WO2019175635A1 - Nouveau biocatalyseur à cellules entières pour la production de tréhalulose - Google Patents

Nouveau biocatalyseur à cellules entières pour la production de tréhalulose Download PDF

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WO2019175635A1
WO2019175635A1 PCT/IB2018/051736 IB2018051736W WO2019175635A1 WO 2019175635 A1 WO2019175635 A1 WO 2019175635A1 IB 2018051736 W IB2018051736 W IB 2018051736W WO 2019175635 A1 WO2019175635 A1 WO 2019175635A1
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sucrose
gene
recombinant
host cell
cells
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PCT/IB2018/051736
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Saravanakumar IYAPPAN
Karthikeyan VENKATA NARAYANAN
Murali Thumala
Banibrata Pandey
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Petiva Private Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99011—Isomaltulose synthase (5.4.99.11)

Definitions

  • the present invention pertains to the field of enzyme engineering. More particularly, the invention relates to a novel whole cell biocatalyst for production of trehalulose from sucrose and the process for development thereof.
  • Trehalulose (a-D-glucosylpyranosyl-l,l-D fmctofuranose) is a structural isomer of sucrose which is composed of glucose and fructose joined by an alpha (1-1) glycosidic bond. It is naturally present in honey in very low quantities. In addition to sweetness, trehalulose shows physical and organoleptic characteristics that are very similar to sucrose. Further, trehalulose is non-cariogenic, has low glycemic index and is a low-insulinemic sugar. Absence of toxicity, mutagenicity and other side effects makes trehalulose a suitable substitute for sugar in various foods and beverages. Trehalulose has been reported to be suitable for consumption by public.
  • Enzymatic bioconversion is a preferred method for the production of trehalulose because of the complexity in its chemical synthesis.
  • sucrose isomerase activity The current methods used for conversion of sucrose into isomers, particularly trehalulose by using sucrose isomerase activity involves following approaches:
  • sucrose isomerase production of sucrose isomerase in recombinant host
  • the inventors have identified the above issues and addressed the same by employing a multidimensional approach wherein recombinant yeast strains have been created by inactivating sucrose hydrolyzing genes in a host cell combined with the expression of sucrose isomerase on the surface of the host by way of fusion protein.
  • the host organism In the absence of SUC2 and AGT1 gene, the host organism is unable to utilize the disaccharide sucrose for metabolism. Subsequently, the yield of trehalulose is high and the downstream processing cost is low.
  • the present invention thus addressed the drawbacks of existing approaches to solve a long-standing problem of providing an efficient, cheap and industrially-scalable means for production of trehalulose.
  • the present invention relates to a novel whole cell biocatalyst for production of trehalulose from sucrose.
  • the recombinant biocatalyst has been developed by deletion of SUC2 and AGT1 encoding genes, which are responsible for hydrolyzing sucrose in a host cell combined with the expression of sucrose isomerase on the surface of the host by way of fusion protein.
  • the biocatalyst shows dramatic decrease in utilization of the disaccharide sucrose for metabolism.
  • the sucrose remained available for the bioconversion in to trehalulose and the recombinant cells exhibits a high rate of bioconversion.
  • the invention discloses a modified open reading frame encoding for sucrose isomerase enzyme fused to the C-terminus of GPI anchor protein.
  • the invention also discloses vectors comprising the modified open reading frame under the control of constitutive or inducible promoter.
  • the invention provides for a fusion protein which is a sucrose isomerase enzyme fused to C-terminus of a cell surface anchor protein.
  • the invention also provides for an efficient bioconversion process for production of trehalulose using the recombinant biocatalyst.
  • the one-step production process can be performed under a wide range of physical and chemical conditions to obtain optimum yield of trehalulose.
  • the biocatalyst used in the process is reusable and provides for efficient and cheap downstream processing.
  • Fig. 1 shows the vector map of recombinant plasmid pGH-SI_R3 which shows the gene construction for constitutive expression of recombinant fusion protein.
  • Fig. 2 shows the vector map of recombinant plasmid pGL-SI_R3 which shows the gene construction for inducible expression of recombinant fusion protein.
  • Fig. 3 shows the sucrose and residual sugars after growth of wild type strains (shown as wild type) and modified strains (shown as NY -EM2 and NY -YM2) in the presence of synthetic growth media containing sucrose, glucose and fructose.
  • Fig. 4 shows the residual sucrose after growth of wild type strains compared with NY- EM2 and NY-YM2 strains in synthetic growth media containing 20 g/L sucrose, 20 g/L sucrose supplemented with 20 g/L glucose or 20 g/L sucrose supplemented with 20 g/L fructose.
  • Lig. 5 shows the residual invertase activity of NY -EM2 and NY -YM2 strains compared to respective wild type strains.
  • Lig. 6 shows the microscopy of immunofluorescence-labeled recombinant yeast cells.
  • Lig. 7 shows the expression profile of cell surface displayed sucrose isomerase by immunoblot analysis from different fractions of cell lysate from constitutive and inducible expression strains compared with the native strains.
  • Lig. 8 shows the fermentation kinetics and cell surface displayed sucrose isomerase activity by recombinant constitutive expression strain NY-YM2 (pGH-SI_R3).
  • Lig. 9 shows the fermentation kinetics and cell surface displayed sucrose isomerase activity by recombinant inducible expression strain NY-EM2 (pGL-SI_R3).
  • Lig. 10 shows the expression profile of CSD-SIase by immunoblot analysis from samples collected from constitutive [NY-YM2 (pGH-SI)] and inducible expression [NY-EM2 (pGL-SI)] strains at different fermentation time points.
  • Lig. 11 shows the product formation kinetics using different amount of CSD-SIase cells.
  • Lig. 12 shows the product formation kinetics using different amount of sucrose as substrate with CSD-SIase.
  • Lig. 13 shows the chromatogram of samples after bioconversion of sucrose into trehalulose by recombinant strains constitutively producing CSD-SIase.
  • Lig. 14 shows the bioconversion kinetics of CSD-SIase produced by constitutive expression strain [NY-YM2 (pGH-SI)].
  • Fig. 15 shows the bioconversion kinetics of CSD-SIase produced by inducible expression strain [NY-EM2 (pGL-SI)].
  • Fig. 16 shows the temperature optima profiles of CSD-SIase and native Slase.
  • Fig. 17 shows the pH profiles of CSD-SIase and native Slase.
  • Fig. 18 shows the residual activity of CSD-SIase enzymes compared to native Slase enzyme.
  • the present invention discloses a genetically modified host cell, which can be used as a whole cell biocatalyst for the production of trehalulose from sucrose.
  • the invention contemplates a multidimensional approach for achieving a high rate of bioconversion of sucrose into trehalulose using a whole cell biocatalyst.
