Polyketides serve
as rich source of therapeutically relevant drug
leads. The manipulation of polyketide synthases (PKSs) for generating
derivatives with improved activities usually results in substantially
reduced yields. Growing evidence suggests that type I PKS thioesterase
(TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics,
such as pimaricin and amphotericin, and their unnatural derivatives.
Herein, we elucidate the structure of the 26-membered macrolide-complexed
TE domain from the pimaricin pathway (Pim TE), which specifies a spacious
bifunnel-shaped substrate channel with a highly hydrophobic cleft
proximal to the catalytic triad and a hydrophilic loop I region specific
for the cyclization of amphiphilic polyene macrolide. Notably, the
natural intermediate with C12-COOH is stabilized by a hydrogen-bond
network, as well as by interactions between the polyene moiety and
the hydrophobic cleft. Moreover, the bottleneck in processing the
unnatural intermediate with C12-CH3 is attributed to the
unstable and mismatched docking of the curved substrate in the channel.
Aided by an in vitro assay with a fully elongated
linear polyene intermediate as the substrate, multiple strategies
were adopted, herein, to engineer Pim TE, including introducing H-bond
donors, enhancing hydrophobic interactions, and modifying the catalytic
center. Efficient TE mutations with increased substrate conversion
up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the
less toxic decarboxylated pimaricin derivatives with C12-CH3. Our work uncovers the mechanism of TE-catalyzed polyene macrolide
formation and highlights TE domains as targets for PKS manipulation
for titer increases in engineered unnatural polyketide derivatives.
Pimaricin is an important polyene antifungal antibiotic that binds ergosterol and extracts it from fungal membranes. In previous work, two pimaricin derivatives (1 and 2) with improved pharmacological activities and another derivative (3) that showed no antifungal activity were produced by the mutant strain of Streptomyces chattanoogensis, in which the P450 monooxygenase gene scnG has been inactivated. Furthermore, inactivation of the DH12 dehydratase domain of the pimaricin polyketide synthases (PKSs) resulted in specific accumulation of the undesired metabolite 3, suggesting that improvement of the corresponding dehydratase activity may reduce or eliminate the accumulation of 3. Accordingly, the DH12-KR12 didomain within the pimaricin PKS was swapped with the fully active DH11-KR11 didomain. As predicted, the mutant was not able to produce 3 but accumulated 1 and 2 in high yields. Moreover, the effect of the flanking linker regions on domain swapping was evaluated. It was found that retention of the DH12-KR12 linker regions was more critical for the processivity of hybrid PKSs. Subsequently, high-yield production of 1 or 2 was obtained by overexpressing the scnD gene and its partner scnF and by disrupting the scnD gene, respectively. To our knowledge, this is the first report on the elimination of a polyketide undesired metabolite along with overproduction of desired product by improving the catalytic efficiency of a DH domain using a domain swapping technology.
Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7-O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.
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