Enhancing the biosynthesis of taxadien-5α-yl-acetate in Escherichia coli by combinatorial metabolic engineering approaches.

Biosynthesis of paclitaxel (Taxol™) is a hot topic with extensive and durable interests for decades. However, it is severely hindered due to the very low titers of intermediates. In this study, Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel, taxadien-5α-yl-acetate (T5OAc). Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production. The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply. Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step. Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system. In order to achieve metabolic balance, the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering. Finally, in a 5-L bioreactor, the T5OAc titer was enhanced to reach 10.9 mg/L. This represents an approximately 272-fold increase in production compared to the original strain, marking the highest yield of T5OAc ever documented in E. coli, which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.

Spiroluchuene A Synthase: A Cyclase from Aspergillus luchuensis Forming a Spirotetracyclic Diterpene.

The diterpene synthase AlTS was identified from Aspergillus luchuensis. AlTS catalyses the formation of the diterpene hydrocarbon spiroluchuene A, which exhibits a novel skeleton characterised by a spirocyclic ring system. The cyclisation mechanism towards this compound was elucidated through isotopic labelling experiments in conjunction with DFT calculations and metadynamic simulations. The biosynthetic intermediate luchudiene, besides the derivative spiroluchuene B, was captured from an enzyme variant obtained through site-directed mutagenesis. With its 10-membered ring luchudiene is structurally related to germacrenes and can undergo a Cope rearrangement to luchuelemene.

Engineering a norcoclaurine synthase for one-step synthesis of (S)-1-aryl-tetrahydroisoquinolines.

Tetrahydroisoquinoline alkaloids (THIQAs) are ubiquitous compounds with important pharmaceutical and biological activity. Their key N-heterocyclic structural motifs are synthesised via Pictet-Spengler (P-S) reaction by norcoclaurine synthases (NCS) in plants. The synthesis of 1-aryl-tetrahydroisoquinoline alkaloids has attracted increasing attention due to their antitumor and antivirus activities. Herein, the L68T/M97V mutant of NCS from Thalictrum flavum with improved activity was developed by semi-rational design. This mutant not only showed higher catalytic performance (> 96% conversion) toward benzaldehyde and dopamine over the wild-type enzyme, but also catalysed the P-S reaction of the bulky substrate 4-biphenylaldehyde and dopamine with high conversion (> 99%) for the effective synthesis of 1-aryl-THIQA. In terms of stereoselectivity, all products synthesised by the L68T/M97V mutant showed high optical purity (92-99% enantiomeric excess).

Hydroxylases involved in terpenoid biosynthesis: a review.

Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism.

Facile Production of (+)-Aristolochene and (+)-Bicyclogermacrene in Escherichia coli Using Newly Discovered Sesquiterpene Synthases from Penicillium expansum.

Penicillium expansum, producer of a wide array of secondary metabolites, has the potential to be a source of new terpene synthases. In this work, a platform was constructed with Escherichia coli BL21(DE3) by enhancing its endogenous 2-methyl-d-erythritol-4-phosphate pathway to supply sufficient terpenoid precursors. Using this precursor-supplying platform, we discovered two sesquiterpene synthases from P. expansum: PeTS1, a new (+)-aristolochene synthase, and PeTS4, the first microbial (+)-bicyclogermacrene synthase. To enhance the sesquiterpene production by PeTS1, we employed a MBP fusion tag to improve the heterologous protein expression, resulting in the increase of aristolochene production up to 50 mg/L in a 72 h flask culture, which is the highest production reported to date. We also realized the first biosynthesis of (+)-bicyclogermacrene, achieving 188 mg/L in 72 h. This work highlights the great potential of this microbial platform for the discovery of new terpene synthases and opens new ways for the bioproduction of other valuable terpenoids.

Regiospecific C-H amination of (-)-limonene into (-)-perillamine by multi-enzymatic cascade reactions.

