Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of β-Ionone.

Biotransformation of ethylene glycol by engineered Escherichia coli

The solar horizontal bubbling fluidized bed concept developed within the SOLPART research project can be used as a solar receiver-reactor. This application offers a considerable industrial potential, as illustrated in the paper further to different experiments and industrial contacts. The most demanding application is the calcination of limestone, either as pure calcite, or as 85% mix in cement raw meal. The decomposition temperature exceeds 850 °C (nearly the application limits of refractory steel alloys). Other calcinations (e.g. dolomite, gypsum, phosphate rock, meta-kaolin, clays, etc.) are less demanding since occurring at a lower calcination temperature and with an endothermic reaction heat that is significantly lower than the reaction heat of CaCO3, which is therefore considered as a relevant test case.The solar horizontal bubbling fluidized bed concept developed within the SOLPART research project can be used as a solar receiver-reactor. This application offers a considerable industrial potential, as illustrated in the paper further to different experiments and industrial contacts. The most demanding application is the calcination of limestone, either as pure calcite, or as 85% mix in cement raw meal. The decomposition temperature exceeds 850 °C (nearly the application limits of refractory steel alloys). Other calcinations (e.g. dolomite, gypsum, phosphate rock, meta-kaolin, clays, etc.) are less demanding since occurring at a lower calcination temperature and with an endothermic reaction heat that is significantly lower than the reaction heat of CaCO3, which is therefore considered as a relevant test case.

BackgroundAmphotericin B (AmB) is widely used against fungal infection and produced mainly by Streptomyces nodosus. Various intracellular metabolites of S. nodosus were identified during AmB fermentation, and the key compounds that related to the cell growth and biosynthesis of AmB were analyzed by principal component analysis (PCA) and partial least squares (PLS).ResultsRational design that based on the results of metabolomics was employed to improve the AmB productivity of Streptomyces nodosus, including the overexpression of genes involved in oxygen-taking, precursor-acquiring and product-exporting. The AmB yield of modified strain S. nodosus VMR4A was 6.58 g/L, which was increased significantly in comparison with that of strain S. nodosus ZJB2016050 (5.16 g/L). This was the highest yield of AmB reported so far, and meanwhile, the amount of by-product amphotericin A (AmA) was decreased by 45%. Moreover, the fermentation time of strain S. nodosus VMR4A was shortened by 24 h compared with that of strain. The results indicated that strain S. nodosus VMR4A was an excellent candidate for the industrial production of AmB because of its high production yield, low by-product content and the fast cell growth.ConclusionsThis study would lay the foundation for improving the AmB productivity through metabolomics analysis and overexpression of key enzymes.

BackgroundThe overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. However, the lack of quality data often makes it difficult to correctly identify key enzymes through control analysis. Here, we proposed a method combining in vitro metabolic pathway analysis and proteomics measurement to find the key enzymes in threonine synthesis pathway.ResultsAll enzymes in the threonine synthesis pathway were purified for the reconstruction and perturbation of the in vitro pathway. Label-free proteomics technology combined with APEX (absolute protein expression measurements) data analysis method were employed to determine the absolute enzyme concentrations in the crude enzyme extract obtained from a threonine production strain during the fastest threonine production period. The flux control coefficient of each enzyme in the pathway was then calculated by measuring the flux changes after titration of the corresponding enzyme. The isoenzyme LysC catalyzing the first step in the pathway has the largest flux control coefficient, and thus its concentration change has the biggest impact on pathway flux. To verify that the key enzyme identified through in vitro pathway analysis is also the key enzyme in vivo, we overexpressed LysC in the original threonine production strain. Fermentation results showed that the threonine concentration was increased 30% and the yield was increased 20%.ConclusionsIn vitro metabolic pathways simulating in vivo cells can be built based on precise measurement of enzyme concentrations through proteomics technology and used for the determination of key enzymes through metabolic control analysis. This provides a new way to find gene overexpression targets for industrial strain improvement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0275-8) contains supplementary material, which is available to authorized users.

Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

The nutritional and health-promoting properties of tomatoes (Solanum lycopersicum), a highly significant crop, are attributed to their abundance of beneficial components, such as flavonoids, phenolic compounds, and carotenoids (including lycopene and β-carotene). The occurrence of these bioactive molecules is influenced by genetic, environmental, and agronomic factors, with ripening playing a critical role in their accumulation. This abstract delves into the molecular machinery controlling phytochemical accumulation, with a specific focus on the regulation of lycopene biosynthesis. The RIPENING-INHIBITOR (RIN) transcription factor, a master regulator of fruit maturation, exerts direct control over lycopene accumulation by binding to the promoters of critical biosynthetic genes. RIN directly activates the expression of PHYTENE SYNTHASE 1 (PSY1), the key rate limiting enzyme committing metabolic flux to the carotenoid pathway, and PDS, encoding phytocene desaturase, thereby orchestrating the massive lycopene synthesis characteristic of the ripening transition. Strategies for the biofortification of tomato fruits have leveraged this understanding through targeted genetic manipulation. Overexpression of key enzymes, such as the bacterial CrtB (phytoene synthase) or manipulation of the endogenous PSY1, has successfully enhanced lycopene flux. More profoundly, the manipulation of transcription factors offers a powerful multi-gene approach. For instance, the overexpression of fruit-specific promoters driving RIN or other regulators like HYR (High Pigment) can simultaneously improve the entire pathway, leading to substantial increases in lycopene content. Flavonoids and phenolic compounds are produced by the phenylpropanoid pathway, which is regulated by enzymes such as chalcone synthase (CHS) and phenylalanine ammonialyase (PAL). Gene regulation of these pathways involves a complex interplay of transcription factors (e.g., RIN, NOR, and HY5) and phytohormones (e.g., ethylene and abscisic acid), which modulate expression patterns during fruit development and stress responses. Phytochemical levels are also significantly influenced by environmental factors; for instance, optimal lycopene synthesis occurs at 20-25 °C, while higher temperatures above 30 °C inhibit lycopene accumulation and promote beta-carotene synthesis, a shift mediated by the temperature-sensitive expression of key genes, including those regulated by RIN. Naturally occurring or induced mutations in genes such as DET1 and HP2, which are negative regulators of light signal transduction, result in high pigment phenotypes with dramatically increased lycopene and flavonoid content. The successful application of metabolic engineering and transcription factor manipulation for biofortification holds immense promises for developing next-generation tomato cultivars with amplified health-promoting properties, directly linking agricultural science to improved human health outcomes through the mitigation of chronic diseases like cancer and cardiovascular disorders.

High yield production of L-isoleucine through readjusting the ratio of two direct precursors in Escherichia coli

BackgroundEthylene is an important industrial compound for the production of a wide variety of plastics and chemicals. At present, ethylene production involves steam cracking of a fossil-based feedstock, representing the highest CO2-emitting process in the chemical industry. Biological ethylene production can be achieved via expression of a single protein, the ethylene-forming enzyme (EFE), found in some bacteria and fungi; it has the potential to provide a sustainable alternative to steam cracking, provided that significant increases in productivity can be achieved. A key barrier is determining factors that influence the availability of substrates for the EFE reaction in potential microbial hosts. In the presence of O2, EFE catalyzes ethylene formation from the substrates α-ketoglutarate (AKG) and arginine. The concentrations of AKG, a key TCA cycle intermediate, and arginine are tightly controlled by an intricate regulatory system that coordinates carbon and nitrogen metabolism. Therefore, reliably predicting which genetic changes will ultimately lead to increased AKG and arginine availability is challenging.ResultsWe systematically explored the effects of media composition (rich versus defined), gene copy number, and the addition of exogenous substrates and other metabolites on the formation of ethylene in Escherichia coli expressing EFE. Guided by these results, we tested a number of genetic modifications predicted to improve substrate supply and ethylene production, including knockout of competing pathways and overexpression of key enzymes. Several such modifications led to higher AKG levels and higher ethylene productivity, with the best performing strain more than doubling ethylene productivity (from 81 ± 3 to 188 ± 13 nmol/OD600/mL).ConclusionsBoth EFE activity and substrate supply can be limiting factors in ethylene production. Targeted modifications in central carbon metabolism, such as overexpression of isocitrate dehydrogenase, and deletion of glutamate synthase or the transcription regulator ArgR, can effectively enhance substrate supply and ethylene productivity. These results not only provide insight into the intricate regulatory network of the TCA cycle, but also guide future pathway and genome-scale engineering efforts to further boost ethylene productivity.

