Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.     

Molecular breeding of carotenoid biosynthetic pathways

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.     

Engineering natural products using combinatorial biosynthesis and biocatalysis

Many biologically active natural products are produced by the host organisms using dedicated biosynthetic pathways. The programming rules of these pathways may be rationally manipulated through combinatorial biosynthesis to produce natural products that contain structural variations or enhanced pharmacological properties. Furthermore, these pathways contain enzymes that can be harvested as powerful biocatalysts for the synthesis of both new drugs and existing blockbuster therapeutics. This review will highlight recent advances in exploring natural product biosynthetic pathways for new compounds, novel enzymes and useful biocatalysts.     

Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Carotenoid metabolism: biosynthesis, regulation, and beyond

Carotenoids are Indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied.Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis,regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.     

Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways

Phosphonate natural products have proven to be a rich source of useful pharmaceutical, agricultural, and biotechnology products, whereas study of their biosynthetic pathways has revealed numerous intriguing enzymes that catalyze unprecedented biochemistry. Here we review the history of phosphonate natural product discovery, highlighting technological advances that have played a key role in the recent advances in their discovery. Central to these developments has been the application of genomics, which allowed discovery and development of a global phosphonate metabolic framework to guide research efforts. This framework suggests that the future of phosphonate natural products remains bright, with many new compounds and pathways yet to be discovered.     

Insights into the evolution of lanthipeptide biosynthesis

Lanthipeptides are a group of posttranslationally modified peptide natural products that contain multiple thioether crosslinks. These crosslinks are formed by dehydration of Ser/Thr residues followed by addition of the thiols of Cys residues to the resulting dehydroamino acids. At least four different pathways to these polycyclic natural products have evolved, reflecting the high efficiency and evolvability of a posttranslational modification route to generate conformationally constrained peptides. The wealth of genomic information that has been made available in recent years has started to provide insights into how these remarkable pathways and their posttranslational modification machineries may have evolved. In this review, we discuss a model for the evolution of the lanthipeptide biosynthetic enzymes that has recently been developed based on the currently available data.     

Microbial genomics for the improvement of natural product discovery

The quest for the discovery of novel natural products has entered a new chapter with the enormous wealth of genetic data that is now available. This information has been exploited by using whole-genome sequence mining to uncover cryptic pathways, or biosynthetic pathways for previously undetected metabolites. Alternatively, using known paradigms for secondary metabolite biosynthesis, genetic information has been 'fished out' of DNA libraries resulting in the discovery of new natural products and isolation of gene clusters for known metabolites. Novel natural products have been discovered by expressing genetic data from uncultured organisms or difficult-to-manipulate strains in heterologous hosts. Furthermore, improvements in heterologous expression have not only helped to identify gene clusters but have also made it easier to manipulate these genes in order to generate new compounds. Finally, and perhaps the most crucial aspect of the efficient and prosperous use of the abundance of genetic information, novel enzyme chemistry continues to be discovered, which has aided our understanding of how natural products are biosynthesized de novo, and enabled us to rework the current paradigms for natural product biosynthesis.     

Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery

Microorganisms have a long track record as important sources of novel bioactive natural products, particularly in the field of drug discovery. While microbes have been shown to biosynthesize a wide array of molecules, recent advances in genome sequencing have revealed that such organisms have the potential to yield even more structurally diverse secondary metabolites. Thus, many microbial gene clusters may be silent under standard laboratory growth conditions. In the last ten years, several methods have been developed to aid in the activation of these cryptic biosynthetic pathways. In addition to the techniques that demand prior knowledge of the genome sequences of the studied microorganisms, several genome sequence-independent tools have been developed. One of these approaches is microorganism co-culture, involving the cultivation of two or more microorganisms in the same confined environment. Microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature. Within these communities, microbes interact through signaling or defense molecules. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Microorganism co-culture can be achieved in either solid or liquid media and has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry methods and metabolomics) are key for the successful detection and identification of co-culture-induced metabolites.     

细菌中倍半萜生物合成的研究进展

萜类化合物是一大类小分子天然产物,在生物体内扮演重要的角色。植物和真菌中萜类化合物的生物合成已被广泛研究,但是在真核生物中克隆或改造萜类化合物生物合成途径还有较大难度。许多细菌同样可以产生萜类化合物。在过去十多年间细菌萜类合酶的研究进展为我们对萜类化合物生物合成的理解做出了显著的贡献。这里我们主要关注细菌中合成的倍半萜化合物,概述其化学结构、倍半萜合酶对法尼基焦磷酸环化的机制、后修饰酶特别是氧化还原酶所参与的后修饰、代谢调控以及合成途径中尚未解决的问题等。     

Combinatorial biosynthesis of antimicrobials and other natural products

Combinatorial biosynthesis utilizes the enzymes from antibiotic (and other natural product) biosynthetic pathways to create novel chemical structures. The manipulation of modular polyketide synthases (PKSs) has been the major focus of this effort and has led to the production of, for example, several erythromycin analogs. Many new tools for manipulating and studying these multifunctional enzymes have been developed. These include multiple hosts and expression systems, enzymology tools for in vitro study, and ways to engineer pre-PKS and post-PKS pathways. The result is more rational and faster methods of engineering new compounds for the development of chemotherapeutic agents from natural products. The most significant recent advances in combinatorial biosynthesis are outlined.     

Manipulation of hormone biosynthetic genes in transgenic plants

Modification of plant hormone biosynthesis through the introduction of bacterial genes is a natural form of genetic engineering, which has been exploited in numerous studies on hormone function. Recently, biosynthetic pathways have been largely elucidated for most of the plant hormone classes, and genes encoding many of the enzymes have been cloned. These advances offer new opportunities to manipulate hormone content in order to study their mode of action and the regulation of their biosynthesis. Furthermore, this technology is providing the means to introduce agriculturally useful traits into crops.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

Synthesis and trafficking of alkaloid biosynthetic enzymes

The biosynthesis of plant natural products involves a large number of enzymes that create and elaborate a bewildering array of chemical structures, which are generally involved in ecophysiological interactions. Alkaloids are one of the largest groups of natural products and are generally produced through an assortment of intricate pathways. The application of molecular biochemical approaches to investigate the cell biology of alkaloid pathways has revealed a paradigm for the complex, yet highly ordered, organization of biosynthetic enzymes at both the cellular and subcellular levels. Many different cell types have been implicated in alkaloid formation and storage, in one case suggesting the intercellular transport of enzymes. The localization of enzymes to numerous cellular compartments shows the importance of protein targeting in the assembly of alkaloid pathways. Recent studies have also pointed to the possible interaction of biosynthetic enzymes in multi-enzyme complexes. These processes must be considered to be integral components of the mechanisms that regulate alkaloid biosynthesis and perhaps other natural product pathways.     

Natural products discovery from micro-organisms in the post-genome era†

With the decision to award the Nobel Prize in Physiology or Medicine to Drs. S. ōmura, W.C. Campbell, and Y. Tu, the importance and usefulness of natural drug discovery and development have been revalidated. Since the end of the twentieth century, many genome analyses of organisms have been conducted, and accordingly, numerous microbial genomes have been decoded. In particular, genomic studies of actinomycetes, micro-organisms that readily produce natural products, led to the discovery of biosynthetic gene clusters responsible for producing natural products. New explorations for natural products through a comprehensive approach combining genomic information with conventional methods show great promise for the discovery of new natural products and even systematic generation of unnaturally occurring compounds.     

Characterization of a novel carotenoid cleavage dioxygenase from plants

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.     

Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.