Chemoinformatic-Guided Engineering of Polyketide Synthases

Abstract

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemo­informatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemo­similarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemo­similarity and production, establishing a new chemo­informatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

A bimodular PKS platform that expands the biological design space

“…(since acquired by Ginkgo Bioworks). They were cloned through Golden Gate assembly into the LipPKS1 module with an inserted RL from NanA2 from Zargar et al (Zargar et al, 2020).…”

“…Figure 2A, we aimed to produce ethyl ketones through an RL exchange in LipPKS1 and an AT swap in LipPKS2. Previously, we had performed an RL exchange in LipPKS1 with a DEBS1-TE, and found a correlation between successful production of the desired product and the chemical similarity of the donor and recipient reductive loops with the most production through the chimera of LipPKS1 with an inserted donor loop form NanA2 J o u r n a l P r e -p r o o f (nanchangamycin, module 2) (Zargar et al, 2020). In the work presented here, we re-engineer this chimera of LipPKS1 with NanA2 RL by replacing the fused DEBS (6-deoxyerythronolide) thioesterase with the native docking domain of LipPKS1.…”

“…This loss of production is partially reflected in the amount of side product generated in the bimodular system. Previously, we found that the unimodular LipPKS with NanA2 reductive loops and a fused TE produced the DH-stalled product, 3-hydroxydimethylpentanoic acid (Zargar et al, 2020). We therefore suspected that incomplete β-carbon reduction by the engineered module 1 could cause premature hydrolysis of the product from the Lip1 ACP, resulting in production of the DH-stalled product, 3-hydroxy 2,4dimethylpentanoic acid, which was produced at a titer of 36.9 mg/L, considerably higher than that produced by the negative control (Figure 2Bi).…”

“…An important fragrant compound class is δ-lactones (Gupta, 2015), and while not commercially used, 3-isopropyl-6-methyltetrahydropyranone has been synthesized previously as a potentially fragrant δ-lactone (Plessis and Derrer, 2001). In a bimodular system with native AT domains, we previously engineered LipPKS1 and LipPKS2 to produce 3-isopropyl-4,6-dimethyltetrahydropyranone (Zargar et al, 2020), and in this work, we sought to incorporate AT-engineering to produce 3-isopropyl-6-methyltetrahydropyranone as an initial proof-of-concept production into this broad category of fragrant molecules.…”

“…1) an inserted TE to produce 3-hydroxy acids (Yuzawa et al, 2017a), 2) a KR knockout and AT domain swap to produce short chain ketones (Yuzawa et al, 2018a(Yuzawa et al, , 2017b, and 3) reductive loop (RL) exchanges to produce saturated, short chain carboxylic acids and triketide lactones (Zargar et al, 2020). With significant engineering efforts, we have shown that Streptomyces albus as our chassis In this work, we seek to capitalize on the expanded design space of multiple modules by combining multiple PKS manipulations (KR knockouts, reductive loop (RL) swaps, AT swaps, fused TE) to produce novel biomolecules, namely biofuels and specialty J o u r n a l P r e -p r o o f chemicals.…”