniprot entries listed herein are incorporated by reference in their entireties.
Additional homologs of SNF3 are known in the art and such embodiments are within the purview of the present disclosure. For example, the homologs of SNF3 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6.
SEQ ID 6:
1 mdpnsnssse tlrqekqgfl dkalqrvkgi alrrnnsnkd httddttgsi rtptslqrqn
61 sdrqsnmtsv ftddistidd nsilfseppq kqsmmmsicv gvfvavggfl fgydtglins
121 itsmnyvksh vapnhdsfta qqmsilvsfl slgtffgalt apfisdsygr kptiifstif
181 ifsignslqv gaggitlliv grvisgigig aisavvplyq aeathkslrg aiistyqwai
241 twgllvssav sqgtharnda ssyripiglq yvwssflaig mfflpespry yvlkdkldea
301 akslsflrgv pvhdsgllee lveikatydy easfgssnfi dcfissksrp kqtlrmftgi
361 alqafqqfsg infifyygvn ffnktgvsns ylvsfityav nwfnvpglf fveffgrrkv
421 lwggvimti anfivaivgc slktvaaakv miaficlfia afsatwggw wvisaelypl
481 gvrskctaic aaanwlvnfi calitpyivd tgshtsslga kiffiwgsln amgviwylt
541 vyetkgltle eidelyikss tgwspkfnk direralkfq ydplqrledg kntfvakrnn
601 fddetprndf rntisgeidh spnqkevhsi pervdiptst eilespnkss gmtvpvspsl
661 qdvpipqtte paeirtkyvd lgnglglnty nrgppslssd ssedytedei ggpssqgdqs
721 nrstmndind ymarlihsts tasnttdkfs gnqstlryht asshsdttee dsnlmdlgng
781 lalnaynrgp psilmnssde eanggetsdn lntaqdlagm kermaqfaqs yidkrgglep
841 etqsnilsts lsvmadtneh nneilhssee natnqpvnen ndlk An example of RGT2 is provided by the sequence of SEQ ID NO: 7, which is RGT2 from S. cerevisiae. Homologs of RGT2 from organisms other than S. cerevisiae, particularly, from yeast, can be used in the microorganisms and methods of the present disclosure. Non limiting examples of the homologs of RGT2 are represented by Uniprot entries: A0A0FHMAJ7, N4TG48, A0A1Q8RPY1, N4U7I0, A0A1L7SSQ2, A0A1L7VB15, A0A0C4E497,
A0A1L7UAN6, A0A0J0CU17, A0A1L7VMA9, S0ED22, A0A1L7SD48, N1R8L8,
A0A1L7V0N4, S3BYD3, E4UUU6, N4UPT5, N4U030, A0A0I9YK83, S0DJS4,
A0A0U1LWH9, A0A0K6FSJ2, N1 S6K7, A0A0J6F3E5, A0A1E4RS51, N4UTN2,
A0A0G2E6D5, A0A1J9R914, A0A0F4GQX7, A0A1 S9RLB9, A3M0N3, J9PF54,
A0A074WC52, A0A0K6GI66, N1QHS4, G2WXK0, B2WL4, B2WDK7, A0A1J9S6A1, G4N0E9, L7JEU7, L7INA5, A0A0L1HE99, A0A0J8QL36, A0A0H5CKW2, A0A0J6Y4E2, W0VMG0, G2WQD8, A0A1C1WV61, A0A1S9RL33, C9SBA9, A0A0G2HY75, J3P244, N1QK04, A0A0N0NQR9, A0A1 S7UJ19, G2XFE7, C9SWZ3, R8BUY9, M7SYH1,
A0A1E1MIV2, A0A1E1LLK3, A0A1E1LJE1, L7J4Y3, L7I304, A0A1L7XU29, A0A136JCY3, A0A0J8RG81, A0A177DW33, A0A1L7X792, W9C8U1, B2VXL1, A0A0L1HMG8,
A0A178DQW4, A0A167V6F7, A0A166WR60, A0A162KLT6, A0A1L7X3D1,
G3JQX8,Q7S9U8, E9F7A6, A0A1S7HPX9, A0A0G2G564, A0A0W0D0B3, A6ZXI9, Q12300, C7GKZ0, G2WC23, A0A0H5CAT9, J4U3Y8, A0A0L8RL54. The Umprot entries listed herein are incorporated by reference in their entireties.
Additional homologs of RGT2 are known in the art and such embodiments are within the purview of the present disclosure. For example, the homologs of RGT2 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7.
