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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
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • 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
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01005—Dextransucrase (2.4.1.5)

Definitions

• polysaccharide relates to production of poly (a 1 , 3 glucan) via an enzymatic reaction. More specifically, it relates to increasing the titer of poly (a 1 , 3 glucan) formed during the enzymatic reaction.
• Cellulose a polysaccharide formed from glucose via ⁇ (1 , 4) glycoside linkages by natural processes (Applied Fiber Science, F. Happey, Ed., Chapter 8, E. Atkins, Academic Press, New York, 1979), has achieved commercial prominence as a fiber as a consequence of the many useful products derived therefrom.
• cotton a highly pure form of naturally occurring cellulose, is well-known for its beneficial attributes in textile applications.
• Cellulose exhibits sufficient chain extension and backbone rigidity in solution to form liquid crystalline solutions (U.S. Patent No. 4,501 ,886).
• sufficient polysaccharide chain extension has hitherto been achieved primarily in ⁇ (1 , 4) linked polysaccharides. Any significant deviation from that backbone geometry in the glucan polysaccharide family lowers the molecular aspect ratio below that required for the formation of an ordered lyotropic phase.
• important commercial cellulosic fibers such as cotton and rayon increasingly present sustainability issues with respect to land use and environmental imprint.
• polymers offer materials that are environmentally benign throughout their entire life cycle.
• Poly (a 1 , 3 glucan) a glucan polymer characterized by having a (1 , 3) glycoside linkages, has been isolated by contacting an aqueous solution of sucrose with a glucosyltransferase (gtfJ) enzyme isolated from Streptococcus salivarius (Simpson et al., Microbiology, 141 : 1451 -1460, 1995).
• Glucan refers to a polysaccharide composed of D-glucose monomers linked by glycosidic bonds. Films prepared from poly ( ⁇ 1 , 3 glucan) tolerated temperatures up to 150 °C and provided an advantage over polymers obtained from ⁇ (1 , 4) linked
• U.S. Patent 7,000,000 disclosed preparation of a polysaccharide fiber comprising hexose units, wherein at least 50% of the hexose units within the polymer were linked via a (1 , 3) glycoside linkages using the glucosyltransferase enzyme gtfJ of Streptococcus salivarius.
• the disclosed polymer formed a liquid crystalline solution when it was dissolved above a critical concentration in a solvent or in a mixture comprising a solvent. From this solution continuous, strong, cotton-like fibers highly suitable for use in textiles were spun and used either in a derivatized form or as a non-derivatized (regenerated) form.
• the poly (a 1 , 3 glucan) in U.S. Patent 7,000,000 was made in a batch process wherein the poly (a 1 , 3 glucan) titers were typically less than 25 grams of poly (a 1 , 3 glucan) per liter of the reactor volume.
• This invention is a process for production of poly (a 1 , 3 glucan) from a renewable feedstock, for applications in fibers, films, and pulps.
• the polymer is made directly in a one step enzymatic reaction using a recombinant
• the disclosed invention is a process for producing poly (a 1 , 3 glucan) in a reaction system comprising two chambers separated by a semipermeable membrane, wherein:
• a first chamber comprises an enzyme reaction solution comprising: i) sucrose;
• a second chamber separated from the first chamber by a semipermeable membrane in contact with the enzyme reaction solution wherein the semi-permeable membrane is permeable to fructose and other low molecular weight moieties but impermeable to poly (a 1 , 3 glucan), facilitates continuous removal of fructose and other low molecular weight moieties while retaining poly (a 1 , 3 glucan) and the at least one glucosyltransferase enzyme inside the first chamber.
• the disclosed invention is a process wherein poly (a 1 , 3 glucan), at a titer of 30 - 200 grams per liter, is produced from sucrose by at least one glucosyltransferase enzyme.
• SEQ NO. 1 is the sequence of the synthesized gene of the mature glucosyltransferase which has been codon optimized for expression in E. coli.