  • the biocatalyst created for efficient, cheap and industrially scalable bioconversion of sucrose to trehalulose is characterized by the following:
  • sucrose hvdrolvzation - Sucrose is utilized by a large array of host organisms for metabolism. Therefore, most of the sucrose present in the substrate is utilized by the host organism and is not available for bioconversion.
  • the present invention overcomes this issue.
  • host cell includes an individual cell or cell culture which can be, or has been, a recipient for the subject of expression constructs.
  • Host cells include progeny of a single host cell.
  • Host cell can be any expression host including prokaryotic cell such as but not limited to Escherichia coli, Bacillus subtilis, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.
  • recombinant strain refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.
  • expression cassette denotes a gene sequence used for cloning in expression vectors or in to integration vectors or integrated in to coding or noncoding regions of chromosome of the host cell in a single or multiple copy numbers, where the expression cassette directs the host cell's machinery to make RNA and protein encoded by the expression cassette.
  • expression construct is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant proteins/peptides, when introduced into an appropriate host cell, can be transcribed and translated into a fusion protein which is displayed on the cell wall.
  • promoter refers a DNA sequences that define where transcription of a gene begins. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription.
  • constitutive promoter is more commonly defined the promoter which allows continual transcription of its associated genes as their expression is normally not conditioned by environmental and developmental factors. Constitutive promoters are very useful tool in genetic engineering because constitutive promoters drive gene expression under inducer-free conditions and often show better characteristics than commonly used inducible promoters.
  • inducible promoter refers the promoters that are induced by the presence or absence of biotic or abiotic and chemical or physical factors. Inducible promoters are a very powerful tool in genetic engineering because the expression of genes operably linked to them can be turned on or off at certain stages of development or growth of an organism or in a particular tissue or cells.
  • RNA messenger RNA
  • translation refers the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis.
  • mRNA messenger RNA
  • the genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes.
  • the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.
  • terminal sequence refers to a nucleotide sequence that is required for the termination reaction of the transcription process. Termination involves recognition of the point at which no further bases should be added to a growing RNA chain.
  • fusion protein refers to a polypeptide which comprises protein domains from at least two different proteins.
  • the fusion protein is sucrose isomerase fused to C- terminus of a cell surface anchor protein.
  • sucrose hydrolyzing genes refers to genes which are responsible for sucrose hydrolysis.
  • sucrose hydrolyzing genes refers to SUC2 invertase and AGT1 alpha-glucoside transporter.
  • inducible excision system refers to site-specific recombinase technologies which can be used for efficiently regulating the excision or deletion of genes.
  • the inducible excision system employed in the invention is cre-lox excision system.
  • replication origin refers to the site on a nucleic acid sequence at which replication is initiated. Bacterial or yeast replication origins may be required in a recombinant vector for significant expression of the desired polypeptide.
  • selection marker gene refers to a gene determinant that, when expressed in the cell, confers a specific set of characteristics upon the cell that allows such a cell to be distinguished, or selected out, from other cells not carrying or expressing said gene determinant.
  • anchor protein or "cell surface anchor protein” is used to describe proteins or peptides which are anchored to the external surface of the plasma membrane generally by covalent bonding to glycans containing phosphatidyl inositol.
  • the structures to which the anchor protein or peptide is bonded are often referred to as glycosylphosphatidylinositol or GPIs.
  • GPIs glycosylphosphatidylinositol
  • anchor proteins covalently bonded to GPIs are found on the external face of the plasma membrane of cells or on the lumenal surface of secretory vesicles.
  • the anchor protein is a GPI anchor protein, AGA2 which is fused in frame to the N-terminus of sucrose isomerase.
  • modified sucrose isomerase is used to refer to a fusion protein containing GPI anchor protein such as AGA2 fused in frame to the N-terminus of sucrose isomerase.
  • GPI anchor protein such as AGA2 fused in frame to the N-terminus of sucrose isomerase.
  • the N- terminus region of the fusion protein will anchor to the cell surface of the host cell and display the free sucrose isomerase over the cell wall for bioconversion of sucrose into trehalulose.
  • specific activity is defined as the micromoles of product formed per minute per milligram of enzyme.
  • NY-YM denotes the wild type yeast strain ATCC 208352 and“NY-YM2” denotes the modified yeast strain after deletion of invertase (SUC2) and symporter (AGT1) genes.
  • SUC2 invertase
  • AGT1 symporter
  • NY-EM strain denotes the wild type yeast strain ATCC 208289 and“NY-EM2” denotes the modified yeast strain after deletion of invertase (SUC2) and symporter (AGT1) genes.
  • SUC2 invertase
  • AGT1 symporter
  • GPI-SI nucleotide sequence designates fusion protein of sucrose isomerase fused in frame with cell surface anchor protein.
  • NY-YM2 pGH-SI_R3 designates the final transformed yeast strain having the artificially synthesized gene encoding for sucrose isomerase of Pseudomonas mesoacidophila under constitutive promoter control.
  • NY-EM2 (pGL-SI_R3) designates the final transformed yeast strain having the artificially synthesized gene encoding for sucrose isomerase of Pseudomonas mesoacidophila under inducible promoter control.
  • the present invention discloses novel whole cell biocatalysts for production of trehalulose from sucrose and the process for development thereof. Further, the invention also nucleic acids which encode sucrose isomerase enzyme fused to a cell surface anchor protein. Further, the invention discloses a recombinant cell in which the invertase and sucrose permease encoding genes have been made inoperative, and the recombinant cell has been engineered to display sucrose isomerase enzyme on the cell surface.
  • the present invention provides a modified nucleic acid encoding sucrose isomerase gene fused in frame with cell surface anchor proteins.
  • the modified nucleic acid may employ native genes or synthetic genes optimized as per the codon preference of the host organism.
  • the modified nucleic acid is the nucleic acid encoding for sucrose isomerase (Slase) of Pseudomonas mesoacidophila, fused in frame with cell surface anchor proteins, such as, but not limited to GPI proteins like AGA2 protein.
  • Slase sucrose isomerase
  • the modified nucleic acid is represented by SEQ ID NO: 1.
  • the invention provides for a fusion protein, which was constructed by the cell surface anchor protein of Saccharomyces cerevisiae being fused in-frame with the N- terminus of sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX-45.
  • the fusion protein is represented by SEQ ID NO:6.
  • the present invention provides a modified expression cassette comprising a promoter, a modified open reading frame encoding for sucrose isomerase enzyme fused to the C-terminus of GPI anchor protein and a terminator sequence.
  • the promoter chosen may either be for constitutive expression or for inducible expression.
