Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering.

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified.  • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

XylR Overexpression in Escherichia coli Alleviated Transcriptional Repression by Arabinose and Enhanced Xylitol Bioproduction from Xylose Mother Liquor.

Xylitol is a polyol widely used in food, pharmaceuticals, and light industries. It is currently produced through the chemical catalytic hydrogenation of xylose and generates xylose mother liquor as a substantial byproduct in the procedure of xylose extraction. If xylose mother liquor could also be efficiently bioconverted to xylitol, the greenness and atom economy of xylitol production would be largely improved. However, xylose mother liquor contains a mixture of glucose, xylose, and arabinose, raising the issue of carbon catabolic repression in its utilization by microbial conversion. Targeting this challenge, the transcriptional activator XylR was overexpressed in a previously constructed xylitol-producing E. coli strain CPH. The resulting strain CPHR produced 16.61 g/L of xylitol in shake-flask cultures from the mixture of corn cob hydrolysate and xylose mother liquor (1:1, v/v) with a xylose conversion rate of 90.1%, which were 2.23 and 2.15 times higher than the starting strain, respectively. Furthermore, XylR overexpression upregulated the expression levels of xylE, xylF, xylG, and xylH genes by 2.08-2.72 times in arabinose-containing medium, suggesting the alleviation of transcriptional repression of xylose transport genes by arabinose. This work lays the foundation for xylitol bioproduction from xylose mother liquor.

Multisite Mutation of the Escherichia coli cAMP Receptor Protein: Enhancing Xylitol Biosynthesis by Activating Xylose Catabolism and Improving Strain Tolerance.

The bioproduction of xylitol from hemicellulose hydrolysate has good potential for industrial development. However, xylitol productivity has always been limited due to corncob hydrolysate toxicity and glucose catabolic repression. To address these challenges, this work selected the S83 and S128 amino acid residues of the cyclic AMP receptor protein (CRP) as the modification target. By introducing multisite mutation in CRP, this approach successfully enhanced xylose catabolism and improved the strain's tolerance to corncob hydrolysate. The resulting mutant strain, designated as CPH (CRP S83H-S128P), underwent fermentation in a 20 L bioreactor with semicontinuous feeding of corncob hydrolysate. Remarkably, xylitol yield and xylitol productivity for 41 h fermentation were 175 and 4.32 g/L/h, respectively. Therefore, multisite CRP mutation was demonstrated as an efficient global regulatory strategy to effectively improve xylitol productivity from lime-pretreated corncob hydrolysates.

[Immobilizing engineered Escherichia coli cells into zeolitic imidazolate framework 8 for efficient biosynthesis of Ala-Gln].

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.

Genetically Engineering Escherichia coli to Produce Xylitol from Corncob Hydrolysate without Lime Detoxification.

Before fermentation with hemicellulosic hydrolysate as a substrate, it is generally necessary to detoxify the toxic substances that are harmful to microorganism growth. Cyclic AMP receptor protein (CRP) is a global regulator, and mutation of its key sites may have an important impact on E. coli virulence tolerance. Using corncob hydrolysate without ion-exchange or lime detoxification as the substrate, shake flask fermentation experiments showed that CRP mutant IS5-dG (I112L, T127G, A144T) produced 18.4 g/L of xylitol within 34 h, and the OD600 was 9.7 at 24 h; these values were 41.5% and 21.3% higher than those of the starting strain, IS5-d, respectively. This mutant produced 82 g/L of xylitol from corncob hydrolysate without ion-exchange or lime detoxification during fed-batch fermentation in a 15-L bioreactor, with a productivity of 1.04 g/L/h; these values were 173% and 174% higher than the starting strain, respectively. To our knowledge, this is the highest xylitol concentration and productivity produced by microbial fermentation using completely non-detoxified hemicellulosic hydrolysate as the substrate to date. This study also showed that alkali neutralization, high temperature sterilization, and fermentation of the hydrolysate had important effects on the xylose loss rate and xylitol production.

Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in industrial yeast.

