Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant natural products with diverse pharmacological properties. Microbial platforms can produce BIAs through heterologous biosynthesis ...

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-β-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass (“DpsC type”) of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.

In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

Arixanthomycins are pentangular polyphenols (PP) with potent antiproliferative activities that were discovered through the heterologous expression of environmental DNA-derived gene clusters. The biosynthesis of arixanthomycin and other PPs is unusual because it requires several novel type II polyketide synthase (PKS) enzymes for its complete maturation. Most type II PKSs contain a ketoreductase (KR) that mediates the C7-C12 first ring cyclization and C-9 reduction. In contrast, based on previous studies of product analysis and genome mining, the arixanthomycin (ARX) gene cluster harbors a C-11 reducing KR (ARX 27), a C9-C14 first-ring aromatase/cyclase (ARX 19), and an unprecedented C-17 and C-19 reducing KR (ARX 21). While bioinformatics is useful for predicting novel enzymes, the functions of ARX 19, ARX 21, and ARX 27 have yet to be confirmed. Further, the structural features that predispose the ARX biosynthetic enzymes to process atypical poly-β-ketone scaffolds remain unknown. We report the crystal structure of ARX 21, the first structure of an enzyme involved in PP biosynthesis and likely a C-17 and C-19 reducing-KR, which is structurally similar to C-15 reducing KRs. Structural comparison of ARX 21 and other C-9 reducing KRs revealed a difference in the enzyme active site that may enlighten the molecular basis of KR substrate specificity. In addition, we report the successful in vitro reconstitution of ARX 19. The structural characterization of ARX 21 in conjunction with the in vitro results of ARX 19 lays the groundwork toward a complete in vitro and structural characterization of type II PKS enzymes involved in PP biogenesis.

Significance Polyketides are a class of diverse natural products with well-documented bioactivity and medicinal importance. Enzymes known as aromatase/cyclases (ARO/CYCs) catalyze regiospecific cyclization and aromatization during type II polyketide biosynthesis. Understanding how ARO/CYCs catalyze cyclization and aromatization is critical for developing strategies for engineering biosynthetic pathways. This is the first study, to our knowledge, to use X-ray crystallography, bioinformatic and structural analysis, and in vitro functional assays to critically compare a reducing di-domain ARO/CYC (BexL) and a nonreducing di-domain ARO/CYC (StfQ). Together, these results fill in a missing link in the structural enzymology of polyketide biosynthesis and will have a direct effect on future biosynthetic engineering efforts and bioinformatic analysis of type II PKS gene clusters.