An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters

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.

CariogenicStreptococcus mutansproduces strain-specific antibiotics that impair commensal colonization

“…Mutanobactins are compounds of hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) origin that inhibit the morphological transition of Candida albicans (22). Previous bioinformatics efforts identified an orphan hybrid PKS/NRPS BGC (recently designated muc )(20, 23), that is distributed among a subset of S. mutans strains. Within muc , five biosynthetic proteins are highly homologous (48%-69%) to cognates involved in the biosynthesis of reutericyclin (RTC)(24) ( Supplementary Fig.…”

“…2-22 ). During preparation of this manuscript, Chen and colleagues published the identification of 4 (designated mutanocyclin (MUC)) using a new heterologous genetic system(23); however, production of 1 - 3 in both heterologous host and wild type producers was not reported.…”

“…As S. mutans is an exceptionally productive biofilm-former, higher numbers of S. mutans are likely to increase plaque biofilm formation and promote the dysbiosis which leads to the formation of caries lesions. Interestingly, Chen and colleagues showed that 4 can significantly suppress the infiltration of leukocytes (CD45 + cells) into the Matrigel plug in a mice model, suggesting an anti-inflammatory activity(23).…”

“…In summary, we describe a versatile biosynthetic pathway from an oral pathogen S. mutans B04Sm5, which can produce three types of compounds with divergent biological activities. These include three N -acyl tetramic acids ( 1 - 3 ) that display antibacterial properties against oral commensal bacteria, a new organic acid ( 4 ) with a reported anti-inflammatory activity in a mice model(23), and a previously characterized SDSF with the ability to interact with pathogenic oral fungi(31). Although two antibiotics have been discovered from the commensal bacteria of human, including lactocillin(13) and lugdunin(35), RTCs, to our knowledge, are the first group of low molecular weight antibacterial molecules identified from a human opportunistic pathogen.…”

Mutanofactin promotes bacterial adhesion and biofilm formation of cariogenic Streptococcus mutans

“…Recently, genomic analysis of various S. mutans isolates demonstrated that these facultative anaerobes collectively harbored a plethora of biosynthetic gene clusters (BGCs) for polyketide/non-ribosomal peptide biosynthesis [14][15][16] . Some of the associated products were also reported, including mutanobactin with an inhibitory activity against the morphological transition of Candida albicans 17,18 , mutanocyclin that suppressed the infiltration of leukocytes 19 , and reutericyclin that inhibited the growth of oral commensal bacteria 20 (Fig. 1a).…”