Brassinolide (BL) is the most biologically active compound among natural brassinosteroids. However, the agricultural applications are limited by the extremely low natural abundance and the scarcity of synthetic precursors. Here, we employ synthetic biology to construct a yeast cell factory for scalable production of 24-epi-ergosterol, an un-natural sterol, proposed as a precursor for BL semi-synthesis. First, we construct an artificial pathway by introducing a Δ24(28) sterol reductase from plants (DWF1), followed by enzyme directed evolution, to enable de novo biosynthesis of 24-epi-ergosterol in yeast. Subsequently, we manipulate the sterol homeostasis (overexpression of ARE2, YEH1, and YEH2 with intact ARE1), maintaining a balance between sterol acylation and sterol ester hydrolysis, for the production of 24-epi-ergosterol, whose titer reaches to 2.76 g L-1 using fed-batch fermentation. The sterol homeostasis engineering strategy can be applicable for bulk production of other economically important phytosterols.

Sequence and Structure-Guided Engineering of Urethanase from Agrobacterium tumefaciens d3 for Improved Catalytic Activity.

The amidase from Agrobacterium tumefaciens d3 (AmdA) degrades the carcinogenic ethyl carbamate (EC) in alcoholic beverages. However, its limited catalytic activity hinders practical applications. Here, multiple sequence alignment was first used to predict single variants with improved activity. Afterward, AlphaFold 2 was applied to predict the three-dimensional structure of AmdA and 21 amino acids near the catalytic triad were randomized by saturation mutagenesis. Each of the mutation libraries was then screened, and the improved single variants were combined to obtain the best double variant I97L/G195A that showed a 3.1-fold increase in the urethanase activity and a 1.5-fold increase in ethanol tolerance. MD simulations revealed that the mutations shortened the distance between catalytic residues and the substrate and enhanced the occurrence of a critical hydrogen bond in the catalytic pocket. This study displayed a useful strategy to engineer an amidase for the improvement of urethanase activity, and the variant obtained provided a good candidate for applications in the food industry.

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via a dual-enzyme screening system.

D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 °C improvement in T505, the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4- and 9.47- fold improvement in the half life at 60 °C, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in kcat/Km compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D­allulose.

Increasing NADPH Availability for Xylitol Production via Pentose-Phosphate-Pathway Gene Overexpression and Embden-Meyerhof-Parnas-Pathway Gene Deletion in Escherichia coli.

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.

Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae.

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

Expression, isolation, and identification of an ethanol-resistant ethyl carbamate-degrading amidase from Agrobacterium tumefaciens d3.

Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d3 exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni-NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the Km was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5-20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

Vibrio natriegens as a pET-Compatible Expression Host Complementary to Escherichia coli.

Efficient and novel recombinant protein expression systems can further reduce the production cost of enzymes. Vibrio natriegens is the fastest growing free-living bacterium with a doubling time of less than 10 min, which makes it highly attractive as a protein expression host. Here, 196 pET plasmids with different genes of interest (GOIs) were electroporated into the V. natriegens strain VnDX, which carries an integrated T7 RNA polymerase expression cassette. As a result, 65 and 75% of the tested GOIs obtained soluble expression in V. natriegens and Escherichia coli, respectively, 20 GOIs of which showed better expression in the former. Furthermore, we have adapted a consensus "what to try first" protocol for V. natriegens based on Terrific Broth medium. Six sampled GOIs encoding biocatalysts enzymes thus achieved 50-128% higher catalytic efficiency under the optimized expression conditions. Our study demonstrated V. natriegens as a pET-compatible expression host with a spectrum of highly expressed GOIs distinct from E. coli and an easy-to-use consensus protocol, solving the problem that some GOIs cannot be expressed well in E. coli.

Analysis of nicotine-induced metabolic changes in Blakeslea trispora by GC-MS.

Blakeslea trispora is a natural source of carotenoids, including ß-carotene and lycopene, which have industrial applications. Therefore, classical selective breeding techniques have been applied to generate strains with increased productivity, and microencapsulated ß-carotene preparation has been used in food industry (Li et al., 2019). In B. trispora, lycopene is synthesized via the mevalonate pathway (Venkateshwaran et al., 2015). Lycopene cyclase, which is one of the key enzymes in this pathway, is a bifunctional enzyme that can catalyze the cyclization of lycopene to produce ß-carotene and exhibit phytoene synthase activity (He et al., 2017).

Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates.

The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

Effects of His-tag on Catalytic Activity and Enantioselectivity of Recombinant Transaminases.

