Biotechnology for Biofuels
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2021
·Vol. 14
·期 1
·1-13
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IF 6.04
Zhao Yu,Liu Shiqi,Lu Zhihui,Zhao Baixiang,Wang Shuhui,Zhang Cuiying,Xiao Dongguang,Foo Jee Loon,Yu Aiqun
Background In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.
研究领域:
微生物学
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生物技术
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代谢工程
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合成生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2022
·Vol. 23
·期 12
·6588
·
IF 6.208
Jiafeng Niu,Ruxue Yan,Juan Shen,Xiaoyu Zhu,Fanqiang Meng,Zhaoxin Lu,Fengxia Lu
Type I L-asparaginase fromBacillus licheniformisZ-1 (BlAase) was efficiently produced and secreted inBacillus subtilisRIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis inB. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.Keywords:L-asparaginase;Bacillus subtilis;promoter;dual-promoter systems;ribosome binding site;scale-up fermentation;acrylamide
研究领域:
生物技术
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分子生物学
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食品科学
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微生物学
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PubMed
高影响因子
Journal of Fungi
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2022
·Vol. 8
·期 2
·119
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IF 5.724
Larissa Balabanova,Aleksandra Seitkalieva,Yulia Yugay,Tatiana Rusapetova,Lubov Slepchenko,Anna Podvolotskaya,Margarita Yatsunskaya,Elena Vasyutkina,Oksana Son,Liudmila Tekutyeva,Yury Shkryl
An efficientAgrobacterium-mediated genetic transformation based on the plant binary vector pPZP-RCS2 was carried out for the multiple heterologous protein production in filamentous fungusThermothelomycesthermophilusF-859 (formerlyMyceliophthorathermophilaF-859). The engineered fungusTh.thermophiluswas able to produce plant storage proteins ofZeamays(α-zein Z19) andAmaranthushypochondriacus(albumin A1) to enrich fungal biomass by valuable nutritional proteins and improved amino acid content. The mRNA levels ofz19anda1genes were significantly dependent on their driving promoters: the promoter of tryptophan synthase (PtrpC) was more efficient to expressa1, while the promoter of translation elongation factor (Ptef) provided much higher levels ofz19transcript abundance. In general, the total recombinant proteins and amino acid contents were higher in the Ptef-containing clones. This work describes a new strategy to improve mycoprotein nutritive value by overexpression of plant storage proteins.Keywords:filamentous fungi;Myceliophthorathermophila;mycoprotein;recombinant protein;maize alpha-zein;amaranth albumin A1
研究领域:
微生物学
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分子生物学
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生物技术
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食品科学
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PubMed
高影响因子
BIOCHEMICAL PHARMACOLOGY
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2025
·
IF 5.6
Dapeng Lu,Lin Ma,Qingyue Zhang,Chengzhong Xing,Yanke Li
5-Fluorouracil (5-FU) is an effective drug in the treatment of colorectal cancer (CRC); however, the development of acquired resistance to 5-FU poses a great challenge in clinical practice. Through the analysis of public datasets, we found that reduced 3-Hydroxybutyrate Dehydrogenase 2 (BDH2) expression in CRC tissues predicted poor survival in CRC patients. Low expression of BDH2 in clinical CRC tissue samples predicted a higher degree of malignancy. In addition, public dataset analysis showed that GLI Pathogenesis Related 1 (GLIPR1) expression was elevated in CRC patients following 5-FU treatment, contrasting with the expression pattern of BDH2. We reported that in 5-FU-resistant CRC cell lines (LOVO/5-FU and HCT15/5-FU), BDH2 was downregulated, while the expression of GLIPR1 was increased. To explore the regulatory relationship between BDH2 and GLIPR1 in 5-FU resistance, BDH2 and GLIPR1 overexpression plasmid vectors were constructed to transfect 5-FU resistant CRC cell lines. BDH2 led to enhanced 5-FU sensitivity in 5-FU-resistant CRC cell lines and inhibited the malignant behavior of CRC-resistant cells in vitro . In nude mice with subcutaneous tumors and intraperitoneal injections of 5-FU, tumor tissues formed by BDH2-overexpressing cells exhibited slower growth and increased apoptosis. Mechanistically, the upregulation of BDH2 increases GLIPR1 promoter methylation, mediated by DNA methyltransferases, thereby inhibiting GLIPR1 expression. High expression of GLIPR1 reduces 5-FU sensitivity in 5-FU-resistant CRC cell lines, which abolished the impact of BDH2 expression. These results suggest that BDH2 inhibits GLIPR1 expression by increasing GLIPR1 promoter methylation, thereby enhancing 5-FU sensitivity of 5-FU-resistant CRC cell lines and inhibiting CRC progression.