BACKGROUND: (-)-Limonene, one of cyclic monoterpenes, is an important renewable compound used widely as a key building block for the synthesis of new biologically active molecules and fine chemicals. (-)-Perillamine, as derived from (-)-limonene, is a highly useful synthon for constructing more complicated and functionally relevant chemicals. AIM: We aimed to report a more sustainable and more efficient method for the regiospecific C-H amination of (-)-limonene into (-)-perillamine. RESULTS: Here, we report an artificial penta-enzymatic cascade system for the transformation of the cheap and easily available (-)-limonene into (-)-perillamine for the first time. This system is composed of cytochrome P450 monooxygenase, alcohol dehydrogenase and w-transaminase for the main reactions, as well as formate dehydrogenase and NADH oxidase for cofactor recycling. After optimization of the multi-enzymatic cascade system, 10 mM (-)-limonene was smoothly converted into 5.4 mM (-)-perillamine in a one-pot two-step biotransformation, indicating the feasibility of multi-enzymatic C7-regiospecific amination of the inert C-H bond of (-)-limonene. This method represents a concise and efficient route for the biocatalytic synthesis of derivatives from similar natural products.

Construction of an Escherichia coli cell factory to synthesize taxadien-5α-ol, the key precursor of anti-cancer drug paclitaxel.

Paclitaxel (Taxol™), an alkaloid of diterpenoid family, is one of the most widely used anti-cancer drugs due to its effectiveness against a variety of tumors. Rather than directly extraction and chemical synthesis of paclitaxel or its intermediates from yew plants, construction of a microbial cell factory for paclitaxel biosynthesis will be more efficient and sustainable. The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol. In this study, we report a recombinant Escherichia coli strain constructed with a heterologous mevalonate pathway, a taxadiene synthase from yew, and a cytochrome P450-mediated oxygenation system for the de novo production of taxadien-5α-ol, the first product of the multi-step taxadiene oxygenation metabolism. The key enzymes including taxadiene synthases and cytochrome P450 reductases were screened, and the linker for fusing taxadiene-5α-hydroxylase with its reductase partner cytochrome P450 reductase was optimized. By reducing the metabolic burden and optimizing the fermentation conditions, the final production of total oxygenated taxanes was raised up to 27 mg L-1 in a 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L-1, representing approximately a 12-fold and 23-fold improvements, respectively, as compared with the initial titers. The engineered MVA pathway for the overproduction of terpenoid precursors can serve as an efficient platform for the production of other valuable terpenoids.

Mining methods and typical structural mechanisms of terpene cyclases.

Terpenoids, formed by cyclization and/or permutation of isoprenes, are the most diverse and abundant class of natural products with a broad range of significant functions. One family of the critical enzymes involved in terpenoid biosynthesis is terpene cyclases (TCs), also known as terpene synthases (TSs), which are responsible for forming the ring structure as a backbone of functionally diverse terpenoids. With the recent advances in biotechnology, the researches on terpene cyclases have gradually shifted from the genomic mining of novel enzyme resources to the analysis of their structures and mechanisms. In this review, we summarize both the new methods for genomic mining and the structural mechanisms of some typical terpene cyclases, which are helpful for the discovery, engineering and application of more and new TCs.

Delayed graft function is correlated with graft loss in recipients of expanded-criteria rather than standard-criteria donor kidneys: a retrospective, multicenter, observation cohort study.

BACKGROUND: Although the use of expanded-criteria donors (ECDs) alleviates the problem of organ shortage, it significantly increases the incidence of delayed graft function (DGF). DGF is a common complication after kidney transplantation; however, the effect of DGF on graft loss is uncertain based on the published literature. Hence, the aim of this study was to determine the relationship between DGF and allograft survival. METHODS: We conducted a retrospective, multicenter, observation cohort study. A total of 284 deceased donors and 541 recipients between February 2012 and March 2017 were included. We used logistic regression analysis to verify the association between clinical parameters and DGF, and Cox proportional hazards models were applied to quantify the hazard ratios of DGF for kidney graft loss. RESULTS: Among the 284 deceased donors, 65 (22.8%) donors were ECD. Of the 541 recipients, 107 (19.8%) recipients developed DGF, and this rate was higher with ECD kidneys than with standard-criteria donor (SCD) kidneys (29.2% vs. 17.1%; P = 0.003). The 5-year graft survival rate was not significantly different between SCD kidney recipients with and without DGF (95.8% vs. 95.4%; P = 0.580). However, there was a significant difference between ECD kidney recipients with and without DGF (71.4% vs. 97.6%; P = 0.001), and the adjusted hazard ratio (HR) for graft loss for recipients with DGF was 1.885 (95% confidence interval [CI] = 1.305-7.630; P = 0.024). Results showed that induction therapy with anti-thymocyte globulin was protective against DGF (odds ratio = 0.359; 95% CI = 0.197-0.652; P = 0.001) with all donor kidneys and a protective factor for graft survival (HR = 0.308; 95% CI = 0.130-0.728; P = 0.007) with ECD kidneys. CONCLUSION: DGF is an independent risk factor for graft survival in recipients with ECD kidneys, but not SCD kidneys.

