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Metabolic Engineering Jan 2024Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as...
Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.
Topics: Monoterpenes; Corynebacterium glutamicum; Mevalonic Acid; Terpenes; Metabolic Engineering
PubMed: 38056688
DOI: 10.1016/j.ymben.2023.11.009 -
Research Square Nov 2023Muscle wasting is a serious complication in heart failure patients, and oxidative stress is involved in the pathogenesis of muscle wasting. Oxidative stress leads to the...
BACKGROUND
Muscle wasting is a serious complication in heart failure patients, and oxidative stress is involved in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) and acrolein, which causemuscle wasting. In tissues, these toxic aldehydes are metabolically removed by enzymes such asaldo keto reductases and endogenous nucleophiles, such as glutathione and carnosine. Whether these metabolic pathways could be affected in skeletal muscle during heart failure has never been studied.
METHODS
Male wild-type C57BL/6J mice were subjected to a pressure overload model of hypertrophy by transaortic constriction (TAC) surgery, and echocardiography was performed after 14 weeks. Different skeletal muscle beds were weighed and analyzed for atrophic and inflammatory markers, and and , respectively, by RT-PCR. Levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, aldose reductase (AKR1B1) and aldehyde dehydrogenase 2 (ALDH2) were measured by Western blotting, and histidyl dipeptides and histidyl dipeptide aldehyde conjugates were analyzed by LC/MS-MS in the gastrocnemius and soleus muscles of sham- and TAC-operated mice. Furthermore, histidyl dipeptide synthesizing enzyme carnosine synthase (CARNS) and amino acid transporters (PEPT2 and TAUT)wasmeasured in the gastrocnemius muscles of the sham and TAC-operated mice.
RESULTS
TAC-induced heart failure decreases body weight and gastrocnemius and soleus muscle weights. The expression of the atrophic and inflammatory markers and TNF-α, respectively, wasincreased (~1.5-2-fold), and the formation of HNE and acrolein-protein adducts was increased in the gastrocnemius muscle of TAC-operated mice. The expression of AKR1B1 remained unchanged, whereas ALDH2 was decreased, in the gastrocnemius muscle of TAC mice. Similarly, in the atrophic gastrocnemius muscle, levels of total histidyl dipeptides (carnosine and anserine) and, in particular,carnosine were decreased. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, the expression of CARNS and TAUT wasdecreased in the atrophic gastrocnemius muscle.
CONCLUSIONS
Collectively, these results show that metabolic pathways involved in the removal of lipid peroxidation products and synthesis of histidyl dipeptides are diminished in atrophic skeletal muscle during heart failure, which could contribute to muscle atrophy.
PubMed: 38045249
DOI: 10.21203/rs.3.rs-3621159/v1 -
JACS Au Nov 2023Photocatalysis offers tremendous opportunities for enzymes to access new functions. Herein, we described a redox-neutral photocatalysis/enzymatic catalysis system for...
Photocatalysis offers tremendous opportunities for enzymes to access new functions. Herein, we described a redox-neutral photocatalysis/enzymatic catalysis system for the asymmetric synthesis of chiral 1,2-amino alcohols via decarboxylative radical C-C coupling of -arylglycines and aldehydes by combining an organic photocatalyst, eosin Y, and carbonyl reductase RasADH. Notably, this protocol avoids using any sacrificial reductants. A possible reaction mechanism proposed is that the transformation proceeds through sequential photoinduced decarboxylative radical addition to an aldehyde and a photoenzymatic deracemization pathway. This redox-neutral photoredox/enzymatic strategy is promising not only for effective synthesis of a series of chiral amino alcohols in a green and sustainable manner but also for the design of other novel C-C radical coupling transformations for the synthesis of bioactive molecules.
PubMed: 38034963
DOI: 10.1021/jacsau.3c00366 -
Metabolites Nov 2023Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a...
Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.
PubMed: 37999222
DOI: 10.3390/metabo13111126 -
Microbial Cell Factories Nov 2023(Hydroxy)cinnamyl alcohols and allylphenols, including coniferyl alcohol and eugenol, are naturally occurring aromatic compounds widely utilised in pharmaceuticals,...
BACKGROUND
(Hydroxy)cinnamyl alcohols and allylphenols, including coniferyl alcohol and eugenol, are naturally occurring aromatic compounds widely utilised in pharmaceuticals, flavours, and fragrances. Traditionally, the heterologous biosynthesis of (hydroxy)cinnamyl alcohols from (hydroxy)cinnamic acids involved CoA-dependent activation of the substrate. However, a recently explored alternative pathway involving carboxylic acid reductase (CAR) has proven efficient in generating the (hydroxy)cinnamyl aldehyde intermediate without the need for CoA activation. In this study, we investigated the application of the CAR pathway for whole-cell bioconversion of a range of (hydroxy)cinnamic acids into their corresponding (hydroxy)cinnamyl alcohols. Furthermore, we sought to extend the pathway to enable the production of a variety of allylphenols and allylbenzene.