研究领域:
肿瘤学
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分子生物学
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药理学
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表观遗传学
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PubMed
高影响因子
DALTON TRANSACTIONS
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2025
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IF 3.3
Yulu Ru,Jiahui Chen,Qinxin Li,Qiyao Chen,Sihui Huang,Jiahuan Zhou,Dandan Wang,Xiaoshun Zhou,Yong Shao
Folding of nucleic acids is strongly dependent on metal ions. For example, G-quadruplex (G4) structures are usually determined by the specific K+/Na+ coordination. However, G4 constructs highly selective to high-valent metal ions (for instance, trivalent metal ions) have not been found due to their strong and nonspecific phosphate backbone interactions. Herein, a higher-order G4 structure having a heptad junction formed by GGA repeats (for example, GGA8 with eight repeats) of the human c-myb promoter region was found to stoichiometrically recruit three Tb3+ ions. The junction binding of Tb3+ did not affect the global G4 structure of GGA8 because of its high stability in K+. The three context A/A contacts with a proper stacking orientation and an appropriate interbase distance were proved to be the specific binding sites of Tb3+ (A/Tb3+/A). Other high-valent metal ions were unable to interfere with this specific Tb3+ binding, suggestive of the potential of GGA8 serving as an aptamer-like DNA for Tb3+. This A/Tb3+/A binding was also found in another KRAS promoter G4 structure with the same A/A contact, indicating the potential of Tb3+ in selectively probing G4 structures. This work will also inspire more interest in searching higher-order G4 structures for selectively coordinating high-valent metal ions with the purpose of their detection, enrichment, and recovery.
研究领域:
生物化学
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分子生物学
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无机化学
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结构生物学
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核酸研究
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高影响因子
PLoS One
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2016
·Vol. 11
·期 10
·e0163340
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IF 3.057
Hong Zhou,Yapei Wang,Qiongying Lv,Juan Zhang,Qing Wang,Fei Gao,Haoli Hou,Hao Zhang,Wei Zhang,Lijia Li
The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer.
研究领域:
分子生物学
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肿瘤学
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表观遗传学
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DOI
PubMed
高影响因子
JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY
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2011
·Vol. 87
·期 4
·553-558
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IF 1.818
Xiao-Feng Wang,Xu-Guang Shen,Yong-Chuan Sun,He-Yun Zhao,Li Xu,Yun Liu,Yun-Jun Yan
BACKGROUND: Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter ( pAOX1 ), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter ( pFLD1 ) is especially attractive, since little is known about its application in methanol-free culture strategies. RESULTS: Three fed-batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1 -based system. When methanol was used as the fed-batch feeding substrate, the maximum YlLIP2 activity obtained in a 10-L bioreactor was 30 000 U mL −1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL −1 by adopting a co-induction strategy with methanol and methylamine as a mixed fed-batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL −1 after 80 h of cultivation in a methanol-free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1 -based system were comparable with those in a pAOX1 -based system. The results suggest that pFLD1 is an attractive alternative to pAOX1 , and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry
研究领域:
生物技术
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分子生物学
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高影响因子
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
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2021
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IF 4.556
Yue-Hong Pang,Yi-Ying Wang,Meng-Meng Sun,Xiao-Fang Shen
The cauliflower mosaicvirus 35S (CaMV35S), as the most common promoter, has a usage rate of 70 % in transgenic crops, especially in dicots. Herein, a visual detection method of rolling circle amplification (RCA) combined with DNAzyme technology was developed for the determination of CaMV35S gene. A template sequence of the G-quadruple gene was designed for combining with the target gene CaMV35S. When the target gene existed, the RCA reaction occurred through the action of the enzyme, and the product combined with hemin to generate DNAzyme, which catalyzed the color reaction of 2′-hydrazine-bis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS). Under the optimal experimental conditions, the linear range is 1 × 10 −13 ∼1 × 10 -8 mol/L, and the detection limit is 3 × 10 -14 mol/L (S/N = 3). The method has been successfully applied to standard samples and practical samples (soybean and tofu). For verifying the feasibility and reliability, real-time fluorescent quantitative PCR method was used to detect the target gene CaMV35S, and the results were in good agreement with those of our method, indicating that our developed method can be used for the determination of actual samples.