SEQ ID 7:
1 mndsqnclrq reenshlnpg ndfghhqgae ctinhnnmph rnaytestnd teaksivmcd
61 dpnayqisyt nnepagdgai ettsillsqp lplrsnvmsv lvgifvavgg flfgydtgli
121 nsitdmpyvk tyiapnhsyf ttsqiailvs flslgtffga liapyisdsy grkptimfst
181 avifsignsl qvasgglvll ivgrvisgig igiisawpl yqaeaaqknl rgaiissyqw
241 aitigllvss avsqgthskn gpssyripig lqyvwssila vgmiflpesp ryyvlkdeln
301 kaakslsflr glpiedprll eelveikaty dyeasfgpst lldcfktsen rpkqilrift
361 giaiqafqqa sginfifyyg vnffnntgvd nsylvsfisy avnvafsipg mylvdrigrr
421 pvllaggvim aianlviaiv gvsegktwa skimiaficl fiaafsatwg gwwvvsael 481 yplgvrskct aicaaanwlv nftcalitpy ivdvgshtss mgpkiffiwg glnwaviw
541 yfavyetrgl tleeidelfr kapnsvissk wnkkirkrcl afpisqqiem ktniknagkl
601 dnnnspivqd dshniidvdg flenqiqsnd hmiaadkgsg slvniidtap ltstefkpve
661 hppvnyvdlg nglglntynr gppsiisdst defyeendss yynnnterng ansvntymaq
721 linsssttsn dtsfspshns nartssnwts dlaskhsqyt spq
An example of GPR1 is provided by the sequence of SEQ ID NO: 8, which is GPR1 from S. cerevisiae. Homologs of GPR1 from microorganisms other than S. cerevisiae, particularly, from yeasts, can be used in the microorganisms and methods of the present disclosure. Non limiting examples of the homologs of GPR1 are represented by Uniprot entries: A0A1S3ALF0, A0A0Q3MD25, A0A146RBQ8, A0A0P5SHA9, A2ARI4, Q9BXB1, Q9Z2H4, F1MLX5, U3DQD9, 12CVT9, 10FI44, K7D663, K7ASZ6, A0A1U7Q769, U3ESI5, T1E5B8,
A0A0F7ZA01, J3RZW5, A0A094ZHC9, W6UF90, A0A0P6J7Q8, F5KYC3, B7P6N0, B0BFW3, A2AHQ2, A0A151N8W7, A0A146RCW3, A0A0X3NYB9, A0A0P5Y3G9, W5UAB2, A0A0P5IC44, A0A090XF51, A0A146NRV7, A0A0X3Q0R0, A0A0P6IRD7, F9JFB7, A0A146YGG2, A0A146WG88, Q12361, B3FGT6, A0A0N8A6F9, P0DM44, W6JM29, A0A1A8FC80, A0A0N8A4D4, Q7Z7M1, A0A1S3G1Q8, A0A1U7QGH1, A6ZXT8, A0A1U8C0F6, D3ZJU9, A0A1 S3KGF3, G5B385, F9KNY9, A0A1 S3AQM3, A0A087UXX9, A0A0F8VW24, A0A0P6AR08, Q9HBX8, Q3FTVD5, A0A1U7UEF2, A0A146XMF9,
A0A146QTV1, A0A1 S3ID45, F5KTU9, A0A1A8EFT4, A0A0N7ZMX8, A0A0P5Q3T8, A0A1A8N9Z4, A0A1A8D807, A0A1A8CVG1, A0A1A8UMB1, A0A1A8JQ07,
A0A1A8P7N2, A0A1A8HF38, E7FE13, A0A1 S3FZF3, A0A0P7WFQ9, H2KQN3,
A0A1 S3WJA9, A0A146PKA1, F5FFQ3, F1Q989, A0A0F8AKY3, A0A0P7VR95,
A0A1U8C8I3, A0A034VIM3, A0A0N8BFD4, A0A146XMJ1, A0A0N8BDM1, A0A1A8KTJ1, A0A1A7X706, A0A0R4ITE3, A0A1U7S4H0, A0A1S3AQ94, A0A1U7UCP2, F8HMA8, A0A0Q3P3V6, A0A1A8CDG3, D6W7N2, A0A1E1XMY8, A0A1A8ACF5, A0A1S3WNY2, T0MHY5, A0A1 S3G113, V8P2X5, A0A1 S3KV51, A0A1 S3G018, A0A1S3PUP5,
A0A1U8C7X5, S9WP18, A0A1S3AQF8, A0A0N8ENF1, K7CIG0, A0A147BFY7,
A0A1 S3FZK9, A0A1U7TUH0, A0A1U8BX93, A0A091DKN5, A0A146W919, A0A147B2K7, A0A146XNF4, A0A091DTX9, A0A0Q3UQB0, A0A146WH37, E9QDD1, Q58Y75,
A0A096MKI0, A0A1S3S901, Q14BH6, A0A1 S3AQ42, A0A0P5SV49, A0A0P5P299, A0A0P5WCR4, K7CHT8, A0A1U7U0Q5, A0A1 S3EXD4, A0A146Y6G0, A0A061HXQ0, A0A1 S3AQ84, A0A1S2ZNQ3, A0A1U7UEE6, A0A1S3G013, A0A1U7QJG4, S7N7M1, A0A1 S3G108, A0A1U8C8H8, and A0A1U8C7X0.
Additional homologs of GPR1 are known in the art and such embodiments are within the purview of the present disclosure. For example, the homologs of GPR1 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8.