RESULTS
By screening the activity of several heterologously expressed enzymes in crude cell lysates, we identified the combination of Segniliparus rugosus CAR (SrCAR) and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) as the most efficient enzymatic cascade for the two-step reduction of ferulic acid to coniferyl alcohol. To optimise the whole-cell bioconversion in Escherichia coli, we implemented a combinatorial approach to balance the gene expression levels of SrCAR and MsCAD2. This optimisation resulted in a coniferyl alcohol yield of almost 100%. Furthermore, we extended the pathway by incorporating coniferyl alcohol acyltransferase and eugenol synthase, which allowed for the production of eugenol with a titre of up to 1.61 mM (264 mg/L) from 3 mM ferulic acid. This improvement in titre surpasses previous achievements in the field employing a CoA-dependent coniferyl alcohol biosynthesis pathway. Our study not only demonstrated the successful utilisation of the CAR pathway for the biosynthesis of diverse (hydroxy)cinnamyl alcohols, such as p-coumaryl alcohol, caffeyl alcohol, cinnamyl alcohol, and sinapyl alcohol, from their corresponding (hydroxy)cinnamic acid precursors but also extended the pathway to produce allylphenols, including chavicol, hydroxychavicol, and methoxyeugenol. Notably, the microbial production of methoxyeugenol from sinapic acid represents a novel achievement.
CONCLUSION
The combination of SrCAR and MsCAD2 enzymes offers an efficient enzymatic cascade for the production of a wide array of (hydroxy)cinnamyl alcohols and, ultimately, allylphenols from their respective (hydroxy)cinnamic acids. This expands the range of value-added molecules that can be generated using microbial cell factories and creates new possibilities for applications in industries such as pharmaceuticals, flavours, and fragrances. These findings underscore the versatility of the CAR pathway, emphasising its potential in various biotechnological applications.
Topics: Eugenol; Pharmaceutical Preparations
PubMed: 37980525
DOI: 10.1186/s12934-023-02246-4 -
Molecules (Basel, Switzerland) Oct 2023This research aimed to determine the target protein and molecular mechanism of -(±)-kusunokinin ((±)-KU) derivatives (-(±)-ARC and -(±)-TTPG-B). Molecular docking...
This research aimed to determine the target protein and molecular mechanism of -(±)-kusunokinin ((±)-KU) derivatives (-(±)-ARC and -(±)-TTPG-B). Molecular docking was used to predict potential synthesized (±)-KU targets among 22 proteins. The (±)-TTPG-B bound HSP90α better than EC44, native (±)-KU and (-)-KU, and (±)-KU and (-)-ARC. In contrast, (-)-ARC bound PI3K more strongly than any other test compound. CSF1R and AKR1B1 were not supposed to be the target of (±)-TTPG-B and (±)-ARC, unlike native (±)-KU. The (±)-TTPG-B bound Tyr139 and Trp162 of HSP90α. Moreover, (-)-ARC bound PI3K via hydrogen bonds and π-π stacking at distinct amino acids, which was different from the other tested compounds. Using half of the IC concentration, (±)-TTPG-B, (±)-KU and (±)-ARC enhanced cell cycle arrest at the G0/G1 phase after 12 h and 24 h on KKU-M213 (CCA) cells. The (±)-TTPG-B showed a stronger inhibitory effect than (±)-ARC and (±)-KU on HSP90α, PI3K, HSP90β, c-Myc, AKT, MEK1, CyclinB1, CyclinD1, and CDK1 for 24 and 48 h after treatment with the same concentration (0.015 µM). Thus, -(±)-TTPG-B, a newly synthesized compound, has pharmacological potential for development as a target therapy for CCA treatment.
Topics: Humans; Molecular Docking Simulation; Cholangiocarcinoma; Cell Proliferation; Cell Division; Bile Ducts, Intrahepatic; Bile Duct Neoplasms; Phosphatidylinositol 3-Kinases; Cell Line, Tumor; Apoptosis; Cell Cycle; Aldehyde Reductase
PubMed: 37959760
DOI: 10.3390/molecules28217342 -
Nature Communications Nov 2023Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional...
Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.
Topics: Triterpenes; Transcriptome; Saponaria; Vaccaria; Plants; Saponins; Uridine Diphosphate; Glucose; Sugars
PubMed: 37925486
DOI: 10.1038/s41467-023-42877-0 -
Microbial Cell Factories Oct 2023Enantio-pure α-hydroxy amides are valuable intermediates for the synthesis of chiral pharmaceuticals. The asymmetric reduction of α-keto amides to generate chiral...
Enantio-pure α-hydroxy amides are valuable intermediates for the synthesis of chiral pharmaceuticals. The asymmetric reduction of α-keto amides to generate chiral α-hydroxy amides is a difficult and challenging task in biocatalysis. In this study, iolS, an aldo-keto reductase from Bacillus subtilis 168 was exhibited as a potential biocatalyst, which could catalyze the reduction of diaryl α-keto amide such as 2-oxo-N, 2-diphenyl-acetamide (ONDPA) with moderate S-selectivity (76.1%, ee) and 60.5% conversion. Through semi-rational engineering, two stereocomplementary variants (I57F/F126L and N21A/F126A) were obtained with ee value of 97.6% (S) and 99.9% (R) toward ONDPA (1a), respectively, delivering chiral α-hydroxy amide with > 98% conversions. Moreover, the excellent S- and R-preference variants displayed improved stereoselectivities toward the other α-keto amide compounds. Molecular dynamic and docking analysis revealed that the two key residues at 21 and 126 were identified as the "switch", which specifically controlled the stereopreference of iolS by regulating the shape of substrate binding pocket as well as the substrate orientation. Our results offer an effective strategy to obtain α-hydroxy amides with high optical purity and provide structural insights into altering the stereoselectivity of AKRs.
