摘要：
Cis -13, 16-docosadienoic acid (DDA) is an omega -6 polyunsaturated fatty acid with great potential for application in medicine and health. Using microbial cell factories for DDA production is considered a viable alternative to extracting DDA from plant seeds. In this study, using Yarrowia lipolytica Po1f ( Delta ku70) as a chassis, firstly, the adaptation of three elongases in Po1f ( Delta ku70) were explored. Secondly, the DDA biosynthetic pathway was redesigned, resulting in a DDA content of 0.046 % of total fatty acids (TFAs). Thirdly, through the " push-pull " strategy, the DDA content increased to 0.078 % of TFAs. By enhancing the supply of acetyl-CoA, the DDA production in the engineered strain YL-7 reached 0.391 % of the TFAs (3.19 mg/L). Through optimizing the fermentation conditions, the DDA titer of YL-7 reached 29.34 mg/L. This research achieves the sustainable biological production of DDA in Y. lipolytica .
期刊：
Current Microbiology,2024年81(1):1-8 ISSN：0343-8651
通讯作者：
Pan, H;Pan, Hu;Tian, Y
作者机构：
[Dai, Yan-na; Pan, H; Zhang, Yi-fan; Pan, Hu; Pu, Ji-feng] Tibet Acad Agr & Anim Husb Sci, Inst Agr Qual Stand & Testing, Lhasa 850032, Peoples R China.;[Tian, Yun; Liu, Hu-hu; Tian, Y; Pan, Hu; Pan, H] Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha 410128, Peoples R China.;[Dai, Yan-na; Pan, H; Zhang, Yi-fan; Pan, Hu; Pu, Ji-feng] Agr & Livestock Prod Engn Technol Res Ctr Tibet Au, Lhasa 850032, Peoples R China.;[Yang, Xiao-feng] Sichuan Acad Agr Sci, Inst Agr Qual Stand & Testing Technol, Chengdu 610066, Peoples R China.
通讯机构：
[Pan, H ] T;[Tian, Y ; Pan, H] H;Tibet Acad Agr & Anim Husb Sci, Inst Agr Qual Stand & Testing, Lhasa 850032, Peoples R China.;Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha 410128, Peoples R China.;Agr & Livestock Prod Engn Technol Res Ctr Tibet Au, Lhasa 850032, Peoples R China.
摘要：
A novel Gram-stain-negative, aerobic, rod-shaped bacterium named T808(T) was isolated from an alpine soil in Qamdo, Tibet, PR China. Strain T808(T) grew at 5-30degree celsius, pH 5.0-9.0 (optimum, 25degree celsius and pH 7.0-8.0) with 0-2% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequences of strain T808(T) showed the highest similarity with Pararhizobium herbae CCBAU83011(T) (98.8%), followed by Pararhizobium polonicum F5.1(T) (98.7%), Pararhizobium giardinii H152(T) (98.5%), Rhizobium gei ZFJT-2( T) (98.4%), and Pararhizobium antarcticum NAQVI59(T) (97.5%). The highest digital DNA-DNA hybridization (dDDH), core-proteome average amino acid identity (cpAAI) and average nucleotide identity (ANI) values between strain T808(T) and related strains were estimated as 28.0%, 92.1% and 84.4%, respectively. Phylogenetic analysis based on 16S rRNA, core-proteome and whole-genome indicated that strain T808(T) belonged to the genus Pararhizobium. The genome size was 6.24 Mbp with genomic DNA G + C content of 60.1%. The major cellular fatty acids were Summed feature 8 (C-18:1 omega 7c or C-18:1 omega 6c), C-16:0 and C-19:0 cyclo omega 8c. The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and unidentified aminophospholipid. The isoprenoid quinone were ubiquinone-10 and ubiquinone-9. Based on phenotypic, phylogenetic, and genotypic data, strain T808(T) is considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium qamdonense sp. nov. is proposed. The type strain is T808(T) (= JCM 36247( T) = CICC 25216( T)). According to phylogenetic coherence based on 16S rRNA, core-proteome and whole-genome, it is also proposed that the type strain Rhizobium gei Shi et al. 2016 should be reclassified as Pararhizobium gei comb. nov., the type strain is ZFJT-2( T) (= CCTCC AB 2013015( T) = KCTC 32301( T) = LMG 27603( T)).