  • the modified expression cassette can express sucrose isomerase on the surface of a wide range of host organisms, such as, but not limited to Saccharomyces cerevisiae.
  • the construct carries a constitutive promoter, such as, but not limited to, GAPDH promoter.
  • the construct carries an inducible/regulated promoter, such as, but not limited to GAL1, pGALlO, pSUC2, pXYL or pADH promoter.
  • an inducible/regulated promoter such as, but not limited to GAL1, pGALlO, pSUC2, pXYL or pADH promoter.
  • the expression cassette for constitutive expression comprises the modified nucleic acid represented by SEQ ID NO: l, a GAPDH promoter represented by SEQ ID NO:2 and a GAPDH terminator represented by SEQ ID NO:4.
  • the expression cassette for inducible expression comprises the modified nucleic acid represented by SEQ ID NO: l, a GAL1 promoter represented by SEQ ID NOG and a MFa terminator represented by SEQ ID NO: 5.
  • the invention provides for expression vectors comprising the expression cassette.
  • the modified expression cassettes are cloned into expression vectors for recombinant expression.
  • the expression vectors can be used in both prokaryotic as well as eukaryotic organisms.
  • the shuttle vector used provides for a number of selectable markers such as, but not limited to, ampicillin resistance marker, uracil selectable marker or tryptophan selectable maker.
  • the vector also comprises of bacterial origin of replication, 2m for replication in yeast or CEN for replication in yeast.
  • the expression vector for constitutive expression is a pYEP plasmid, more specifically a pGH plasmid.
  • the recombinant plasmid contains the open reading frame, a constitutive GAPDH promoter, GAPDH terminator, ampicillin resistance marker for bacterial selection, pBR322 for bacterial origin of replication, URA (uracil) auxotrophic markers for selection in yeast and 2m for replication in yeast.
  • the modified sequence encoding for the fusion protein was cloned in to pGH yeast expression vector using Nhel and Xhol sites in frame with GPI anchor which is under control of constitutive promoter yielding pGH_GPI-SI_R3 (also referred to as pGH-SI_R3).
  • GPI-sucrose isomerase (GPI-SI) gene in pGH-SI_R3 plasmid is flanked by EcoRI at 5’end and Hindlll at 3’end. Sall and Xhol were lost during the cloning procedure.
  • the modified vector is depicted in Figure 1.
  • the expression vector for inducible expression is a pRS3 l4 plasmid, more specifically, pGL plasmid.
  • the recombinant plasmid contains an inducible promoter, MFa (Alpha-factor) terminator, Ampicillin resistance marker for bacterial selection, fl for bacterial origin of replication, TRP (tryptophan) auxotrophic markers for selection in yeast and CEN for replication in yeast.
  • the modified sequence was cloned in to pGL yeast expression vector using Nhel and Bam HI sites in frame with GPI anchor which is under control of inducible promoter yielding pGL_GPI-SI_R3 (also referred to as pGL-SI_R3).
  • GPI-sucrose isomerase (GPI-SI) gene in pGL-SI_R3 plasmid is flanked by EcoRI at 5’end, and BamHI at 3’end.
  • the modified vector is depicted in Figure 2.
  • the invention provides host cells in which sucrose hydrolyzing genes have been inactivated by knock-out or deletion.
  • the invention provides yeast strains which lacks expression of both SUC2 invertase and AGT1 alpha-glucoside transporter.
  • the strains are characterized by extremely low sucrose hydrolysis ability. Therefore, the problem of rapid depletion of sucrose due to metabolism of cells is avoided and the complete substrate is available for bioconversion to desirable products by cell surface display of recombinant enzymes.
  • the deletion of genes encoding for SUC2 and AGT1 in the host strain is done using an inducible gene excision system, such as, cre-lox system.
  • Gene disruption cassettes designed with the heterologous dominant kanamycin resistant marker with a Cre/loxP mediated marker removal procedure is used.
  • the loxP-Marker gene-loxP gene disruption cassettes are generated by PCR amplification using pUG6 vector as template. Flanking homology sequences to the AGT1 gene upstream and downstream to the target gene are added to the disruption cassette primers.
  • the PCR product was used as a template for further long flanking homology ends using a 2nd set of primers to add more nucleotide sequence for an accurate homologous recombination.
  • the amplified products were used as deletion cassettes and transformed into yeast strain, such as, but not limited to, W303-1A or BJ5465 strains.
  • yeast strain such as, but not limited to, W303-1A or BJ5465 strains.
  • the invention provides for engineered strain which can grow in media with glucose or fructose. But the engineered strains show severe growth defect when grown in media with sucrose alone.
  • sucrose hydrolysis is dramatically decreased in engineered strains compared to their wild type strains. Additionally, the engineered strains NY-EM2 and NY-YM2 hydrolyzes sucrose very slowly in the presence of additional glucose or fructose in growth media when compared to its wild type strains.
  • the invention provides for the development of recombinant host cells.
  • the recombinant plasmids encoding a cell surface anchor protein of Saccharomyces cerevisiae being fused in-frame with the N-terminus of sucrose isomerase (Slase) of Pseudomonas mesoacidophila are transformed into a yeast host strains which lacks both SUC2 invertase and AGT1 alpha-glucoside transporter to create a whole cell biocatalyst with a high rate of bioconversion.
  • the biocatalyst shows dramatic decrease in utilization of the disaccharide sucrose for metabolism.
  • the sucrose remained available for the bioconversion in to trehalulose and the recombinant cells exhibits a high rate of bioconversion.
  • the engineered strain may also contain additional modifications for preproduction of AGA1 protein under control of GAL1 promoter and/or deletion of major Pir (Pir 1-3) proteins for enhanced display of cell surface anchor proteins.
  • the invention relates to recombinant host cell which has been developed by deletion of SUC2 and AGT1 encoding genes in a host cell combined with the expression of sucrose isomerase on the surface of the host by way of fusion protein.
  • the recombinant host cell has been engineered for constitutive expression.
  • the cells were treated to prepare electrocompetent cells.
  • the recombinant plasmids pGH-SI_R3 were transformed in to electrocompetent cells by standard electroporation method.
  • the strains transformed with pGH-SI_R3 were selected on SD-Ura plates.
  • the invention relates to recombinant host cell which has been developed by deletion of SUC2 and AGT1 encoding genes in a host cell combined with the expression of sucrose isomerase on the surface of the host by way of fusion protein.
  • the recombinant host cell has been engineered for inducible expression.
  • the cells were treated to prepare electrocompetent cells.
  • the recombinant plasmids pGL-SI_R3 were transformed in to electrocompetent cells by standard electroporation method.
  • the strains transformed with pGH-SI_R3 were selected on SD-Trp plates.
