Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics

Abstract

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 μg/mL, respectively.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library

“…A 10 g soil sample was processed for high molecular weight (HMW) DNA isolation and purification as previously described (Liles et al, 2008), and the metagenomic DNA was randomly sheared. The fragmented DNA was endrepaired, and BstXI adaptors ligated on to the DNA, followed by gel-fractionation and ligation into the pSmartBAC-S vector as previously described (Nasrin et al, 2018). The vector:insert ligation was transformed by electroporation into BAC-optimized E. coli DH10B (Lucigen Corp, Middleton, WI, United States), and transformants were selected on LB agar (10 mg/mL of Tryptone, 10 mg/mL of NaCl, 5 mg/mL of Yeast Extract, 15 mg/mL of Agar in 1L of water) containing 12.5 µg/ml chloramphenicol.…”

“…Despite these developments in uncovering BGC diversity, direct sequencing does not lend itself to heterologous expression of BGCs in order to identify the encoded secondary metabolites. In order to access and express complete BGCs, bacterial artificial chromosome (BAC) vectors can be used to clone contiguous genomic fragments that can exceed 300 Kb, thereby increasing the probability of obtaining intact BGCs from metagenomic sourced DNA, while also enabling inducible copy number in E. coli and conjugal transfer to heterologous expression hosts (Wild et al, 2002;Aakvik et al, 2009;Liu et al, 2010;Kakirde et al, 2011;Liu et al, 2016;Nasrin et al, 2018). Functional screening of metagenomic clones typically results in very low hit rates for enzymatic activities (Healy et al, 1995;Thies et al, 2016;Lewin et al, 2017) and even lower rates for discovering bioactive metabolites (Tulp and Bohlin, 2005); thus, it is desirable to first identify clones containing the target gene or BGC of interest.…”

“…In this study we screened a large-insert soil metagenomic BAC library hosted in E. coli (Nasrin et al, 2018) using multiple sequence-based approaches, including PCR amplification of 16S rRNA genes and KS domains associated with PKS clusters, as well as using a novel next-generation sequencing (NGS) strategy to mine the entire library for BGCs. A multiplexed pooling strategy was used to allow bioinformatic identification of sequences associated with each of the 19,200 clones formatted in 384-well plates.…”

Data_Sheet_1.PDF

“…A large-insert metagenomic library was constructed in the shuttle vector pSMART-BAC-S using high molecular weight DNA isolated from the Auburn University's Cullars Rotation agricultural soil (Nasrin et al, 2018). A total of 19,200 independent clones were picked as isolated E. coli DH10B colonies into 50 plates in 384-well format.…”

“…A total of 19,200 independent clones were picked as isolated E. coli DH10B colonies into 50 plates in 384-well format. The insert size of 215 randomly selected BAC clones was 12 Kb to >200 Kb, with an average insert size of 113 Kb (Nasrin et al, 2018).…”