SEQ ID 8:
1 mitegfppnl nalkgsslle krvdslrqln tttvnqllgl pgmtstftap qllqlriiai
61 tasavsliag clgmfflskm dkrrkvfrhd liafliicdf lkafilmiyp miilinnsvy
121 atpaffntlg wftafaiega dmaimifaih failifkpnw kwrnkrsgnm egglykkrsy
181 iwpitalvpa ilaslafiny nklnddsdtt iildnnnynf pdsprqggyk pwsawcylpp
241 kpywykivls wgpryfiiif ifavylsiyi fitseskrik aqigdfnhnv leeekekkkl
301 fglghwgkak wyfrsyfklp llhllrnlkn fftisfidpn eetddsgssn gtfnfgessn
361 eiptlfrktn tgsdenvsas ggvrlldyns akpldmskya mseqpdlern npfdcendit
421 lnpselvskq kehkvtfsve negldtrkss mlghqtfscq nslesplamy dnkndnsdit
481 snikekggii nnnsnndddd nnnnndndnd nnnsnnnnnn nnnnnnnnnn nnnnnnnnnn
541 nnnnsnnikn nvdnnntnpa dniptlsnea ftpsqqfsqe rvnnnadrce nssftnvqqh
601 fqaqtykqmk krraqiqknl raifiyplsy igiwlfpiia dalqynheik hgptmwvtyi
661 dtcvrplscl vdvivylfke kpwnyswakt eskyliekyi lkgelgekei lkfchsnwgk
721 rgwyyrgkwk krkcwkystn plkrilwfve rffkqlfelk lhfsfydncd dfeywenyys
781 akdsndnkrt esdetktnss drslpsnsle lqamlnnita eevevplfwr iihhipmlgg
841 idldelnrll kirynndhfs lpglkfalnq nkshdkhqdv stnsmvkssf fssnivtndd
901 ensieedknl rysdasasen ylvkptipgt tpdpiieaqn dndssdssgi dliaflrngp
961 1
Substrates for production of HMOs
In certain embodiments, the present disclosure provides microorganisms comprising one or more genetic modifications that provide for import and/or enhanced uptake of one or more substrates that can be used by the microorganism to make an HMO. For example, a
microorganism can include:
i) a genetic modification that introduces a substrate transporter gene LAC 12, or its
analogues which increases the uptake of lactose and/or other substrate into the microorganism;
ii) a genetic modification that introduces a transporter which can both import a substrate, such as lactose and export a produced HMO, such as the wild type cellodextrin transporter gene ceil- 1 or a variant of the cellodextrin transporter gene ceil- 1 such as those described herein (for example, CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, CDT-l N209S F262W).
Lactose transporter (Lacl2) [Kluyveromyces lactis] SEQ ID NO:4l
1 MADHSSSSSS LQKKPINHE HKDTLGNDRD HKEALNSDND NTSGLKINGV PIEDAREEVL
61 LPGYLSKQYY KLYGLCFITY LCATMQGYDG ALMGSIYTED AYLKYYHLDI NSSSGTGLVF
121 SIFNVGQICG AFFVPLMDWK GRKPAILIGC LGVVIGAIIS SLTTTKSALI
GGRWF V AFF A
181 TIANAAAPTY CAEVAPAHLR GKVAGLYNTL WSVGSIVAAF STYGTNKNFP NSSKAFKIPL
241 YLQMMFPGLV CIFGWLIPES PRWLVGVGRE EEAREFIIKY HLN GDRTHPL LDMEMAEIIE
301 SFHGTDLSNP LEMLDVRSLF RTRSDRYRAM LVILMAWFGQ FSGNNYCSYY LPTMLRNVGM
361 KS V SLNVLMN GVYSIVTWIS SICGAFFIDK IGRREGFLGS ISGAALALTG LSICTARYEK
421 TKKKSASNGA LVFIYLFGGI FSFAFTPMQS MYSTEVSTNL TRSKAQLLNF WSGVAQFVN
481 QFATPKAMKN IKYWFYVFYV FFDIFEFIVI YFFFVETKGR SLEELEVVFE APNPRKASVD
541 QAFLAQVRAT L V QRND VRV A NAQNLKEQEP LKSDADHVEK LSEAESV
Production, Separation and Isolation of HMOs
In some embodiments, the microorganisms described herein are capable of producing HMOs such as 2’-FL. In some embodiments, the microorganisms are capable of converting lactose into 2’-FL. In particular embodiments, the microorganisms described herein have higher capacity, compared to the parental microorganisms, of converting lactose into 2’-FL. In specific embodiments, the conversion of lactose into 2’-FL occurs in the cytosol of the microorganisms.
In still another aspect, methods of producing products of interest by culturing the microorganisms described herein in appropriate media containing an appropriate oligosaccharide under appropriate conditions for an appropriate period of time and recovering an oligosaccharide from the culture media, is provided.