  • the yeast strains used were procured from ATCC (American Type Culture Collection: Global Bioresource Centre) headquartered in Manassas, Virginia, USA.
  • ATCC 208352 (Strain Designation: W303-la) has been used to develop MTCC 5985 recombinant host cell for constitutive CSD-SIase expression
  • ATCC 208289 (Strain designation: BJ5465) has been used to develop MTCC 5987 recombinant host cell for inducible CSD-SIase expression. Both the strains were deposited on 2lst January, 2015.
  • CSD-SIase shows significant bioconversion activity not only at the optimum temperature but even at lower temperatures in the range of l5-40°C as compared to the native enzyme.
  • the invention provides that the optimum temperature for CSD sucrose isomerase is in the range of l5°C to 40°C, more precisely 25°C compared to native enzyme which is in the range of l5°C to 40°C, more precisely 30°C.
  • a further aspect of the present invention is that the optimum pH for CSD sucrose isomerase is in the range of 4 to 6.5, more precisely 6.2 as compared to native enzyme which is in the range of 5.0 to 6.7, more precisely 6.5.
  • the cell surface displayed sucrose isomerase exhibits broader pH tolerance higher than that of the native enzyme.
  • the invention provides for an industrially scalable process for production of trehalulose.
  • the process includes culturing the recombinant Saccharomyces cerevisiae host cells in defined media.
  • the culture medium has pH ranging between 4 to 6.5 and temperature maintained between l5°C to 30°C. Further, the recombinant host cells are contacted with 10% to 45% sucrose solution and trehalulose is harvested from the solution.
  • bioconversion depends upon the amount of substrate used.
  • the amount of substrate can be increased upto 45% for efficient conversion of sucrose into trehalulose.
  • Another aspect of the invention provides that cell surface displayed sucrose isomerase is stable and retains more than 50% of its activity for up to 120 hrs at optimum pH and temperature when compared to native enzyme.
  • Example 1 Gene construction for constitutive expression of recombinant fusion protein GPI-SI in Saccharomyces cerevisiae
  • sucrose isomerase (SIase) was modified for expression of sucrose isomerase on the surface of Saccharomyces cerevisiae cells.
  • the modified gene contains a constitutive promoter, a modified open reading frame encoding for sucrose isomerase enzyme fused to the C-terminus of GPI anchor protein and a terminator sequence.
  • the sequence of the modified open reading frame encoding for sucrose isomerase enzyme fused to the C-terminus of GPI anchor protein is represented by SEQ ID NO: 1.
  • the constitutive promoter used is GAPDH promoter sequence which is represented by SEQ ID NO: 2.
  • the terminator used is a GAPDH terminator which is represented by SEQ ID NO: 4.
  • This modified open reading frame has been artificially synthesized by using the sequence for sucrose isomerase of Pseudomonas mesoacidophilaMX-4-5 and the sequence of AGA2 protein from Saccharomyces cerevisiae.
  • the plasmid used in the process was a pYEP plasmid, more specifically pGH plasmid.
  • the recombinant plasmid contains the open reading frame, a constitutive GAPDH promoter, GAPDH terminator, Ampicillin resistance marker for bacterial selection, pBR322 for bacterial origin of replication, UR A (uracil) auxotrophic markers for selection in yeast and 2m for replication in yeast.
  • the modified sequence encoding for the fusion protein was cloned in to pGH yeast expression vector using Nhel and Xhol sites in frame with GPI anchor which is under control of constitutive promoter yielding pGH_GPI-SI_R3(also referred to as pGH-SI_R3).
  • the vector map is represented in Figure 1.
  • GPI-sucrose isomerase (GPI-SI) gene in pGH-SI_R3 plasmid is flanked by EcoRI at 5’end and Hindlll at 3’end. Sall and Xhol were lost during the cloning procedure. Recombinant plasmids were confirmed by restriction digestion analysis and followed by DNA sequencing.
  • Example 2 Gene construction for inducible expression of recombinant fusion protein GPI- SI in Saccharomyces cerevisiae
  • sucrose isomerase was modified for expression of sucrose isomerase on the surface of Saccharomyces cerevisiae cells.
  • the modified gene contains an inducible promoter, a modified open reading frame encoding for sucrose isomerase fused to the C-terminus of GPI anchor protein and a terminator sequence.
  • the sequence of the modified open reading frame encoding for sucrose isomerase enzyme fused to the C-terminus of GPI anchor protein is represented by SEQ ID NO: 1.
  • the inducible promoter used is GAL1 promoter sequence which is represented by SEQ ID NO: 3.
  • the terminator used is a MFa (Alpha-factor) terminator which is represented by SEQ ID NO: 5.
  • the plasmid used in the process was a pRS3l4 plasmid, more specifically pGL plasmid.
  • the recombinant plasmid contains an inducible promoter, MFa (Alpha-factor) terminator, Ampicillin resistance marker for bacterial selection, fl for bacterial origin of replication, TRP (tryptophan) auxotrophic markers for selection in yeast and CEN for replication in yeast.
  • the modified sequence was cloned in to pGL yeast expression vector using Nhel and Bam HI sites in frame with GPI anchor which is under control of inducible promoter yielding pGL_GPI-SI_R3 (also referred to as pGL-SI_R3).
  • the vector map is represented in Figure 2.
  • GPI-sucrose isomerase (GPI-SI) gene in pGL-SI_R3 plasmid is flanked by EcoRI at 5’end, and BamHI at 3’end. Recombinant plasmids were confirmed by restriction digestion analysis and followed by DNA sequencing.
  • Example 3 Polynucleotide sequence for expression of sucrose isomerase and corresponding polypeptide sequence
  • a modified open reading frame represented by SEQ ID NO: 1 was artificially synthesized for encoding a fusion protein wherein a polynucleotide sequence coding for cell surface anchor protein was fused in-frame with a polynucleotide sequence encoding sucrose isomerase (Slase) enzyme.
  • Slase sucrose isomerase
  • the fusion protein obtained by translating the gene encoding for sucrose isomerase fused to the C-terminus of GPI anchor protein is represented by SEQ ID NO:6.
  • the fusion protein is a cell surface anchor protein of Saccharomyces cerevisiae being fused in-frame with the N- terminus of sucrose isomerase (Slase) of Pseudomonas mesoacidophilaMX-4-5.
  • Example 4 Development of strain for reduced ability to hydrolyze sucrose by deletion of AGT1 and SUC2 gene
  • yeast cells were grown in rich media such as Yeast Extract Peptone Dextrose (YEPD) broth and 50 mL of the yeast cells at log-phase were collected by centrifugation at 2000 g, washed with phosphate buffer twice and resuspended in 1M chilled sorbitol. The cells were washed once in 1M chilled sorbitol, resuspended in 5 mL of chilled sorbitol and used for the electro-transformation with the disruption cassettes for AGT1.
