Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins

Abstract

The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites − a reduction in chain length of 14 carbons relative to the 51-membered parental compounds − but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

“…Modular PKS are arranged into the required assembly-line order via the docking domains to ensure efficient polyketide chain elongation and intermodular transfer. Thus, rewiring the PKS assembly-line by exchanging and recombining these recognition subunits is a promising strategy for generating novel polyketide products 43 . In the case of AZL PKS, we demonstrated an intermodular recognition to effect cross-module enoylreduction and achieved the gain-of-function of engineered module 6 in the recruitment of ER 1/2 , thus realizing cross-module enoylreduction.…”

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering

“…While our lab has employed the updated module boundary to engineer trimodular synthases that outcompete equivalent synthases engineered using the traditional module boundary 7,8,13 , and other labs have used it to modify larger synthases 9,32,33 , the utility of the updated module boundary has not been systematically examined. In this study, we sought to test the limits of PKS engineering with updated modules and identify impediments to programming synthases that biosynthesize designer polyketides.…”

Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

“…Over the last two decades, diverse approaches were explored to generate new polyketide compounds by engineering PKSs 68 , with many focused on swapping and replacing PKS modules and functional domains, or modifying the active sites to induce higher substrate promiscuity or produce novel polyketides 37 , 44 – 47 . However, there is a lack of in-depth understanding for suitably applying the DDs isolated from PKSs.…”

“…which could be orthogonal interaction with each other 34 , 41 – 43 . These PKSs contain different numbers of domains and subunits, with the potential interchangeability of the PKSs catalytic modules making them an appealing prospective toolkit for combinatorial biosynthesis of new polyketide products 37 , 44 – 47 , rational computational design for new catalytic modules 48 , or to act interactions between fluorescent protein variants 49 .

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