摘要：
Squalene is an important triterpene with a wide range of applications. Given the growing market demand for squalene, the development of microbial cell factories capable of squalene production is considered a sustainable method. This study aimed to investigate the squalene production potential of Yarrowia lipolytica. First, HMG-CoA reductase from Saccharomyces cerevisiae and squalene synthase from Y. lipolytica was co-overexpressed in Y. lipolytica. Second, by enhancing the supply of NADPH in the squalene synthesis pathway, the production of squalene in Y. lipolytica was effectively increased. Furthermore, by constructing an isoprenol utilization pathway and overexpressing YlDGA1, the strain YLSQ9, capable of producing 868.1 mg/L squalene, was obtained. Finally, by optimizing the fermentation conditions, the highest squalene concentration of 1628.2 mg/L (81.0 mg/g DCW) in Y. lipolytica to date was achieved. This study demonstrated the potential for achieving high squalene production using Y. lipolytica.
摘要：
<jats:sec><jats:title>Background and Purpose</jats:title><jats:p>The P2X3 receptor, a trimeric ionotropic purinergic receptor, has emerged as a potential therapeutic target for refractory chronic cough (RCC). Nevertheless, gefapixant/AF‐219, the only marketed P2X3 receptor antagonist, might lead taste disorders by modulating the human P2X2/3 (hP2X2/3) heterotrimer. Hence, in RCC drug development, compounds exhibiting strong affinity for the hP2X3 homotrimer and a weak affinity for the hP2X2/3 heterotrimer hold promise. An example of such a molecule is sivopixant/S‐600918, a clinical Phase II RCC candidate with a reduced incidence of taste disturbance compared to gefapixant. Sivopixant and its analogue, (3‐(4‐([3‐chloro‐4‐isopropoxyphenyl]amino)‐3‐(4‐methylbenzyl)‐2,6‐dioxo‐3,6‐dihydro‐1,3,5‐triazin‐1(2<jats:italic>H</jats:italic>)‐yl)propanoic acid (DDTPA), exhibit both high affinity and high selectivity for hP2X3 homotrimers, compared with hP2X2/3 heterotrimers. The mechanism underlying the druggable site and its high selectivity remains unclear.</jats:p></jats:sec><jats:sec><jats:title>Experimental Approach</jats:title><jats:p>To analyse mechanisms that distinguish this drug candidate from other inhibitors of the P2X3 receptors we used a combination of chimera construction, site covalent occupation, metadynamics, mutagenesis and whole‐cell recording.</jats:p></jats:sec><jats:sec><jats:title>Key Results</jats:title><jats:p>The high affinity and selectivity of sivopixant/DDTPA for hP2X3 receptors was determined by the tri‐symmetric site located close to the upper vestibule. Substitution of only four amino acids inside the upper body domain of hP2X2 with those of hP2X3, enabled the hP2X2/3 heterotrimer to exhibit a similar level of apparent affinity for sivopixant/DDTPA as the hP2X3 homotrimer.</jats:p></jats:sec><jats:sec><jats:title>Conclusion and Implications</jats:title><jats:p>From the receptor‐ligand recognition perspective, we have elucidated the molecular basis of novel RCC clinical candidates' cough‐suppressing properties and reduced side effects, offering a promising approach to the discovery of novel drugs that specifically target P2X3 receptors.</jats:p></jats:sec>
摘要：
As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.