In certain embodiments, the disclosure provides methods of producing 2’-FL by culturing the microorganisms described herein in culture media containing lactose under appropriate conditions for an appropriate period of time and recovering 2’-FL from the culture media.
In preferred embodiments, the microorganisms belong to Saccharomyces spp. In even more preferred embodiments, the microorganisms are S. cerevisiae.
In certain embodiments, the media contains about 10 g/L yeast extract, 20 g/L peptone, and about 40 g/L oligosaccharide, particularly, lactose or sucrose. In particular embodiments, the microorganisms, particularly, yeast, are grown at 30 °C.
Additional culture media, conditions appropriate for culturing the microorganisms, and the methods of recovering the products of interest from the culture media are well known in the art and such embodiments are within the purview of the invention.
In certain aspects, the present disclosure provides methods for producing
oligosaccharides by culturing the microorganisms described herein in the presence of appropriate oligosaccharides and recovering the products of interest. In some embodiments, an HMO is separated from the cells (microorganism) that produce the HMO. In some cases, an HMO can be further isolated from other constituents of the culture media (fermentation broth) in which the HMO-producing cells are grown.
In some embodiment, an HMO is recovered from the fermentation broth (also referred to a culture medium). Many methods are available for separation of cells and/or cell debris and other broth constituents from the produced HMO.
For example, cell/debris separation can be achieved through centrifugation and/or filtration. The filtration can be microfiltration or ultrafiltration or a combination thereof.
Separation of charged compounds can be achieved through ion exchange chromatography, nanofiltration, electrodialysis or combinations thereof. Ion exchange chromatography can be cation or anion exchange chromatography, and can be performed in normal mode or as simulated moving bed (SMB) chromatography. Other types of chromatography may be used to separate based upon size (size exclusion chromatography) or affinity towards a specific target molecule (affinity chromatography). For example, US 2019/0119314 Al, GRAS applications
GRN0005718 and GRN 000749. Drying or concentration steps can be achieved with evaporation, lyophilization, reverse osmosis, or spray drying. Crystallization can serve as a concentration and separation step and can be done with for example evaporative or temperature-based crystallization, or induced by modification of pH or increase in ionic strength. For example, US20170369920A1,
WO2018164937A1.
Absorption techniques, such as adsorption using activated charcoal, can also be used as a separation step and in particular is useful for removal of color bodies or separation of oligosaccharides from monomers.
An HMO product can also be pasteurized, filtered, or otherwise sterilized for food quality purposes.
Products and compositions
The microorganisms and methods described herein can be used to produce a variety of products and compositions containing one or more HMOs. In some embodiments, a product suitable for animal consumption includes one or more HMO produced by the microorganisms or methods herein. The product can include one or more additional consumable ingredients, such as a protein, a lipid, a vitamin, a mineral or any combination thereof. The product can be suitable for mammalian consumption, human consumption or consumption as an animal feed or supplement for livestock and companion animals. In some embodiments, the product is suitable for mammalian consumption, such as for human consumption and is an infant formula, an infant food, a nutritional supplement or a prebiotic product. Products can have 1 , 2, 3 or more than 3 HMOs, and one or more of the HMOs can be produced by the microorganisms or by the methods described herein. In some cases, the HMO is 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I), or any combinations thereof.
Exemplary embodiments
In some embodiment, an engineered microorganism for production of an HMO comprises one or more of the following genetic modifications
a) a genetic modification producing a GFS enzyme;
b) a genetic modification producing a GMD enzyme; c) a genetic modification producing a FT enzyme;
d) genetic modifications producing any combination of GFS, GMD and FT enzymes;
e) a genetic modification producing a transporter for export of an HMO, for example CDT-l or a variant of CDT-l such as one of CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411 A, CDT-l N209S F262W, one or more ammo acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket and/or the PESPR motif such as positions G336, Q337, N341, and G471 ;
f) genetic modifications producing any combination of GMD, GFS and FT enzymes and a transporter for export of an HMO, for example CDT-l or a variant of CDT-l such as one of
CDT-l N209S F262Y, CDT-l G91A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, CDT-l N209S F262W;
g) a genetic modification of any of the embodiments (a)-(f) and the CDT-l can have one or more amino acid changes that correspond to one or more of positions predicted to be near the sugar substrate binding pocket the PESPR motif such as positions G336, Q337, N341, and
G471.
