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AMB Express (1)
BMC Biology (1)
Bioengineered Bugs (1)
Goss, Rebecca JM (3)
Grüschow, Sabine (2)
Barke, Jörg (1)
Bibb, Mervyn J (1)
Drou, Nizar (1)
Hackett, Louise (1)
Heavens, Darren (1)
Hutchings, Matthew I (1)
Overton, Tim W (1)
Perni, Stefano (1)
Rackham, Emma J (1)
Seipke, Ryan F (1)
Simmons, Mark J (1)
Yu, Douglas W (1)
Year of Publication
Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans
Simmons, Mark J
Overton, Tim W
Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.
E. coli; Biofilm; Biotransformation; Haloindole; Halotryptophan
Revealing the first uridyl peptide antibiotic biosynthetic gene cluster and probing pacidamycin biosynthesis
Rackham, Emma J
There is an urgent need for new antibiotics with resistance continuing to emerge toward existing classes. The pacidamycin antibiotics possess a novel scaffold and exhibit unexploited bioactivity rendering them attractive research targets. We recently reported the first identification of a biosynthetic cluster encoding uridyl peptide antibiotic assembly and the engineering of pacidamycin biosynthesis into a heterologous host. We report here our methods toward identifying the biosynthetic cluster. Our initial experiments employed conventional methods of probing a cosmid library using PCR and Southern blotting, however, it became necessary to adopt a state-of-the-art genome scanning and in silico hybridization approach to pinpoint the cluster. Here we describe our “real” and “virtual” probing methods and contrast the benefits and pitfalls of each approach.
pacidamycin; antibiotic; uridyl peptide antibiotic; translocase I inhibitor; MraY; biosynthesis; nonribosomal peptide synthetase; nucleoside; genome scan; genome mining
A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus
Seipke, Ryan F
Bibb, Mervyn J
Yu, Douglas W
Hutchings, Matthew I
Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive.
In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus.
Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.
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