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(12) Patent Application Publication (10) Pub. No.: US 2017/0211105 A1 HN S

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(12) Patent Application Publication (10) Pub. No.: US 2017/0211105 A1 HN S US 20170211105A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0211105 A1 Anderson et al. (43) Pub. Date: Jul. 27, 2017 (54) BIOSYNTHETIC PRODUCTION OF Publication Classification CARNOSINE AND BETA-ALANNE (51) Int. Cl. Applicant: 20n Labs, Inc., Washington, DC (US) CI2P I3/00 (2006.01) (71) CI2P I3/06 (2006.01) (72) Inventors: John Christopher Anderson, Berkeley, CI2R L/865 (2006.01) CA (US); Saurabh Srivastava, San (52) U.S. Cl. Francisco, CA (US); Mark T. Daly, CPC ............ CI2P 13/005 (2013.01): CI2R 1/865 Oakland, CA (US) (2013.01); CI2P 13/06 (2013.01) (21) Appl. No.: 15/408,317 (57) ABSTRACT (22) Filed: Jan. 17, 2017 Related U.S. Application Data The present disclosure provides compositions and methods (60) Provisional application No. 62/281,621, filed on Jan. for the biosynthetic production of carnosine and beta-ala 21, 2016. nine. HN s Na- Oh Nav NH w O usNH histicine N ce- N w sul ^. OH os- O MscMr. ---lO A Carrosie O OH NH co, H2N O asparate beta-alarine Patent Application Publication Jul. 27, 2017 Sheet 1 of 3 US 2017/0211105 A1 Figure 1 HNC ^^'oh N Nh Is NH histicite \, Y O s • O O - -- HN Y N O l w8w-aa-e-4-4-4-4-4-4-3a O * C S-S-S-proh ~ss: --- X w C3OShe co, H:N O aspa?tate beta-aafire Patent Application Publication Jul. 27, 2017 Sheet 2 of 3 US 2017/0211105 A1 Figure 2 Relative carnosine titer in cell pellet 3OOOOOOO .........................................................................-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 2SOOOOOO 2000000.O 15OOOOOO 1000000.0 ----------------------------------- SOOOOOO . OO . - i.e. ...8.................. 8. '', nego ca2 ca8 ca10 ca14 nego call ca19 ca2O ca22 ca23 ca24 ca28 Patent Application Publication Jul. 27, 2017 Sheet 3 of 3 US 2017/0211105 A1 3OOOOO 3. .8. 20000.0 - - - - i 100000 O. ...--8 8.............'.88888........... 88888.’.................................w ... 8 --- XXXX ----- ...8 ---. nego Ca2 Ca8 ca10 ca14 nego ca1 ca19 ca20 Ca22 ca23 ca24 Ca28 US 2017/0211105 A1 Jul. 27, 2017 BIOSYNTHETIC PRODUCTION OF pressures. It is also natively produced in bacteria and yeasts CARNOSINE AND BETA-ALANNE in Small quantities. In bacteria, beta-alanine is produced by decarboxylation of aspartate. Lysates of bacteria have been CROSS REFERENCE TO RELATED used in biocatalytic production from aspartate (Patent APPLICATIONS CN104531796A). 0001. This application claims priority to, and benefit of 0011. There remains a need in the industry for a safer, U.S. Provisional Application No. 62/281,621 filed Jan. 21, more economical system for the production of carnosine and 2016, the contents of which are incorporated herein by beta-alanine. reference in its entirety. SUMMARY OF THE INVENTION INCORPORATION-BY-REFERENCE OF 0012. The present disclosure provides compositions and SEQUENCE LISTING methods for the biosynthetic production of nutritional 0002. The contents of the text file named “NLAB 002 Supplements such as beta-alanine and carnosine. 01 US ST25.txt” submitted electronically herewith which 0013 Embodiments of the present invention comprise was created on Jan. 5, 2017 and is 80 KB in size, are engineered organisms that produce beta-alanine and carnos incorporated herein by reference in their entirety. ine. The engineered organisms may include genetically tractable organisms such as plants, animals, bacteria, or FIELD OF THE INVENTION fungi. 0014. The present invention comprises methods of pro 0003. The present disclosure relates compositions and ducing carnosine. The methods comprise providing a recom methods for the biosynthetic production of nutritional binant microorganism comprising an engineered carnosine Supplements such as beta-alanine and carnosine. In particu biosynthesis pathway. The engineered microorganism may lar, the disclosure features recombinant microorganisms be used for the commercial production of carnosine. Accord comprising an engineered carnosine biosynthesis pathway. ingly, in one embodiment the invention provides growing in Suitable conditions, a recombinant microbial host cell com BACKGROUND OF THE INVENTION prising at least one DNA molecule encoding an enzyme(s) 0004 Carnosine is a dipeptide of the amino acids beta that catalyze a Substrate to product conversion selected from alanine and histidine. It is highly concentrated in muscle and the group consisting of: brain tissues. 