h) a genetic modification producing a transporter for import of a substrate, such as lactose, for production of an HMO, for example Lacl2, CDT-l or a variant or analog thereof;
i) genetic modifications producing any combination of GMD, GFS and FT enzymes and a transporter for import of a substrate, such as lactose, for production of an HMO, for example
Lac 12, CDT-l or a variant or analog thereof;
j) genetic modifications producing any combination of GMD, GFS and FT enzymes, a transporter for import of a substrate, such as lactose, for production of an HMO, for example Lac 12, CDT-l or a variant or analog thereof, and a transporter for export of an HMO, for example CDT-l or a variant of CDT-l such as one of CDT-l N209S F262Y, CDT-l G91 A, CDT-l F213A, CDT-l L256V, CDT-l F335A, CDT-l S411A, CDT-l N209S F262W; k) supplying a substrate to a genetically modified microorganism for production of an HMO, such as lactose, and one or more of the modifications of a)-j);
l) production of an HMO in a genetically engineered microorganism, wherein the HMO is a non-branched HMO comprised of a lactose core, such as 2'-fucosyllactose (2'-FL), 3'- fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) or lacto-N-fucopentaose I (LNFP I)
m) any of a)-l) wherein the microorganism is a Ascomycetes fungus, including but not limited to, a Sacharomyces spp., a Schizosaccharomyces spp., a Pichia spp., Trichoderma,
Kluyveromyces, Yarrowia, Aspergillus, and Neurospora.
EXAMPLES
Example 1: Improved 2’-FL production in Saccharomyces cerevisiae expressing GMD, GFS, and/or FT
Expression vectors conferring well known activities for the enzymes GMD, GFS and FT (named GMD t, GFS t and FT_t) were generated for expression in the yeast Saccharomyces cerevisiae. Under selection pressure, these expression vectors are believed to occur in multiple tens of copies per cell and thus expression of a plasmid born gene is likely higher than from a single genomic locus if comparable promoters are used.
Constructs expressing heterologous GMD, GFS or FT genes were then co-transformed with plasmids containing all but the genes for which enzymatic activity was to be tested. The acceptor strain was a genetically modified Saccharomyces cerevisiae strain producing low titers of 2’-FL if grown on lactose. The strain also expresses Lad 2 from Kluyveromyces lactis for improved import of lactose and an engineered oligosaccharide transporter for improved export of 2’-FL as indicated in Fig. 8.
After introduction of the plasmids GMD t, GFS t and FT_t, higher levels of 2’-FL were produced. The base strain was auxotrophic for the synthesis of Leucine, Histidine and Uracil while plasmids carried individual gene cassettes restoring auxotrophy for the respective compounds, respectively.
Omitting one of the plasmids restored 2’-FL production rates similar to the acceptor strain and, vice versa, additional expression of a gene encoding a protein that can functionally compensate for the lack of such enzymatic activity will increase 2’-FL production.
Putative GFSs were tested by transforming an expression construct comprising the putative GFS gene together with expression constructs containing GMD t and FT_t. After transformation, cells were selected on respective media omitting the compound for which transformed plasmids conferred auxotrophy for.
Colonies forming after the transformation were grown in drop out medium (omitting the compound the transformed plasmids conferred auxotrophy for) overnight at 30°C and 250 rpm shaking. Cells were then washed and then transferred into YP4D0.4L medium, which is YPD medium with 0.4 g/L lactose and 4 g/L Glucose, and grown for 6 days under identical conditions. Supernatants were analyzed by HPLC analysis.
Fig. 9 shows 2’-FL production by introducing a heterologous fucosyltransferase (FT) from different organisms to a yeast strain which also expresses CDT-l M7, GMD and WcaG from plasmids. Ctrl is control strain without FT expression.
Fig. 10 shows 2’-FL formation compared to the base strain, which was capable of producing lower amounts of 2’-FL with integrated 2’-FL pathway consist of GMD, WcaG and WbgL. Strains expressing plasmids with a GMD, a FT and a plasmid expressing a GFS selected from SEQ ID NOs: 20, 21, 22, and 23, respectively, produced significantly more 2’-FL than the base strain.
Likewise, putative FTs were tested by preparing expression constructs containing GMD t and GFS t. An additional plasmid carrying each one of the Fucose transferase genes from SEQ ID NO: 38, 29, 30, 31, 32, and 40 was included in each of these transformations. Cells were transformed with expression plasmids GMD t, GFS t and expression plasmids carrying each one of the FTs genes from SEQ ID NO: 38, 29, 30, 31, 32, and 40 and then selected, grown an analyzed as indicated above.
Fig. 11 shows that strains expressing various FTs accumulate more 2’-FL compared to the base strain.
The activity of an enzyme represented by SEQ ID NO: 24 was tested. This enzyme consists of 2 modules, one that has homology to GDP-Mannose-Dehydratases and one that shares homology with GDP fucose synthases. An enzyme comprising both, GMD and GFS, activities would hence be able to produce GDP fucose from GDP Mannose, NADPFEFE and GTP.
A base strain capable of low level 2’-FL biosynthesis as described above was
transformed with plasmids expressing i) a GMD, a FT and SEQ ID NO: 24 and ii) a FT and SEQ ID NO: 24 only. Cells were transformed, selected and grown as described above. Compared to the base strain, both combinations yielded higher 2’-FL production when compared to the base strain without expression of additional plasmids. The addition of plasmids expressing SEQ ID NO: 24 in absence of an additional plasmids expressing a fucose synthase significantly increases 2’-FL production compared to the base strain. Expression of a plasmid carrying a GMD gene in addition to plasmids carrying a FT and SEQ ID NO: 24 further 2’-FL production.
