蛋白质组学和代谢组学在微生物代谢工程中的应用

构建微生物细胞工厂是化学品、生物能源以及药物分子可持续生产的可行性策略。然而，微生物的代谢复杂、调控严谨，制约着目标产物高效合成。蛋白质组学和代谢组学可以从系统生物学角度分析酶和代谢物组分，从而理解复杂的生物系统，为微生物代谢工程改造提供重要线索。该文介绍了蛋白质组学和代谢组学在微生物代谢工程中的应用，包括基因组尺度代谢模型构建、菌株生物合成优化、指导菌株耐受性改造、限速步骤预测、植物次级代谢途径挖掘，从而为微生物合成天然产物提供新的基因或途径。在此基础上，该文还展望了生物大数据未来的发展方向。     

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.     

Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol

Methylotrophic yeasts, which can use methanol as carbon and energy source, have been wildly used as microbial cell factories for biomanufacturing. Methanol derived from diverse sources could be transformed into precursor, such as pyruvate and acetyl-CoA, for the production of valuable chemicals through genetic engineering of methylotrophic yeast.     

Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review

Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, pharmaceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review.     

Enabling Heterologous Synthesis of Lupulones in the Yeast Saccharomyces cerevisiae

Lupulones, naturally produced by glandular trichomes of hop (Humulus lupulus), are prenylated phloroglucinol derivatives that contribute the bitter flavor of beer and demonstrate antimicrobial and anticancer activities. It is appealing to develop microbial cell factories such that lupulones may be produced via fermentation technology in lieu of extraction from limited plant resources. In this study, the yeast Saccharomyces cerevisiae transformants harboring a synthetic lupulone pathway that consisted of five genes from hop were constructed. The transformants accumulated several precursors but failed to accumulate lupulones. Overexpression of 3-hydroxy-3-methyl glutaryl co-enzyme A reductase, the key enzyme in precursor formation in the mevalonate pathway, also failed to achieve a detectable level of lupulones. To decrease the consumption of the precursors, the ergosterol biosynthesis pathway was chemically downregulated by a small molecule ketoconazole, leading to successful production of lupulones. Our study demonstrated a combination of molecular biology and chemical biology to regulate the metabolism for heterologous production of lupulones. The strategy may be valuable for future engineering microbial process for other prenylated natural products.

     

Plant cell culture technologies: A promising alternatives to produce high-value secondary metabolites

Plants have been used for its medicinal values since ancient time. The medicinal properties of plants are based on their phytochemical constituent particularly secondary metabolites which are produced in low amounts by plants. Secondary metabolites have been used as medicines, flavors, colors, and fragrances. In recent time, these natural compounds are gaining enormous attention in pharmaceutical, cosmetics, and nutraceutical industries and are regarded economically valuable products. The production of plant secondary metabolites in plant is largely dependent on the plant species, environmental factors and geographical regions. In addition, the main challenges in their mass production is reported to be the quality and quantity issues during their synthesis. Therefore, enthusiasm has grown for increasing the production of secondary metabolites by employing in vitro plant cell culture technology and bioengineering methods. Such technological advancement, has led to production of a huge number of medicinal herbs and high-value secondary metabolites that are mostly used in pharmaceuticals, cosmetics and nutraceuticals industries. The current mini-review article focuses on applications of plant cell culture system for the production secondary metabolites and recent techniques used to improve metabolite contents. Furthermore, our review emphasizes safety issues of plant cell culture derived products.     

Structural biology of plant sulfur metabolism: From assimilation to biosynthesis

Covering: 1966 to 2012Sulfur is an essential element that must be assimilated by all organisms; however, the metabolic pathways for this task vary significantly, even among individual genera of bacteria, and especially so among eukaryotes. While all organisms require sulfurous amino acids, plants require specialized sulfur-containing metabolites, such as glucosinolates and allylsulfur compounds, for protection from herbivory and microbial infection; and the synthesis of specialized peptides (i.e., glutathione and phytochelatins) for protection against reactive oxygen species and exposure to transition metals, such as cadmium. In order to provide the complex array of sulfur-containing metabolites essential to plant viability, flux through the sulfur assimilatory pathway must be tightly regulated by controlling enzymatic activity. The X-ray crystal structures of several primary sulfur assimilatory enzymes, complemented by kinetics, have revealed mechanisms of enzymatic regulation (i.e., via redox state and protein-protein interaction) in these biosynthetic pathways, in addition to the chemical mechanisms of catalysis. This review summarizes the state of our structural knowledge of primary and secondary sulfur assimilatory enzymes from plants.     

Functional Diversification and Structural Origins of Plant Natural Product Methyltransferases

In plants, methylation is a common step in specialized metabolic pathways, leading to a vast diversity of natural products. The methylation of these small molecules is catalyzed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which are categorized based on the methyl-accepting atom (O, N, C, S, or Se). These methyltransferases are responsible for the transformation of metabolites involved in plant defense response, pigments, and cell signaling. Plant natural product methyltransferases are part of the Class I methyltransferase-superfamily containing the canonical Rossmann fold. Recent advances in genomics have accelerated the functional characterization of plant natural product methyltransferases, allowing for the determination of substrate specificities and regioselectivity and further realizing the potential for enzyme engineering. This review compiles known biochemically characterized plant natural product methyltransferases that have contributed to our knowledge in the diversification of small molecules mediated by methylation steps.     

