Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

Menzella, Hugo G.; Reid, Ralph; Carney, John R.; Chandran, Sunil S.; Reisinger, Sarah J.; Patel, Kedar G.; Hopwood, David A.; Santi, Daniel V.

doi:10.1038/nbt1128

Cited by 303 publications

(249 citation statements)

References 29 publications

Supporting

Mentioning

243

Contrasting

Unclassified

Order By: Relevance

“…The UCS's functionality clearly demonstrated the portability of involved COM domains by the simultaneous formation of the expected di-and tripeptide products. Most recently, Menzella et al (14) reported on a very similar goal in the analogous polyketide synthases. By exploiting a compatible set of interpeptide linkers (also referred to as docking domains, the counterparts of NRPS COM domains), Menzella et al investigated the productive interaction between 154 bimodular combinations of donor and acceptor modules and found that nearly half of the combinations successfully mediated the biosynthesis of the desired triketide lactones.…”

Section: Tycb3mentioning

confidence: 79%

Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides

Hahn

Stachelhaus

2006

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

The interaction between enzymes of a nonribosomal peptide synthetase (NRPS) complex relies on the interplay of compatible sets of donor and acceptor communication-mediating (COM) domains. Hence, these domains are essential for the formation of a defined biosynthetic template, thereby directing the synthesis of a specific peptide product. Without the selectivity provided by different sets of COM domains, NRPSs should form random biosynthetic templates, which would ultimately lead to combinatorial peptide synthesis. This study aimed to exploit this inherent combinatorial potential of COM domains. Based on sequence alignments between COM domains, the crosstalk between different biosynthetic systems was predicted and experimentally proven. Furthermore, key residues important for maintaining (or preventing) NRPS interaction were identified. Point mutation of one of these key residues within the acceptor COM domain of TycC1 was sufficient to shift its selectivity from the cognate donor COM of TycB3 toward the noncognate donor COM domain of TycB1. Finally, an artificial NRPS complex was constructed, constituted of enzymes derived from three different biosynthetic systems. By virtue of domain fusions, the interactions between all enzymes were established by the same set of COM domains. Because of the abrogated selectivity, this universal communication system was able to simultaneously form two biosynthetic complexes that catalyzed the combinatorial synthesis of different peptide products.nonribosomal peptide synthetase ͉ peptide antibiotics ͉ protein-protein interaction

show abstract

Section: Tycb3mentioning

confidence: 79%

Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides

Hahn

Stachelhaus

2006

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

show abstract

“…Library approaches can provide a direct route for obtaining and refining functional in vivo systems (39)(40)(41). In the field of metabolic engineering, for example, researchers have repeatedly improved natural product yields and synthesized analogs by searching collections of isozymes (42,43), mutant biosynthetic enzymes (3,44), or promoters and regulatory regions that modulate the expression levels of genes that alter pathway flux (45,46). Testing these multiple variables in the context of a pathway causes library sizes to rapidly swell (e.g., testing 100 mutants of enzyme A against 100 mutants of enzyme B is already Fig.…”

Section: Discussionmentioning

confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

show abstract

“…All these platforms have dramatically improved their performance in recent years to cope with to the expected shortage of recombinant protein manufacturing capacity [2,3]. For instance, the modifications introduced in prokaryotic systems have made them increasingly able to produce and display complex proteins [4], or have facilitated the introduction of whole metabolic pathways [5]; similarly, yeast strains have been engineered to display nearlyhuman glycosylation profiles [6]; CHO cells have multiplied their yields, reaching g/L levels [7]; alternative animal systems as chicken eggs have arisen [8], and baculovirusbased systems have been adapted to the production of multi-subunit protein complexes [9].…”

Section: Introductionmentioning

confidence: 99%

Manufacturing antibodies in the plant cell

Orzáez

Granell

Blázquez

2009

Biotechnology Journal

View full text Add to dashboard Cite

International audiencePlants have long been considered advantageous platforms for large-scale production of antibodies due to their low cost, scalability, and the low chances of pathogen contamination. Much effort has therefore been devoted to efficiently produce mAbs (from nanobodies to secretory antibodies) in plant cells. Several technical difficulties have been encountered and are being coped with. Improvements in production levels have been achieved by manipulation of gene expression and, more efficiently, of cell targeting and protein folding and assembly. Differences in mAb glycosylation patterns between animal and plant cells are being successfully addressed by the elimination and introduction of the appropriate enzyme activities in plant cells. Another relevant battlefield is the dichotomy between production capacity and speed. Classically, stably transformed plant lines have been proposed for large scale mAb production, whereas the use of transient expression systems has always provided production speed at the cost of scalability. However, recent advances in transient expression techniques have brought impressive yield improvements, turning speed and scalability highly compatible assets. In the era of personalized medicines, the combination of yield and speed, and the advances in glyco-engineering have made the plant cell a serious contender in the field of recombinant antibody production

show abstract

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

Cited by 303 publications

References 29 publications

Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides

Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Manufacturing antibodies in the plant cell

Contact Info

Product

Resources

About