Shikimate is a key intermediate in the synthesis of neuraminidase inhibitors. Compared with traditional methods, microbial production of shikimate has the advantages of environmental friendliness, low cost, feed stock renewability, and product selectivity and diversity. Despite these advantages, shikimate kinase I and II respectively encoded by aroK and aroL are inactivated in most shikimate microbial producers, thus requiring the addition of aromatic compounds during the fermentation process. To overcome this problem, we constructed a non-auxotrophic, shikimate-synthesising strain of Escherichia coli. By inactivation of repressor proteins, blocking of competitive pathways and overexpression of key enzymes, we increased the shikimate production of wild-type E. coli BW25113 to 1.73 g/L. We then designed a tunable switch that can conditionally decrease gene expression and substituted it for the original aroK promoters. Expression of aroK in the resulting P-9 strain was maintained at a high level during the growth phase and then reduced at a suitable time by addition of an optimal concentration of inducer. In 5-L fed-batch fermentation, strain P-9 produced 13.15 g/L shikimate without the addition of any aromatic compounds. The tunable switch developed in this study is an efficient tool for regulating indispensable genes involved in critical metabolic pathways.

Our previous study demonstrated that apurinic/apyrimidinic endonuclease 1 (APE1) and miR-514a are significantly overexpressed in the cytoplasm of drug-resistant neuroblastoma (NB) cells. Furthermore, we have developed a novel strategy for monitoring drug resistance in NB by targeting cytoplasmic APE1 and miR-514a. The overexpression of key enzymes in the mitochondrial base excision repair pathway, along with dysregulated miRNAs, is closely associated with chemotherapy resistance in tumors. Therefore, this study leverages cytochrome c (cyt c), located in the inner mitochondrial membrane as a targeting agent and the mitochondria-specific expression of 16S rRNA as a response switch to develop a spatially resolved, sequential activation system for an allosteric DNA nanomachine (AP-miR-tFNA), enabling in vivo detection of APE1 and miR-514a within mitochondria and facilitating molecular imaging of NB. AP-miR-tFNA sequentially responds to cyt c, 16S rRNA, miR-514a, and APE1, thereby undergoing a conformational change that efficiently achieves progressive dissociation of the fluorophore from the quencher through a sequential mechanism, ultimately generating a detectable fluorescence signal. Experimental results demonstrate that AP-miR-tFNA enables in vivo monitoring of drug resistance in NB, providing an innovative and dependable approach for monitoring therapeutic resistance in NB. In particular, AP-miR-tFNA enables in situ detection of APE1 and miR-514a within NB plasma exosomes, thereby allowing non-invasive differentiation between high-risk and low-to-intermediate-risk NB, as well as between drug-resistant NB and non-drug-resistant NB.

