WO2019175635A1

WO2019175635A1 - Novel whole-cell biocatalyst for the production of trehalulose

Info

Publication number: WO2019175635A1
Application number: PCT/IB2018/051736
Authority: WO
Inventors: Saravanakumar IYAPPAN; Karthikeyan VENKATA NARAYANAN; Murali Thumala; Banibrata Pandey
Original assignee: Petiva Private Limited
Priority date: 2018-03-15
Filing date: 2018-03-15
Publication date: 2019-09-19

Abstract

The present invention provides for a novel whole cell biocatalyst for the production of trehalulose. The invention represents an advancement in the field of enzyme engineering and discloses a novel double deletion mutant whose invertase and permease encoding genes have been made inoperative. Further, the mutant host cell comprises an expression construct encoding sucrose isomerase enzyme fused to a cell surface anchor protein. The recombinant cell can be used as a biocatalyst for effective conversion of sucrose to trehalulose.

Description

NOVEL WHOLE-CELL BIOCATALYST FOR THE PRODUCTION OF

TREHALULOSE

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

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.

The overconsumption of sugar is potentially linked to many diseases which creates a growing need for sugar substitutes. Trehalulose, being one of the most potent sugar substitute has a huge demand in the healthy lifestyle segment wherein the consumers demand a suitable alternative to sucrose for following a low glycemic diet and avoidance of significant blood sugar variation.

Enzymatic bioconversion is a preferred method for the production of trehalulose because of the complexity in its chemical synthesis.

The current methods used for conversion of sucrose into isomers, particularly trehalulose by using sucrose isomerase activity involves following approaches:

1. usage of non- viable organism which converts sucrose in to trehalulose

2. production of sucrose isomerase in recombinant host

3. isolation and purification of native or recombinant sucrose isomerase

4. immobilization of sucrose isomerase for bioconversion

But, the various approaches which have been used till date suffers from some limitations and drawbacks such as low expression level of sucrose isomerase, additional cost in isolating the enzyme produced, difficulties in identifying appropriate immobilization method for retaining the activity of the enzyme, instability of the enzyme, recycling of the enzymes and the bioconversion activity is restricted to a narrow range of pH and temperature. Further, the bioconversion activity is limited to a narrow range of pH and temperature.

For the first time, 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. 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.

Thus, 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.

SUMMARY OF THE INVENTION

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.

Further, 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.

Further, 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. Further, the biocatalyst used in the process is reusable and provides for efficient and cheap downstream processing.

BRIEF DESCRIPTION OF THE DRAWINGS 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.

DETAILED DESCRIPTION OF THE INVENTION

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:

i. Deletion of the genes involved in 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.

ii. Transformation of the host cell for expression of sucrose isomerase on the surface of the host cells - By this approach, the substrate to be processed has free access to the enzyme and does not need to cross a membrane barrier. Further, yield increases and downstream processing cost decreases as a result.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

The term“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.

The term“recombinant strain” refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.

The term“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.

The term“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. The term“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.

The term“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.

The term“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.

The term“transcription” refers the process of making an RNA copy of a gene sequence. This copy, called a messenger RNA (mRNA) molecule, leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein, which it encodes.

The term“translation” refers the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes. In the cell cytoplasm, the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.

The term“terminator 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.

The term "fusion protein" refers to a polypeptide which comprises protein domains from at least two different proteins. As used herein, the fusion protein is sucrose isomerase fused to C- terminus of a cell surface anchor protein.

The term“sucrose hydrolyzing genes” refers to genes which are responsible for sucrose hydrolysis. As used herein, the term sucrose hydrolyzing genes refers to SUC2 invertase and AGT1 alpha-glucoside transporter. The term“inducible excision system” refers to site-specific recombinase technologies which can be used for efficiently regulating the excision or deletion of genes. As used herein, the inducible excision system employed in the invention is cre-lox excision system.

The term“replication origin” as used herein 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.

The term“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.

The term "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. In all cells, 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. As used herein, the anchor protein is a GPI anchor protein, AGA2 which is fused in frame to the N-terminus of sucrose isomerase.

