Synthetic biology approaches and combinatorial biosynthesis towards heterologous lipopeptide production

Yan, Fu; Burgard, Christian; Popoff, Alexander; Zaburannyi, Nestor; Zipf, Gregor; Maier, Jutta; Bernauer, Hubert S.; Wenzel, Silke C.; Müller, Rolf

doi:10.1039/c8sc02046a

Cited by 42 publications

(33 citation statements)

References 56 publications

(51 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The most important consideration, and therefore restriction, of this strategy is the substrate specificity of the downstream C-domain, which must be respected. The Mueller group also utilised A–T–C XUs to successfully modify lipopeptide BGCs in Myxococcus xanthus, suggesting their general applicability [53], but the downstream C-domain restriction calls for a very large number of available building blocks if new-to-nature NRPs are to be designed in appreciable numbers.…”

Section: Nrps: Challenging Dogmamentioning

confidence: 99%

Engineering enzymatic assembly lines to produce new antibiotics

Bozhüyük

Micklefield

Wilkinson

2019

Current Opinion in Microbiology

View full text Add to dashboard Cite

HighlightsMany clinical antibiotics are natural products produced by thiotemplate-based assembly line biosynthetic pathways.Assembly line pathways provide an opportunity for rational bioengineering to modify complex natural product structures.New, rule-based mix and match strategies facilitate the engineering of non-ribosomal peptide assembly line synthetases.Evolutionary guided approaches highlight new avenues for polyketide synthase assembly line reprogramming.

show abstract

Section: Nrps: Challenging Dogmamentioning

confidence: 99%

Engineering enzymatic assembly lines to produce new antibiotics

Bozhüyük

Micklefield

Wilkinson

2019

Current Opinion in Microbiology

View full text Add to dashboard Cite

show abstract

“…The modified BGC was chemically synthesized in fragments, because of the size, GC content, and repetitive sequence segments in the cluster. Assembly of the cluster fragments was done by a combination of in vivo transformation-associated recombination (TAR) cloning in yeast 23 and a previously described in vitro three-step restriction/ligation cloning strategy 24 using Bsa I (cloning steps are summarized in Supplementary Method 4, Supplementary Figs. 4 and 5 and Supplementary Table 6 ).…”

Section: Resultsmentioning

confidence: 99%

In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

et al. 2021

Self Cite

View full text Add to dashboard Cite

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-l-isoasparagine or a β-methoxy-l-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound l-asparagine. Furthermore, CysQ O-methylates hydroxyl-l-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

show abstract

“…The presented data suggest that the artificial DD splitting approach between E-and C-domain modules can be incorporated into the expanding efforts of de novo design of NRPSs. [6,12,13,25] Being able to create smaller NRPS genes of a cluster without losing enzymatic activity, increases engineering flexibility, for example, by enabling the coding of smaller de novo engineered subunits [12,13,26] on different vectors, thereby allowing any de novo NRPS subunit on one vector to be combined with any de novo change on the other. It has not slipped our attention that adding N-and C-terminal protein tags to the NRPS led to a 2.5-fold increase in XFP production and this approach will be applied and further explored in the future.…”

Section: Angewandte Chemiementioning

confidence: 99%

Artificial Splitting of a Non‐Ribosomal Peptide Synthetase by Inserting Natural Docking Domains

Kegler

Bode

2020

Angewandte Chemie

View full text Add to dashboard Cite

The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme.

show abstract

Synthetic biology approaches and combinatorial biosynthesis towards heterologous lipopeptide production

Abstract: Synthetic biology techniques coupled with heterologous secondary metabolite production offer opportunities for the discovery and optimisation of natural products.

Cited by 42 publications

References 56 publications

Engineering enzymatic assembly lines to produce new antibiotics

Engineering enzymatic assembly lines to produce new antibiotics

In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

Artificial Splitting of a Non‐Ribosomal Peptide Synthetase by Inserting Natural Docking Domains

Contact Info

Product

Resources

About