US 20060277.632A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0277.632 A1 Carr et al. (43) Pub. Date: Dec. 7, 2006 (54) METHODS FOR PRODUCTION OF CHITIN Publication Classification AND CHITOSAN (51) Int. Cl. AOIH I/00 (2006.01) (75) Inventors: Brian Carr, Raleigh, NC (US); Philip COSB 37/08 (2006.01) E. Hammer, Cary, NC (US) C7H 2L/04 (2006.01) CI2P 19/28 (2006.01) CI2N I/2 (2006.01) Correspondence Address: CI2N L/6 (2006.01) ALSTON & BIRD LLP CI2N 15/74 (2006.01) BANK OF AMERICA PLAZA (52) U.S. Cl. ...................... 800/284; 435/85; 435/252.33; 101 SOUTH TRYON STREET, SUITE 4000 435/254.3: 435/.484; 435/488; CHARLOTTE, NC 28280-4000 (US) 536/20, 536/23.2 (57) ABSTRACT Compositions and methods for producing chitin and chito Assignee: Athenix Corporation, Durham, NC (US) san are provided. The compositions comprise genetically (73) modified organisms, including fungi, yeast, bacterial and plant organisms that have been engineered to express het erologous genes involved in chitin and chitosan synthesis. (21) Appl. No.: 11/434,526 Microorganisms and plants that have been modified for production of chitin and/or chitosan within the vacuole of a cell are encompassed. Methods for production of chitin also (22) Filed: May 15, 2006 comprise culturing the genetically engineered organisms in conditions that allow for chitin production. Further methods include converting the chitin to chitosan by a chemical Related U.S. Application Data process. Production of chitosan also comprises culturing organisms that are genetically modified to produce chitosan (60) Provisional application No. 60/680.94 2, filed on May without the need for chemical modification. Methods for in 13, 2005. vitro chitosan production are also encompassed. SH AHSYSE:REAH Glutamine Glutamate SN-2 Fructose-6-phosphate > Glucosamine-6-phosphate Dedication Step Acetylate N-AcGln-6-phosphate Acetylate > Gln-1-phosphate 4. N-AcGln-1-phosphate Dcacctylase (known) G UTP ppi . UDP-Gln-1-phosphate 1Hs UDP-N-AcGln-1-phosphate Deacetylase (known) Directed Evolution Chitosa Citi Chitin Synthetase Synthetase Synthetase (Km -30-50uM) (Km 30-50uM) (Km ~1-2mm) HAEF33A VISSEYERSH Chitosan Chitin Alkaline deacetylation Chitosan Patent Application Publication Dec. 7, 2006 US 2006/0277.632 A1 YSE:RYAH Glutamine Glutamate Fructose-6-phosphate SNZ6 D Glucosamine-6-phosphate Dedication Step Acetylate N-AcGln-6-phosphate Acetylate l -> Gln-1-phosphate t N-AcGln-1-phosphate Deacetylase (known) G UTP UTP ppi g ppi UDP-Gln-1-phosphate an UDP-N-AcGln-1-phosphate Deacetylase (known?) Directed Evolution Chitosan Chitin Chitin Synthetase Synthetase Synthetase (Km 30-50uM) (Km ~1-2m M) (Km -30-50uM) Alkaline deacetylation Chitosan FIG. 1 US 2006/0277.632 A1 Dec. 7, 2006 METHODS FOR PRODUCTION OF CHTIN AND when chitin/chitosan are to be produced as a value-added CHTOSAN product in an existing fermentation system, care must be taken to ensure that the yield of the primary fermentation CROSS REFERENCE TO RELATED product is not reduced and that the processing of the primary APPLICATION fermentation product is not altered in the process. An attractive approach would be to develop a cell expression 0001) This application claims the benefit of U.S. Provi system that is dedicated to production of chitin/chitosan. sional Application Ser. No. 60/680,942, filed May 13, 2005, the contents of which are herein incorporated by reference in 0007 Numerous publications report use of fungal biom its entirety. ass for production and recovery of chitosan. Methods of chitosan production from microbial biomass. Such as fila FIELD OF THE INVENTION mentous fungi, were disclosed in U.S. Pat. No. 4,806,474, International application No. WO 01/68714, and other pub 0002 This invention relates to the field of molecular lications (Synowiecki and Al-Khateeb (2003) Crit Rev Food biology. Provided are compositions and methods for pro Sci Nutr 43(2): 145-171; Pochanavanich and Suntomsuk ducing chitin and/or chitosan. (2002) Lett Appl Microbiol 35(1):17-21). However, these processes yield relatively expensive chitosan as compared to BACKGROUND OF THE INVENTION product extracted from shellfish. The yield of extracted 0003 Chitin is a natural polysaccharide present in vari chitosan is limited by the chitin and chitosan contents in the ous marine and terrestrial organisms, including crustacea, biomass. insects, mollusks, and microorganism or plants, such as 0008. Other methods of producing chitosan involve fungi. Chitin is typically an amorphous solid that is largely recovery from microbial biomass, such as the methods insoluble in water, dilute acids, and alkali. Although chitin taught by U.S. Pat. No. 4,806,474 and U.S. Patent Applica has various commercial applications, greater commercial tion No. 20050042735, herein incorporated by reference. utility is found by converting chitin to the deacetylated Another method, taught by U.S. Pat. No. 4,282,351, teaches product chitosan. only how to create a chitosan-beta-glucan complex. 