Fig. 12 shows relative production of 2’-FL in yeast cells expressing plasmids with (lst column) GMD, a FT and SEQ ID NO: 24 and (2nd column) plasmids with a FT and SEQ ID NO: 24 only, relative to a base strain that contains a set of genomic GMD, GFS and FT genes. Fermentation and metabolite analysis
Triplicates of single colonies were inoculated in 10 mL of YPD and incubated at 30 °C overnight. The final fermentation volume was 10 mL in YPDL medium. The cells were incubated at 30 °C and 250 rpm for l20h. Lactose concentration was determined by high performance liquid chromatography on a Prominence HPLC (Shimazu, Kyoto, Japan) equipped with Rezex ROA-Organic Acid H 10 x 7.8 mm column. The column was eluted with 0.005 N of sulfuric acid at a flow rate of 0.6 mL/min, 50 °C. 2’-FL concentration as determined using an ICS-3000 Ion Chromatography System (Dionex, Sunnyvale, CA, EISA) equipped with CarboPac PA20 column. The column was eluted with KOH gradient at a flow rate of 0.4 mL/min, 30 °C. Example 2: 2’-FL production in Saccharomyces cerevisiae, which lacks 2’-FL biosynthesis, by expressing GMD, GFS, and/or FT
A base strain only carrying Lacl2 for improved lactose import and an engineered membrane transporter for improved 2’-FL export as indicated in Fig. 8 was prepared. However, while this strain lacks any genes for 2’-FL biosynthesis it also had not been improved for 2’-FL biosynthesis. This base strain was transformed with plasmids expressing the GMDs encoded by SEQ ID NOs i) 17, ii) 18, and iii) 19. 2’-FL was produced in all these strains indicating that GMDs encoded by SEQ ID NOs: 17, 18, and 19, respectively all confer GMD activity if expressed in yeast cells.
Fig. 13 shows production of 2’-FL by expression of plasmids in a control strain otherwise not capable of 2’-FL production (Ctrl). Strains were transformed with plasmids expressing a GFS and a FT along with a plasmid carrying either SEQ ID NO: 17, 18, or 19, respectively. The control strain carrying no plasmids does not produce any 2’-FL. Example 3: Increase in 2’-FL production in Saccharomyces cerevisiae expressing CDT-1 N209S/F262Y
Strains and Media
S. cerevisiae was grown and maintained on YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 30 °C. All genes were expressed chromosomally. The cdt-lsy gene (encoding CDT-l N209S/F262Y) was expressed within a background strain producing 2’-FL and 2’-FL accumulation in the growth medium was during a fermentation experiment was compared to the 2’-FL accumulation produced from the same strain without the cdt-l-sy gene.
The 2’-FL producing utilizing strain contains GDP-mannose-4, 6-dehydratase ( gmdl ), GDP-L-fucose synthase (wcaG), lactose permease {LAC 12) and two fucosyltransferases ( FucT2 , wbgL ).
The experiments were conducted in YPDL medium (10 g/L yeast extract, 20 g/L peptone, 30 g/L glucose 2 g/L lactose) at 30 °C.
Fermentation and metabolite analysis
Triplicates of single colonies were inoculated in 10 mL of YPD and incubated at 30 °C overnight. The final fermentation volume was 10 mL in YPDL medium. The cells were incubated at 30 °C and 250 rpm for l20h. Lactose concentration was determined by high performance liquid chromatography on a Prominence HPLC (Shimazu, Kyoto, Japan) equipped with Rezex ROA-Organic Acid H 10 x 7.8 mm column. The column was eluted with 0.005 N of sulfuric acid at a flow rate of 0.6 mL/min, 50 °C. 2’-FL concentration as determined using an ICS-3000 Ion Chromatography System (Dionex, Sunnyvale, CA, USA) equipped with CarboPac PA20 column. The column was eluted with KOH gradient at a flow rate of 0.4 mL/min, 30 °C.
The cdt-lsy gene (encoding CDT-l N209S/F262Y) was expressed within a background strain producing 2’-FL and 2’-FL accumulation in the growth medium was during a fermentation experiment was compared to the 2’-FL accumulation produced from the same strain without the cdt-l-sy gene.
Unexpectedly, the expression of CDT-l N209S/F262Y significantly increased the accumulation of 2’-FL within the growth medium (Fig. 2), indicating that CDT-l SY can act as an efficient substrate exporter. Example 4: Increase in 2’-FL production in Saccharomyces cerevisiae expressing CDT-1 mutants
Strains and Media
The 2’-FL producing S. cerevisiae strain contains genome integrated Lacl2 or CDT-l mutants as transporter and 2’-FL producing pathway on pRS424, and pRS426 plasmids consist of GDP-mannose-4, 6-dehydratase ( gmdl ), GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4- reductase (wcaG), and fucosyltransferases (wbgL).