  • rich media such as Yeast Extract Peptone Dextrose (YEPD) broth
  • 50 mL of the yeast cells at log-phase were collected by centrifugation at 2000 g, washed with phosphate buffer twice and resuspended in 1M chilled sorbitol.
  • the cells were washed once in 1M chilled sorbitol, resuspended in 5 mL of chilled sorbitol and used for the electro-transformation with the disruption cassettes for A
  • the gene disruption cassette was designed with the heterologous dominant Kan R resistant marker with a Cre/loxP mediated marker removal procedure.
  • the loxP-Marker gene-loxP gene disruption cassettes were generated by PCR amplification using pUG6 deletion cassette plasmid as template and primers having flanking homology sequences to the AGT1 gene (30 bp) upstream and downstream to the target gene followed by the sequence homologous to universal loxP-marker amplification sequence.
  • the disruption cassette used for deletion of AGT1 gene is represented by SEQ ID NO: 7.
  • the amplified PCR product was purified and was reused as template for second PCR amplification and primers having long flanking homology sequences to the AGT1 gene further upstream and downstream to the target gene followed by the sequence homologous to first set of deletion cassette amplification primer sequence.
  • the amplified loxP-marker gene-loxP gene disruption cassette with long (60 bp) flanking ends homology to AGT1 gene sequence was purified and were transformed into electrocompetent yeast cells by electroporation using the rapid DNA transformation protocol as described in Methods in Enzymology, Vol 191.
  • the transformants were plated on YPD plates containing 200pg/mL G418 and incubated for 4 days at 28°C. Putative transformants obtained were restreaked on YPD agar plated containing 200 pg/mL G418. The transformants were verified by PCR with appropriate primers for confirmation of deletion of the AGT1 gene in the chromosome by comparing the amplified product with the PCR product obtained from the wild type genomic DNA. Among transformants, the strains NY-EM1 and NY-YM1 showed promising results with sucrose uptake studies which is used as host strain for further development.
  • SUC2 knockout cassette was designed using loxP flanking end with BLE (phleomycin) marker which confers resistance to phleomycin in yeast for the selection.
  • the disruption cassette was PCR amplified using pUG66 deletion cassette plasmid as template and further amplified using the primary PCR product as template for long flanking homology ends with SUC2 upstream and downstream sequences in the primers.
  • the disruption cassette used for deletion of SUC2 gene is represented by SEQ ID NO: 8.
  • the PCR product was purified and transformed into electrocompetent NY-EM1 and NY- YM1 yeast cells and selected on YEPD plates containing 7.5 pg/mL phleomycin. The transformants were verified by PCR with respective primers for confirmation of deletion of the SUC2 gene in the chromosome and by comparing the amplified product with the PCR product obtained from the wild type genomic DNA.
  • the developed double deletion strain was designated as NY -EM2 and NY -YM2.
  • Example 5 Sucrose utilization studies by recombinant NY-EM2 and NY-YM2 strains
  • the double mutant strains NY -EM2 and NY -YM2 were tested for their ability to grow in media containing sucrose alone or in combination with glucose or fructose.
  • the growth profile and the residual sugars present in growth media at different time points were analyzed for utilization of sugars for cell growth and the presence of residual invertase activity for sucrose hydrolysis respectively.
  • the mutant strains showed slow growth behavior when grown in sucrose compared to their wild type background due to reduced ability of sucrose uptake and hydrolysis.
  • the uptake and utilization of sucrose, glucose and fructose were individually studied.
  • the wild type strains and modified strains were grown. Samples were collected at different time points and the availability of residual sucrose, glucose and fructose were analyzed by HPLC analysis with appropriate standards.
  • Both NY -YM2 and NY -EM2 strains show dramatic reduction in sucrose hydrolysis when grown on synthetic growth media containing sucrose as carbon source compared to their wild type strains NY-YM and NY-EM, respectively.
  • the NY-YM2 strain hydrolyzed only 22% of sucrose. Hence 78% of sucrose remains in the medium after 24hrs incubation whereas the wild type NY-YM hydrolyzed 52% of sucrose. Hence, only 48% of sucrose remains in the medium after 24hrs incubation. This result highlights that the NY-YM2 strain dramatically lost the ability to hydrolyze the sucrose for its growth and the use of this stain makes 30% more sucrose available for bioconversion in to trehalulose by the whole cell biocatalyst.
  • the NY-EM2 strain hydrolyzed only 33% of sucrose. Hence, 67% of sucrose remains in the medium after 24hrs incubation.
  • the wild type NY-EM hydrolyzed 68% of sucrose and hence, only 32% of sucrose remains in the medium after 24hrs incubation.
  • This result highlights that the NY-EM2 strain also dramatically lost the ability to hydrolyze the sucrose for its growth and makes 35% more sucrose available for bioconversion in to trehalulose by sucrose isomerase. The results of this study are depicted in Fig.3.
  • sucrose individually and in combination with glucose and fructose was studied.
  • Wild type strains (NY-EM and NY-YM) and the recombinant strains (NY-EM2 and NY-YM2) were grown in synthetic growth media containing 20 g/L sucrose alone or 20 g/L sucrose supplemented with 20 g/L glucose or fructose. After 24 hrs of growth the residual sucrose was analyzed by HPLC analysis.
  • Both NY-YM2 and NY-EM2 strains show the presence of residual sucrose in the media after 24 hrs of incubation due to their inability to hydrolyze the sucrose, while the wild type strain was found to be hydrolyzing the sucrose completely.
  • glucose or fructose hydrolysis products of sucrose
  • both NY-YM2 and NY-EM2 strain show better accumulation of sucrose compared to wild type strain.
  • This result highlights that the glucose can be used as carbon source for growth of NY -YM2 and NY-EM2 strains for whole cell biocatalyst preparation.
  • Glucose can also be used while bioconversion of sucrose in to trehalulose is being done by the recombinant strains.
  • NY-YM2 and NY-EM2 strains grow slowly compared to their wild type strains in growth media containing sucrose as sole carbon source due to their inability to hydrolyze sucrose, whereas the growth behavior of NY-YM2 and NY-EM2 stains are similar to wild type strains when grown with glucose.
  • the residual invertase activity of NY-EM2 and NY-YM2 strains compared to the respective wild type strains was studied.
  • the cells were incubated with 9.5 g/L sucrose in sucrose isomerase assay buffer at 30°C for up to 12 hrs and samples were analyzed for residual sucrose after formation of glucose and fructose (not shown) by action of residual invertase activity.