The term“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. 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.

The term“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. NY-YM2 is used for transformation of construct pGH-SI_R3 under constitutive promoter.

“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. NY-EM2 is used for transformation of construct pGL-SI_R3 under inducible promoter.

“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.

Before the modified expression cassette, vectors, recombinant hosts, peptides and methods for production of trehalulose are described in greater detail, it is to be understood that the invention is not limited to particular embodiments and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

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.

In one aspect, 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.

In one embodiment, 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.

In a preferred embodiment, the modified nucleic acid is represented by SEQ ID NO: 1.

In another aspect, 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. In a preferred embodiment, the fusion protein is represented by SEQ ID NO:6. In another aspect, 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.

In one embodiment, the construct carries a constitutive promoter, such as, but not limited to, GAPDH promoter.

In yet another embodiment, the construct carries an inducible/regulated promoter, such as, but not limited to GAL1, pGALlO, pSUC2, pXYL or pADH promoter.

In a preferred embodiment, 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.

In another preferred embodiment, 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.

In another aspect, 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. Hence, 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.

In one embodiment, 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.

In a preferred embodiment, 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.

In another embodiment, 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.

In a preferred embodiment, 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.

In another aspect, the invention provides host cells in which sucrose hydrolyzing genes have been inactivated by knock-out or deletion.

In one embodiment, 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.

In a preferred embodiment, 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.

In the preferred embodiment, the amplified products were used as deletion cassettes and transformed into yeast strain, such as, but not limited to, W303-1A or BJ5465 strains. In another aspect, 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.

In one embodiment, it has been exhibited that the 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.

In another embodiment, it is exhibited that the residual invertase activity of the engineered strains are reduced when compared to their wild type strains as depicted by sucrose isomerase assay conditions.

In yet another aspect, the invention provides for the development of recombinant host cells. In an embodiment, 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.

In yet another aspect, 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.

In one embodiment, 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.

In a preferred embodiment, 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.

In another embodiment, 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.

In a preferred embodiment, 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.

In the preferred embodiments, 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 and 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.

In another aspect of the invention, 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.

In one embodiment, 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. Thus, the cell surface displayed sucrose isomerase exhibits broader pH tolerance higher than that of the native enzyme.

In another aspect, the invention provides for an industrially scalable process for production of trehalulose.

In one embodiment, 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.

Yet another aspect of the invention provides that when cells are contacted with sucrose solution for the formation of trehalulose, the bioconversion increases depending on the amount of cell used. In one embodiment, it is exhibited that when 5 OD units of stationary phase cell surface displayed-sucrose isomerase cells (1 OD = 3xl0⁷ cells/ml) were used, upto 40% product is formed between 1 to 6 hrs of reaction time.

In another aspect of the invention, it is exhibited that the bioconversion depends upon the amount of substrate used.

In one embodiment, it is exhibited that 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.

EXAMPLES

The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.

Example 1: Gene construction for constitutive expression of recombinant fusion protein GPI-SI in Saccharomyces cerevisiae

Gene encoding for 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

Gene encoding for sucrose isomerase (Slase) 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 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.

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

The 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.

For deletion of AGT1 in the chromosome of S. cerevisiae, 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

Among developed recombinant strains NY -EM2 and NY -YM2 showed promising results with sucrose uptake studies compared to their wild type and/or single mutant 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. In the first instance, the uptake and utilization of sucrose, glucose and fructose were individually studied. In the presence of synthetic growth media containing of 37 g/L sucrose, 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.

Similarly, 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.

In the second instance, the uptake and utilization of 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. When glucose or fructose (hydrolysis products of sucrose) is supplemented in the synthetic growth media the hydrolysis of sucrose is reduced in wild type strain itself since the presence of glucose can regulate the invertase enzymes expression but 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.

Further, it was observed that the 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.

When glucose or fmctose was supplemented with sucrose the wild type strain showed reduced hydrolysis of sucrose due to regulated expression of invertase gene expression by glucose. However, the double mutant strain showed remarkable reduction in hydrolysis of sucrose compared to the respective wild type strains. The results of the studies are depicted in Fig.4.

