Pedro A. Baldera-Aguayo

Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered Saccharomyces cerevisiae (baker's yeast) and Saccharomyces boulardii (probiotic yeast) to produce the nonantibiotic fungal anhydrotetracycline, TAN-1612, in synthetic defined media─necessary for clean purifications─through heterologously expressing TAN-1612 genes mined from the fungus, Aspergillus niger ATCC 1015. This was accomplished via (i) a promoter library-based combinatorial pathway optimization of the biosynthetic TAN-1612 genes coexpressed with a putative TAN-1612 efflux pump, reducing TAN-1612 toxicity in yeasts while simultaneously increasing supernatant titers and (ii) the development of a medium-throughput UV-visible spectrophotometric assay that facilitates TAN-1612 combinatorial library screening. Through this multipronged approach, we optimized TAN-1612 production, yielding an over 450-fold increase compared to previously reported S. cerevisiae yields. TAN-1612 is an important tetracycline analogue precursor, and we thus present the first step toward generating novel tetracycline analogue therapeutics to combat current and emerging antibiotic resistance. We also report the first heterologous production of a fungal polyketide, like TAN-1612, in the probiotic S. boulardii. This highlights that engineered S. boulardii can biosynthesize complex natural products like tetracyclines, setting the stage to equip probiotic yeasts with synthetic therapeutic functionalities to generate living therapeutics or biocontrol agents for clinical and agricultural applications.

The fluorescence polarization (FP) assay has been widely used to study enzyme kinetics, antibody–antigen interactions, and other biological interactions. We propose that the FP assay can be adapted as a high-throughput and potentially widely applicable screen for small molecules. This is useful in metabolic engineering, which is a promising approach to synthesizing compounds of pharmaceutical, agricultural, and industrial importance using bioengineered strains. There, the development of high-yield strains is often a costly and time-consuming process. This problem can be addressed by generating and testing large mutant strain libraries. However, a current key bottleneck is the lack of high-throughput screens to detect the small molecule products. The FP assay is quantitative, sensitive, fast, and cheap. As a proof of principle, we established the FP assay to screen for FK506 (tacrolimus) produced by Streptomyces tsukubaensis, which was cultivated in 96-well plates. An ultraviolet mutagenized library of 160 colonies was screened to identify strains showing higher FK506 productivities. The FP assay has the potential to be generalized to detect a wide range of other small molecules.

Mycobacterium tuberculosis pyrazinamidase (PZAse) is a key enzyme to activate the pro-drug pyrazinamide (PZA). PZAse is a metalloenzyme that coordinates in vitro different divalent metal cofactors in the metal coordination site (MCS). Several metals including Co²⁺, Mn²⁺, and Zn²⁺ are able to reactivate the metal-depleted PZAse in vitro. We use quantum mechanical calculations to investigate the Zn²⁺, Fe²⁺, and Mn²⁺ metal cofactor effects on the local MCS structure, metal–ligand or metal–residue binding energy, and charge distribution. Results suggest that the major metal-dependent changes occur in the metal–ligand binding energy and charge distribution. Zn²⁺ shows the highest binding energy to the ligands (residues). In addition, Zn²⁺ and Mn²⁺ within the PZAse MCS highly polarize the O–H bond of coordinated water molecules in comparison with Fe²⁺. This suggests that the coordination of Zn²⁺ or Mn²⁺ to the PZAse protein facilitates the deprotonation of coordinated water to generate a nucleophile for catalysis as in carboxypeptidase A. Because metal ion binding is relevant to enzymatic reaction, identification of the metal binding event is important. The infrared vibrational mode shift of the C═Nε (His) bond from the M. tuberculosis MCS is the best IR probe to metal complexation.