Topics: Aldo-Keto Reductases; Amides; Substrate Specificity; Biocatalysis; Catalysis; Aldehyde Reductase
PubMed: 37840127
DOI: 10.1186/s12934-023-02225-9 -
The Turkish Journal of Gastroenterology... Dec 2023Gastric cancer is a prevalent malignancy with unfavorable prognosis partially resulting from its high metastasis rate. Clarifying the molecular mechanism of gastric...
BACKGROUND/AIMS
Gastric cancer is a prevalent malignancy with unfavorable prognosis partially resulting from its high metastasis rate. Clarifying the molecular mechanism of gastric cancer occurrence and progression for improvement of therapeutic efficacy and prognosis is needed. The study tended to delineate the role and regulatory mechanism of aldo-keto reductase 1B10 (AKR1B10) in gastric cancer progression.
MATERIALS AND METHODS
The relationship of AKR1B10 expression with survival rate in gastric cancer was analyzed through Kaplan-Meier analysis. The mRNA levels of AKR1B10 and integrin subunit alpha 5 (ITGA5) in gastric cancer tissues and cell lines were measured by real-time quantitative polymerase chain reaction. Protein levels of AKR1B10 and integrin subunit alpha 5 were assayed via western blot. The molecular relationship between AKR1B10 and ITGA5 was analyzed by co-immunoprecipitation assay. Cell viability was assayed through Cell Counting Kit-8, invasion and migration of tumor cells was assessed through wound healing and transwell assays. Transwell assay was utilized to detect invasion. The adhesion of gastric cancer cells was detected using cell adhesion assays.
RESULTS
The results unveiled that integrin subunit alpha 5 was upregulated, while AKR1B10 was downregulated in gastric cancer tissues and cells. Overexpressing AKR1B10 hindered gastric cancer cell proliferation, migration, invasion and adhesion. It was striking that we certified the inhibitory effect of AKR1B10 on integrin subunit alpha 5 expression and their (AKR1B10 and ITGA5)) negative relationship via bioinformatics method, real-time quantitative polymerase chain reaction, and co-immunoprecipitation assays. Via rescue experiments, it was concluded that AKR1B10 served as tumor suppressor potentially by ITGA5 expression in gastric cancer.
CONCLUSION
Our results indicated that AKR1B10 inhibited migration, invasion, and adhesion of gastric cancer cells via modulation of ITGA5.
Topics: Humans; Aldehyde Reductase; Aldo-Keto Reductases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Integrins; Stomach Neoplasms
PubMed: 37823316
DOI: 10.5152/tjg.2023.22555 -
Journal of Translational Medicine Oct 2023Nonalcoholic steatohepatitis (NASH) is a progressive and inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatocellular injury,...
BACKGROUND
Nonalcoholic steatohepatitis (NASH) is a progressive and inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatocellular injury, inflammation, and fibrosis in various stages. More than 20% of patients with NASH will progress to cirrhosis. Currently, there is a lack of clinically effective drugs for treating NASH, as improving liver histology in NASH is difficult to achieve and maintain through weight loss alone. Hence, the present study aimed to investigate potential therapeutic drugs for NASH.
METHODS
BMDMs and THP1 cells were used to construct an inflammasome activation model, and then we evaluated the effect of epalrestat on the NLRP3 inflammasome activation. Western blot, real-time qPCR, flow cytometry, and ELISA were used to evaluate the mechanism of epalrestat on NLRP3 inflammasome activation. Next, MCD-induced NASH models were used to evaluate the therapeutic effects of epalrestat in vivo. In addition, to evaluate the safety of epalrestat in vivo, mice were gavaged with epalrestat daily for 14 days.
RESULTS
Epalrestat, a clinically effective and safe drug, inhibits NLRP3 inflammasome activation by acting upstream of caspase-1 and inducing ASC oligomerization. Importantly, epalrestat exerts its inhibitory effect on NLRP3 inflammasome activation by inhibiting the activation of aldose reductase. Further investigation revealed that the administration of epalrestat inhibited NLRP3 inflammasome activation in vivo, alleviating liver inflammation and improving NASH pathology.
CONCLUSIONS
Our study indicated that epalrestat, an aldose reductase inhibitor, effectively suppressed NLRP3 inflammasome activation in vivo and in vitro and might be a new therapeutic approach for NASH.
Topics: Humans; Mice; Animals; Non-alcoholic Fatty Liver Disease; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Aldehyde Reductase; Inflammation; Fibrosis; Mice, Inbred C57BL
PubMed: 37805545
DOI: 10.1186/s12967-023-04380-4