Vitro Biosynthesis

One of the ultimate goals in the investigation of membrane protein assembly is to develop a cell-free assembly system of a particular 

The purified mRNA was 7 S in size and separated into two closely associated bands. Their sizes were estimated to be about 250 and 230 nucleotides long. At present, it has not yet been established which band has the mRNA activity. It is, however, possible that one RNA is processed into the other. 

---

S. Shojima, N. Nishizawa, S. Fushiya, S. Nozoe, T. Irifune, and S. Mori, Biosynthesis of phytosiderophores in vitro biosynthesis of 2 -deoxymugineic acid from L-methionine and nicotianamine. Plant Phy.siol. 93 1491 (1990). 

Flurkey WH. In vitro biosynthesis of Vicia faba polyphenoloxidse. Plant Physiol 1985 79 564-567. 

Kambampati R, Lauhon CT. 2000. Evidence for the transfer of sulfane snlfnr from IscS to Thil during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. J Biol Chem 275 10727-30. 

Funk C, Brodelius P (1992) Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr. IV Induction of vanillinic acid formation. Plant Physiol 99 256-262 Funk C, Brodelius P (1994) Vanilla planifolia Andrews in vitro biosynthesis of vanillin and other phenylpropanoids derivatives. In Bajaj YPS (ed) Biotechnology in agriculture and forestry. Medicinal and aromatic plants VI, vol 26. Springer, Berlin Heidelberg New York, pp 377-402... 

Liu, S.J. Steinbuchel, A. Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid). Appl. Microbiol. Biotechnol., 53, 545-552 (2000)... 

Fig. 6 Four enzymes (one salicylate-AMP ligase YbtE/PchD, two NRPS and/or NRPS/PKS enzymes HMWPl/PchE and HMWP2/PchF, and one reductase YbtU/PchG) are required for the in vitro biosynthesis of (a) yersiniabactin (24) and (b) pyochelin (25). (c) The initial stages of 24 and 25 biosynthesis proceed via a similar mechanism from chorismic acid 26 to the salicylate-bisthiazole intermediate 33...

Fig. 18 (a) Total in vitro biosynthesis of 65 requires three enzymes, MxcE, MxcF, and MxcG. (b) Biosynthesis of 65 and 103 from two ArCP-bound DHB units (79) and one PCP-bound lysine (80b)... 

Fig. 34 Selection of novel wailupemycin 202—204 and enterocin analogs 205-207 generated by in vitro biosynthesis using unnatural starter units 199-201...

The experiments related here represent only some of the successful reengineering of the erythromycin PKS and other related synthases. In the future, this technology should provide many hybrid proteins for in vitro biosynthesis. 

Despite this limitation, in vitro biosynthesis using purified biocatalysts promises to be a simple and inexpensive means to access a wealth of complex natural product structures under clean and controlled conditions. The versatility of this technology can be significantly enhanced by the application of genetic engineering to create novel proteins. 

A Lawen, R Traber, D. Geyl. In vitro biosynthesis of [Thr2, Leu5, D-Hiv8, Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activities by a multienzyme polypeptide. J Biol Chem 266 15567-15570, 1991. 

Hosel, W., Berlin, J., Hanzlik, T.N. and Conn, E.E. (1985) In vitro biosynthesis of l-(4 -hydroxyphenyl)-2-nitroethane and production of cyanogenic compounds in osmotically stressed cell suspension cultures of Eschscholtzia californica. Planta, 166, 176-81. 

Hosel, W. and Nahrstedt, A. (1980) In vitro biosynthesis of the cyanogenic glucoside, taxiphyllin, in Triglochin maritima. Arch. Biochem. Biophys., 203, 753-7. 

McFarlane, I.J., Less, E.M. and Corm, E.E. (1975) The in vitro biosynthesis of dhurrin, the cyanogenic glucoside of Sorghum bicolor.. Biol. Chem., 250, 4708-13. 

Fig.9. Polyketide products generated via in vitro biosynthesis by the fusion protein DEBS 1+TE. 1 and 2, C-labeled alternate starter unit substrates were used to synthesize the Cj- and CiQ-lactones, respectively. 3 and 4, (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine (NAC) thioester was used as an intermediate to synthesize the C9-lactone and the 3-oxo analog, respectively. 5, In the absence of NADPH, a pyran-2-one is generated using propionyl-CoA and C-labeled methylmalonyl CoA...

McClerren AL, Cooper LE, Quan C, Thomas PM, Kelleher NL, van der Donk WA. Discovery and in vitro biosynthesis of halo-duracin, a new two-component lantibiotic. Proc. Natl. Acad. Sci. U.S.A. 2006 103 17243-17248. 

Delongcamp, D., Lubet, P. and Drosdowsky, M. (1974) The in vitro biosynthesis of steroids by the gonad of the mussel (Mytilus edulis). Gen. Comp. Endocrinol., 22, 116-127. 

Rachmel, A. Hazelton, G.A. Yergey, A.L. Llberato, D.J. Furosemide 1-0-acyl Glucuronide. In Vitro Biosynthesis and pH-dependent Isomerization to P -Glucuronidase-resistant Forms, Drug Metab. and Dispos. 13(6), 705-710 (1985). 

Kobayashi, S., Uyama, H., and Namekawa, S. (1998) In vitro biosynthesis of polyesters with isolated enzymes in aqueous systems and organic solvents. Polym. Degrad. Stab., 59 (1-3), 195-201. 

Jossek, R., Reichelt, R., and Steinbiichel, A. (1998) In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum. Appl. Microbiol. Biotechnol., 49, 258-266. 

---