  • the mutant strains were able to grow identical to their wild type strain when grown in presence of glucose or fructose. Sucrose remained unaltered up to 8hrs when incubated with double mutant strains whereas the wild type strain exhibited that sucrose was getting depleted gradually. Approximately 70 - 75 % sucrose remained unaltered when incubated with double mutant strain(s) for 24 - 48 hrs whereas sucrose was completely depleted by wild type strain at similar conditions. Both NY-YM2 and NY-EM2 strains show high amount of residual sucrose in the sucrose isomerase assay buffer at optimum temperature even after 12 hrs of incubation, whereas the sucrose is depleted when incubated with wild type strain due presence of high invertase activity.
  • NY-YM2 and NY-EM2 strains are very suitable host strains for the development of recombinant strains for production of cell surface displayed sucrose isomerase, which can be used as whole cell biocatalyst for bioconversion of sucrose in to trehalulose.
  • the results of the studies are depicted in Fig.5.
  • Example 6 Development of recombinant yeast strains by transformation with recombinant gene constructs
  • the strains were grown in YEPD broth and supplemented with 2% glucose and the cells were treated with ice cold sorbitol to prepare electrocompetent cells.
  • the recombinant constructs pGH-SI_R3 and pGL-SI_R3 were transformed in to electrocompetent cells by standard electroporation method.
  • strains transformed with pGH-SI_R3 were selected on SD-Uraplates and the strains transformed with pGL-SI_R3were selected on SD-Trp or at 30°C.
  • Transformed strains NY-YM(pGH-SI_R3), NY- YM2 (pGH-SI_R3), NY-EM (pGL-SI_R3), and NY-EM2 were verified for the presence of plasmid by subsequent selections and restriction digestion analysis.
  • CSD-SIase in the modified recombinant strain NY-EM2 (pGL-SI_R3) and control recombinant strain NY-EM (pGL-SI_R3) were grown in Synthetic Dropout Media without tryptophan.
  • Cells were grown in SD Trp- medium with 2% glucose up to late logarithmic phase and induced by addition of 2% galactose for production of CSD-SIase.
  • Example 7 Microscopy of immunofluorescence labelled yeast cells
  • yeast strains NY-EM2 pGL-empty
  • NY-EM2 pGL-SI_R3
  • NY-YM2 pGH-empty
  • NY-YM2 pGH-YM2
  • the cells were washed with phosphate buffered saline (PBS) and treated with 3.7 % formaldehyde in PBS at room temperature for 15 min. After fixation, the cells were washed with PBS and attached to a polylysine coated slide. Fixed cells were coated with 5% bovine serum albumin (BSA) in PBS at room temperature for 20 minutes in order to avoid non-specific binding of antibodies. Coated cells were washed with PBS containing 1% BSA (PBS-B) and incubated with anti-sucrose isomerase primary antibody developed in rabbit in PBS-B for 30 min at room temperature.
  • BSA bovine serum albumin
  • Antibody bound cells were washed thrice in PBS-B and incubated with biotinylated anti-rabbit secondary antibody for another 30-60 minutes at room temperature. The cells were washed with PBS-B for thrice to remove the unbound antibodies and incubated with streptavidin (AlexaFluor488, Invitrogen) in PBS at room temperature for 30-60 minutes. The cells were washed and a drop of DAPI in an aqueous mounting medium was added for the staining the DNA of mounted cells and for fixing the cover-slip.
  • Immunofluorescence analysis was performed using excitation which gives a Cyan-Green color at absorbance 495nm and excitation at 5l9nm wavelength for Alexa-488 staining and 345nm absorbance spectra and 455nm emission spectra for DAPI staining.
  • Lane 1 and 2 depict control strain and strain expressing CSD-SIase constitutively, respectively.
  • Lane 3 and 4 depict uninduced strain carrying inducible construct and induced strain for expression of CSD-SIase, respectively.
  • Cell surface displayed GPI-fused Slase were detected in the I st row by using anti-Sucrose isomerase antibody produced in rabbit, biotinylated goat anti -rabbit IgG and Streptavidin Alexa Fluor® 488 which is a biotin-binding protein (streptavidin) covalently attached to a fluorescent label.
  • DAPI stain was used to visualize the nucleus in the 2 nd row.
  • the normal images of the cells were viewed by Nomarski differential interference contrast microscopy in the 4 th row.
  • the images were taken with an inverted Nikon Eclipse 50i fluorescent microscope under 200x magnification using different filters.
  • Overlay of both Alexa and DAPI staining in the 3 rd row shows the number of yeast cells stained with cell surface displayed recombinant sucrose isomerase.
  • yeast strains NY-EM pGL-SI_R3
  • NY-EM2 pGL-SI_R3
  • NY-YM pGH-SI_R3
  • NY-YM2 pGH-SI_R3
  • Equal amount of cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 1% DMSO, 50-200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 pg/ml leupeptin, 1 pg/ml pepstatin A) using acid washed glass beads.
  • lysis buffer 50 mM Tris-HCl pH 8.0, 1% DMSO, 50-200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 pg/ml leupeptin, 1 pg/ml pepstatin A
  • the cell lysates were subject to SDS-PAGE and Western blot analysis.
  • Anti-sucrose isomerase antibody developed in rabbit was used as primary antibody and goat anti rabbit AP-conjugate (alkaline phosphatase conjugate) was used as secondary antibody.
  • the blots were developed using BCIP/NBT (5-bromo-4-chloro-3- indolylphosphate/nitro blue tetrazolium) substrate for the detection.
  • CSD-SIase The activity of CSD-SIase was examined by incubating cells with 500mM sucrose solution in 20 mM citrate acetate buffer (pH 6.5) with I OmM CaCFas an additive and the reaction mixture was incubated at l2°C for 8 hrs. The enzymatic reaction was stopped by deactivating the enzyme at 95 °C in a boiling water bath for 10 min.
  • Fig. 7 shows the expression profile of CSD-SIase by immunoblot analysis from different fractions of cell lysate from constitutive or inducible expression strains. Equal amount of cells were lysed and the whole cell lysate (lane 1 and 5), supernatant (lane 2 and 6) and pellet (lane 3 and 7) fractions were separated on SDS-PAGE and transferred to nitrocellulose membrane for Western blot analysis. Rabbit anti-SIase antibody and Goat anti-rabbit secondary antibody were used for detection. Lane 8 depicts protein molecular weight marker and Lane 9 depicts purified recombinant Slase.
  • sucrose isomerase displayed on the yeast cell wall was confirmed by Western blot analysis (in lane 5 - 7), whereas there is no signal for sucrose isomerase in control strain (lane 1 - 3). Moreover, the cell membrane fraction (lane 7) showed that the majority of sucrose isomerase is present on the cell wall.
