Determination of Enantiomeric Excess and Diastereomeric Excess via Optical Methods. Application to α-methyl-ß-hydroxy-carboxylic acids.

Characterization of chiral molecules in solution is paramount for measuring reaction success. However, techniques to distinguish between chiral molecules containing more than one stereocenter through the use of optical techniques remains a challenge. Herein, we report a techique using a series of circular dichroism spectra to train multivariate regression models that are capable of predicting the complete speciation of 3-hydroxy-2-methylbutanoic acid stereoisomers. From this, it is possible to rapidly and accurately determine the enantiomeric excess and diastereomeric excess of the solution without the need for chiral chromatography.

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2-6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.

Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L-1 (627 mg L-1 isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L-1 to 100 mg L-1, in the titer of its tetraketide product was achieved.

Crystal structures reveal the framework of cis -acyltransferase modular polyketide synthases.

Although the domains of cis -acyltransferase ( cis -AT) modular polyketide synthases (PKS's) have been understood at atomic resolution for over a decade, the domain-domain interactions responsible for the architectures and activities of these giant molecular assembly lines remain largely uncharacterized. The multimeric structure of the α 6 ß 6 fungal fatty acid synthase (FAS) provides 6 equivalent reaction chambers for its acyl carrier protein (ACP) domains to shuttle carbon building blocks and the growing acyl chain between surrounding, oriented enzymatic domains. The presumed homodimeric oligomerization of cis -AT assembly lines is insufficient to provide similar reaction chambers; however, the crystal structure of a ketosynthase (KS)+AT didomain presented here and three already reported show an interaction between the AT domains appropriate for lateral multimerization. This interaction was used to construct a framework for the pikromycin PKS from its KS, AT, and docking domains that contains highly-ordered reaction chambers. Its AT domains also mediate vertical interactions, both with upstream KS domains and downstream docking domains.

Functional Studies and Revision of the NFAT-133/TM-123 Biosynthetic Pathway in Streptomyces pactum.

The biosynthetic gene cluster of NFAT-133, an inhibitor of the nuclear factor of activated T cells, was recently identified in Streptomyces pactum ATCC 27456. This cluster is conspicuous by its highly disordered noncollinear type I modular polyketide synthase (PKS) genes that encode PKSs with one module more than those expected for the heptaketide NFAT-133 biosynthesis. Thus, the major metabolite NFAT-133 was proposed to derive from an octaketide analogue, TM-123. Here, we report that further bioinformatic analysis and gene inactivation studies suggest that NFAT-133 is not derived from TM-123 but rather a product of programmed KS7 extension skipping of a nascent heptaketide from the PKS assembly line that produces TM-123. Furthermore, identification of NFAT-133/TM-123 analogues from mutants of the ATCC 27456 strain suggests that NftN (a putative dehydrogenase), NftE (a cytochrome P450), and NftG (a putative hydrolase/decarboxylase) function "in trans" during the polyketide chain assembly processes.

Priming enzymes from the pikromycin synthase reveal how assembly-line ketosynthases catalyze carbon-carbon chemistry.

The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KSQ, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KSQ and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KSQ and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KSQ active site, while an AT-swapped version of Pik127 demonstrated that KSQ can also decarboxylate malonyl groups. Mechanisms for how KSQ and KS domains catalyze carbon-carbon chemistry are proposed.

Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.

How cis-Acyltransferase Assembly-Line Ketosynthases Gatekeep for Processed Polyketide Intermediates.

With the redefinition of polyketide synthase (PKS) modules, a new appreciation of their most downstream domain, the ketosynthase (KS), is emerging. In addition to performing its well-established role of generating a carbon-carbon bond between an acyl-CoA building block and a growing polyketide, it may gatekeep against incompletely processed intermediates. Here, we investigate 739 KSs from 92 primarily actinomycete, cis-acyltransferase assembly lines. When KSs were separated into 16 families based on the chemistries at the α- and ß-carbons of their polyketide substrates, a comparison of 32 substrate tunnel residues revealed unique sequence fingerprints. Surprisingly, additional fingerprints were detected when the chemistry at the γ-carbon was considered. Representative KSs were modeled bound to their natural polyketide substrates to better understand observed patterns, such as the substitution of a tryptophan by a smaller residue to accommodate an l-α-methyl group or the substitution of four smaller residues by larger ones to make better contact with a primer unit or diketide. Mutagenesis of a conserved glutamine in a KS within a model triketide synthase indicates that the substrate tunnel is sensitive to alteration and that engineering this KS to accept unnatural substrates may require several mutations.

Preparative production of an enantiomeric pair by engineered polyketide synthases.

