Human-associated bacteria adopt an unusual route for synthesizing 3-acetylated tetramates for environmental adaptation
Authors: Zhang Yuwei , Liao Ge , Wang Min , Zhang Zhao , Liu Liwei , Song Yuqin , Wang Dacheng , Hao Tingting , Feng Jie , Xia Bin , Wang Yixiang , Tang Xiaoyu , Chen Yihua 
Published: May 2023

Abstract
Background
Tetramates or tetramic acid-containing compounds (TACs) are a group of bioactive natural products featuring a pyrrolidine-2,4-dione ring acknowledged being closed via Dieckmann cyclization. The cariogenic Streptococcus mutans strains bearing a muc biosynthetic gene cluster (BGC) can synthesize mutanocyclin (MUC), a 3-acetylated TAC that can inhibit both leukocyte chemotaxis and filamentous development in Candida albicans. Some strains can also accumulate reutericyclins (RTCs), the intermediates of MUC biosynthesis with antibacterial activities. However, the formation mechanism of the pyrrolidine-2,4-dione ring of MUC and the distribution of muc-like BGCs along with their ecological functions has not been explored extensively.

Results
We demonstrated that a key intermediate of MUC biosynthesis, M-307, is installed by a hybrid nonribosomal peptide synthetase-polyketide synthase assembly line and its pyrrolidine-2,4-dione ring is closed via an unprecedented lactam bond formation style. Subsequent C-3 acetylation will convert M-307 to RTCs, which is then hydrolyzed by a deacylase, MucF, to remove the N-1 fatty acyl appendage to generate MUC. Distribution analysis showed that the muc-like BGCs distribute predominantly in human-associated bacteria. Interestingly, most of the muc-like BGCs possessing a mucF gene were isolated from human or livestock directly, indicating their involvement in alleviating the host’s immune attacks by synthesizing MUC; while those BGCs lacking mucF gene distribute mainly in bacteria from fermented products, suggesting that they tend to synthesize RTCs to compete with neighboring bacteria. It is noteworthy that many bacteria in the same habitats (e.g., the oral cavity) lack the muc-like BGC, but possess functional MucF homologues to “detoxify” RTCs to MUC, including several competitive bacteria of S. mutans. We also comparably studied the distribution of TAS1, a fungal enzyme responsible for the production of phytotoxic tenuazonic acids (TeAs), a class of 3-acetylated TACs with similar structure but distinct biosynthetic mechanism to MUC, and found that it mainly exists in plants or crops.

Conclusions
The in vivo and in vitro experiments revealed that the pyrrolidine-2,4-dione ring of MUC is closed via lactam bond formation, which may be adopted by many TACs without 3-acyl decorations. Besides, we found that muc-like BGCs are widespread in human-associated bacteria and their shapes and main products can be influenced by the habitat environment and vice versa. By comparing with TeAs, we provided thought-provoking insights into how ecological and evolutionary forces drive bacteria and fungi to construct a common 3-acetylated pyrrolidine-2,4-dione core through different routes, and how the biosynthetic processes are delicately controlled to generate diverse 3-acetylated TACs for environmental adaptation.