Microbial biosynthesis of plant natural products from simple building blocks is a promising approach toward scalable production and modification of high-value compounds. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine was recently elucidated as a 10-gene cluster from opium poppy. Here we demonstrate the de novo production of noscapine in , through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to ∼2.2 mg/L. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain we further demonstrated microbial production of halogenated benzylisoquinoline alkaloids. This work highlights the potential for microbial biosynthetic platforms to support the synthesis of valuable and novel alkaloid compounds, which can advance alkaloid-based drug discovery and development.

A paradigm-shift lithium-ion battery recycling method based on defect-targeted healing can fully recover the composition, structure, and electrochemical performance of spent LiFePO4 cathodes with various degradation conditions to the same levels as that of the pristine materials. Such a direct recycling approach can significantly reduce energy usage and greenhouse gas emissions, leading to significant economic and environmental benefits compared with today's hydrometallurgical and pyrometallurgic methods. This work may pave the way for industrial adoption of directly recycled lithium-ion battery materials.

The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains, and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonist respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn2Cys6-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase (FMO) that hydroxylates the α-carbon of an ACP-bound polyketide and a bifunctional metallo-β-lactamase-type thioesterase (MβL-TE). The bifunctional MβL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, while performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MβL-TE is a dimanganese enzyme and requires both Mn2+ cations for the observed activities. The MβL-TE is the first example of TE in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/β hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKSWJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKSWJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.cyclase ͉ ketosynthase ͉ megasynthase

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