In the third instance, 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.

This result highlights that the 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

Both recombinant yeast strains developed in this invention NY-YM2 and NY-EM2 and respective wild type strains NY - YM and NY -EM were transformed with recombinant constructs pGH-SI_R3 and pGL-SI_R3, respectively. 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. The strains transformed with pGH-SI_R3were 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 (pGL-SI_R3) were verified for the presence of plasmid by subsequent selections and restriction digestion analysis.

For verification of constitutive expression of CSD-SIase in the modified recombinant strain NY-YM2(pGH-SI_R3) and control recombinant strain NY-YM(pGH-SI_R3) were grown in Synthetic Dropout Medium without uracil.

Similarly, for verification of inducible expression 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

For immunofluorescence analysis, the yeast strains NY-EM2 (pGL-empty), NY-EM2 (pGL-SI_R3), NY-YM2 (pGH-empty) and NY-YM2 (pGH-SI_R3) were collected after induction or constitutive expression of CSD-SIase, respectively.

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. 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.

Images were taken on inverted Nikon Eclipse 50i fluorescent microscope under 200 x magnifications and analyzed.

The results of the microscopy of immunofluorescence-labeled yeast cells are depicted in Figure 6. 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.

Almost all recombinant NY-YM2 (pGH-SI_R3) yeast strains displaying the modified sucrose isomerase on their cell surface is confirmed by immunofluoresence in Lane2, whereas the control NY-YM2 (pGH-empty) yeast strains do not display any modified sucrose isomerase (lane 1, column 1).

For the induced expression of GPI-SIase in NY-EM2 (pGH-SI_R3), more than 50% cells are displaying the modified sucrose isomerase on their cell surface as depicted in Lane 4, whereas the control NY-YM2 (pGH-empty) yeast strains do not display any modified sucrose isomerase (lane 3, column 1). Example 8: Immunoblot analysis from different fractions of cell lysate

For immunoblot analysis the yeast strains NY-EM (pGL-SI_R3), NY-EM2 (pGL-SI_R3), NY-YM (pGH-SI_R3) and NY-YM2 (pGH-SI_R3) were collected after induction or constitutive expression of CSD-SIase, respectively. 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. 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.

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.

The results of the immunoblot analysis is depicted in Fig. 7, which 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.

Identity of 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 (pGH-SI_R3) 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 OD₆OO is roughly 3x 10⁷ 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).

This result highlights that the NY-YM2 (pGH-SI_R3) strain is constitutively producing modified sucrose isomerase (GPI-SIase), which is GPI anchor protein fused to 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 OD₆oo is roughly 3x 10⁷ 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).

This result highlights that upon induction the NY-EM2 (pGL-SI_R3) strain starts producing modified sucrose isomerase (GPI-SIase), which is GPI anchor protein fused to sucrose isomerase. The produced GPI-SIase is subsequently displayed on the cell surface of the NY- EM2 (pGL-SI_R3) strain and was able to convert sucrose in to trehalulose when the 34 - 70 hrs fermentation cells were tested for sucrose isomerase activity. Example 11: Immunoblot analysis of samples collected from constitutive or inducible expression strains at different fermentation times

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.

This result shows the presence and identity of sucrose isomerase on the cell surface of both NY-YM2 (pGL-SI_R3) and NY-EM2 (pGL-SI_R3) strains as shown in figure A and B, respectively. 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