Levetiracetam inhibits THP-1 monocyte chemotaxis and adhesion via the synaptic vesicle 2A.

Long-term therapy with older antiepileptic drugs (AEDs), but not levetiracetam (LEV), may increase the risk of atherosclerosis (AS), suggesting that LEV may have a potential anti-AS effect. The synaptic vesicle 2A (SV2A) is known to the specific binding site of LEV. Numerous studies have documented that SV2A is a membrane protein specifically expressed in nervous system. Interestingly, our previous research showed that SV2A also existed in human CD8+ T lymphocytes. Therefore, we hypothesized that LEV was associated with decreased risk of AS by regulating monocytes chemotaxis and adhesion. We showed that SV2A protein were detected in THP-1 human monocytic leukemia cells. LEV (300 µM) inhibited the chemotaxis and adhesion of THP-1 cells after transfection with plasmids expressing SV2AWT, but not SV2AR383Q which was a known functional mutation site of human SV2A. Furthermore, RT-PCR and western blot analysis demonstrated that LEV (300 µM) decreased the expression level of chemokine-related receptors (CX3CL1, CCR1, CCR2, and CCR5),and reduced levels of phosphorylated AKT (p-AKT) in THP-1 cells with SV2AWT expressing plasmids. Taken together, these findings indicated that LEV has an inhibitory effect on THP-1 monocyte adhesion and chemotaxis, suggesting that SV2A may serve as a novel therapeutic target to prevent AS.

[Spatial Distribution, Sources and Bioavailability of Heavy Metals in the Surface Sediments of Longjiang River, Southern China].

In order to evaluate the pollution status, possible sources, and bioavailability of heavy metals (As, Cd, Pb, Sb, Zn, and Tl), 33 surface sediments were collected from Longjiang River, Southern China. The total concentrations and potential bioavailable concentrations of the heavy metals were analyzed using ICP-MS. Enrichment factors (EFs), Pearson correlation analysis, and principal component analysis (PCA) were used to further assess their pollution degree and potential sources. Results showed that the surface sediments of Longjiang River have been suffering heavy metal (As, Cd, Pb, Sb, and Zn) pollution to different degrees. The maximum concentrations of As, Cd, Pb, Sb, and Zn were 67.0, 7.42, 227, 229, and 807 mg·kg-1, respectively, while the Tl concentration were very low, with little variation. Moreover, the polluted sites were mostly located in the mid-lower of the main stem and in tributaries (Dongxioajiang and downstream of Dahuanjiang), and the pollution degree of the heavy metals, in a descending order, were Cd > Sb > Zn > Pb > As > Tl. Pearson correlation analysis and PCA indicated that As, Cd, Pb, Sb, and Zn predominantly originated from anthropogenic inputs, including nonferrous metal mining and smelting, municipal sewage, and agricultural activities, and Tl mostly derived from natural rock weathering. The bioavailability of heavy metals in the sediments tended to be controlled by their sources. The percentages of bioavailable heavy metals (As, Cd, Pb, Sb, and Zn) in the highly anthropogenic impacted areas (the mid-lower of the main stem and downstream of Dongxiaojiang tributary) were also high, with the average percentages of bioavailable As, Cd, Pb, Sb, and Zn of 26%, 51%, 49%, 38%, and 47%, respectively. High EF values and high bioavailable percentages of heavy metals easily and greatly cause high ecological risk of Longjiang River.

Influence of PTGS1, PTGFR, and MRP4 genetic variants on intraocular pressure response to latanoprost in Chinese primary open-angle glaucoma patients.

