Structure, function, and engineering of enzymes in isoflavonoid biosynthesis

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.     

Scent engineering: toward the goal of controlling how flowers smell

Floral scent has an important role in the reproductive processes of many plants and a considerable economic value in guaranteeing yield and quality of many crops. It also enhances the aesthetic properties of ornamental plants and cut flowers. Many floral scent volatiles fall into the terpenoid or phenylpropanoid/benzenoid classes of compounds. Although the biochemistry of floral scent is still a relatively new field of investigation, in the past decade investigators have begun to identify 'scent genes'. Several of these genes, most of which, but not all, encode enzymes that directly catalyze the formation of volatile terpenoid or phenylpropanoid/benzenoid compounds, have now been used to manipulate, through genetic engineering techniques, the mix of volatiles emitted from the flowers of several plant species. The outcomes of these experiments, which are discussed here, have indicated that the genetic engineering approach to altering floral scents has potential; however, they have also revealed the limitations that result from our inadequate knowledge of the metabolic pathways responsible for scents and their regulation.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently.With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis path way are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosnthesis are discussed.     

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1-deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.     

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.     

大豆异黄酮生物合成关键酶及其代谢工程研究进展

异黄酮是一类具有C-6/C-3/C-6骨架的二次代谢产物,具有抗氧化和抗肿瘤活性。异黄酮与黄酮类物质具有相似的苯丙烷生物合成途径。天然的绝大部分异黄酮分布在豆科植物中,目前在大豆中已经发现了超过12个异黄酮(苷)。大豆异黄酮的生物合成主要涉及三个关键的酶查尔酮合酶(CHS)、查尔酮异构酶(CHI)和异黄酮合酶(IFS)。总结了大豆异黄酮的提取分离方法和生物合成途径,着重综述了CHI、CHS、IFS生物学特征和功能及异黄酮的代谢工程研究。     

Engineering isoflavone metabolism with an artificial bifunctional enzyme

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route, represented by a metabolic grid for which most of the genes involved have been sequenced in several plants, mainly in the model-plants Arabidopsis thaliana and Populus. Plants are exposed to different stresses, which may change lignin content and composition. In many cases, particularly for plant-microbe interactions, this has been suggested as defence responses of plants to the stress. Thus, understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall. This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.     

Pathway engineering for functional isoprenoids

Pathway engineering is to engineer biosynthetic pathways for compounds of interests in heterologous organisms such as microbes and higher plants, which has also been one of the most important fields in metabolic engineering and synthetic biology. This review focuses on pathway engineering researches for the production of functional isoprenoids containing monoterpenes, sesquiterpenes, diterpenes, and triterpenes as well as carotenoids and for the elucidation of relevant biosynthesis genes and enzymes, which have been performed in the last two years. As microbial hosts, Escherichia coli and Saccharomyces cerevisiae have often been employed, since they, specifically the former, are fully amenable to genetic manipulations with extensive molecular resources. Various crops have also been used as the hosts for engineering pathways of functional isoprenoids of the plant origin, particularly carotenoids.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Chlorogenic Acid Metabolism: The Evolution and Roles in Plant Response to Abiotic Stress

During the evolution, plants acquired the ability to synthesize different phenylpropanoid compounds like chlorogenic acid (CGA), which plays vital roles in resistance mechanisms to abiotic stresses. These environmental factors, including heavy metal, cold, heat, ultraviolet (UV) light, drought, and salinity affect the plant physiological processes, resulting in massive losses of agriculture production. As plants evolve from green algae to bryophytes, ferns, gymnosperms and angiosperms, phenylpropanoids are produced and accumulated in different tissues, giving the plant the capacity to counteract the harmful effects of the adverse environments. Studies have been performed on the metabolic evolution of rosmarinic acid, flavonoids and lignin, showing that the biosynthesis of phenylpropanoids begins in green algae until the emersion of genes found in angiosperms; however, the evolution of the CGA pathway has not yet been reviewed. We hypothesize that CGA could also be synthesized from algae to angiosperms. In the present review, the evolutionary analysis of CGA pathway and the function of this compound in plant tolerance to abiotic stresses are summarized. Bioinformatics analyzes were carried out on CGA-related genes across 37 plant species and revealed that the metabolic pathway starts in algae and gradually increases until it becomes complete in angiosperms. The key genes exhibited different expression patterns in stress and plant tissues. Interestingly, some genes accumulated rapidly during evolution and were more sensitive to environmental stresses, while others appeared only later in angiosperms. Further studies are needed to better understand the evolution of the CGA metabolic pathway in plants under environmentally stressed conditions.     

Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or ‘metabolons’, which represent the molecular level of organization. Here, we have confirmed the existence of the long‐postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4–hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non‐consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.