0004 Chitosan can be created by N-deacetylation of the 0009 Methods for reducing the cost of chitosan produc chitin polymer. It is an amorphous solid that is largely tion are needed in order to realize the industrial potential for insoluble in water, but is soluble in aqueous organic acids, this polymer. Such as formic and acetic acids. Chitosan has many indus trial, medical, pharmaceutical, and nutritional uses, includ ing those requiring a biodegradable, non-toxic polymer. For BRIEF SUMMARY OF THE INVENTION example, chitosan is used as a polyelectrolytic coagulant and 0010 Compositions and methods for producing chitin a sludge dewatering aid in wastewater treatment. Medical, and chitosan are provided. The compositions comprise pharmaceutical, and nutritional uses often require a higher genetically modified organisms, including fungi, yeast, and quality chitosan for functional and aesthetic reasons. These bacterial organisms that have been mutated or engineered to uses include applications as anticoagulants, antiviral agents, express heterologous genes involved in chitin and chitosan drug carriers, cosmetic additives, dialysis membranes, synthesis. Genes including chitin synthase, glutamine-fruc orthopedic materials, wound dressings, food Stabilizers and tose-6-phosphate aminotransferase (GFA), and chitin thickeners, flavor and nutrient carriers, and dietary fiber. deacetylase can be mutated, for example, to allow produc 0005 Commercially produced chitosan is currently har tion of chitin as an insoluble polymer within a cell (including vested from shellfish by a lengthy extraction process in within vacuole compartments), to have improved processiv which chitin is chemically deacetylated to chitosan using ity, increased reaction velocity, a modified K for a Sub strong alkali treatment. The resulting chitosan is then iso strate, a substrate preference for UDP-glucosamine, or to lated as an acid soluble material. Though chitosan has allow for secretion of chitin from the cell. Alternatively, numerous industrial uses, the requirement for raw material heterologous genes from viral, fungal, insect, or other organ and the lengthy extraction process contribute to high pro isms may be expressed in the organism of choice to produce duction costs that limit actual industrial use of this polymer. increased amounts of chitin, or to produce chitosan directly, In fact, the potential industrial use of chitosan exceeds the without the need for chemical modification of chitin. production capacity of many traditional production schemes. 0011 Compositions also include polynucleotides encod Given the industrial high demand for chitosan, methods ing enzymes or polypeptides involved in the production of aimed at improving its production are needed. chitin and/or chitosan (“chitin/chitosan-related sequences”), 0006 Chitin and chitosan are related polymers that are vectors comprising those polynucleotides, and host cells produced by several types of fungi and yeasts as part of their comprising the vectors. Compositions comprising a coding cell wall. One approach to improving commercial chitosan sequence for one or more polypeptides involved in the production methods would be to engineer existing fermen production of chitin and/or chitosan are provided. Compo tation Strains to produce more chitin/chitosan as a value sitions of the present invention further include synthetic added product. For example, Rhizopus Oryzae or Aspergillus polynucleotides encoding enzymes or polypeptides involved niger Strains utilized for citric acid production contain in the production of chitin and/or chitosan. The coding Substantial amounts of chitin/chitosan, and thus are an sequences can be used in DNA constructs or expression attractive source for a value-added approach. Alternatively, cassettes for transformation and expression in organisms, a fermentative yeast can be engineered to produce chitosan including microorganisms and plants. Compositions also as a component of its cell walls. Regardless of the approach, comprise transformed fungi, bacteria, plants, plant cells, US 2006/0277.632 A1 Dec. 7, 2006 tissues, and seeds. In addition, methods are provided for been modifed to improve expression or activity or both, producing the polypeptides encoded by the synthetic nucle and/or can include a chitin synthase that has been modifed otides of the invention. to catalyze the polymerization of both chitin and/or chitosan. 0012. In particular, isolated polynucleotides correspond 0016 Chitin is produced by the polymerization