• SEQ NO. 2 is the DNA sequence for the plasmid pMP52.
• SEQ NO. 3 is the DNA sequence of the mature glucosyltransferase (gtfJ enzyme; EC 2.4.1 .5; GENBANK® AAA26896.1 ) from Streptococcus salivarius (ATCC 25975).
• Poly (a 1 , 3 glucan) is a potentially low cost polymer which can be enzymatically produced from renewable resources such as sucrose using the gtfJ enzyme of Streptococcus salivarius. It has been shown that selected polymers comprising hexose units with a (1 , 3) glycoside linkages can form ordered liquid crystalline solutions when the polymer is dissolved in a solvent under certain conditions (U. S. Patent No. 7,000,000). Moreover such solutions can be spun into continuous, high strength, cotton-like fibers. In U. S. Patent No.
• glucose refers to a disaccharide consisting of glucose and fructose, linked by an a (1 , 5) bond.
• glycosidic bond can join two monosaccharides to form a disaccharide.
• the glycosidic bonds can be in the a or ⁇ configuration and can generate, for example, a (1 , 2), a (1 , 3), a (1 , 4), a (1 , 6), ⁇ (1 , 2), ⁇ (1 , 3), ⁇ (1 , 4) or ⁇ (1 , 6) linkages.
• ⁇ (1 ,3) glycoside linkage refers to a type of covalent bond that joins glucose molecules to each other through the ring carbons 1 and 3 on adjacent glucose rings.
• poly (a 1 , 3 glucan) refers to high molecular weight, linear polymers obtained from polysaccharide molecules resulting from linking glucose units via a (1 ,3) glycosidic linkages.
• the present invention relates to a process for increasing the titer of the polysaccharide, poly (a 1 , 3 glucan), produced from sucrose in an enzymatic reaction using one or more gtf enzymes.
• the term "enzymatic reaction” refers to a reaction that is performed by the gtf enzyme.
• An "enzyme reaction solution” of the present invention generally refers to a reaction mixture comprising at least one gtf enzyme in a buffer solution comprising sucrose and possibly one or more primers to convert sucrose to poly (a 1 , 3 glucan).
• the glucosyltransferase enzyme used in the present invention can be any gtf enzyme.
• the gtf enzyme used can be from any streprococci.
• Suitable gtf enzymes can be, for example, the gtfJ of Streptococcus salivarius, the gtfB and the gtfC from Streptococcus mutans, and the gtfl from Streptococcus downei.
• the Streptococcus species can be Streptococcus salivarius.
• the gtf enzyme can be the gtfJ (E.C. 2.4.1 .5) enzyme of
• the enzyme reaction solution can comprise only one gtf enzyme as described herein. In another embodiment, the enzyme reaction solution can comprise a combination of more than one type of gtf enzyme.
• sufficient quantities of the gtfJ enzyme can be produced using a recombinant E. coli strain for gtfJ production as described in the Examples. Methods for designing the codon optimized genes and expression in E. coli are well known in the art.
• Recombinant microorganisms expressing the desired gtf enzyme to perform the instant reaction can be grown in any container, such as, for example: various types of flasks with and without indentations; any autoclavable container that can be sealed and temperature-controlled; or any type of fermenter.
• production of the gtfJ enzyme for poly (a 1 , 3 glucan) production in the present invention can be achieved by growing the recombinant E. coli MG1655/pMP52, expressing the gtfJ enzyme, in a fermenter.
• the gtfJ enzyme of Streptococcus salivarius used as the catalyst for conversion of sucrose to poly (a 1 , 3 glucan) in the current invention, is a primer- dependent gtf enzyme.
• a primer-dependent gtf enzyme as referenced in the present application refers to a gtf enzyme that requires the presence of an initiating molecule in the enzyme reaction solution to act as a primer for the enzyme during poly (a 1 , 3 glucan) synthesis.
