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United States Patent Application

View the Complete Application at the US Patent & Trademark Office
Lawrence Berkeley National Laboratory - Visit the Technology Transfer and Intellectual Property Management Department Website
To produce a bacterial microcompartment shell, or a designed shell based on naturally occurring bacterial microcompartment shells in a new host organism, a synthetic operon is constructed that contains the desired shell protein genes and translation efficiency is controlled by host specific ribosomal binding sites. Proteins or other molecules can be encapsulated in the microcompartment shells by various methods described herein. The constructs can also be used to express self-assembling sheets comprised of shell proteins.
Kerfeld, Cheryl A. (Walnut Creek, CA), Lassila, Jonathan K. (San Francisco, CA), Kinney, James N. (Clayton, CA)
The Regents of the University of California (Oakland CA)
14/ 214,172
March 14, 2014
STATEMENT OF GOVERNMENTAL SUPPORT [0001] This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy, under Contract No. DE-0000200 awarded by the Department of Energy ARPA-E, and under Grant Nos. MCB0851094 and MCB1160614 awarded by the National Science Foundation. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application is a nonprovisional of and claims priority to U.S. Provisional Patent Application No. 61/800,118, filed on Mar. 15, 2013, which is hereby incorporated by reference in its entirety for all purposes. [0003] This application is related to co-pending nonprovisional application U.S. patent application Ser. No. 13/564,676, filed on Aug. 1, 2012, hereby incorporated by reference in its entirety. This application is related to and incorporates by reference U.S. patent application Ser. No. 13/367,260, filed on Feb. 6, 2012 in its entirety for all purposes. REFERENCE TO SEQUENCE LISTING AND TABLES [0004] This application also incorporates by reference the attached sequence listings which is also found in computer-readable form in a *.txt file entitled, "IB3335US_seqlisting_ST25.txt", created on Mar. 14, 2014. BACKGROUND OF THE INVENTION [0005] 1. Field of the Invention [0006] The present invention relates to synthetic biology, especially using operons and synthetic constructs to produce microcompartments and bacterial microcompartment shells and to integrate molecules and proteins into these microcompartments, or on the microcompartment surface. [0007] 2. Related Art [0008] Bacterial microcompartments (BMCs) encapsulate enzymes and metabolic pathways. The most well known type of BMC is the carboxysome, which fixes CO.sub.2 in cyanobacteria. Several other types of BMC gene clusters have been identified in prokaryotes, including the propanediol utilization and ethanolamine utilization microcompartment gene clusters. [0009] The shells of BMCs are composed of multiple paralogs of proteins containing BMC domains pfam00936 and pfam03319. Three types of shell proteins have been identified: single pfam00936 domains ("hexamer"), fusion proteins composed of two pfam00936 domains ("tandem domain"), and single pfam03319 domains ("pentamer"). Hexamer and tandem domain proteins are the major components of known microcompartment shells, while pentamer proteins are minor components. Natural BMC gene clusters vary widely in composition and gene arrangement and are defined by genes that encode shell proteins. Three types of BMC shell proteins exist, identified here as hexamers or BMC-H, tandem domains or BMC-T, and pentamers or BMC-P, that together form polyhedral shells (FIG. 1). BMC-H polypeptides contain a single domain of the pfam00936 family from the pfam database (Punta, M., Coggill, P. C., Eberhardt, R. Y., Mistry, J. & Tate, J. e. a. (2012). The Pfam protein families database. Nucleic Acids Research 40, D290-D301), about 90 amino acids, that assembles into a six-fold symmetric hexamer in crystal structures. This type of subunit represents the most abundant component of characterized BMC shells. Tandem domains (BMC-T) contain two pfam00936 domains in a single polypeptide. These proteins form trimers with a pseudo-hexameric configuration that are sometimes found stacked into a double layer in crystal structures. (Klein, M. G., Zwart, P., Bagby, S. C., Cai, F., Chisholm, S. W., Heinhorst, S., Cannon, G. C. & Kerfeld, C. A. (2009). Identification and structural analysis of a novel carboxysome shell protein with implications for metabolite transport. Journal of Molecular Biology 392, 319-333; Cai, F., Sutter, M., Cameron, J. C., Stanley, D. N., Kinney, J. N. & Kerfeld, C. A. (2013). The structure of CcmP, a tandem bacterial microcompartment domain protein from the .beta.-carboxysome forms a subcompartment within a microcompartment. Journal of Biological Chemistry 288, 16055-16063). The third type, referred to here as pentamers or BMC-P, contain a single domain of the pfam03319 family. The five-fold symmetric assemblies formed by these proteins are presumed to occupy the vertices of icosahedral shells. Accordingly, they are a minor component of characterized BMC shells; only 60 copies of the gene product (12 pentamers) are required to close an icosahedral shell. [0010] Previously others have expressed only naturally existing microcompartment operons or partial operons in heterologous hosts. See Bonacci W, Teng P K, Afonso B, Niederholtmeyer H, Grob P, Silver P A, Savage D F, Modularity of a carbon-fixing protein organelle, Proc. Natl. Acad. Sci. USA 2012 Jan. 10; 109(2):478-83.Epub 2011 Dec. 19. However, a general approach for producing synthetic microcompartment shell operons, synthetic microcompartment shells and integrating molecules into microcompartments has not been described.