Recombinant proteins were often expressed with His-tag to simplify the purification process. Among them, transaminase was mostly expressed with fusion tags and widely used in the production of numerous amino moieties. However, the existence of the His-tag has been reported to affect various properties of different recombinant enzymes, while the effect on transaminase was rarely studied. In this paper, we investigated the effect of His-tag on transaminase based on the various activities of 4-aminobutyrate-2-oxoglutarate transaminase (GabT) when it was expressed in vector pETDuet-1. We found that His-tag did not affect the enantioselectivity, but decreased the catalytic activity to different extents according to its existence and location. Native GabT maintained the highest catalytic activity; GabT with C-terminal His-tag showed slightly lower activity than native GabT but about 2.2-fold higher than GabT with N-terminal His-tag. Besides, other fusion tags like T7-tag and S-tag inserted between N-His-tag and GabT can relieve the decreasing effect of His-tag on GabT activity. Furthermore, whole cell catalytic activity of several transaminases was improved by deleting the N-terminal His-tag. This study provided a strategy for the efficient expression of recombinant transaminase with improved catalytic activity and might attract attention to the effect of His-tag on other enzymatic properties.

Microencapsulated ß-carotene preparation using different drying treatments.

ß-Carotene is one of the most abundant natural pigments in foods; however, usage of ß-carotene is limited because of its instability. Microencapsulation techniques are usually applied to protect microencapsulated ß-carotene from oxidization. In this study, ß-carotene was microencapsulated using different drying processes: spray-drying, spray freeze-drying, coating, and spray granulation. The properties of morphology, particle size, water content, thermal characteristic, and chemical stability have been explored and compared. Scanning electron microscopy measurements showed that the coated powder had a dense surface surrounded by starch and suggested that the coating process gave a microencapsulated powder with the smallest bulk density and the best compressibility among the prepared powders. The chemical stabilities of microcapsules were evaluated during six months of storage at different temperatures. The coated powder had the highest mass fraction of ß-carotene, which indicated that the coating process was superior to the three other drying processes.

Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity.

S-adenosylmethionine synthetase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and L-methionine. SAM is the major methyl donor for more than 100 transmethylation reactions. It is also a common cosubstrate involved in transsulfuration and aminopropylation. However, product inhibition largely restrains the activity of MAT and limits the enzymatic synthesis of SAM. In this research, the product inhibition of MAT from Escherichia coli was reduced via semi-rational modification. A triple variant (Variant III, I303 V/I65 V/L186 V) showed a 42-fold increase in Ki,ATP and a 2.08-fold increase in specific activity when compared to wild-type MAT. Its Ki,ATP was 0.42 mM and specific acitivity was 3.78 ±0.19 U/mg. Increased Ki,ATP means reduced product inhibition which enhances SAM accumulation. The SAM produced by Variant III could reach to 3.27 mM while SAM produced by wild-type MAT was 1.62 mM in the presence of 10 mM substrates. When the residue in 104th of Variant III was further optimized by site-saturated mutagenesis, the specific activity of Variant IV (I303 V/I65 V/L186 V/N104 K) reached to 6.02 ±0.22 U/mg at 37 °C, though the SAM concentration decreased to 2.68 mM with 10 mM substrates. Analysis of protein 3D structure suggests that changes in hydrogen bonds or other ligand interactions around active site may account for the variety of product inhibition and enzyme activity. The Variant III and Variant IV with reduced inhibition and improved enzyme activity in the study would be more suitable candidates for SAM production in the future.

Efficient production of xylitol by the integration of multiple copies of xylose reductase gene and the deletion of Embden-Meyerhof-Parnas pathway-associated genes to enhance NADPH regeneration in Escherichia coli.

Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6 g L-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09 g L-1 h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.

Advancement of multi-target drug discoveries and promising applications in the field of Alzheimer's disease.

Complex diseases (e.g., Alzheimer's disease) or infectious diseases are usually caused by complicated and varied factors, including environmental and genetic factors. Multi-target (polypharmacology) drugs have been suggested and have emerged as powerful and promising alternative paradigms in modern medicinal chemistry for the development of versatile chemotherapeutic agents to solve these medical challenges. The multifunctional agents capable of modulating multiple biological targets simultaneously display great advantages of higher efficacy, improved safety profile, and simpler administration compared to single-targeted agents. Therefore, multifunctional agents would certainly open novel avenues to rationally design the next generation of more effective but less toxic therapeutic agents. Herein, the authors review the recent progress made in the discovery and design processes of selective multi-targeted agents, especially the successful application of multi-target drugs for the treatment of Alzheimer's disease.