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All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene 1 . This approach is fundamentally different from the biosynthesis of short-chain (C 10 –C 25 ) terpenes that are formed from polyisoprenyl diphosphates 2 – 4 . In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature. Chimeric triterpene synthases are identified that catalyse non-squalene-dependent triterpene biosynthesis.

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica . To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products. Substrate inhibition has not been widely studied in the context of synthetic biology and metabolic engineering. Here, the authors report removal of lycopene substrate inhibition by two different strategies and enable high carotenoid productivity in Yarrowia lipolytica .

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics 1 . Here we show that particular lineages of methicillin-resistant Staphylococcus aureus —a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development. Methicillin-resistant strains of Staphylococcus aureus appeared in European hedgehogs in the pre-antibiotic era as a co-evolutionary adaptation to antibiotic-producing dermatophytes and have spread within the local hedgehog populations and between hedgehogs and secondary hosts.

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L −1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids. Plants synthesize more than 3000 tetrahydroisoquinoline (THIQ) alkaloids, but only a few of them have been produced by engineered microbes and titers are very low. Here, the authors increase ( S )-reticuline titer to 4.6 g/L and repurpose the yeast Ehrlich pathway to synthesize a diverse array of THIQ scaffolds.

Key Points This Review covers the recent advances in synthetic biology and how these advances will affect the field of natural products. There has been an emphasis on creating genetic parts, such as promoters, that generate precise levels of gene expression. The generation of large libraries of well-characterized parts and the development of biophysical and bioinformatic models to predict the behaviour of genetic parts in different organisms will aid in the transfer of biosynthetic gene clusters between hosts. The capacity of DNA synthesis has exploded over the past decade and it is routine to synthesize the 20–100 kb required for a large gene cluster. In addition, new DNA assembly methods enable the rapid construction of different genetic part permutations or to substitute many genetic parts in a single step. With regard to synthetic regulation, genetic circuits have been constructed that function as logic gates, timers, switches and oscillators. Sensors have also been developed that respond to many inducible inputs as well as metabolite levels. These could be incorporated into natural product pathways to control the timing of expression of different genes or to implement feedback in response to a toxic intermediate. It is often desirable to make many simultaneous genomic changes. Methods such as CRISPR–Cas9 can target essentially any region of the genome and have been shown to function in many species, including several host species that are well suited for the industrial-scale production of small molecules. Advances in synthetic biology have simplified the characterization and production of biologically active molecules from various organisms. In this Review, Voigt and colleagues outline the design and construction of pathways used for the synthesis of such natural products in host microorganisms. Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products.

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9 α -hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1 β -dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5 α -ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol. Despite intensive investigation, stepwise reactions from diol to Taxol tetracyclic core skeleton remain unclear. Here, authors fill this gap by identifying two P450s and confirming the reaction order.

Six selected weedy Amaranthus genotypes (three accessions from each species of A. viridis and A. spinosus ) were evaluated in terms of nutrients, minerals, antioxidant constituents and antioxidant activity for the possibilities of weedy species as a vegetable cultivar in a randomized complete block design with three replications. As leafy vegetable, Weedy Amaranthus has remarkable protein, dietary fiber, carbohydrates, Ca, K, Mg, P, S, Fe, Mn, Cu, Zn, Na, Mo, B, chlorophylls, β-cyanins, β-xanthins, betalains, β-carotene, vitamin C, TPC, TFC, and TAC (DPPH and ABTS + ) compared to any cultivated species. The A. viridis genotype WAV7 and A. spinosus genotype WAS13 had the highest nutrients, pigments, vitamins, phenolics, flavonoids, and antioxidant. Hence, these two weedy accessions could be used as an antioxidant profile enriched cultivar with high nutritional and antioxidant activity. Pigments, β-carotene, vitamin C, phenolics, and flavonoids had strong antioxidant activity and played a vital role in the antioxidant activity of weedy Amaranthus genotypes. Weedy species are an excellent source of phenolics, flavonoids, and antioxidants that have many pharmacological and medicinal effects of their traditional applications and detoxify ROS and offered huge prospects for feeding the antioxidant-deficient community to cope with the hidden hunger and attaining nutritional and antioxidant sufficiency.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction 1 – 4 . However, biologically ‘rare’ functional groups, such as the fluoro 5 , chloro 6 , 7 , bromo 7 , 8 , phosphonate 9 , enediyne 10 , 11 , cyano 12 , diazo 13 , alkene 14 and alkyne 15 – 17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu( i )-catalysed azide-alkyne cycloaddition ‘click’ reaction 18 . Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya . We found that l -lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C–C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications. Microbial generation of a terminal-alkyne-containing amino acid can be encoded into E. coli and provides the potential for in vivo generation of proteins and natural products for click chemistry.

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris , Plectranthus barbatus , and Leonotis leonurus . We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites. A diterpenoid biosynthetic gene cluster (BGC) has been identified in a few species in the Lamiaceae (mint) family, but its origin and evolution remain unclear. Here, the authors report assembly of genomes of three species within the family and reveal the dynamic evolutionary trajectory of the BGC.