Fundamental Research
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2024
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IF 5.7
Xuewen Wang,Song Hong,Guirong Tang,Chengshu Wang
Filamentous fungi frequently degenerate during subculturing, which manifests as the reduction or loss of conidiation, sexuality, secondary metabolite production, and/or virulence against hosts. The underlying mechanism of spontaneous fungal degeneration is still elusive. In this study, the fluffy mycelium-type sector variants formed by three ascomycete fungi were transferred and found to show the typical features of culture degeneration. The variant cells were evidenced with the accumulation of reactive oxygen species (ROS), and the ROS-associated formation of hyphal coils. Genome resequencing of these sector cultures identified substantial random mutation sites in each variant in a trend associated with fungal reproduction style. The high bias towards transversions over transitions was similarly detected in degenerate genomes. Otherwise, a higher number of mutations were accumulated in the intergenic regions of the Metarhizium robertsii and Cordyceps militaris sector genomes, whereas the exonic regions of the Aspergillus nidulans variant genes were detected with a higher mutation rate. Unexpectedly, none of those mutated genes had orthologous relationships among the three sectors, while only a few of them were shared between two fungi. A few transcription factor genes with frameshift mutations in sectors were selected for deletions in parental strains, and the null mutants demonstrated the varied degrees of degenerate phenotypes. In addition to reasoning the causal mechanism of fungal degeneration, our data provide insights to better maintain and monitor fungal culture stability.
研究领域:
微生物学
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遗传学
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分子生物学
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DOI
高影响因子
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
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2024
·Vol. 34
·期 9
·1912
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IF 2.5
Sang-Hun Do,Tae-Gi Lee,Sun-Ki Kim
Single-cell protein (SCP) derived from microorganisms is widely recognized as a viable alternative protein source for the future. Nevertheless, the commercialization of yeast-based SCP is hampered by its relatively low protein content. Therefore, this study aimed to enhance the protein content of Saccharomyces cerevisiae via random mutagenesis. To achieve this, S. cerevisiae KCCM 51811, which exhibited the highest protein concentration among 20 edible S. cerevisiae strains, was selected as a chassis strain. Subsequently, a KCCM 51811 mutant library was constructed (through UV irradiation) and screened to isolate mutants exhibiting high protein content and/or concentration. Among the 174 mutant strains studied, the #126 mutant exhibited a remarkable 43% and 36% higher protein content and concentration, respectively, compared to the parental strain. Finally, the #126 mutant was cultured in a fed-batch system using molasses and corn-steep liquor, resulting in a protein concentration of 21.6 g/l in 100 h, which was 18% higher than that produced by the parental strain. These findings underscore the potential of our approach for the cost-effective production of food-grade SCP.
研究领域:
生物技术
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食品科学
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2025
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IF 3.8
Daoyang Zhu,Qiuya Gu,Xiaobin Yu
Evolutionary engineering requires the development of high-throughput screening techniques to identify and isolate target products or phenotypes of interest from extensive libraries. This study aimed to establish a high-throughput screening strategy for high-yielding single-cell strains of pyrroloquinoline quinone (PQQ) and combined it with random mutagenesis approaches to enhance PQQ accumulation. Initially, the fluorescence properties of PQQ were analyzed for the first time, leading to the development of a fluorescence-based detection method for PQQ and a fluorescence-activated cell sorting-based screening approach for high-yield strains of PQQ. Secondly, the library constructed via random mutagenesis confirmed the feasibility of the screening methodology, resulting in the identification of a mutant exhibiting a 1.9-fold enhancement in PQQ titer. Ultimately, omics tools found multiple candidate genes for improving PQQ production. In summary, the high-throughput screening model established provides practical guidance for the screening of other natural products.
研究领域:
微生物学
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合成生物学
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代谢工程
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产品关联
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2015
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IF 2.871
Yue-De Cao,Yu-Cai He,Hao Li,Guo-Yin Kai,Jian-He Xu,Hui-Lei Yu
Hyoscyamine 6 β -hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (−)-hyoscyamine and the subsequent epoxidation of 6 β -hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus ( Aa H6H). A double mutant, Aa H6H M1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34 h cultivation of Escherichia coli cells harboring Aa h6h m1 in a 5-L bioreactor with a working volume of 3 L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500 mg L −1 (−)-hyoscyamine in 97% conversion, and 1.068 g of the product were isolated, corresponding to a space-time yield of 251 mg L −1 d −1 . This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.
研究领域:
生物化学
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生物技术
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微生物学
·
酶学
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产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
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2020
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IF 4.872
Jiarong Qiu,Haiyan Yang,Yuting Shao,Longxiang Li,Shengwei Sun,Luyao Wang,Yuzhi Tan,Zhihong Xin
Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.
研究领域:
生物化学
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酶工程
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生物技术
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环境科学
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DOI
PubMed
高影响因子
STAR Protocols
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2024
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IF 1.3
Shihao Song,Binbin Cui,Xiaohan Kong,Yinyue Deng
Summary Here, we present a protocol for identifying the components of a quorum sensing signaling system in bacteria. We describe the steps for characterizing the novel response regulator and receptor of the cis -2-dodecenoic acid (BDSF) quorum sensing signaling system in Burkholderia cenocepacia . The technical assays of this protocol include generating a random mutant library, chromatin immunoprecipitation sequencing (ChIP-seq), electrophoretic mobility transfer assay (EMSA), microscale thermophoresis (MST), and molecular simulation docking. For complete details on the use and execution of this protocol, please refer to Li et al. 1 and Yang et al. 2
研究领域:
微生物学
·
分子生物学
·
遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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