Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from enzyme complexes named type I polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides with enhanced or new properties. We have repurposed PKSs, employing up to three modules to produce a number of short-chain molecules that could have applications as fuels or industrial chemicals. Examining the enzymatic functions in vitro of these repurposed PKSs, we have uncovered a number of expanded substrate specificities and requirements of various PKS domains not previously reported and determined an unexpected difference in the order of enzymatic reactions within a module. In addition, we were able to efficiently change the stereochemistry of side chains in selected PKS products.

Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. We have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

Complex reduced polyketides represent the largest class of natural products that have applications in medicine, agriculture, and animal health. This structurally diverse class of compounds shares a common methodology of biosynthesis employing modular enzyme systems called polyketide synthases (PKSs). The modules are composed of enzymatic domains that share sequence and functional similarity across all known PKSs. We have used the nomenclature of synthetic biology to classify the enzymatic domains and modules as parts and devices, respectively, and have generated detailed lists of both. In addition, we describe the chassis (hosts) that are used to assemble, express, and engineer the parts and devices to produce polyketides. We describe a recently developed software tool to design PKS system and provide an example of its use. Finally, we provide perspectives of what needs to be accomplished to fully realize the potential that synthetic biology approaches bring to this class of molecules.

Polyketide natural products have broad applications in medicine. Exploiting the modular nature of polyketide synthases to alter stereospecificity is an attractive strategy for obtaining natural product analogues with altered pharmaceutical properties. We demonstrate that by retaining a dimerization element present in LipPks1+TE, we are able to use a ketoreductase domain exchange to alter α-methyl group stereochemistry with unprecedented retention of activity and simultaneously achieve a novel alteration of polyketide product stereochemistry from anti to syn. The substrate promiscuity of LipPks1+TE further provided a unique opportunity to investigate the substrate dependence of ketoreductase activity in a polyketide synthase module context.

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

Streptomyces genomes have a high G + C content and typically use an ATG or GTG codon to initiate protein synthesis. Although gene-finding tools perform well in low GC genomes, it is known that the accuracy in predicting a translational start site (TSS) is much less for high GC genomes. LipPks1 is a Streptomyces-derived, well-characterized modular polyketide synthase (PKS). Using this enzyme as a model, we experimentally investigated the effects of alternative TSSs using a heterologous host, Streptomyces venezuelae. One of the TSSs employed boosted the protein level by 59-fold and the product yield by 23-fold compared to the originally annotated start codon. Interestingly, a structural model of the PKS indicated the presence of a structural motif in the N-terminus, which may explain the observed different protein levels together with a proline and arginine-rich sequence that may inhibit translational initiation. This structure was also found in six other modular PKSs that utilize noncarboxylated starter substrates, which may guide the selection of optimal TSSs in conjunction with start-codon prediction software.

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5-C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l^-1 of C6 and C7 ethyl ketones and several hundred mg · l^-1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.

Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA and n-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4-9 μM; methylmalonyl-CoA at 0.5 μM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 μM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in S. cerevisiae. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore S. cerevisiae as a heterologous production host for novel secondary metabolites.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.