  • Example 9 Fermentation kinetics and cell surface display for recombinant transformed strains exhibiting constitutive expression
  • Fermentation of yeast strain NY-YM2 was carried out in defined media without uracil at 30°C for 42 hrs. Samples were collected at different time points and tested for growth and CSD-SIase activity.
  • the enzyme assay was carried out in 20 mM citrate acetate buffer (pH 6.5) with 10 mM CaCk , 8.5% sucrose solution and 5 OD units of CSD-SIase cells (1 OD 6 OO is roughly 3x 10 7 cells/ml).
  • the reaction mixture was incubated at l2°C for 4 hrs.
  • the enzymatic reaction was stopped by deactivating the enzyme at 95°C in a boiling water bath for 10 min.
  • the reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation.
  • Fig. 8 shows the fermentation kinetics and cell surface displayed Slase activity of recombinant strain producing CSD-SIase by constitutive expression strain NY-YM2 (pGH- SI_R3).
  • the NY-YM2 (pGH-SI_R3) strain is constitutively producing modified sucrose isomerase (GPI-SIase), which is GPI anchor protein fused to sucrose isomerase.
  • GPI-SIase modified sucrose isomerase
  • the produced GPI-SIase is subsequently displayed on the cell surface of the NY- YM2 (pGH-SI_R3) strain which enabled it to convert sucrose in to trehalulose when cells were tested for sucrose isomerase activity throughout the fermentation.
  • Example 10 Fermentation kinetics and cell surface display for recombinant transformed strains exhibiting inducible expression
  • Fermentation of NY-EM2 (pGL-SI_R3) yeast strain was carried out in defined media without uracil at 30 °C for 42 hrs. Samples were collected at different time points and tested for growth and CSD-SIase activity. The enzyme assay was carried out in 100 mM citrate acetate (pH 6.5) with 150 mM NaCb , 8.5% sucrose solution and 5 OD units of CSD-SIase cells (1 OD 6 oo is roughly 3x 10 7 cells/ml). The reaction mixture was incubated at l4°C for 4 hrs. The enzymatic reaction was stopped by deactivating the enzyme at 95 °C in a boiling water bath for 10 min. The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation with appropriate standards.
  • Fig. 9 shows the fermentation kinetics and cell surface displayed sucrose isomerase activity of recombinant strain producing CSD-SIase by inducible expression strain NY-EM2 (pGL-SI_R3).
  • Fig. 10 shows the expression profile of CSD-SIase by immunoblot analysis from samples collected from constitutive [NY-YM2 (pGH-SI)] or inducible expression [NY-EM2 (pGL-SI)] strains at different fermentation time points. Equal amount of cells were collected at different fermentation time points and analyzed for the expression profile of CSD-SIase. The cells were lysed using glass beads in yeast cell lysis buffer and total cell lysate was separated on 10 % SDS- PAGE and were analyzed by Western blot. Rabbit anti-sucrose isomerase antibody and goat anti-rabbit secondary antibody was used for detection. The abbreviations used in the figure are C for purified Slase as positive control, M for protein molecular weight marker, kDa for Kilo Dalton and Ab for Antibody.
  • sucrose isomerase is produced constitutively from the beginning in NY-YM2 (pGH-SI_R3), whereas in NY-EM2 (pGL-SI_R3) it is produced only after induction.
  • Example 12 Production of whole cell biocatalysts with cell surface display
  • the cells were grown in defined media comprised of base medium components (10 g/L (NH 4)2 S0 4 , 10 g/L KH 2 P04, 0.5 g/L CaCl 2 .2FbO, 0.5 g/L NaCb and 3 g/L MgSo 4. 7H 2 0), trace elements (278 mg/L FeS0 4. 7H 2 0, 288 mg/L ZnS0 4. 7H 2 0, 80 mg/L CuS0 4.
  • base medium components (10 g/L (NH 4)2 S0 4 , 10 g/L KH 2 P04, 0.5 g/L CaCl 2 .2FbO, 0.5 g/L NaCb and 3 g/L MgSo 4. 7H 2 0
  • trace elements (278 mg/L FeS0 4. 7H 2 0, 288 mg/L ZnS0 4. 7H 2 0, 80 mg/L CuS0 4.
  • Example 13 Production of trehalulose using CSD-SIase cell
  • CSD-SIase cells produced in previous example was used as whole cell biocatalyst.
  • the optimization of process parameters for the production of trehalulose was carried out with varying pH and temperature, which were used for the production of trehalulose.
  • trehalulose from sucrose was carried out by using 5-50 OD units more precisely, 10 OD units of CSD-SIase cells (1 OD 6 oo is roughly 3 x 10 7 cells/ml).
  • the cells were contacted with varying concentration of sucrose, more particularly 10%, 20%, 30%, 40% and 45% sucrose solution, in 20 mM citrate acetate buffer (pH 6.5) with 10 mM CaCFas an additive atl2°C for 12-24 hrs.
  • the sugar solution was subjected to cation and anion exchange resins to remove salt and ions present in buffer solutions. Furthermore, the sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC and ions contaminations were analyzed in ion chromatography.
  • the enzyme assay was carried out using 1 OD, 3 OD and 5 OD units of CSD-SIase cells (1 OD 6 oo is roughly 3 x 10 7 cells/ml) in 20 mM citrate acetate buffer (pH 6.5) with 10 mM CaCF and 8.5% sucrose solution.
  • the reaction mixture was incubated at l2°C for 8 hrs.
  • the enzymatic reaction was stopped by deactivating the enzyme at 95°C in a boiling water bath for 10 min.
  • the reaction mixtures were subjected to HPLC analysis to confirm the residual substrate and product formation with appropriate standards.
  • Fig. 11 shows the product formation kinetics using different amount of CSD-SIase cells.
  • the recombinant strain exhibiting constitutive expression was used.
  • This result shows the product formation kinetics by different amount of CSD-SIase whole cell biocatalyst.
  • the results show that the production of trehalulose increases proportionately related to the amount of CSD-SIase cells used in given reaction time with same amount of substrate.
  • 5 OD cells were used, 11% to 45% product is formed between 0.5 to 24 hrs of reaction time, while only 6% to 19% product is formed in the same time when used 1 OD cells are used. Therefore, amount of whole cell biocatalyst can be increased up to 50 OD units for the bioconversion reaction for efficient conversion of sucrose in to trehalulose.
  • Example 15 Product formation kinetics using different amounts of sucrose
  • the cells were harvested and used for analysis of product formation kinetics by CSD-SIase cells.
  • the enzyme assay was carried out in 20 mM sodium acetate (pH 6.5) with 10 mM CaCb as an additive with different concentration of substrate and 5 OD units of CSD-SIase (1 OD 6 oo is roughly 3 x 10 7 cells/ml).