Using the updated module boundary of polyketide assembly lines, modules from the pikromycin synthase were recombined into engineered synthases that furnish an enantiomeric pair of 2-stereocenter triketide lactones at >99% ee with yields up to 0.39 g per liter of E. coli K207-3 in shake flasks.

Insights into modular polyketide synthase loops aided by repetitive sequences.

The loops of modular polyketide synthases (PKSs) serve diverse functions but are largely uncharacterized. They frequently contain amino acid repeats resulting from genetic events such as slipped-strand mispairing. Determining the tolerance of loops to amino acid changes would aid in understanding and engineering these multidomain molecule factories. Here, tandem repeats in the DNA encoding 949 modules within 129 cis-acyltransferase PKSs were cataloged, and the locations of the corresponding amino acids within the module were identified. The most frequently inserted interdomain loop corresponds with the updated module boundary immediately downstream of the ketosynthase (KS), while the loops bordering the dehydratase are nearly intolerant to such insertions. From the 949 modules, no repetitive sequence loop insertions are located within ACP, and only 2 reside within KS, indicating the sensitivity of these domains to alteration.

An in vitro platform for engineering and harnessing modular polyketide synthases.

To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.

General chemoenzymatic route to two-stereocenter triketides employing assembly line ketoreductases.

Modular polyketide synthases (PKSs) are enzymatic assembly lines that fuse carbon fragments into complex chiral products. Here, their synthetic logic is employed to chemoenzymatically generate two-stereocenter triketides. Each of the four stereoisomers was constructed in a stereocontrolled manner using C-acylation and two PKS ketoreductases possessing opposite stereoselectivities.

Employing 25-Residue Docking Motifs from Modular Polyketide Synthases as Orthogonal Protein Connectors.

The proteins of trans-acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of ∼25 residues at the C- and N-termini of these polypeptides (CDDs and NDDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins. Here, the ability of six CDD/NDD pairs to link non-PKS proteins is examined using green fluorescent protein (GFP) variants. As observed through size-exclusion chromatography and Förster resonance energy transfer (FRET), matched but not mismatched pairs of Venus+CDD and NDD+mTurquoise2 fusion proteins associate with low micromolar affinities.

Seven-enzyme in vitro cascade to (3R)-3-hydroxybutyryl-CoA.

Economical and environmentally-friendly routes to convert feedstock chemicals like acetate into valuable chiral products such as (R)-3-hydroxybutyrate are in demand. Here, seven enzymes (CoaA, CoaD, CoaE, ACS, BktB, PhaB, and GDH) are employed in a one-pot, in vitro, biocatalytic synthesis of (3R)-3-hydroxybutyryl-CoA, which was readily isolated. This platform generates not only chiral diketide building blocks but also desirable CoA derivatives.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on ß-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase.

Ketoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereochemical control of PKS KR domains.

The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein.

Here, the term "module" is redefined for trans-acyltransferase (trans-AT) assembly lines to agree with how its domains cooperate and evolutionarily co-migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans-AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co-evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not-in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering.

Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line.

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cis-acyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and six-membered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine.

Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases.

In an effort to uncover the structural motifs and biosynthetic logic of the relatively uncharacterized trans-acyltransferase polyketide synthases, we have begun the dissection of the enigmatic dehydrating bimodules common in these enzymatic assembly lines. We report the 1.98 Å resolution structure of a ketoreductase (KR) from the first half of a type A dehydrating bimodule and the 2.22 Å resolution structure of a dehydratase (DH) from the second half of a type B dehydrating bimodule. The KR, from the third module of the bacillaene synthase, and the DH, from the tenth module of the difficidin synthase, possess features not observed in structurally characterized homologs. The DH architecture provides clues for how it catalyzes a unique double dehydration. Correlations between the chemistries proposed for dehydrating bimodules and bioinformatic analysis indicate that type A dehydrating bimodules generally produce an α/ß-cis alkene moiety, while type B dehydrating bimodules generally produce an α/ß-trans, γ/δ-cis diene moiety.

The Uncommon Enzymology of Cis-Acyltransferase Assembly Lines.

The enzymology of 135 assembly lines containing primarily cis-acyltransferase modules is comprehensively analyzed, with greater attention paid to less common phenomena. Diverse online transformations, in which the substrate and/or product of the reaction is an acyl chain bound to an acyl carrier protein, are classified so that unusual reactions can be compared and underlying assembly-line logic can emerge. As a complement to the chemistry surrounding the loading, extension, and offloading of assembly lines that construct primarily polyketide products, structural aspects of the assembly-line machinery itself are considered. This review of assembly-line phenomena, covering the literature up to 2017, should thus be informative to the modular polyketide synthase novice and expert alike.