Exploiting an unusual polyketide chain release mechanism for production of novel enacyloxin antibiotic analogues

Project Information

NOVENA

Grant agreement ID: 656067

DOI

10.3030/656067

Project closed

EC signature date 12 March 2015

Start date 1 June 2015

End date 31 May 2017

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

UNIVERSITY OF WARWICK
United Kingdom

Periodic Reporting for period 1 - NOVENA (Exploiting an unusual polyketide chain release mechanism for production of novel enacyloxin antibiotic analogues)

Reporting period: 2015-06-01 to 2017-05-31

Summary of the context and overall objectives of the project

Enacyloxin IIa is a polyketide antibiotic with activity against Gram-positive and Gram-negative bacteria that targets ribosomal elongation factor Tu. It has recently been identified as metabolite of Burkholderia ambifaria and shown to have clinically-relevant activity against Acinetobacter baumannii, a problematic panresistant Gram-negative pathogen that is estimated to account for 2-10% of all Gram-negative bacterial infections in intensive care units in Europe and for the death of thousands of European hospital patients every year. Despite its promising biological activity, enacyloxin IIa has not been used in the clinic, presumably due to stability issues. Preliminary experiments have provided evidence for an unusual mechanism of modular polyketide synthase chain release in enacyloxin biosynthesis, involving intermolecular condensation of an acyl carrier protein (ACP)-bound thioester with the C-3 hydroxyl group of (1S, 3R, 4S)-3,4- dihydroxycyclohexane carboxylic acid (DHCCA). This project aimed to elucidate and engineer the biosynthetic pathway to enacyloxin. It also aimed to illuminate the structure-activity relationship of the antibiotic by studying the characteristics of the enacyloxin analogues generated.

The specific objectives of the project were:
(1) Identify the enzyme responsible for epimerisation of C-1 of the DHCCA moiety
(2) Explore the ability of the Bamb_5915 chain release enzyme to catalyse the acylation of diverse DHCCA analogues
(3) Create a Burkholderia ambifaria mutant blocked in DHCCA biosynthesis
(4) Produce novel enacyloxin IIa derivatives via a mutasynthesis approach involving feeding of DHCCA analogues to a B. ambifaria mutant blocked in DHCCA biosynthesis.

The project was highly successful in its aims and fully reached its objectives. The unusual mechanism for polyketide chain release in the biosynthesis of enacyloxin was biochemically characterized and the role of several tailoring enzymes was elucidated. Based on these results, different biosynthetic engineering approaches were developed which afforded over a dozen enacyloxin derivatives, with the potential to yield many more.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work Package 1: Identify the enzyme responsible for epimerisation of C-1 of the DHCCA moiety.
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At the start of the project, it was believed that C-1 of the DHCCA moiety in enacyloxin undergoes epimerisation after the Bamb_5915-catalysed esterification reaction. However, this hypothesis was refuted by in vitro biochemical assays which showed that, contrary to initial observations, the Bamb_5915 protein can utilise (1S, 3R, 4S)-DHCCA as a substrate. This finding led us to conclude that there is no epimerase involved in enacyloxin biosynthesis.

Work package 2: Explore the ability of the Bamb_5915 chain release enzyme to catalyse the acylation of diverse DHCCA analogues
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To probe its substrate specificity, purified 5915 protein was incubated with acetyl-ACP and a range of commercially-available and chemically-synthesized DHCCA analogues. These experiments revealed that the chain release enzyme has a relaxed substrate specificity and is able to utilise a wide range of DHCCA analogues in vitro.

Work package 3: Create a Burkholderia ambifaria mutant blocked in DHCCA biosynthesis
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A mutant of B. ambifaria blocked in DHCCA biosynthesis was constructed by simultaneously deleting three DHCCA biosynthetic genes in the enacyloxin biosynthetic gene cluster. LC-MS analysis of extracts from agar cultures confirmed that enacyloxin IIa production is abolished in this mutant and showed that it is possible to restore enacyloxin biosynthesis by feeding chemically-synthesised (1S,3R,4S)-DHCCA.

Work Package 4: Produce novel enacyloxin IIa derivatives via a mutasynthesis approach involving feeding of DHCCA analogues to a B. ambifaria mutant blocked in DHCCA biosynthesis.
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Over a dozen enacyloxin derivatives with altered DHCCA moieties were produced by feeding the DHCCA analogues identified as substrates of the Bamb_5915 protein to the mutant of B. ambifaria blocked in DHCCA biosynthesis. By studying the activity of the purified analogues against A. baumannii, insight was obtained into the structure-activity relationship for the DHCCA moiety of the antibiotic.


The novel analogues are currently being protected through patent applications (1 filed, 1 in preparation) to facilitate exploitation and commercial development. Collaborations with two industrial partners have been established under the protection of a confidentiality agreement, focused on carrying out preclinical evaluation of the analogues generated. Once IP rights are secured, the results of this project will also be disseminated to the scientific community through publication in international peer-reviewed journals.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Progress beyond the state-of-the-art:

• Novel enacyloxin analogues (2 patent applications are pending)
• New methodology for biosynthetic engineering of polyketide synthases in Gram-negative bacteria: Although mutasynthesis is a well-established methodology for the production of polyketide analogues in Gram-positive Streptomycetes, its use in Gram-negative Burkholderia species is virtually unexplored. Thus, the mutasynthetic production of enacyloxin analogues achieved in this project represents a significant advance for polyketide biosynthetic engineering.


Expected potential impact:
The research project has had a positive impact on European competitiveness and scientific excellence because the enacyloxin IIa analogues generated by this project can serve as useful starting points for the development of novel antibiotics with the potential to tackle infections caused by multi-drug resistant Gram- negative pathogens such as A. baumannii. Thus, they are of benefit to European pharmaceutical and biotechnology companies actively engaged in the quest to bring new antibiotics to the market. Indeed, a collaboration has been initiated with a European biotech company. Tangible economic benefit to Europe would result if this companies decided to take one of the enacyloxin analogues into development, through the employment of scientists and other personnel to develop the drug. Moreover, should this result in a marketable drug, the benefit to the European economy in the medium to long term could be significant. It would also have a significant impact on the everyday lives of people living in Europe. A. baumannii is a highly antibiotic-resistant bacterium that is estimated to account for 2-10% of all Gram-negative bacterial infections in intensive care units in Europe and for the death of thousands of European hospital patients every year. Thus, this project might ultimately allow patients to benefit from improved antibiotics. Even if the project does not ultimately lead to the creation of a commercial product, it will have generated new tools (e.g. mutasynthesis) for the manipulation of polyketide antibiotic biosynthetic pathways that are useful to industry.

Structure of enacyloxin IIa and X-ray crystal structure of an EF-Tu/GTP analogue/enacyloxin complex