通讯机构：
[Xiangyang Lu; Yun Tian] C;College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, China
关键词：
Cordycepin;Microbial sources;De novo biosynthesis;Metabolic engineering;Synthetic biology
摘要：
Background: Cordycepin is one of the nucleoside compounds with various nutraceutical and health-promoting functions, which makes it widely used in nutraceutical and pharmaceutical fields. Traditionally, cordycepin is mainly extracted from the natural fruiting bodies of Cordyceps spp.. However, the collection of natural Cordyceps willlead to the depletion of wild resources and the decline of biodiversity, as well as damage to the local fragile ecological environment.Scope and approach: This review provides the microbial resources of cordycepin and introduces the relationship between key metabolic pathways, transcription factors, and cordycepin biosynthesis in artificial cultured cordycepin-producing strains. In addition, the methods of strain improvement and genetic engineering to in-crease cordycepin production were reviewed, and the possibility of using synthetic biology to construct cell factories to increase cordycepin production was discussed.Key findings and conclusions: The analysis of the cordycepin biosynthesis pathway in fungi is of great guiding significance for the improvement of cordycepin production capacity. The construction of cell factories using synthetic biology and systematic metabolic engineering can be used as a green and energy-saving alternative method for the industrial production of cordycepin.
期刊：
Archives of Microbiology,2022年204(6):345 ISSN：0302-8933
通讯作者：
Tian, Y.;Yu, Y.-B.
作者机构：
[Zhou, Jie; Tian, Yun; Liu, Hu-Hu; Pan, Hu; He, Guo-Wei; Wang, Chong; Yang, Hui] Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha 410128, Peoples R China.;[Pan, Hu; Yu, Yao-Bin] Tibet Acad Agr & Anim Husb Sci, Inst Agr Prod Qual Stand & Testing Res, Lhasa 850032, Peoples R China.;[Zhou, Zi-Qiong] Tibet Inst Agr & Anim Husb, Sch Food Sci, Nyingchi 860000, Peoples R China.;[Jin, Kai] Agr Technol Extens Serv Ctr Tibet, Lhasa 850032, Peoples R China.
通讯机构：
[Yao-Bin Yu] I;[Yun Tian] C;Institute of Agricultural Product Quality Standard and Testing Research, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa, China<&wdkj&>College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, China
摘要：
<jats:p>Watermelon (<jats:italic>Citrullus lanatus</jats:italic>) is one of the most popular fruit crops. However, Fusarium wilt (FW) is a serious soil-borne disease caused by <jats:italic>Fusarium oxysporum</jats:italic> f. sp. <jats:italic>niveum</jats:italic> (FON) that severely limits the development of the watermelon industry. <jats:italic>Trichoderma</jats:italic> spp. is an important plant anti-pathogen biocontrol agent. The results of our previous study indicated that <jats:italic>Trichoderma asperellum</jats:italic> M45a (<jats:italic>T</jats:italic>. <jats:italic>asperellum</jats:italic> M45a) could control FW by enhancing the relative abundance of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of watermelon. However, there are few studies on its mechanism in the pathogen resistance of watermelon. Therefore, transcriptome sequencing of <jats:italic>T</jats:italic>. <jats:italic>asperellum</jats:italic> M45a-treated watermelon roots combined with metabolome sequencing of the rhizosphere soil was performed with greenhouse pot experiments. The results demonstrated that <jats:italic>T</jats:italic>. <jats:italic>asperellum</jats:italic> M45a could stably colonize roots and significantly increase the resistance-related enzymatic activities (e.g., lignin, cinnamic acid, <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://fanyi.so.com/?src=onebox#peroxidase" xlink:type="simple">peroxidase</jats:ext-link> and <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://fanyi.so.com/?src=onebox#peroxidase" xlink:type="simple">peroxidase</jats:ext-link>) of watermelon. Moreover, the expression of defense-related genes such as MYB and PAL in watermelon roots significantly improved with the inoculation of <jats:italic>T</jats:italic>. <jats:italic>asperellum</jats:italic> M45a. In addition, KEGG pathway analysis showed that a large number of differentially expressed genes were significantly enriched in phenylpropane metabolic pathways, which may be related to lignin and cinnamic acid synthesis, thus further inducing the immune response to resist FON. Furthermore, metabolic analysis indicated that four differential metabolic pathways were enriched in M45a-treated soil, including six upregulated compounds and one down-regulated compound. Among them, galactinol and urea were significantly positively correlated with <jats:italic>Trichoderma</jats:italic>. Hence, this study provides insight into the biocontrol mechanism of <jats:italic>T</jats:italic>. <jats:italic>asperellum</jats:italic> M45a to resist soil-borne diseases, which can guide its industrial application.</jats:p>
