Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase.

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.

Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for o-Dialkylbenzene Biosynthesis.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

Synthetic biology of polyketide synthases.

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.

Alteration of Polyketide Stereochemistry from anti to syn by a Ketoreductase Domain Exchange in a Type I Modular Polyketide Synthase Subunit.

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.

Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation.

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.

In vitro analysis of carboxyacyl substrate tolerance in the loading and first extension modules of borrelidin polyketide synthase.

The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates. However, the loading module has a requirement for substrates containing a carboxy moiety, which are not decarboxylated in situ. Thus, the loading module is the basis for the observed specific incorporation of carboxylated starter units by the borelidin PKS.

Complete genome sequence of Kitasatospora aureofaciens Tü117.

Actinobacteria produce about two-thirds of all naturally derived antibiotics currently in clinical use. Kitasatospora aureofaciens Tü117 is a species of Actinobacteria and produces α-lipomycin. We report the complete genome sequence of K. aureofaciens, composed of a single linear chromosome of 8,717,539 Mbp with a G + C content of 72.0%.

Broad substrate specificity of the loading didomain of the lipomycin polyketide synthase.

LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in Streptomyces aureofaciens Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries.

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.

Construction of a part of a 3-hydroxypropionate cycle for heterologous polyketide biosynthesis in Escherichia coli.

Polyketides, an important class of natural products with complex chemical structures, are widely used as antibiotics and other pharmaceutical agents. A clear barrier to heterologous polyketide biosynthesis in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common substrate of multimodular polyketide synthases. Here we report a route for synthesizing (2S)-methylmalonyl-CoA from malonyl-CoA with a 3-hydroxypropionate cycle in thermoacidophilic crenarchaeon. The engineered E. coli strain produced both propionyl-CoA and methylmalonyl-CoA at intracellular levels similar to those of acetyl-CoA and succinyl-CoA, respectively. This approach may open a way to produce a variety of polyketide drugs in E. coli from renewable carbon sources.

Role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular polyketide synthases.

The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain-linker interactions in assembly line polyketide biosynthesis.

Complete Genome Sequence of Streptomyces albus Strain G153.

The genus Streptomyces is a promising source of biologically active secondary metabolites. Here, we report the complete genome sequence of Streptomyces albus strain G153. The assembled genome comprised a single linear chromosome of 6.9 Mbp with a G+C content of 73.3%.

Nasal immunization with a fusion protein consisting of the hemagglutinin A antigenic region and the maltose-binding protein elicits CD11c(+) CD8(+) dendritic cells for induced long-term protective immunity.

We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4(+) T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-γ), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c(+) CD8α(+), but not CD11c(+) CD11b(+) or CD11c(+) B220(+), dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans.

A Reporter System for Cytosolic Protein Aggregates in Yeast.

Protein misfolding and aggregation are linked to neurodegenerative diseases of mammals and suboptimal protein expression within biotechnology. Tools for monitoring protein aggregates are therefore useful for studying disease-related aggregation and for improving soluble protein expression in heterologous hosts for biotechnology purposes. In this work, we developed a promoter-reporter system for aggregated protein on the basis of the yeast native response to misfolded protein. To this end, we first studied the proteome of yeast in response to the expression of folded soluble and aggregation-prone protein baits and identified genes encoding proteins related to protein folding and the response to heat stress as well as the ubiquitin-proteasome system that are over-represented in cells expressing an aggregation-prone protein. From these data, we created and validated promoter-reporter constructs and further engineered the best performing promoters by increasing the copy number of upstream activating sequences and optimization of culture conditions. Our best promoter-reporter has an output dynamic range of approximately 12-fold upon expression of the aggregation-prone protein and responded to increasing levels of aggregated protein. Finally, we demonstrate that the system can discriminate between yeast cells expressing different prion precursor proteins and select the cells expressing folded soluble protein from mixed populations. Our reporter system is thus a simple tool for diagnosing protein aggregates in living cells and should be applicable for the health and biotechnology industries.

Commodity Chemicals From Engineered Modular Type I Polyketide Synthases.

Reduced polyketides are a subclass of natural products that have a variety of medical, veterinary, and agricultural applications and are well known for their structural diversity. Although these compounds do not resemble each other, they are all made by a class of enzymes known as modular polyketide synthases (PKSs). The commonality of PKS domains/modules that compose PKSs and the understanding of the relationship between the sequence of the PKS and the structure of the compound it produces render modular PKSs as excellent targets for engineering to produce novel compounds with predicted structures. Here, we describe experimental protocols and considerations for modular PKS engineering and two case studies to produce commodity chemicals by engineered PKSs.

Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae.

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.

Probing the Flexibility of an Iterative Modular Polyketide Synthase with Non-Native Substrates in Vitro.

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules.

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus.

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.

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

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.