Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid

Abstract

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[238] Small chemicals that have been the aim of conventional metabolic engineering can also be produced by means of recombinant PKSs, such as in the case of adipic acid production described in Figure 14d. [239] Production of short-chain ketones was also successful by swapping the acyltransferase domain, thus altering the substrate specificity. [240] These design-build-test models with the PKS engineering strategies have opened a new era of novel value-added chemical production through systems metabolic engineering.…”

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

“…Interestingly, we found that the DH domain from the second module of the borrelidin PKS (BorA3) was required for efficient production of adipic acid in vitro (Figure 2b). 17 These results indicated that DH domains generally disfavor carboxylated substrates, and that only specialized DH domains will employ a distally carboxylated 3-OH acyl-ACP chain as a substrate for dehydration. The KR and ER domains from a variety of PKS modules appear to be tolerant to acidic substrates.…”

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges

“…Interestingly, we found that the DH domain from the adjacent downstream module of the borrelidin PKS (BorA3) greatly enhanced production of adipic acid in vitro. 27 We speculate the presence of the terminal carboxyl on the acyl chain undergoing dehydration might negatively influence binding to DH domains that do not normally employ substrates with terminal carboxyls, whereas the native substrate of the DH domain of BorA3 carries a terminal carboxyl group. Going beyond adipic acid, further swapping of the AT domain may enable us to produce adipic acid analogs carrying side chains that have a terminal double bond, a chloro-or a keto group, each of which can be used to crosslink polymer chains, giving the polymers unique properties that are useful in industrial applications.…”

“…25 This enzyme-bound product was released as a 3-hydroxyadipic acid by linking the erythromycin PKS TE domain immediately downstream of BorA2. 27 As BorA2 carries only a KR domain, production of adipic acid required engineering of BorA2 to carry the full complement of reductive domains: KR, dehydratase (DH) and enoyl reductase (ER) domains. Hence, we replaced the native KR domain of BorA2 with a contiguous segment containing the KR-DH-ER tridomains from a number of PKS modules, and subsequently introduced additional replacements of the DH domain.…”