PURPOSE: The purpose of this study is to evaluate the association between variants in prostaglandin-endoperoxide synthase 1 (PTGS1), prostaglandin F (2α) receptor (PTGFR), and multidrug resistance protein 4 (MRP4) genes and intraocular pressure (IOP) response to latanoprost in Chinese patients with primary open-angle glaucoma (POAG). METHODS: The IOP response to latanoprost was evaluated by percent IOP reduction (%ΔIOP) in the treated eye with the formula %ΔIOP = (Baseline IOP values - IOP values posttreatment) / Baseline IOP values × 100 %. Polymorphisms in PTGS1 (rs3842787 and rs10306114), PTGFR (rs3753380 and rs3766355), and MRP4 (rs11568658 and rs11568668) genes were detected by direct DNA sequencing. The differences among %ΔIOP of genotypes or haplotypes were obtained by use of the Mann-Whitney U test. Association analyses were performed by multiple linear regression analysis. RESULTS: Latanoprost were prescribed to 63 subjects, 60 of which met the inclusion/exclusion criteria for the current study. Notably, the %ΔIOP in the rs11568658 GT heterozygous genotype was 10.4 %ΔIOP lower than that of GG homozygous wild-type on day 7 (15.7 ± 2.52 vs. 26.1 ± 2.88, P=0.003), and the corresponding results in the rs10306114 AG heterozygous genotype and AT haplotype constructed by rs3753380 and rs3766355 on day 7 were 7.2 and 10.3 %ΔIOP (P<0.05). Interestingly, similar results were also observed on day 30 (P=0.008, P=0.006, and P=0.002, respectively). Multiple regression analysis showed that heterozygous genotypes of rs10306114, rs11568658, and carrier of AT haplotype were significantly correlated with the lower %ΔIOP. On day 30, the above variations explained 9.9, 10.7, and 17.7 % of the total variability of %ΔIOP in the Chinese POAG patients, respectively. CONCLUSION: rs10306114, rs3753380, rs3766355, and rs11568658 single-nucleotide polymorphisms (SNPs) correlate with a response to latanoprost treatment in patients with POAG. These SNPs may be important determinants of variability in response to latanoprost.

Morphology, molecular stacking, dynamics and device performance correlations of vacuum-deposited small-molecule organic solar cells.

The "all carbon" organic solar cells (OSCs) based on the homocyclic molecule tetraphenyldibenzoperiflanthene (DBP) as a donor and C60 as an acceptor were comprehensively characterized. The optimized planar-mixed heterojunction device with a DBP:C60 mixture ratio of DBP : C60 (1 : 2) exhibited a power conversion efficiency of 4.47%. To understand why DBP possesses such advantageous characteristics, the correlations of the morphology, molecular stacking, carrier dynamics and performance of DBP:fullerene-based devices have been systematically studied. First, the face-on stacked DBP molecules could enhance both the absorption of light and the charge carrier mobility. Second, DBP : C60 (1 : 2) thin films with optimized domain sizes and partially interconnected acceptor grains led to the most balanced carrier mobility and the lowest bimolecular recombination in devices. Finally, the DBP molecules were found to stack closely using grazing incidence wide-angle X-ray scattering measurements, with a π-π stacking spacing of 4.58 Å, indicating an effective molecular orbital overlap in DBP. The study not only reveals the promising characteristics of DBP as a donor in OSCs but the clear correlations of the thin-film nano-morphology, molecular stacking, carrier mobility and charge recombination found here could also provide insights into the characterization methodology and optimization of the small molecule OSCs.

Spontaneous formation of light-trapping nano-structures for top-illumination organic solar cells.

By introduction of nano-structured crystallite capping layers, the power conversion efficiency of top-illumination organic solar cells is improved from 4.2 ± 0.1% to 6.0 ± 0.2%, representing a 44% enhancement. This is caused by the increase in JSC and led by the enhancement in the local E distribution inside the active layers. Comprehensive finite-difference time domain simulation reveals two main reasons for this enhancement: (1) the nano-structured capping layers can be treated as gradient-index films that effectively increase the light entering the devices. (2) The nano-structured capping layers can also diffract the light from original normal-incident paths, hence increasing the absorption length inside the active layers.

Microcavity-embedded, colour-tuneable, transparent organic solar cells.