For large scale production of CSD-SIase, the cells were grown in defined media comprised of base medium components (10 g/L (NH₄₎₂S0₄, 10 g/L KH₂P04, 0.5 g/L CaCl₂.2FbO, 0.5 g/L NaCb and 3 g/L MgSo_4.7H₂0), trace elements (278 mg/L FeS0_4.7H₂0, 288 mg/L ZnS0_4.7H₂0, 80 mg/L CuS0_4.5H₂0, 242 mg/L Na₂Mo0₄.2H₂0, 238 mg/L C0CI₂.6H₂O and 198 mg/L MnCbAFLO), vitamins (10 mg/L biotin and 120 mg/L Thiamine HC1), liquor ammonia and 1% yeast extract, which was used as an alkali and nitrogen source. The temperature of the fermentation was maintained at 28 °C at a pH 5.5 and oxygen level was maintained not less than 40%, throughout the fermentation. The fermentation process for constitutive production of CSD-SIase at 2L scale was carried out for 24 - 48 hrs and which yields 40 - 80 g/L biomass. For production of inducible CSD-SIase strain the cells were grown in defined media with 10 - 20 % glucose for generation of biomass and after depletion of glucose the cells were induced with 2 - 5% galactose for production of CSD-SIase. At each time points the cells were collected and monitored for the expression of CSD-SIase activity. The cells were collected after fermentation and washed with phosphate saline buffer and used as whole cell biocatalyst for production of trehalulose. Example 13: Production of trehalulose using CSD-SIase cell

For production of trehalulose from sucrose, 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.

Production of trehalulose from sucrose was carried out by using 5-50 OD units more precisely, 10 OD units of CSD-SIase cells (1 OD₆oo is roughly 3 x 10⁷ 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.

After bioconversion of sucrose to trehalulose using CSD-SIase, 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.

Physico-chemical properties and purity of the product were checked by using standard techniques to confirm the safety aspects of produced trehalulose in this process. The percentage of bioconversion of sucrose to trehalulose was about 50-60 %. From total produced sugars, trehalulose was found to be 90-95 % with trace amounts of isomaltulose, glucose and fructose. Example 14: Product formation kinetics using different amount of CSD-SIase cells

After fermentation of CSD-SIase expressing strains, the cells were harvested and used for sucrose isomerase activity analysis. The enzyme assay was carried out using 1 OD, 3 OD and 5 OD units of CSD-SIase cells (1 OD₆oo is roughly 3 x 10⁷ 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. For this experiment, 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. When 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

After fermentation of CSD-SIase expressing strains, 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 OD₆oo is roughly 3 x 10⁷ cells/ml). For this experiment, 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.

This result shows the product formation kinetics with different amount of substrate for bioconversion. The results shows that the production of trehalulose increases proportionately to the amount of substrate used in given reaction time with same amount of CSD-SIase cells. When 150 g/L, 300 g/L and 450 g/L sucrose were used, 49 g/L, 120 g/L and 189 g/L trehalulose were produced respectively when contacted with 5 OD cells for 16 hrs. Therefore, amount of substrate can be increased up to 45% with appropriate whole cell biocatalyst and reaction time for efficient conversion of sucrose in to trehalulose.

Example 16: Chromatogram of bioconversion of sucrose to trehalulose by recombinant strain exhibiting constitutive expression

HPLC analysis of substrate (sucrose) blank, bioconversion of substrate in to products by recombinant strain and substrate incubated with wild type strain were performed. Substrate and products were identified with appropriate standards. 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.

In 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. However, 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. Hence 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.

This result shows the bioconversion kinetics of 5 OD of NY-YM2 (pGL-SI_R3) cells when contacted with 8.5% sucrose. Within 2.5 hrs, 1% trehalulose is formed and increased up to 2.2% when incubated for 4 hrs. Therefore, amount of whole cell biocatalyst up to 40-50 OD cells (as shown in Fig. 11) and substrate up to 45 - 50% (as shown in Fig. 12) in combination with reaction time up to 24 to 48 hrs can be used for efficient conversion of sucrose in to trehalulose.

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)].

This result shows the bioconversion kinetics of 5 OD of NY-EM2 (pGL-SI_R3) cells when contacted with 8.2% sucrose. Within 2 hrs,2% trehalulose is formed and increased up to 3.2% when incubated for 4 hrs.

Therefore, amount of whole cell biocatalyst up to 40-50 OD cells (as shown in Fig. 11) and substrate up to 45-50% (as shown in Fig. 12) in combination with reaction time up to 24 hrs can be used for efficient conversion of sucrose in to trehalulose.

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.

This result shows the optimum temperature for bioconversion of sucrose in to trehalulose by CSD-SIase cells or whole cell biocatalyst is 30°C whereas the native enzyme has temperature optima at 35°C. Moreover, the CSD-SIase cells or whole cell biocatalyst remains active with slight decrease at lower temperatures as well which might me an advantage to operate the bioconversion at lower temperatures.