  • the recombinant strain exhibiting constitutive expression was used.
  • the reaction mixture was incubated at l2°C for 8 hrs.
  • the enzymatic reaction was stopped by deactivating the enzyme at 95°C in a boiling water bath for 10 min.
  • the reaction mixtures were subjected to HPLC analysis to confirm the residual substrate and product formation with appropriate standards.
  • Fig. 12 shows the product formation kinetics using different amount of sucrose as substrate with CSD-SIase.
  • Example 16 Chromatogram of bioconversion of sucrose to trehalulose by recombinant strain exhibiting constitutive expression
  • FIG. 13 depicts the results of HPLC analysis of bioconversion of sucrose into trehalulose by CSD-SIase strain.
  • Figure A presents the chromatogram of blank substrate (sucrose). Chromatograms of bioconversion performed using NY-YM2 (pGH-SI_R3) and NY-YM (pGH-SI_R3) strains are shown as figure B and C, respectively.
  • NY-YM pGH-SI_R3 strain constitutively producing CSD-SIase is able convert sucrose in to trehalulose but besides the formation of trehalulose the fructose and glucose were also formed due the invertase activity of the yeast strain S. cerevisiae.
  • the NY-YM2 (pGH-SI_R3) strain show dramatic decrease in fructose and glucose level as the major sucrose invertase and symporter, SUC2 and AGT1 are deleted in this strain.
  • the sucrose (substrate) was mainly converted to trehalulose by CSD-SIase.
  • Example 17 Bioconversion kinetics of CSD-SIase produced by recombinant strain exhibiting constitutive expression
  • CSD-SIase yeast cells [NY-YM2 (pGH-SI_R3)] were incubated with 8.2% sucrose in 10 mM calcium acetate (pH 6.5) with 150 mM NaCFand incubated at l2°C for different time points. The enzymatic reaction was stopped by deactivating the enzyme at 95°C in a boiling water bath for 10 min. The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation with appropriate standards.
  • Fig. 14 depicts the bioconversion kinetics of CSD-SIase produced by constitutive expression strain [NY -YM2 (pGH-SI)] .
  • Example 18 Bioconversion kinetics of CSD-SIase produced by recombinant strain exhibiting inducible expression
  • CSD-SIase yeast cells [NY-EM2 (pGL-SI_R3)] were incubated with 8.2% sucrose in 10 mM calcium acetate (pH 6.5) with 150 mM NaCFand incubated at l2°C for different time points. The enzymatic reaction was stopped by deactivating the enzyme at 95°C in a boiling water bath for 10 min. The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation with appropriate standards.
  • Fig. 15 shows the bioconversion kinetics of CSD-SIase produced by inducible expression strain [NY-EM2 (pGL- SI_R3)].
  • Example 19 Temperature optima profile of CSD-SIase and native enzyme
  • Temperature optimum was determined by incubating the reaction mixture with constitutive produced CSD-SIaseand the respective native Slase at various temperatures ranging from l0°C - 70°C in 10 mM calcium acetate (pH 6.5) with 150 mM NaCEand incubated for 12 hrs. Enzyme activities were determined at pH 6.5 and the relative activity was calculated by assuming that the activity observed at 30°C for CSD-SIase and 40°C for native Slase was 100%.
  • Fig. 16 shows the temperature optima profiles of CSD-SIase and native Slase.
  • Example 20 pH profile of CSD-SIase and native enzyme
  • the pH optimum was determined by incubating the constitutively produced CSD-SIase and native Slase with sucrose in assay buffer with different pH and 10 mM CaCb as an additive. Enzyme activities were measured at l2°C and the relative activity was calculated by assuming that the activity observed at pH 6.5 was 100%.
  • Fig. 17 shows the pH profiles of CSD-SIase and native Slase.
  • CSD-SIase The stability of CSD-SIase were checked by incubating the CSD-SIase cells in sucrose isomerase buffer at optimum temperature and cells were collected at different time points for residual CSD-SIase activity analysis.
  • the enzyme assay was carried out in 10 mM calcium acetate (pH 6.5) with 150 mM NaCh and 8.5% sucrose solution and 5 OD units of CSD-SIase cells (1 OD 6OO is roughly 3 x 10 7 cells/ml).
  • Fig. 18 shows the residual activity of CSD-SIase enzymes compared to native Slase enzyme. This result shows that the CSD-SIase cells or whole cell biocatalyst retains 80%, 70%, 55% and 50% of its activity after 24 hrs, 48 hrs, 96 hrs and 120 hrs respectively in optimum reaction condition, which is slightly lower than the native free sucrose isomerase activity.
  • the stability of native sucrose isomerase falls rapidly after 72 hrs whereas the CSD-SIase activity retains stability for a longer period. Therefore, the CSD-SIase has almost similar stability to the native enzyme for continuous bioconversion.

Abstract

La présente invention concerne un nouveau biocatalyseur à cellules entières pour la production de tréhalulose. L'invention représente une avancée dans le domaine de l'ingénierie enzymatique et concerne un nouveau mutant de délétion double dont les gènes codant l'invertase et la perméase ont été rendus inopérants. En outre, la cellule hôte mutante comprend une construction d'expression codant pour une enzyme saccharose isomérase fusionnée à une protéine d'ancrage de surface cellulaire. La cellule recombinante peut être utilisée comme biocatalyseur pour une conversion efficace du saccharose en tréhalulose.
PCT/IB2018/051736 2018-03-15 2018-03-15 Nouveau biocatalyseur à cellules entières pour la production de tréhalulose WO2019175635A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156940A1 (fr) * 2012-04-17 2013-10-24 Agtive Bio-Sciences Private Limited Procédé de production de disaccharides rares
US20170204394A1 (en) * 2014-05-30 2017-07-20 Braskem S.A. Modified microorganisms comprising an optimized system for oligosaccharide utilization and methods of using same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013156940A1 (fr) * 2012-04-17 2013-10-24 Agtive Bio-Sciences Private Limited Procédé de production de disaccharides rares
US20170204394A1 (en) * 2014-05-30 2017-07-20 Braskem S.A. Modified microorganisms comprising an optimized system for oligosaccharide utilization and methods of using same

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* Cited by examiner, † Cited by third party
Title
GIL-YONG LEE ET AL.: "Isomaltulose production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae", BIORESOURCE TECHNOLOGY, vol. 102, 2011, pages 9179 - 9184, XP028276325 *
MARQUES WL ET AL.: "Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism", FEMS YEAST RESEARCH, vol. 17, no. 1, 1 January 2017 (2017-01-01), pages 1 - 11, XP055635132 *

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