<i>Streptomyces albus</i>: A New Cell Factory for Non-Canonical Amino Acids Incorporation into Ribosomally Synthesized Natural Products

Most clinical antibiotics are derived from actinomycete natural products 19 (NPs) discovered at least 60 years ago. Repeated rediscovery of known compounds 20 led the pharmaceutical industry to largely discard microbial NPs as a source of new 21 chemical diversity but advances in genome sequencing revealed that these organisms 22 have the potential to make many more NPs than previously thought. Approaches to 23 unlock NP biosynthesis by genetic manipulation of the strain, by the application of 24 chemical genetics, or by microbial co-cultivation have resulted in the identification of 25 new antibacterial compounds. Concomitantly, intensive exploration of coevolved 26 ecological niches, such as insect-microbe defensive symbioses, has revealed these 27 to be a rich source of chemical novelty. Here we report the novel lanthipeptide 28 antibiotic kyamicin generated through the activation of a cryptic biosynthetic gene 29 2 cluster identified by genome mining Saccharopolyspora species found in the obligate 30 domatia-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium. 31 Heterologous production and purification of kyamicin allowed its structural 32 characterisation and bioactivity determination. Our activation strategy was also 33 successful for the expression of lantibiotics from other genera, paving the way for a 34 synthetic heterologous expression platform for the discovery of lanthipeptides that are 35 not detected under laboratory conditions or that are new to nature. 37Importance. The discovery of novel antibiotics to tackle the growing threat of 38 antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic 39 potential of microorganisms. The development of new tools to unlock the biosynthesis 40 of cryptic bacterial natural products will greatly increase the repertoire of natural 41 product scaffolds. Here we report an activation strategy that can be rapidly applied to 42 activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This 43 allowed the discovery of a new lanthipeptide antibiotic directly from the native host and 44 via heterologous expression.45 46Antimicrobial resistance (AMR) is arguably the greatest health threat facing humanity 47 in the 21 st century (1-3). It is predicted that without urgent action, infectious disease 48 will become the biggest killer of humans by 2050 (1). The majority of clinically used 49 antibiotics are based on microbial natural products, isolated mostly from soil-dwelling 50 Streptomyces species and other filamentous actinomycete bacteria, and these 51 organisms remain a promising source of new antibiotics. Although the discovery 52 pipeline began to dry up in the 1960s, blighted by the rediscovery of known 53 compounds, we know from large scale genome sequencing that up to 90% of microbial 54 natural products are not produced under laboratory conditions (4). Thus, there exists 55 a wealth of novel chemistry waiting to be discovered by mining the genomes of these 56 organisms. Bearing in m...

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“…S4). This is consistent with previous work which reported that deoxy versions of lantibiotics have lower biological activity (43).…”

Section: Resultssupporting

confidence: 94%

In situ activation and heterologous production of a cryptic lantibiotic from a plant-ant derived Saccharopolyspora species

Vikeli

Widdick

Batey

et al. 2019

Preprint

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“…The high substrate tolerance of the NisBTC system allows modification not only of RiPPs from different origins ( van Heel et al, 2016 ), but also of the newly synthesized nisin(BocK) variants. With our results for L. lactis and previous works on B. cereus ( Luo et al, 2016 ) and Streptomyces albus ( Lopatniuk et al, 2017 ), SCS has now been successfully implemented in three Gram-positive organisms from different prokaryotic families. In all three cases, expression of an OTS was successfully combined with the natural PTM enzymes for either nisin (current study), thiocillin ( Luo et al, 2016 ), or cinnamycin ( Lopatniuk et al, 2017 ) of the expression host.…”

Section: Discussionmentioning

confidence: 60%

Expanding the Genetic Code of Lactococcus lactis and Escherichia coli to Incorporate Non-canonical Amino Acids for Production of Modified Lantibiotics

et al. 2018

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The incorporation of non-canonical amino acids (ncAAs) into ribosomally synthesized and post-translationally modified peptides, e.g., nisin from the Gram-positive bacterium Lactococcus lactis, bears great potential to expand the chemical space of various antimicrobials. The ncAA Nε-Boc-L-lysine (BocK) was chosen for incorporation into nisin using the archaeal pyrrolysyl-tRNA synthetase–tRNAPyl pair to establish orthogonal translation in L. lactis for read-through of in-frame amber stop codons. In parallel, recombinant nisin production and orthogonal translation were combined in Escherichia coli cells. Both organisms synthesized bioactive nisin(BocK) variants. Screening of a nisin amber codon library revealed suitable sites for ncAA incorporation and two variants displayed high antimicrobial activity. Orthogonal translation in E. coli and L. lactis presents a promising tool to create new-to-nature nisin derivatives.

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“…For example, incorporation of ncAAs at discrete positions on Nisin expanded building blocks that can be biosynthetically incorporated into lanthipeptide and diversified macrocyclic topologies of this antibacterial peptide [ 100 ]. Likewise ncAAs have been successfully introduced into other lanthipeptides [ 101 , 102 ], lasso peptides [ 103 , 104 ], macrocyclic peptides [ 105 ], thiopeptides [ 106 ] as well as in the natural product cinnamycin, recently [ 107 ].…”

Section: Biosynthesized Therapeutic Peptidesmentioning

confidence: 99%

Therapeutic applications of genetic code expansion

Huang

Liu

2018

Synthetic and Systems Biotechnology

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In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.

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Streptomyces albus: A New Cell Factory for Non-Canonical Amino Acids Incorporation into Ribosomally Synthesized Natural Products

Cited by 38 publications

References 53 publications

In situ activation and heterologous production of a cryptic lantibiotic from a plant-ant derived Saccharopolyspora species

In situ activation and heterologous production of a cryptic lantibiotic from a plant-ant derived Saccharopolyspora species

Expanding the Genetic Code of Lactococcus lactis and Escherichia coli to Incorporate Non-canonical Amino Acids for Production of Modified Lantibiotics

Therapeutic applications of genetic code expansion

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