0015 i.aspartate to beta-alanine (pathway step a); 0005 B-Alanine (or beta-alanine) is a naturally occurring 0016 ii. beta-alanine to carnosine (pathway step b); beta amino acid, which is an amino acid in which the amino the at least one DNA molecule is heterologous to said group is at the B-position from the carboxylate group (i.e., microbial host cell and wherein said microbial host cell two atoms away). produces carnosine. The method further includes cultivating 0006 B-Alanine is not used in the biosynthesis of any the microorganism in a culture medium until a recoverable major proteins or enzymes. It is formed in vivo by the quantity of carnosine is produced and recovering the car degradation of dihydrouracil and carnosine. It is a compo nosine. nent of the naturally occurring peptides carnosine and anser 0017. In one aspect, a biotransformation method of pro ine and also of pantothenic acid (vitamin B5), which itself ducing carnosine is provided. The method comprises pro is a component of coenzyme A. Under normal conditions, viding a recombinant microorganism comprising an engi B-alanine is metabolized into acetic acid. neered carnosine synthesis pathway. The engineered 0007 B-Alanine is the rate-limiting precursor of carnos microorganism may be used for the commercial production ine, which is to say carnosine levels are limited by the of carnosine. Accordingly, in one embodiment, the invention amount of available f-alanine, not histidine. Supplementa provides growing in Suitable conditions, a recombinant tion with 3-alanine has been shown to increase the concen microbial host cell comprising at least one DNA molecule tration of carnosine in muscles, decrease fatigue in athletes encoding an exogenous enzyme that catalyzes the joining of and increase total muscular work done. beta-alanine to histidine to produce carnosine wherein the at 0008 Carnosine and beta-alanine are popular dietary least one DNA molecule is heterologous to said microbial Supplements currently produced using chemical methods. host cell, wherein beta-alanine substrate is added to the Beta-alanine is also a synthetic precursor to pantothenic growth culture, and wherein said microbial host cell pro acid, the essential vitamin B5. Beta-alanine can also be used duces carnosine. The method further includes cultivating the as a monomer for the production of a polymeric resin (U.S. microorganism in a culture medium until a recoverable Pat. No. 4,082,730). quantity of carnosine is produced and recovering the car 0009 Naturally, carnosine is produced exclusively in nosine. animals from beta-alanine (via uracil) and histidine. In 0018. In another aspect of the invention, a method of yeasts and animals, beta-alanine is typically produced by producing beta-alanine provided. The method comprises degradation of uracil. Chemically, carnosine can be synthe providing a recombinant microorganism comprising an sized from histidine and beta-alanine derivatives. For engineered beta-alanine biosynthesis pathway. The engi example, the coupling of an N-(thiocarboxy) anhydride of neered microorganism may be used for the commercial beta-alanine with histidine has been described (Vinicket al. production of beta-alanine. Accordingly, in one embodiment A simple and efficient synthesis of L-carnosine. J. Org. the invention provides growing in Suitable conditions, a Chem, 1983, 48(3), pp. 392–393). recombinant microbial host cell comprising at least one 0010 Beta-alanine can be produced synthetically by DNA molecule encoding an enzyme that catalyzes an aspar Michael addition of ammonia to ethyl- or methyl-acrylate. tate to beta-alanine conversion (pathway step a) and wherein This requires the use of the caustic agent ammonia and high the at least one DNA molecule is heterologous to said US 2017/0211105 A1 Jul. 27, 2017 microbial host cell and wherein said microbial host cell impact on the environment, safety of production workers, produces beta-alanine. The method further includes culti and potentially reduced impurities in the final products. Vating the microorganism in a culture medium until a recoverable quantity of beta-alanine is produced and recov BRIEF DESCRIPTION OF THE DRAWINGS ering the beta-alanine. 0024 FIG. 1 shows the biosynthetic pathway encoded by 0019 Embodiments of the present invention comprise engineered organisms that produce beta-alanine, carnosine, strains of the present disclosure. or both. The engineered organisms may include genetically 0025 FIG. 2 shows the relative carnosine titer from tractable organisms such as plants, animals, bacteria, or various strains of the present invention. fungi. Some embodiments of the present invention comprise 0026 FIG. 3 shows the relative beta-alanine titer from genetically engineered strains of yeast. In further embodi various strains of the present invention. ments, the yeast is S. cerevisiae. S. cerevisiae is a preferred organism for biosynthetic production due to favorable con DETAILED DESCRIPTION OF THE Sumer sentiment, the robust experience and infrastructure INVENTION for Scaling up fermentation,
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