Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S-adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH₂ for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH₂-dependent hydroxylation.     

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO₂—but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.     

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products.     

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review

Hydrogen is a potential sustainable energy source and it could become an alternative to fossil fuel combustion, thus helping to reduce greenhouse gas emissions. The biological production of hydrogen, instead of its chemical synthesis, is a promising possibility since this process requires less energy and is more sustainable and eco-friendly. Several microorganisms have been used for this purpose, but Escherichia coli is one of the most widely used in this field. The literature in this area has increased exponentially in the last 10 years and several strategies have been reported in an effort to improve hydrogen production. In this work, the stay of the art of hydrogen biosynthesis by E. coli and metabolic engineering strategies to enhance hydrogen production are reviewed. This work includes a discussion about the hydrogenase complexes responsible for the hydrogen synthesis in this microorganism and the central carbon metabolism pathways connected to this process. The main metabolic engineering strategies applied are discussed, including heterologous gene expression, adaptive evolution and metabolic and protein engineering. On the other hand, culture conditions, including the use of carbon sources such as glycerol, glucose or organic wastes, have also been considered. Yields and productivities of the most relevant engineered strains reported using several carbon sources are also compared.     

Production of fuels and chemicals from renewable resources using engineered Escherichia coli

Biotechnological production of fuels and chemicals from renewable resources is an appealing way to move from the current petroleum-based economy to a biomass-based green economy. Recently, the feedstocks that can be used for bioconversion or fermentation have been expanded to plant biomass, microbial biomass, and industrial waste. Several microbes have been engineered to produce chemicals from renewable resources, among which Escherichia coli is one of the best studied. Much effort has been made to engineer E. coli to produce fuels and chemicals from different renewable resources. In this paper, we focused on E. coli and systematically reviewed a range of fuels and chemicals that can be produced from renewable resources by engineered E. coli. Moreover, we proposed how can we further improve the efficiency for utilizing renewable resources by engineered E. coli, and how can we engineer E. coli for utilizing alternative renewable feedstocks. e.g. C1 gases and methanol. This review will help the readers better understand the current progress in this field and provide insights for further metabolic engineering efforts in E. coli.     

Helicobacter pylori 26695 基因组尺度代谢研究进展

代谢网络在代谢功能研究、生物代谢过程控制、疾病诊断分析和药物靶标设计等方面具有重要理论和实践意义。生物信息学研究利用序列同源、结构模拟、对接等手段与生化实验有效结合促进了生物体代谢网络的进一步完善。本文作者在构建幽门螺杆菌（Helicobacter pylori 26695,H.pylori 26695）代谢网络的工作基础上综合了近年来研究者对H.pylori 26695代谢通路关键酶的研究成果,并结合基因组信息,综述了H.pylori 26695特异性的重要代谢通路。本文从基因组水平阐明代谢通路与基因的关系,并详细分析了关键酶对H.pylori 26695生理的重要作用,最后探讨了重构一个连续、完整的代谢网络面临的困难及其在药物靶标设计方面的研究前景。     

Mycobacterium tuberculosis carbon and nitrogen metabolic fluxes

Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens causing tuberculosis (TB), a devastating infectious disease responsible for the highest human mortality and morbidity. The emergence of drug-resistant strains of the pathogen has increased the burden of TB tremendously and new therapeutics to overcome the problem of drug resistance are urgently needed. Metabolism of Mtb and its interactions with the host is important for its survival and virulence; this is an important topic of research where there is growing interest in developing new therapies and drugs that target these interactions and metabolism of the pathogen during infection. Mtb adapts its metabolism in its intracellular niche and acquires multiple nutrient sources from the host cell. Carbon metabolic pathways and fluxes of Mtb has been extensively researched for over a decade and is well-defined. Recently, there has been investigations and efforts to measure metabolism of nitrogen, which is another important nutrient for Mtb during infection. This review discusses our current understanding of the central carbon and nitrogen metabolism, and metabolic fluxes that are important for the survival of the TB pathogen.     

Biochemical and immunological changes on oral glutamate feeding in male albino rats

 High altitude stress leads to lipid peroxidation and free radical formation which results in cell membrane damage in organs and tissues, and associated mountain diseases. This paper discusses the changes in biochemical parameters and antibody response on feeding glutamate to male albino Sprague Dawley rats under hypoxic stress. Exposure of rats to simulated hypoxia at 7576 m, for 6 h daily for 5 consecutive days, in an animal decompression chamber at 32±2° C resulted in an increase in plasma malondialdehyde level with a concomitant decrease in blood glutathione (reduced) level. Supplementation of glutamate orally at an optimal dose (27 mg/kg body weight) in male albino rats under hypoxia enhanced glutathione level and decreased malondialdehyde concentration significantly. Glutamate feeding improved total plasma protein and glucose levels under hypoxia. The activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) and the urea level remained elevated on glutamate supplementation under hypoxia. Glutamate supplementation increased the humoral response against sheep red blood cells (antibody titre). These results indicate a possible utility of glutamate in the amelioration of hypoxia-induced oxidative stress. Received: 23 March 1998 / Accepted: 19 October 1998     

Camptothecin, over four decades of surprising findings

Camptothecin (CPT) is a modified monoterpene indole alkaloid produced by Camptotheca acuminata (Nyssaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacarpum (Icacinaceae), Ophiorrhiza pumila (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae), species belonging to unrelated orders of angiosperms. From the distribution of CPT and other secondary metabolites, it has been postulated that the genes encoding enzymes involved in their biosynthesis evolved early during evolution. These genes were presumably not lost during evolution but might have been "switched off" during a certain period of time and "switched on" again at some later point. The CPT derivatives, irinotecan and topotecan, are used throughout the world for the treatment of various cancers, and over a dozen more CPT analogues are currently at various stages of clinical development. The worldwide market size of irinotecan/topotecan in 2002 was estimated at about $750 million and at $1 billion by 2003. In spite of the rapid growth of the market, CPT is still harvested by extraction from bark and seeds of C. acuminata and N. foetida. All parts of C. acuminata contain some CPT, although the highest level is found in young leaves (approximately 4-5 mg g(-1) dry weight), approximately 50% higher than in seeds and 250% higher than in bark. The development of hairy root cultures of O. pumila and C. acuminata, and the cloning and characterization of genes encoding key enzymes of the pathway leading to CPT formation in plants has opened new possibilities to propose alternative and more sustainable production systems for this important alkaloid.