Discovery of a highly efficient TylF methyltransferase via random mutagenesis for improving tylosin production.

Macrolides are currently a class of extensively used antibiotics in human and animal medicine. Tylosin is not only one of the most important veterinary macrolides but also an indispensable material for the bio- and chemo-synthesis of new generations of macrolide antibiotics. Thus, improving its production yield is of great value. As the key rate-limiting enzyme catalyzing the terminal step of tylosin biosynthesis in Streptomyces fradiae (S. fradiae), TylF methyltransferase's catalytic activity directly affects tylosin yield. In this study, a tylF mutant library of S. fradiae SF-3 was constructed based on error-prone PCR technology. After two steps of screening in 24-well plates and conical flask fermentation and enzyme activity assay, a mutant strain was identified with higher TylF activity and tylosin yield. The mutation of tyrosine to phenylalanine is localized at the 139th amino acid residue on TylF (TylFY139F), and protein structure simulations demonstrated that this mutation changed the protein structure of TylF. Compared with wild-type protein TylF, TylFY139F exhibited higher enzymatic activity and thermostability. More importantly, the Y139 residue in TylF is a previously unidentified position required for TylF activity and tylosin production in S. fradiae, indicating the further potential to engineer the enzyme. These findings provide helpful information for the directed molecular evolution of this important enzyme and the genetic modification of tylosin-producing bacteria.

Development and Application of Two Inducible Expression Systems for Streptococcus suis.

Streptococcus suis is an important zoonotic bacterial pathogen posing a threat to the pig industry as well as public health, for which the mechanisms of growth and cell division remain largely unknown. Developing convenient genetic tools that can achieve strictly controlled gene expression is of great value for investigating these fundamental physiological processes of S. suis. In this study, we first identified three strong constitutive promoters, Pg, Pt, and Pe, in S. suis. Promoter Pg was used to drive the expression of repressor genes tetR and lacI, and the operator sequences were added within promoters Pt and Pe. By optimizing the insertion sites of the operator sequence, we successfully constructed an anhydrotetracycline (ATc)-inducible expression system and an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible expression system in S. suis. We showed that these two systems provided inducer-concentration- and induction-time-dependent expression of the reporter gene. By using these tools, we investigated the subcellular localization of a key cell division protein, FtsZ, which showed that it could be correctly localized to the midcell region. In addition, we constructed a conditional knockout strain for the glmS gene, which is an essential gene, and showed that our ATc-inducible promoter could provide strictly controlled expression of glmS in trans, suggesting that our inducible expression systems can be used for deletion of essential genes in S. suis. Therefore, for the first time we developed two inducible expression systems in S. suis and showed their applications in the study of an important cell division protein and an essential gene. These genetic tools will further facilitate the functional study of other important genes of S. suis. IMPORTANCE Streptococcus suis is an important zoonotic bacterial pathogen. Studying the mechanisms of cell growth and division is important for the identification of novel antimicrobial drug targets. Inducible expression systems can provide strictly controlled expression of the protein of interest and are useful tools to study the functions of physiologically important proteins. However, there is a lack of convenient genetic tools that can achieve inducible protein expression in S. suis. In this study, we developed two (ATc-inducible and IPTG-inducible) inducible expression systems and showed their applications in a subcellular localization study of a cell division protein and the construction of conditional knockout of essential genes in S. suis. These systems will be useful for functional studies of important proteins of S. suis.

IPA-3: An Inhibitor of Diadenylate Cyclase of Streptococcus suis with Potent Antimicrobial Activity.

Antimicrobial resistance (AMR) poses a huge threat to public health. The development of novel antibiotics is an effective strategy to tackle AMR. Cyclic diadenylate monophosphate (c-di-AMP) has recently been identified as an essential signal molecule for some important bacterial pathogens involved in various bacterial physiological processes, leading to its synthase diadenylate cyclase becoming an attractive antimicrobial drug target. In this study, based on the enzymatic activity of diadenylate cyclase of Streptococcus suis (ssDacA), we established a high-throughput method of screening for ssDacA inhibitors. Primary screening with a compound library containing 1133 compounds identified IPA-3 (2,2'-dihydroxy-1,1'-dinapthyldisulfide) as an ssDacA inhibitor. High-performance liquid chromatography (HPLC) analysis further indicated that IPA-3 could inhibit the production of c-di-AMP by ssDacA in vitro in a dose-dependent manner. Notably, it was demonstrated that IPA-3 could significantly inhibit the growth of several Gram-positive bacteria which harbor an essential diadenylate cyclase but not E. coli, which is devoid of the enzyme, or Streptococcus mutans, in which the diadenylate cyclase is not essential. Additionally, the binding site in ssDacA for IPA-3 was predicted by molecular docking, and contains residues that are relatively conserved in diadenylate cyclase of Gram-positive bacteria. Collectively, our results illustrate the feasibility of ssDacA as an antimicrobial target and consider IPA-3 as a promising starting point for the development of a novel antibacterial.