Nanotechnology involves research and technology development at the atomic, molecular, and macromolecular levels, aimed at creating and using structures, devices, and systems with novel properties and functions based on their small size. It is strategically a very important research field with considerable industrial potential. Nowadays nanotechnology has rapidly become a significant component in the food and agriculture industry. In the food industry, nanotechnology is being used to produce intelligent packages (packages are sensitive to air and moisture changes, also have time temperature indicators, leakage indicators, spoilage indicators), produce healthier foods, improve food quality, as food ingredients, additives and supplements, water treatment and filtration, as waste water treatment chemicals (arsenic, heavy metals). In the agriculture, nanotechnology is being used to pesticides, herbicides, fertilizers and plant growth regulators, veterinary medicines, and animal feed. Food and agriculture-related nanotechnologies may also pose indirect threats to human health. For example, created as a result of free radical production of nano particles damage and toxicity caused by some organisms the behavior of nanomaterials is still the human body unknown (DNA injury, the possibility of cancer cells, destructions of cells).In this chapter, some applications of nanotechnology in food and agriculture industry are discussed.KeywordsConjugate Linoleic AcidPackaging MaterialFood PackagingPrecision FarmingFood SectorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Sugar and dietary fibre components of tamarind (tamarindus indica l.) fruits from Nigeria

Hydroquinone, a widely used phenolic compound, poses significant health and environmental risk and must be removed from wastewater. In this study, a novel adsorbent was developed for the removal of hydroquinone from aqueous solutions through the in-situ polymerisation of 2-(dimethylamino)ethyl methacrylate (DMAEMA) in the presence of zinc oxide nanoparticles (ZnO NPs). The kinetics of equilibrium swelling in deionised water (DI) and the effect of pH on the swelling behaviour were investigated. The structure of the nanocomposite hydrogel was analysed using FTIR, XRD, FESEM, EDX, TEM, and BET analysis. Various factors affecting the adsorption process, such as solution pH (2–12), initial concentration (25–200 mg/L), adsorbent dosage (4.0–80 mg/100 mL), NaCl concentration (0.1–1.0 mol/L), and temperature, were also investigated. The adsorption capacity of hydroquinone and the removal efficiency of PDMAEMA@ZnO reached 455 mg/g and 91%, respectively, at pH 11 within 15 minutes. Furthermore, PDMAEMA@ZnO showed a high removal efficiency of 99.47% in tests with real wastewater. Reusability studies showed that the adsorbent retained its ability to remove hydroquinone after four regeneration cycles. The adsorption data were well described by the pseudo-second order and Langmuir models. The thermodynamic parameters indicated that the adsorption process is spontaneous and endothermic. Therefore, the adsorbent, has considerable industrial potential as it quickly and efficiently adsorbs phenolic impurities such as hydroquinone from real textile wastewater.

Two and three dimensional photoelasticity is an extremely useful stress analysis tool for the design and development of complex engineering structures. Confirmation by analysis of the prototype may become more difficult however for economic reasons. The development of a non-contact method of analysis requiring the minimum surface preparation and plant shutdown is therfore doubly attractive. Electronic Speckle Pattern Interferometry (ESPI) or Tv-Holography using a pulsed laser is a technique which displays considerable industrial potential in this way.

Yarrowia lipolytica (YL) is a “non-conventional” yeast that is capable of producing important metabolites. One of the most important products that is secreted by this microorganism is lipase, a ubiquitous enzyme that has considerable industrial potential and can be used as a biocatalyst in the pharmaceutical, food, and environmental industries. In this work, Yarrowia lipolytica lipase (YLL) was immobilized on Lewatit and Amberlite beads and is used in the enzymatic ring-opening polymerization (ROP) of cyclic esters in the presence of different organic solvents. YLL immobilized on Amberlite XAD7HP had the higher protein adsorption (96%) and a lipolytic activity of 35 U/g. Lewatit VPOC K2629 has the higher lipolytic activity (805 U/g) and 92% of protein adsorption. The highest molecular weight (Mn 10,685 Da) was achieved at 90 °C using YLL that was immobilized on Lewatit 1026 with decane as solvent after 60 h and 100% of monomer conversion.