Abstract
Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.
Highlights
Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity
The rapid acquisition of genome sequence data from anaerobic bacteria has uncovered a plethora of natural product biosynthetic gene clusters (BGCs) encoding polyketides (PKs), non-ribosomal peptides (NRPs), ribosomally synthesized and post-translationally modified peptides (RiPPs), and terpenoids[5,6,7]
An ideal anaerobic host for heterologous expression of natural product BGCs should (i) be safe to manipulate in the laboratory; (ii) grow rapidly under anaerobic conditions; (iii) have clear genetic and metabolic backgrounds; (iv) have versatile genetic tools and readily accept large DNA fragments to accommodate the 10–120 kb size range of most natural product BGCs; and (v) possess the requisite precursor molecules to support the biosynthesis of diverse natural products
Summary
Development of the NabLC technique in S. mutans UA159. We firstly tried to construct a vector-based heterologous expression. The recipient strain S. mutans UA159-RS/BGC1, in which the mutanobactin gene cluster was replaced by the capture cassette CAR1-IFDC2-CAL1, was obtained by transforming pNCL-159/BGC1 into S. mutans UA159 and screening for transformants resistant to erythromycin and sensitive to spectinomycin. The higher screening efficiency of the tetM-based counterselection system was verified by transforming both recipient strains, S. mutans UA159-RS/BGC1 and UA159*-RS/BGC1, with the same amount of S. mutans 35 genomic DNA. After successfully capturing different BGCs on the S. mutans UA159* genome using the NabLC technique, we set out to test the utility of S. mutans UA159* as a host for functionally expressing BGCs as judged by detection of their encoded natural products. To verify mutanocyclin as the bona fide natural product of BGC1 in S. mutans wild-type strains, it was detected by HPLC and HR-LC-MS from cultures of S. mutans 35, B30, B409, and B608 grown in ASS medium (Supplementary Fig. 10). Mutanocyclin was not discovered as a natural product before, it was chemically synthesized as racemate and found to have no considerable cytotoxicity[45]
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