The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional d-xylose utilization pathway (∼9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (∼11 kb DNA consisting of five genes) and a functional combined d-xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of eight genes) with high efficiencies (70–100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.

The mineralized tube of the sandcastle worm Phragmatopoma californica is made from exogenous mineral particles (sand, shell, etc.) glued together with a cement secreted from the "building organ" on the thorax of the worm. The glue is a cross-linked mixture of three highly polar proteins. The complete sequences of Pc-1 (18 kDa) and Pc-2 (21 kDa) were deduced from cDNAs derived from previously reported peptide sequences (Waite, J. H., Jensen, R., and Morse, D. E. (1992) Biochemistry 31, 5733-5738). Both proteins are basic (pI ϳ10) and exhibit Gly-rich peptide repeats. The consensus repeats in Pc-1 and -2 are VGGYGYGGKK (15 times), and HPAVX-HKALGGYG (eight times), respectively, in which X denotes an intervening nonrepeated sequence and Y is modified to 3,4-dihydroxyphenyl-L-alanine (Dopa). The third protein, Pc-3, was deduced from the cement to be about 80 mol % phosphoserine/ serine, and the cDNA was obtained by exploiting the presence of poly-serine repeats. Pc-3 consists of a family of at least seven variants with 60 -90 mol % serine most of which is phosphorylated in the cement. Pc-1, -2, and -3 contain cysteine some of which reacts to form 5-S-cysteinyl-Dopa cross-links during the setting process.The California sandcastle worm, Phragmatopoma californica (Fewkes), is a premier sand mason (1). In common with other sabellariid polychaetes, it exhibits an almost frenzied diligence in the collection, sorting and placement of sand grains for the construction and repair of its tubular home.3 Although each worm builds primarily the tube in which it resides, a colony of worms can coordinate its efforts to erect massive boulder-like concretions that play a pivotal role in reef ecology (3, 4). The cement used by Phragmatopoma and related sabellariids to bind together grains of sand has been of interest for some time in that it adheres irreversibly to wet mineral surfaces and is used with extraordinary speed and economy. Perhaps 4 -7 "spot welds," each about 100 m in diameter, are used to hold each sand grain (diameter 500 m) in place in the natural concrete (3,5).Phragmatopoma cement consists of proteins and significant levels of phosphate, calcium, and magnesium (6, 7). Two of the cement proteins, Pc-1 and Pc-2, 4 known from an earlier partial characterization (8),resemble the byssal adhesives of mussels (9) in that they are basic and contain 3,4-dihydroxyphenyl-L-alanine (Dopa) (5,7,8). Surprisingly, the abundant phosphate was not found to be associated with mineral but rather with serine residues in the cement (7). Indeed, the cement is dominated by phosphoserine and glycine, which together account for nearly 60 mol % of all the residues detected post-hydrolysis. Since the serine content of Pc-1 and -2 is negligible (8), the existence of a third serine-rich precursor is postulated. The aim of the present research was to identify the serine-rich protein, to obtain full-length sequences of Pc-1 and Pc-2, and to gain some insights into the mechanism of cement solidification. MATERIALS AND METHODSWorm Maintenance for Tub...

No abstract

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090 -11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.Mussels inhabit wind and wave swept rocky seashores. Such habitats are deathtraps for exposed organisms lacking a secure attachment. Accordingly, mussels have evolved a robust holdfast known as the byssus, which is essentially a specialized 4 -5-cm long bundle of collagenous fibers that is proximally rooted in the mussel and distally bonded to foreign surfaces underwater by flattened attachment plaques (1).Given that engineering durable adhesive bonds between minerals and organic polymers in the presence of moisture remains a serious technological challenge, fundamental insights into the mechanism of holdfast adhesion in mussels and other sessile marine organisms represent a potential data base of bio-inspired solutions to the moisture problem (1). One popular technique for improving "wet" adhesion on siliceous substrates involves the application of surface-coupling agents or adhesion promoters (2). Organosilanes are the best known synthetic adhesion promoters and typically designed with specific moieties for silica ligation at one end and reactivity toward the organic polymer at the other (2).The use of surface-coupling agents to promote adhesion resonates with the adhesive biochemistry of byssal plaques made by mussels. A recent investigation of plaque footprints in Mytilus californianus has revealed a family of protein variants (mcfp-3) with a 3,4-dihydroxyphenyl-L-alanine (Dopa) 2 content that approaches 25 mol % (3, 4). With its many Dopa residues, mcfp-3 has been compared wi...

No abstract