S. cerevisiae was initially grown and maintained in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 30 °C. Optimized minimum medium (oMM) (See Lin Y. et al, Biotechnol Biofuels. 2014 Aug 27;7(l): 126) with 20 g/L of glucose was used for preculture of yeast cells. Verdyun medium (See Verduyn et al, Yeast. 1992 Jul;8(7):50l-l7, see the World Wide Web at apz-rl.de/002_download/003_mitgeltende_dokumente/0l2_Verduyn- Medium_002.pdf) with 60 g/L glucose and 6 g/L lactose (V60D6L) was used for 2’-FL production.
Lactose uptake test
To measure lactose uptake, yeast strains with different transporters were grown in 4 mL YPD medium overnight at 30 °C and 250 rpm. Wild type yeast strain without transporter was used as control. The cell density was measured by a plate reader and was converted to Dry Cell Weight (DCW)The cell culture was washed in water and resuspended in lactose solution. The supernatant was analyzed by HPLC and lactose uptake was normalized by DCW. The lactose uptake from strains expressing CDT-l mutant was normalized by lactose uptake from strain expressing wild type CDT-l and shown as relative values in Figs. 3 and 4.
Fermentation and metabolite analysis
Triplicates of single colonies were inoculated in 10 mL of oMM medium with 20 g/L glucose and incubated at 30 °C overnight. The cell cultures were centrifuged and resuspended in 10 mL V60D6L medium and incubated at 30 °C and 250 rpm for 48 hours. Extracellular lactose, glucose, and 2’-FL concentration was determined by high performance liquid chromatography (HPLC) equipped with Rezex ROA-Organic Acid H 10 x 7.8 mm column and a refractive index detector (RID). The column was eluted with 0.005 N of sulfuric acid at a flow rate of 0.6 mL/min, 50 °C. To measure total (intracellular and extracellular) 2’-FL, the fermentation broth containing yeast cells was boiled to release all of the intracellular 2’-FL. The supernatant was then analyzed by HPLC.
The extracellular and total 2’-FL titer shown in percentage in Figs. 5-7 was normalized by the titer of strains with wild type CDT-l . Extracellular 2’-FL ratio (%) was calculated as follows: (extracellular 2’-FL titer) / (total 2’-FL titer) x 100%.
INCORPORATION BY REFERENCE
Each of the patents, published patent applications, and non-patent references cited herein are hereby incorporated by reference in their entirety. EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:
1. A microorganism for enhanced production of a human milk oligosaccharide (HMO) comprising a heterologous CDT-l transporter or a variant thereof and at least one heterologous pathway gene for production of the HMO.
2. The microorganism of claim 1 , wherein the microorganism is capable of producing and exporting the HMO.
3. The microorganism of claim 2, wherein the transporter is capable of exporting at least 20%, 30%, 40%, 50%, or 60% of the produced HMO.
4. The microorganism of claim 2 or 3, wherein the microorganism is capable of exporting at least 50% more of the HMO than a parental microorganism lacking the transporter.
5. The microorganism according to any one of claims 1-4, wherein the yeast comprises a transporter that has an amino sequence of SEQ ID NO:4 or a sequence with at least 80%, 85%, 90%, 95%, 98% or 99% homology thereto.
6. The microorganism according to any one of claims 1-5, wherein the transporter comprises a PESPR motif.
7. The microorganism according to any one of claims 1-6, wherein the transporter comprises a sequence having one or more amino acid replacements at positions corresponding to ammo acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO: 4.
8. The microorganism according to any one of claims 1-7, wherein the CDT-l is encoded by a codon optimized nucleic acid.
9. The microorganism according to claim 8, wherein at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast.
10. The microorganism according to any one of claims 7-9, wherein the transporter comprises an amino acid replacement selected from the group consisting of 91A, 209S, 213A, 256V, 262Y, 262 W, 335A, 411 A and any combination thereof.
11. The microorganism according to any one of claims 1-10, wherein the pathway gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an - 1 ,2-fucosyl transferase.
12. The microorganism of claim 11, comprising a second heterologous pathway gene.
13. The microorganism according to any one of claims 1-12, wherein the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N-tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N-fucopentaose I (LNFP I).
14. The microorganism of claim 13, wherein the HMO is 2’- fucosyllactose.
15. The microorganism according to any one of claims 1-14, wherein the microorganism is an Ascomycetes fungus.
16. The microorganism of claim 15, wherein the Ascomycetes fungus is selected from the group consisting of a Sacharomyces spp., a Schizosaccharomyces spp. and a Pichia spp.
17. The microorganism of claim 15, wherein the Ascomycetes fungus is selected from the group consisting of Trichoderma, Kluyveromyces, Yarrowia, Aspergillus, and Neurospora.
18. The microorganism according to any one of claims 1-17, wherein one or both of the heterologous CDT-l transporter and the pathway gene are integrated into the yeast chromosome.
19. The microorganism according to any one of claims 1-17, wherein one or both of the heterologous CDT-l transporter and the pathway gene are episomal.