In this work microcavity-capped colour-tuneable SMOSCs are evaluated. By adopting a microcavity-structured cathode with optical spacer layers of different thicknesses fabricated in a Ag/NPB/Ag structure, the transmission spectra of complete devices can be tuned over the entire visible-light region (400-750 nm). The fabricated semitransparent colour-tuneable solar cells show an average efficiency of 4.78% under 1-sun illumination.

Immature CD4⁺ dendritic cells conditioned with donor kidney antigen prolong renal allograft survival in rats.

BACKGROUND: Allogeneic transplant rejection is currently a major problem encountered during organ transplantation. The dendritic cell (DC) is the most effective powerful known professional antigen-presenting cell, and recent studies have found that DCs can also induce immune tolerance, and avoid or reduce the degree of transplant rejection. The aim of this study was to evaluate the effect of transfused immature CD4(+) DCs on renal allografts in the rat model. METHODS: In this study, we induced CD4(+) immature DCs from rat bone marrow cells by a cytokine cocktail. The immature CD4(+) DCs were identified by morphological analysis and then the suppressive activity of these cells conditioned with donor kidney antigen was evaluated in vitro and in vivo. RESULTS: Immature CD4(+) DCs conditioned with donor kidney antigen possessed immunosuppressive activity in vitro and they were able to prolong renal transplant survival in an allograft rat model in vivo. CONCLUSIONS: Our study provides new information on efficacious renal transplantation, which might be useful for understanding the function of immature CD4(+) DCs in modulating renal transplant rejection and improving clinical outcome in future studies.

Vacuum-deposited small-molecule organic solar cells with high power conversion efficiencies by judicious molecular design and device optimization.

Three new tailor-made molecules (DPDCTB, DPDCPB, and DTDCPB) were strategically designed and convergently synthesized as donor materials for small-molecule organic solar cells. These compounds possess a donor-acceptor-acceptor molecular architecture, in which various electron-donating moieties are connected to an electron-withdrawing dicyanovinylene moiety through another electron-accepting 2,1,3-benzothiadiazole block. The molecular structures and crystal packings of DTDCPB and the previously reported DTDCTB were characterized by single-crystal X-ray crystallography. Photophysical and electrochemical properties as well as energy levels of this series of donor molecules were thoroughly investigated, affording clear structure-property relationships. By delicate manipulation of the trade-off between the photovoltage and the photocurrent via molecular structure engineering together with device optimizations, which included fine-tuning the layer thicknesses and the donor:acceptor blended ratio in the bulk heterojunction layer, vacuum-deposited hybrid planar-mixed heterojunction devices utilizing DTDCPB as the donor and C(70) as the acceptor showed the best performance with a power conversion efficiency (PCE) of 6.6 ± 0.2% (the highest PCE of 6.8%), along with an open-circuit voltage (V(oc)) of 0.93 ± 0.02 V, a short-circuit current density (J(sc)) of 13.48 ± 0.27 mA/cm(2), and a fill factor (FF) of 0.53 ± 0.02, under 1 sun (100 mW/cm(2)) AM 1.5G simulated solar illumination.

Origins of device performance in dicarboxyterpyridine Ru(II) dye-sensitized solar cells.

This study carefully examines carrier dynamics in highly efficient dye-sensitized solar cells (DSSCs) based on a series of recently developed dicarboxyterpyridine Ru(II) dyes (PRT-11-15). Comprehensive spectral response analyses, transient photovoltage and photocurrent measurements, and transient absorption techniques are exploited to investigate the effects of various functionalized terpyridines on carrier injection efficiency (η(inj)), electron diffusion length (L) and dye-regeneration efficiency (η(reg)). The resulting parameters are fully comprehended, which are then correlated with the origins of the device performance such as short circuit current density J(sc), open circuit voltage V(oc) and hence the overall conversion efficiency η in these Ru(II) based DSSCs.

A donor-acceptor-acceptor molecule for vacuum-processed organic solar cells with a power conversion efficiency of 6.4%.

A D-A-A-type molecular donor (DTDCTP) featuring electron-accepting pyrimidine and dicyanovinylene blocks has been synthesized for vacuum-deposited planar-mixed heterojunction solar cells with C(70) as the acceptor, giving a power conversion efficiency as high as 6.4%.