High-Yield Lasso Peptide Production in a <i>Burkholderia</i> Bacterial Host by Plasmid Copy Number Engineering

Fernandez, Hannah N.; Kretsch, Ashley M.; Kunakom, Sylvia; Kadjo, Adjo E.; Mitchell, Douglas A.; Eustáquio, Alessandra S.

doi:10.1021/acssynbio.3c00597

Cited by 4 publications

(1 citation statement)

References 48 publications

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“…FERM BP-3421. This emerging chassis strain is closely related to B. thailandensis E264 and has been shown to significantly enhance the production of RiPP and non-RiPP natural products. − To facilitate expression, we inserted the genomic DNA containing the apy BGC downstream of the rhamnose-inducible promoter in a pSCrhaB2 plasmid used previously for protein expression in Burkholderia (Table S2). Additionally, we introduced an N-terminal hexa-His tag on ApyA to facilitate isolation.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Nguyen,

Zhu,

Gray

et al. 2024

ACS Cent. Sci.

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Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin-and radical Sadenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C−C cross-link between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the β-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.

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Section: Resultsmentioning

confidence: 99%

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Nguyen,

Zhu,

Gray

et al. 2024

ACS Cent. Sci.

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Modulation of major components of bergamot essential oil in Escherichia coli through metabolic engineering strategies

Zhu,

Wang,

Shi

et al. 2024

Industrial Crops and Products

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Evaluation of vectors for gene expression inPseudovibriobacteria and their application inAplysinamarine sponge studies

Dai,

Pita,

Eustáquio

2024

Preprint

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The filter feeding capacity of marine sponges contributes to biogeochemical cycling and they are also involved in habitat formation, properties that are critical to marine ecology. Sponge-associated microbes are crucial to the functional roles provided by sponges. α-Proteobacteria belonging to thePseudovibriogenus have been isolated from many different marine sponge genera and have been proposed to contribute to sponge health. We recently reported specialized metabolites we named pseudovibriamides fromPseudovibrio brasiliensisAb134. The pseudovibriamide encodingpppgene cluster is found in two thirds ofPseudovibriogenomes. Pseudovibriamides coordinate motility and biofilm formation, behaviors that are known to be important for host colonization. Although reverse genetics methods to delete genes via homologous recombination have been established, no self-replicative vectors have been reported forPseudovibrio. We show that plasmid vectors containing three different broad-host-range replicons, RSF1010, RK2, and pBBR1, can be used inP. brasiliensisfor fluorescent protein expression and consequent labeling. We then applied GFP and mCherry expressing strains to answer the question of whether pseudovibriamides affect the uptake ofP. brasiliensisbyAplysina aerophobasponges.P. brasiliensiscell counts decreased in the sponge aquaria at an equivalent rate for wild-type and pseudovibriamide-defective ΔpppAmutant strains, indicating that the sponge filters each strain indiscriminately under the conditions tested. Yet, the filtering capacity varied for each sponge individual tested, stressing the importance of performing experiments with wild-type and mutant bacterial strains in the same aquarium to allow for rigorous conclusions, which is now enabled with the methods established here.ImportanceMarine sponges are ecosystem engineers. They transform nutrients into a bioavailable form for other marine organisms. Microbes are critical to the functional roles provided by sponges because they expand the metabolic capabilities of the sponge host. Yet, most of our knowledge on sponge microbes comes from genomic studies, since cultivability and the ability to perform genetics with sponge bacterial isolates is limited. The genusPseudovibrioof α-Proteobacteria has consistently been isolated from marine sponges and it has been hypothesized to contribute to marine sponge health. Moreover,Pseudovibriobacteria are a source of antibiotics and other secondary metabolites with the potential to be developed into pharmaceuticals. Here we established vectors for the expression of fluorescent proteins inPseudovibriobacteria and demonstrated their utility inin vivostudies with marine sponges. The availability of genetic tools is important to enable us to explore the emerging ecological and biotechnological potential ofPseudovibriobacteria.

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High-Yield Lasso Peptide Production in a Burkholderia Bacterial Host by Plasmid Copy Number Engineering

Cited by 4 publications

References 48 publications

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Modulation of major components of bergamot essential oil in Escherichia coli through metabolic engineering strategies

Evaluation of vectors for gene expression inPseudovibriobacteria and their application inAplysinamarine sponge studies

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