20. The microorganism according to any one of claims 1-19, comprising a set of pathway genes for production of the HMO.
21. The microorganism of claim 20, wherein the set comprises GDP-mannose 4,6- dehydratase (GMD), a GDP-L-fucose synthase (GFS), and a fucosyl transferase (FT).
22. The microorganism of claim 20, wherein the set comprises GDP-mannose 4,6- dehydratase, a GDP-L-fucose synthase, and an -1 ,2-fucosyl transferase and wherein the HMO is 2’-FL.
23. The microorganism of claim 20, wherein the set comprises a bifunctional fucokinase/L- fucose- 1 -P-guany ly ltransf erase.
24. The microorganism of claim 20, wherein the set comprises an enzyme capable of converting fucose and ATP to fucose-l -phosphate and an enzyme capable of converting the fucose-l -phosphate and GTP to GDP-fucose, and a glucosyl transferase.
25. The microorganism of claim 24, wherein the glucosyl transferase is an - 1 ,2-fucosyl transferase and wherein the HMO is 2’-FL.
26. The microorganism of any one of claims 21-22, where the set of pathway genes comprises Gmd, WcaG and WbgL.
27. The microorganism of claim 21, wherein the GDP-mannose 4,6-dehydratase is selected from SEQ ID Nos. 17-19, 42, and 61-63 or a variant having at least 85% homology thereto.
28. The microorganism of claim 21, wherein the GDP-L-fucose synthase is selected from SEQ ID Nos. 20-23 or a variant having at least 85% homology thereto.
29. The microorganism of claim 21, wherein the - 1 ,2-fucosyl transferase is selected from SEQ ID Nos. 26-40 or a variant having at least 85% homology thereto.
30. A method of producing an HMO comprising:
providing a culture medium with at least one carbon source;
providing a microorganism capable of producing and exporting an HMO, wherein the microorganism expresses a heterologous transporter and one or more heterologous genes for the production of the HMO; and
culturing microorganism in the culture medium;
wherein a substantial portion of the HMO is exported into the culture medium.
31. The method of claim 30, further comprising separating the culture medium from the microorganism.
32. The method of claim 31, further comprising isolating the HMO from the culture medium.
33. The method according to any one of claims 30-32, wherein the heterologous transporter is CDT-l, CDT-2 or a variant thereof.
34. The method according to any one of claims 30-33, wherein the HMO is 2’-FL.
35. The method of claim 33, wherein the transporter is a CDT-l variant comprising an amino acid sequence having one or more amino acid replacements at positions corresponding to amino acid positions 91, 209, 213, 256, 262, 335, 411 of SEQ ID NO:4.
36. The method according to any one of claims 30-35 wherein the CDT-l is encoded by a codon optimized nucleic acid.
37. The method of claim 36, wherein at least the first 90 nucleotides of the nucleic acid are codon optimized for yeast or at least 5% of the nucleic acid is codon optimized for yeast.
38. The method of claim 35, wherein the transporter comprises an amino acid replacement selected from the group consisting of 91A, 209S, 213A, 256V, 262Y, 262W, 335A, 411A and any combination thereof.
39. The method according to any one of claims 30-38, wherein the heterologous gene is selected from a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, and an□-l,2-fucosyl transferase.
40. The method according to any one of claims 30-39, wherein the export of the HMO is increased as compared to a parental microorganism that does not contain the heterologous transporter.
41. The method according to any one of claims 30-41 , wherein the heterologous transporter is capable of importing lactose and exporting the HMO.
42. The method according to any one of claims 30-41, wherein the culture medium comprises lactose.
43. The method of claim 30, wherein the ratio of the HMO in the culture medium to total HMO produced by the microorganism is at least about 1 : 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1 or greater than 4: 1.
44. The method according to any one of claims 30-43, wherein the HMO is selected from the group consisting of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3’-sialyllactose (3'-SL), 6’-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), sialyllacto-N- tetraose a (LST a), sialyllacto-N-neotetraose c (LST c), lacto-difucotetraose (LDFT) and lacto-N- fucopentaose I (LNFP I).
45. The method according to any one of claims 30-44, wherein the microorganism is according to any one of claims 1-29.
46. A product suitable for animal consumption comprising the microorganism according to any one of claims 1-29, the HMO produced by the microorganism according to any one of claims 1-29 or according to the method of any one of claims 30-45 and at least one additional consumable ingredient.
47. The product of claim 46, wherein the product is suitable for human consumption.
48. The product of claim 47, wherein the product is an infant formula, an infant food, a nutritional supplement or a prebiotic product.
49. The product of claim 46 suitable for mammalian consumption.
50. The product of claim 46, further comprising at least one additional human milk oligosaccharide.
51. The product of claim 46, wherein the additional consumable ingredient is selected from a protein, a lipid, a vitamin, a mineral or any combination thereof.
52. The product of claim 49 suitable for use as an animal feed.
PCT/US2019/054258 2018-10-02 2019-10-02 Use of substrate importers for the export of oligosaccharides WO2020072617A1 (en)

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