Material and Interface Engineering for High‐Performance Perovskite Solar Cells: A Personal Journey and Perspective

Hybrid organic‐inorganic perovskite solar cells (PSCs) have become a shining star in the photovoltaic field due to their spectacular increase in power conversion efficiency (PCE) from 3.8 % to over 23 % in just few years, opening up the potential in addressing the important future energy and environment issues. The excellent photovoltaic performance can be attributed to the unique properties of the organometal halide perovskite materials, including high absorption coefficient, tunable bandgap, high defect tolerance, and excellent charge transport characteristics. The authors entered this field when pursuing research on dye‐sensitized solar cells (DSCs) by leveraging nanorods arrays for vectorial transport of the extracted electrons. Soon after, we and others realized that while the organometal halide perovskite materials have excellent intrinsic properties for solar cells, interface engineering is at least equally important in the development of high‐performance PSCs, which includes surface defect passivation, band alignment, and heterojunction formation. Herein, we will address this topic by presenting the historical development and recent progress on the interface engineering of PSCs primarily of our own group. This review is mainly focused on the material and interface design of the conventional n‐i‐p, inverted p‐i‐n and carbon electrode‐based structure devices from our own experience and perspective. Finally, the challenges and prospects of this area for future development will also be discussed.     

Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.     

A Biomimetic Approach for Spatially Controlled Cell Membrane Engineering Using Fusogenic Spherical Nucleic Acid

Engineering of the cell plasma membrane using functional DNA is important for studying and controlling cellular behaviors. However, most efforts to apply artificial DNA interactions on cells are limited to external membrane surface due to the lack of suitable synthetic tools to engineer the intracellular side, which impedes many applications in cell biology. Inspired by the natural extracellular vesicle-cell fusion process, we have developed a fusogenic spherical nucleic acid construct to realize robust DNA functionalization on both external and internal cell surfaces via liposome fusion-based transport (LiFT) strategy, which enables applications including the construction of heterotypic cell assembly for programmed signaling pathway and detection of intracellular metabolites. This approach can engineer cell membranes in a highly efficient and spatially controlled manner, allowing one to build anisotropic membrane structures with two orthogonal DNA functionalities.     

Engineering Substrate Topography at the Micro‐ and Nanoscale to Control Cell Function

The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. We review the use of in vitro synthetic cell–nanotopography interactions to control cell behavior and influence complex cellular processes, including stem‐cell differentiation and tissue organization. Future challenges and opportunities in cell–nanotopography engineering are also discussed, including the elucidation of mechanisms and applications in tissue engineering.     

Recent Advances in DNA-Based Cell Surface Engineering for Biological Applications

Due to its excellent programmability and biocompatibility, DNA molecule has unique advantages in cell surface engineering. Recent progresses provide a reliable and feasible way to engineer cell surfaces with diverse DNA molecules and DNA nanostructures. The abundant form of DNA nanostructures has greatly expanded the toolbox of DNA-based cell surface engineering and gave rise to a variety of novel and fascinating applications. In this review, we summarize recent advances in DNA-based cell surface engineering and its biological applications. We first introduce some widely used methods of immobilizing DNA molecules on cell surfaces and their application features. Then we discuss the approaches of employing DNA nanostructures and dynamic DNA nanotechnology as elements for creating functional cell surfaces. Finally, we review the extensive biological applications of DNA-based cell surface engineering and discuss the challenges and prospects of DNA-based cell surface engineering.     

Cellulose-Based Composites as Scaffolds for Tissue Engineering: Recent Advances

Today, numerous studies have focused on the design of novel scaffolds for tissue engineering and regenerative medicine applications; however, several challenges still exist in terms of biocompatibility/cytocompatibility, degradability, cell attachment/proliferation, nutrient diffusion, large-scale production, and clinical translation studies. Greener and safer technologies can help to produce scaffolds with the benefits of cost-effectiveness, high biocompatibility, and biorenewability/sustainability, reducing their toxicity and possible side effects. However, some challenges persist regarding their degradability, purity, having enough porosity, and possible immunogenicity. In this context, naturally derived cellulose-based scaffolds with high biocompatibility, ease of production, availability, sustainability/renewability, and environmentally benign attributes can be applied for designing scaffolds. These cellulose-based scaffolds have shown unique mechanical properties, improved cell attachment/proliferation, multifunctionality, and enhanced biocompatibility/cytocompatibility, which make them promising candidates for tissue engineering applications. Herein, the salient developments pertaining to cellulose-based scaffolds for neural, bone, cardiovascular, and skin tissue engineering are deliberated, focusing on the challenges and opportunities.     

Is Genetic Engineering a Route to Enhance Microalgae-Mediated Bioremediation of Heavy Metal-Containing Effluents?

