共检索到124条,权限内显示50条;
[学术文献 ] Versatile filamentous fungal host highly-producing heterologous natural products developed by genome editing-mediated engineering of multiple metabolic pathways 进入全文
COMMUNICATIONS BIOLOGY
Natural secondary metabolites are medically, agriculturally, and industrially beneficial to humans. For mass production, a heterologous production system is required, and various metabolic engineering trials have been reported in Escherichia coli and Saccharomyces cerevisiae to increase their production levels. Recently, filamentous fungi, especially Aspergillus oryzae, have been expected to be excellent hosts for the heterologous production of natural products; however, large-scale metabolic engineering has hardly been reported. Here, we elucidated candidate metabolic pathways to be modified for increased model terpene production by RNA-seq and metabolome analyses in A. oryzae and selected pathways such as ethanol fermentation, cytosolic acetyl-CoA production from citrate, and the mevalonate pathway. We performed metabolic modifications targeting these pathways using CRISPR/Cas9 genome editing and demonstrated their effectiveness in heterologous terpene production. Finally, a strain containing 13 metabolic modifications was generated, which showed enhanced heterologous production of pleuromutilin (8.5-fold), aphidicolin (65.6-fold), and ophiobolin C (28.5-fold) compared to the unmodified A. oryzae strain. Therefore, the strain generated by engineering multiple metabolic pathways can be employed as a versatile highly-producing host for a wide variety of terpenes.
[学术文献 ] Increased artemisinin production in Artemisia annua L. by co-overexpression of six key biosynthetic enzymes 进入全文
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua, is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes-Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)-in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to nontransgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC-MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment.
[学术文献 ] Engineering lipase TLL to improve its acid tolerance and its biosynthesis in Trichoderma reesei for biodiesel production from acidified oil 进入全文
Bioresource Technology
Lipases catalyze the synthesis of biodiesel, which is an important renewable alternative energy source. Cost-efficient conversion of waste acidified oil to biodiesel entails acid-tolerant lipases which have not been extensively studied. This study showed that the commonly used Thermomyces lanuginosus lipase TLL displayed a weak acid tolerance and an unsatisfactory performance in biodiesel production from acidified oil. Directed evolution of TLL identified one TLL-T3 variant with three residue substitutions (A69S/V150P/N222G). TLL-T3 displayed significantly enhanced acid tolerance, and its application in acidified oil treatment led to a biodiesel yield up to 90 % (w/w). A scaled-up production of TLL-T3 in Trichoderma reesei was further achieved and the highest extracellular lipase activity reached 16,123 U/mL after fermentation optimization. These results provide new insights into structural adaptation to acid tolerance by lipases and show that TLL-T3 holds great potential in commercial biodiesel production from waste acidified oil.
[学术文献 ] Homologous expression, purification, and characterization of a recombinant acetylxylan esterase from Aspergillus nidulans 进入全文
International Journal of Biological Macromolecules
Acetylxylan esterases (AXEs) are essential enzymes that break down the acetyl groups in acetylated xylan found in plant cell walls polysaccharides. They work synergistically with backbone-depolymerizing xylanolytic enzymes to accelerate the degradation of complex polysaccharides. In this study, we cloned the gene axeA, which encodes the acetylxylan esterase from Aspergillus nidulans FGSC A4 (AxeAN), into the pEXPYR expression vector and introduced it into the high protein-producing strain A. nidulans A773. The purified AxeAN, with a molecular weight of 33.5 kDa as confirmed by SDS-PAGE, was found to be active on ρ-nitrophenyl acetate (ρNPA), exhibiting a remarkably high specific activity (170 U mg−1) at pH 7.0 and 55 °C. AxeAN demonstrated stability over a wide pH range (5.5–9.0), retaining >80% of its initial activity after 24 h. The KM and Vmax were 0.098 mmol L−1 and 320 U mg−1, respectively, using ρNPA as a substrate. We also evaluated the synergistic effect of AxeAN with an endo-1,4-β-xylanase from Malbranchea pulchella (MpXyn10) in the hydrolysis of four different xylans (Birchwood, Beechwood, Oat spelt, and Arabinoxylan) to produce xylooligosaccharides (XOS). The best results were obtained using Birchwood xylan as substrate and MpXyn10-AxeAN as biocatalysts after 24 h of reaction (50 °C), with a XOS-yield of 91%, value 41% higher when compared to MpXyn10 (XOS-yield of 63%). These findings showed the potential of the application of AxeAN, together with other xylanases, to produce xylooligosaccharides with high purity and other products with high added value in the field of lignocellulosic biorefinery.
[学术文献 ] Expanding the genome editing toolbox with designer CRISPR–Cas-like transposons 进入全文
NATURE METHODS
Similarly to CRISPR-Cas systems, TnpB proteins from bacterial transposons can be employed as RNA-guided endonucleases for genome editing. By combining rational protein design and machine learning, ISDra2 TnpB variants with enhanced editing efficiency and a broader targeting range were developed, along with a prediction tool to design effective guiding RNAs.
[学术文献 ] New application of a dye-decolorizing peroxidase immobilized on magnetic nanoparticles for efficient simultaneous degradation of two mycotoxins 进入全文
Food Chemistry
Nowadays the enzymatic approaches are the most promising strategies for mycotoxins detoxification in food stuffs. Herein, the dye-decolorizing peroxidase RhDypB from Rhodococcus jostii was studied for its ability to degrade two mycotoxins in both free and the immobilized enzyme forms. This enzyme was recombinantly expressed and purified, while Fe3O4 nanoparticles were prepared and modified with chitosan as the immobilization carrier. The immobilized enzyme Fe3O4@CS@RhDypB demonstrated degradation rate of 85.61 % toward aflatoxin B1, while it was firstly found to be able to degrade zearalenone with the rate of 86.52 %, at pH 4.0 on 30 °C. The degradation products were identified as aflatoxin Q1 and 15-OH-ZEN respectively. After 5 cycles of reuse, Fe3O4@CS@RhDypB still exhibited degradation rates of 38.50 % and 49.76 % toward the mycotoxins, indicating its high reusability. Moreover, Fe3O4@CS@RhDypB exhibited excellent stability after 10 days of storage. This work identified potential applications of nanoparticle-immobilized enzyme for biodegradation of mycotoxins in food industry.