• a "primer”, as the term is used herein, refers to any molecule that can act as the initiator for the primer- dependent glucosyltransferases. Many other glucosyltransferases are primer- independent enzymes.
• primer-independent enzymes do not require the presence of a primer to perform the reaction.
• a primer-independent enzyme, and/or a primer- dependent gtf enzyme can be used in the same enzyme reaction system during poly (a 1 , 3 glucan) synthesis.
• the gtfJ is a primer-dependent enzyme.
• dextran which is a complex, branched glucan was used as a primer for the gtfJ enzyme.
• gtf is a primer-dependent enzyme
• conversion of sucrose to poly (a 1 , 3 glucan) with this enzyme can also occur in the absence of a primer.
• Streptococcus salivarius is inhibited by its by-product, fructose.
• fructose When fructose accumulates in the enzyme reaction solution it can inhibit the production of poly (a 1 , 3 glucan) by the enzyme, presumably by competing for available glucosyl moieties which results in the formation of the disaccharide, leucrose.
• the fructose in the enzyme reaction solution can be continuously removed to prevent its
• the reaction system can comprise a semipermeable membrane that separates the enzyme reaction solution, contained in the first chamber, comprising one or more gtf enzymes, one or more primers and sucrose, from the surrounding buffer contained in the second chamber.
• the term "chamber” as used herein, refers to any container that can hold the enzyme reaction solution or the products of the enzyme reaction solution.
• the chamber can be made of glass, plastic, metal, film, membrane or any other type of inert material that can hold the enzyme reaction solution.
• the term "semi-permeable membrane”, as used herein, refers to a membrane that will allow passage of certain molecules or ions by diffusion while retaining some other molecules.
• any semi-permeable membrane with a molecular cutoff between 12,000 and 100,000 Daltons that will allow fructose and other low molecular weight moieties to pass through while retaining the enzyme and poly (a 1 , 3 glucan) can be suitable for use in the present invention.
• dialysis tubing can be used as the semi-permeable membrane to remove the by-product fructose from the enzyme reaction solution.
• the enzyme reaction solution can be maintained at 20 °C to 25 °C.
• the present invention provides for production of poly (a 1 , 3 glucan), as a low cost material that can be economically obtained from readily renewable sucrose feedstocks for a variety of applications including fibers, films, and pulps.
• poly (a 1 , 3 glucan) fibers for example, will functionally substitute for cotton and regenerated cellulose fibers, leading to new textile fibers with minimal environmental impact and excellent sustainability versus the aforementioned incumbents.
• Dialysis tubing (Spectrapor 25225-226, 12000 molecular weight cut-off) was obtained from VWR (Radnor, PA).
• Dextran and ethanol were obtained from Sigma Aldrich. Sucrose was obtained from VWR. Suppressor 7153 antifoam was obtained from Cognis Corporation
• the seed medium used to grow the starter cultures for the fermenters, contained: yeast extract (Amberx 695, 5.0 grams per liter, g/L), K 2 HPO 4 (10.0 g/L), KH 2 PO 4 (7.0 g/L), sodium citrate dihydrate (1 .0 g/L), (NH 4 ) 2 SO 4 (4.0 g/L), MgSO heptahydrate (1 .0 g/L) and ferric ammonium citrate (0.10 g/L).
• the pH of the medium was adjusted to 6.8 using either 5N NaOH or H 2 SO 4 and the medium was sterilized in the flask. Post sterilization additions included glucose (20 mL/L of a 50% w/w solution) and ampicillin (4 mL/L of a 25 mg/mL stock solution).
• Fermenter medium included glucose (20 mL/L of a 50% w/w solution) and ampicillin (4 mL/L of a 25 mg/mL stock solution).