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.

This result shows the optimum pH for bioconversion of sucrose in to trehalulose by CSD- SIase cells or whole cell biocatalyst is 6.5which is similar to that of the native enzyme. Moreover, the CSD-SIase cells or whole cell biocatalyst remains active with slight decrease at lower pH as well which might be an advantage to operate the bioconversion at decreased pH conditions. Example 21: Residual activity of CSD-SIase enzyme compared to native Slase enzyme

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 10⁷ cells/ml).

For this experiment, the recombinant strain exhibiting constitutive expression was used. 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.

However, 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.

Claims

The claims:

1. A recombinant host cell, wherein the sucrose hydrolyzing genes of the host cell have been deleted and the host cell comprises a fusion protein encoding gene comprising the nucleotide sequence of SEQ ID NO: 1.

2. The recombinant host cell as claimed in claim 1 , wherein the host cell is Saccharomyces cerevisiae.

3. The recombinant host cell as claimed in claim 1, wherein the sucrose hydrolyzing genes are selected from a group comprising SUC2 gene and AGT1 gene.

4. The recombinant host cell as claimed in claim 1 , wherein the host cell is selected from a group comprising MTCC 5985 and MTCC 5987.

5. An expression cassette, comprising:

a. a fusion protein encoding gene comprising the nucleotide sequence of SEQ ID NO: 1, wherein the fusion protein is sucrose isomerase fused to C -terminus of a cell surface anchor protein;

b. a constitutive or an inducible promoter sequence which is operably linked to the fusion protein encoding gene; and

c. a terminator sequence.

6. The expression cassette as claimed in claim 5, wherein the constitutive promoter comprises the nucleotide sequence of SEQ ID NO: 2, the inducible promoter comprises the nucleotide sequence of SEQ ID NO: 3 and the terminator sequence is chosen from a group comprising a GAPDH terminator comprising the nucleotide sequence of SEQ ID NO: 4 and an Alpha Factor terminator comprising the nucleotide sequence of SEQ ID NO: 5.

7. A polypeptide comprising a polypeptide sequence of SEQ ID NO: 6, wherein the polypeptide is sucrose isomerase fused to C-terminus of a cell surface anchor protein.

8. A vector comprising the expression cassette as claimed in claim 5.

9. The vector as claimed in claim 8, further comprising a yeast replication origin, a yeast selection marker gene, a bacterial replication origin and a bacterial selection marker gene.

10. The vector as claimed in claim 9, wherein the yeast replication origin is selected from a group comprising a yeast 2-micron sequence and a yeast centromere sequence.

11. The vector as claimed in claim 9, wherein the bacterial selection marker gene is an ampicillin resistance gene and the yeast selection marker gene is selected from a group comprising URA3 gene and TRP1 gene.

12. A recombinant Saccharomyces cerevisiae host cell, wherein the SUC2 and AGT1 genes have been deleted.

13. The recombinant Saccharomyces cerevisiae host cell as claimed in claim 12, comprising the vector as claimed in claim 8.

14. A process for development of a recombinant Saccharomyces cerevisiae host cell exhibiting high sucrose isomerase activity, said process comprising the steps of:

a. deleting SUC2 and AGT1 genes in the host cell using an inducible excision system; and

b. transforming the host cell with a vector of claim 8.

15. The process as claimed in claim 14, wherein the inducible excision system is cre-lox excision system using a marker selected from a group comprising kanamycin resistance gene and phleomycin resistance gene.

16. A process for production of trehalulose from sucrose, said process comprising the steps of:

a. culturing the recombinant Saccharomyces cerevisiae host cells as claimed in claim 13 in defined media, wherein the culture medium has pH ranging between 4 to 6.5 and temperature maintained between l5°C to 30°C;

b. contacting the recombinant host cells with sucrose solution, wherein the sucrose solution comprises 10% to 45% (w/v) sucrose; and

c. harvesting trehalulose from the solution.