研究领域:
分子生物学
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基因工程
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农业科学
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高影响因子
JOURNAL OF BIOTECHNOLOGY
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2018
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IF 2.533
Ming Zhao,Shui-Ling Wang,Xin-Yi Tao,Guo-Ling Zhao,Yu-Hong Ren,Feng-Qing Wang,Dong-Zhi Wei
Due to the lack of powerful gene regulation elements, the engineering development of Streptomyces is often limited. Here, we disclosed that the heterologous σ 70 -dependent promoters, which have been reported as inefficient tools for gene expression in Streptomyces , could be efficiently recognized by Streptomyces housekeeping factor σ hrdB . Therefore, an effective strategy was developed to engineer these promoters for robust gene expression in Streptomyces by fusing them with optimized 5′-untranslation regions (5′-UTRs). As a proof of concept, the widely used P tac in E. coli was engineered by fusing its core promoter region with the 5′-UTR R15 from a relatively powerful Streptomyces promoter P kasO * R15 and resulted in P tac* , the activity of which was 8.1-fold that of P tac and 1.7-fold that of P kasO * R15 in S. lividans TK24. Next, the 5′-UTR R15 was optimized by randomizing the ribosome binding site (RBS). Based on the base biases of those RBSs with higher activity, eight artificial RBSs were rationally designed, and the optimal resulting promoter P tac * RBS3 showed about 2.1, 3.6, and 17.6 times the activity of P tac *, P kasO * R15 , and P tac , respectively, demonstrating that the heterologous P tac was converted into a type of robust Streptomyces promoters. This study thus greatly expands promoter diversity for the engineering of Streptomyces .
研究领域:
合成生物学
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分子遗传学
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DOI
PubMed
高影响因子
TALANTA
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2025
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IF 6.1
Tiantian Chen,Qian Xiang,Xuemei Sang,Daxiu Li,Wenjiao zhou,Fengfeng Xu
Malaria remains a significant threat to global health, especially with the emergence of drug-resistant strains. Therefore, precise monitoring of antimalarial drug levels is crucial for assessing and managing drug resistance. Here, we report a multiplex aptasensor that enables simultaneous fluorescence detection of two antimalarial drugs (MQ and PQ) via split dsT7 promoter-mediated transcription of two-color light-up RNA aptamers. The detection mechanism relies on specific binding of target antimalarial drugs (MQ/PQ) to their aptamers, each triggering formation of intact dsT7 promoter to induce amplified transcription of its cognate RNA aptamer. The generated RNA aptamers bind their corresponding organic dyes (TO1-Biotin and MG) with high affinity, yielding 7.7-/25-fold fluorescence enhancement to enable ultrasensitive detection of MQ and PQ with limits of 0.71 ng/mL and 0.069 ng/mL, respectively. The method also exhibits high selectivity and low background signal. Furthermore, this label-free system achieves multiplexed detection of antimalarial drugs in 10-fold diluted human serum (recovery: 98.67–106.84 % for PQ and 99.89–106.19 % for MQ), demonstrating its real utility. This label-free system is adaptable to other biomarkers through aptamer replacement.
研究领域:
生物化学
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分子生物学
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医学诊断
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PubMed
高影响因子
PLANT CELL AND ENVIRONMENT
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2024
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IF 6
Shihang Sun,Xiujuan Qi,Zhenzhen Zhang,Leiming Sun,Ran Wang,Yukuo Li,Jinyong Chen,Hong Gu,Jinbao Fang,Miaomiao Lin
Proanthocyanidins (PAs) are important metabolites that enhance freezing tolerance of plants. Actinidia arguta , especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes, KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene ( AaLAR1 ) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementation and coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis -element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a–AaMYB5a– AaLAR1 module shape freezing tolerance in A. arguta . The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.
研究领域:
植物生理学
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分子生物学
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遗传学
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PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Tianyuan Bian,Shitao Gao,Xinyu Sun,Songtao Zhou,Yufeng Pei,Jie Song
G-quadruplexes (G4s) are noncanonical DNA structures that regulate gene expression by serving as mechanical barriers. Telomeric DNA and oncogenic promoter regions (e.g., c-KIT and h-RAS) often contain multiple G-quadruplex structures, which may regulate the transcription of adjacent genes through their interactions. These G4 clusters hold significant therapeutic potential as drug targets for cancer treatment. Thus, understanding the interactions within G4 clusters is of considerable importance. However, current bulk measurements struggle to elucidate the dynamic interactions among multiple intrastranded G4s due to the presence of potential interstrand interactions. Herein, we establish a single-molecule platform to investigate the intrastrand interactions between G4s in the c-KIT promoter region. Our results reveal that these G4 units interact to form higher-order structures, with kit* acting as a key regulator that couples and influences the formation and stability of adjacent G4 units. This single-molecule platform can also detect interactions between small-molecule ligands and consecutive G4 clusters. Compared to CX-5461, PDS is a more effective inhibitor of c-KIT. This single-molecule detection method provides robust support for investigating the intrastrand interactions between consecutive G4 structures.
研究领域:
分子生物学
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生物物理学
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药理学
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DOI
PubMed
高影响因子
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