• the growth medium used in the fermenter contained: KH 2 PO 4 (3.50 g/L), FeSO 4 heptahydrate (0.05 g/L), MgSO 4 heptahydrate (2.0 g/L), sodium citrate dihydrate (1 .90 g/L), yeast extract (Ambrex 695, 5.0 g/L), Suppressor 7153 antifoam (0.25 milliliters per liter, mL/L), NaCI (1 .0 g/L), CaCI 2 dihydrate (10 g/L), and NIT trace elements solution (10 mL/L).
• the NIT trace elements solution contained citric acid monohydrate (10 g/L), MnSO 4 hydrate (2 g/L), NaCI (2 g/L), FeSO 4 heptahydrate (0.5 g/L), ZnSO 4 heptahydrate (0.2 g/L), CuSO 4
• the plasmid pMP52 was used to transform E.
• coli MG1655 ATCC 47076TM to generate the strain identified as MG1655/pMP52. All procedures used for construction of the glucosyltransferase enzyme expression strain are well known in the art and can be performed by individuals skilled in the relevant art without undue experimentation.
• PRODUCTION OF RECOMBINANT gtfJ IN FERMENTATION Production of the recombinant gtfJ enzyme in a fermenter was initiated by preparing a pre-seed culture of the E. coli strain MG1655/pMP52, expressing the gtfJ enzyme, constructed as described in Example 1 .
• a 10 mL aliquot of the seed medium was added into a 125 mL disposable baffled flask and was inoculated with a 1 .0 mL culture of E. coli MG1655/pMP52 in 20% glycerol. This culture was allowed to grow at 37 °C while shaking at 300 revolutions per minute (rpm) for 3 hours.
• glucosyltransferase enzyme activity was initiated, when cells reached an OD 550 of 70, with the addition of 9 ml_ of 0.5 M IPTG (isopropyl ⁇ -D-l -thiogalacto- pyranoside).
• the dissolved oxygen (DO) concentration was controlled at 25% of air saturation.
• the DO was controlled first by impeller agitation rate (400 to 1200 rpm) and later by aeration rate (2 to 10 standard liters per minute, slpm).
• the pH was controlled at 6.8. NH 4 OH (14.5% weight/volume, w/v) and H 2 SO 4 (20% w/v) were used for pH control.
• the back pressure was maintained at 0.5 bars.
• the cell paste obtained above was suspended at 150 g/L in 50 mM potassium phosphate buffer pH 7.2 to prepare a slurry.
• the slurry was
• This Example demonstrates that removal and/or dilution of the by-product fructose, formed during conversion of sucrose to poly (a 1 , 3 glucan), increases poly (a 1 ,3 glucan) titer.
• Dialysis tubing was used as a semi-permeable membrane in this Example since it allows passage of the by-product fructose formed during the enzymatic reaction, from inside the tubing to outside of the dialysis tubes.
• the enzyme reaction solution in this Example contained 8 L of the sucrose stock solution (Table 1 ), 24 g of dextran T-10, as the primer, and 1 .0 volume % of the gtf enzyme.
• test samples in the dialysis tubes were removed from the surrounding buffer, the tubes were cut open and the poly (a 1 , 3 glucan) solids were collected on a Buchner funnel using 325 mesh screen over 40 micometers filter paper.
• the filter cake was resuspended in deionized water and filtered twice more as above to remove residual sucrose, fructose and other low molecular weight, soluble by-products. Finally two additional washes with methanol were carried out. The filter cake was pressed out thoroughly on the funnel and dried under vacuum at room temperature.
• the poly (a 1 , 3 glucan) formed in the control sample was also isolated and weighed. Formation of poly (a 1 , 3 glucan) in the tests and the control samples was confirmed using publically available information (Nakamura, T., et al., Biosci. Biotechnol.
• Example 4 was consistently around 0.6 g after 72 hours, no control samples were used in this experiment.
• the poly (a 1 , 3 glucan) solids formed in the dialysis tubes were isolated as described in Example 4.
• the resulting dry weights of the poly (a 1 , 3 glucan) obtained enzyme reaction solution at various time intervals are shown in Table 3.

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