Bioprocess Development for Lantibiotic Ruminococcin-A Production in Escherichia coli and Kinetic Insights Into LanM Enzymes Catalysis.

Elvis L. Ongey,Sebastian L. Riedel,Peter Neubauer,Lara Santolin,Lorenz Adrian,Saskia Waldburger

doi:10.3389/fmicb.2019.02133

Abstract

Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 μg L–1) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24-microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso® B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L–1 for the chimeric construct and >150 mg L–1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.

Highlights

The accelerated growth of resistant pathogenic bacteria against the existing battery of anti-infective agents has prompted efforts in developing alternative drug candidates that possess multiple mechanisms of action to counter the engrossing effect of disease burdens
Parallelized microwell plate cultivations were used to evaluate the behavior of E. coli W3110 pLEOgrA∗M1 under processrelevant conditions using on-line monitoring of dissolved oxygen concentrations (DO) and pH in EnPresso B cultures
Data recorded for the DO and pH during the first 14 h of cultivation were indicative of a typical E. coli growth profile (Figures 2B,C)

Summary

Introduction

The accelerated growth of resistant pathogenic bacteria against the existing battery of anti-infective agents has prompted efforts in developing alternative drug candidates that possess multiple mechanisms of action to counter the engrossing effect of disease burdens. R. gnavus by its nature as an obligate anaerobic bacterium already poses substantial cultivation challenges that would certainly hinder the development of an efficient and robust production process To overcome these limitations, we reconstructed the biosynthesis pathway of RumA in Escherichia coli and showed that all the desired post-translational modifications (PTMs) were correctly formed in the peptide resulting in a 3-membered ring compound shown in Figure 1D (Ongey et al, 2018). Michael-type addition cyclization reactions between the sulfhydryl group of cysteine and the resulting Dhb and Dha produce a lanthionine (Lan) or a methyllanthionine (MeLan) cross-bridge respectively (Schnell et al, 1988; Arnison et al, 2013; Yang and van der Donk, 2015; Repka et al, 2017) We applied this knowledge in developing the heterologous biosynthesis route. Moderate amounts of modified RumA precursor (6 mg L−1) was obtained with complex media cultivations in shake flasks (Ongey et al, 2018)

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Conclusion