Contamination of the biosphere by heavy metals has been rising, due to accelerated anthropogenic activities, and is nowadays, a matter of serious global concern. Removal of such inorganic pollutants from aquatic environments via biological processes has earned great popularity, for its cost-effectiveness and high efficiency, compared to conventional physicochemical methods. Among candidate organisms, microalgae offer several competitive advantages; phycoremediation has even been claimed as the next generation of wastewater treatment technologies. Furthermore, integration of microalgae-mediated wastewater treatment and bioenergy production adds favorably to the economic feasibility of the former process—with energy security coming along with environmental sustainability. However, poor biomass productivity under abiotic stress conditions has hindered the large-scale deployment of microalgae. Recent advances encompassing molecular tools for genome editing, together with the advent of multiomics technologies and computational approaches, have permitted the design of tailor-made microalgal cell factories, which encompass multiple beneficial traits, while circumventing those associated with the bioaccumulation of unfavorable chemicals. Previous studies unfolded several routes through which genetic engineering-mediated improvements appear feasible (encompassing sequestration/uptake capacity and specificity for heavy metals); they can be categorized as metal transportation, chelation, or biotransformation, with regulation of metal- and oxidative stress response, as well as cell surface engineering playing a crucial role therein. This review covers the state-of-the-art metal stress mitigation mechanisms prevalent in microalgae, and discusses putative and tested metabolic engineering approaches, aimed at further improvement of those biological processes. Finally, current research gaps and future prospects arising from use of transgenic microalgae for heavy metal phycoremediation are reviewed.     

Diversity in Chemical Structures and Biological Properties of Plant Alkaloids

Phytochemicals belonging to the group of alkaloids are signature specialized metabolites endowed with countless biological activities. Plants are armored with these naturally produced nitrogenous compounds to combat numerous challenging environmental stress conditions. Traditional and modern healthcare systems have harnessed the potential of these organic compounds for the treatment of many ailments. Various chemical entities (functional groups) attached to the central moiety are responsible for their diverse range of biological properties. The development of the characterization of these plant metabolites and the enzymes involved in their biosynthesis is of an utmost priority to deliver enhanced advantages in terms of biological properties and productivity. Further, the incorporation of whole/partial metabolic pathways in the heterologous system and/or the overexpression of biosynthetic steps in homologous systems have both become alternative and lucrative methods over chemical synthesis in recent times. Moreover, in-depth research on alkaloid biosynthetic pathways has revealed numerous chemical modifications that occur during alkaloidal conversions. These chemical reactions involve glycosylation, acylation, reduction, oxidation, and methylation steps, and they are usually responsible for conferring the biological activities possessed by alkaloids. In this review, we aim to discuss the alkaloidal group of plant specialized metabolites and their brief classification covering major categories. We also emphasize the diversity in the basic structures of plant alkaloids arising through enzymatically catalyzed structural modifications in certain plant species, as well as their emerging diverse biological activities. The role of alkaloids in plant defense and their mechanisms of action are also briefly discussed. Moreover, the commercial utilization of plant alkaloids in the marketplace displaying various applications has been enumerated.     

Role of Phenylpropanoids and Flavonoids in Plant Resistance to Pests and Diseases

Phenylpropanoids and flavonoids are specialized metabolites frequently reported as involved in plant defense to biotic or abiotic stresses. Their biosynthetic accumulation may be constitutive and/or induced in response to external stimuli. They may participate in plant signaling driving plant defense responses, act as a physical or chemical barrier to prevent invasion, or as a direct toxic weapon against microbial or insect targets. Their protective action is described as the combinatory effect of their localization during the host’s interaction with aggressors, their sustained availability, and the predominance of specific compounds or synergy with others. Their biosynthesis and regulation are partly deciphered; however, a lot of gaps in knowledge remain to be filled. Their mode of action on microorganisms and insects probably arises from an interference with important cellular machineries and structures, yet this is not fully understood for all type of pests and pathogens. We present here an overview of advances in the state of the art for both phenylpropanoids and flavonoids with the objective of paving the way for plant breeders looking for natural sources of resistance to improve plant varieties. Examples are provided for all types of microorganisms and insects that are targeted in crop protection. For this purpose, fields of phytopathology, phytochemistry, and human health were explored.     

Synthesis of [(3S,5R)-3-Hydroxy-5-methylpiperidin-1-yl](2-methylpyridin-3-yl)methanone

Russian Journal of Organic Chemistry - There remain challenges for effectively synthesizing heterocycles containing both piperidine and pyridine rings, mainly due to the inefficient synthetic...     

The phytoalexins from cultivated and wild crucifers: chemistry and biology

Phytoalexins are antimicrobial secondary metabolites produced de novo by plants in response to stress, including microbial attack. In general, phytoalexins are important components of plant defenses against fungal and bacterial pathogens. The phytoalexins of crucifers are indole alkaloids derived from (S)-tryptophan, most of which contain a sulfur atom derived from cysteine. Beside their antimicrobial activity against different plant pathogenic species, cruciferous phytoalexins have shown anticarcinogenic effects on various human cell lines. This review focuses on the phytoalexins produced by cruciferous plants reported to date, with particular emphasis on their chemical synthesis, biosynthesis, metabolism by plant fungal pathogens and biological activities. A summary table containing all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biological activities is provided.