通讯机构：
[Liu, GQ ; Ma, JS] C;Cent South Univ Forestry & Technol, Int Cooperat Base Sci & Technol Innovat Forest Res, Changsha, Peoples R China.;Cent South Univ Forestry & Technol, Hunan Prov Key Lab Forestry Biotechnol, Changsha, Peoples R China.;Yuelushan Natl Lab Seed Ind, Econ Forest Variety Creat Ctr, Changsha, Peoples R China.
关键词：
glycerol dehydratase;expression regulation mechanism;H-NS;Klebsiella pneumoniae;oligomerization state
摘要：
Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.
作者机构：
[郝宏伟; 陈意琪; 何姣; 李娜; 原成林; 刘勇男; 刘高强] Hunan Provincial Key Laboratory of Forestry Biotechnology, International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Microbial Variety Creation Center, Yuelushan Laboratory of Seed Industry, Hunan, Changsha, 410004, China
期刊：
Methods in Molecular Biology,2023年2594:13-28 ISSN：1064-3745
作者机构：
[Wu, Qiang] College of Food and Chemical Engineering, Shaoyang University, Shaoyang, China. qiangwusmile@163.com;[Li, Qiang; Ma, Jiang-Shan; Liu, Gao-Qiang; Wu, Qiang] Hunan Provincial Key Laboratory of Forestry Biotechnology &[Wu, Qiang] International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of Forestry and Technology, Changsha, China. qiangwusmile@163.com;[Wu, Qiang] Microbial Variety Creation Center, National Laboratory of Yuelushan Seed Industry, Changsha, China. qiangwusmile@163.com;[Li, Qiang; Ma, Jiang-Shan; Liu, Gao-Qiang] International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of Forestry and Technology, Changsha, China
关键词：
DNA pull-down;EMSA;Gene knockout;Transcription factor
摘要：
This protocol describes a method for verifying the specific transcription factor regulating glycerol dehydratase (GDH) expression in Klebsiella. DNA pull-down accompanied with mass spectrometry is used to screen and identify the transcription factor interacting with the promoter region of the key gene in Klebsiella. EMSA method is used to validate the specific binding of the transcription factor to the promoter region in vitro. In addition, the target DNA fragments are constructed by fusion PCR to prepare competent cells from Klebsiella for electrical transformation and further transformed to obtain key gene deletion strains to verify the transcription factor responsible for the target gene expression in Klebsiella.
通讯机构：
[Gao-Qiang Liu] H;Hunan Provincial Key Laboratory of Forestry Biotechnology, Central South University of Forestry & Technology, Changsha, China<&wdkj&>International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Changsha, China<&wdkj&>Microbial Variety Creation Center, Yuelushan Laboratory of Seed Industry, Changsha, China
摘要：
Ganoderic acids (GAs) are well recognized as important pharmacological components of the medicinal species belonging to the basidiomycete genus Ganoderma. However, transcription factors directly regulating the expression of GA biosynthesis genes remain poorly understood. Here, the genome of Ganoderma lingzhi is de novo sequenced. Using DNA affinity purification sequencing, we identify putative targets of the transcription factor sterol regulatory element-binding protein (SREBP), including the genes of triterpenoid synthesis and lipid metabolism. Interactions between SREBP and the targets are verified by electrophoretic mobility gel shift assay. RNA-seq shows that SREBP targets, mevalonate kinase and 3-hydroxy-3-methylglutaryl coenzyme A synthetase in mevalonate pathway, sterol isomerase and lanosterol 14-demethylase in ergosterol biosynthesis, are significantly upregulated in the SREBP overexpression (OE::SREBP) strain. In addition, 3 targets involved in glycerophospholipid/glycerolipid metabolism are upregulated. Then, the contents of mevalonic acid, lanosterol, ergosterol and 13 different GAs as well as a variety of lipids are significantly increased in this strain. Furthermore, the effects of SREBP overexpression on triterpenoid and lipid metabolisms are recovered when OE::SREBP strain are treated with exogenous fatostatin, a specific inhibitor of SREBP. Taken together, our genome-wide study clarify the role of SREBP in triterpenoid and lipid metabolisms of G. lingzhi. An integration of genomic, DAP-seq, RNA-seq, metabolomic and genetic approaches identifies SREBP as a key transcription factor regulating triterpenoid and lipid metabolisms in the medicinal fungus Ganoderma lingzhi.
摘要：
<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Background</jats:title>
<jats:p>The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains.</jats:p>
</jats:sec><jats:sec>
<jats:title>Results</jats:title>
<jats:p>In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from <jats:italic>Klebsiella pneumoniae</jats:italic> 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with <jats:italic>K</jats:italic><jats:sub><jats:italic>m</jats:italic></jats:sub> and <jats:italic>V</jats:italic><jats:sub><jats:italic>m</jats:italic></jats:sub> values of 3.42mM and 58.15 nkat mg<jats:sup>−1</jats:sup>, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37°C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe<jats:sup>2+</jats:sup> and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility.</jats:p>
</jats:sec><jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.</jats:p>
</jats:sec>
通讯机构：
[Yong-Nan Liu; Gao-Qiang Liu] A;Authors to whom correspondence should be addressed.<&wdkj&>International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Changsha 410004, China<&wdkj&>Microbial Variety Creation Center, Yuelushan Laboratory of Seed Industry, Changsha 410004, China<&wdkj&>Hunan Provincial Key Laboratory of Forestry Biotechnology, Central South University of Forestry & Technology, Changsha 410004, China
作者机构：
[张婧; 王思贤; 薛菲菲; 王晓玲; 张家顺; 刘高强] Hunan Provincial Key Laboratory of Forestry Biotechnology, International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Hunan, Changsha, 410004, China
通讯机构：
[Chen, Xiulai] J;Jiangnan Univ, State Key Lab Food Sci & Technol, Wuxi 214122, Jiangsu, Peoples R China.;Jiangnan Univ, Key Lab Ind Biotechnol, Minist Educ, Wuxi 214122, Jiangsu, Peoples R China.
摘要：
Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 mu m(-1) and acetoin titer to 67.2g.L-1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 mu m(3) and poly(lactate-co-3-hydroxybutyrate) titer to 14.31g.L-1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories. Manipulation of genes controlling microbial shapes can affect bio-production. Here, the authors employ an optogenetic method to realize dynamic morphological engineering of E. coli replication and division and show the increased production of acetoin and poly(lactate-co-3-hydroxybutyrate).
作者机构：
[刘高强] Hunan Provincial Key Laboratory of Forestry Biotechnology, Central South University of Forestry & Technology, International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province, Central South University of Forestry & Technology, Hunan, Changsha, 410004, China
摘要：
Broken-rice starch nanoparticles with different mean particle diameters for 100, 200, 400 and 800 nm were prepared by nanopredpitation, alkali freezing, cross-linking and H2SO4 hydrolysis methods respectively, and their structural, morphological and physicochemical properties were systematically characterized. The results showed that broken-rice starch nanoparticles had higher water absorption rate, and the maximum water absorption rate was obtained from the 100 nm starch granules being 91.53%, which means an increase about 2.07-fold in water absorption rate as compared with native rice starch. The stability of native rice starch is the worst, but the viscosity characteristic value is always higher than that of starch nanoparticles in the whole gelatinization process. The FT-IR spectrum showed that only starch nanoparticles prepared by cross-linking method showed the characteristic peak of secondary amide structure at 1714 cm(-1), but the structure of was basically the same as native starch. The X-ray diffraction pattern revealed that there were obvious characteristic diffraction peaks near 20 for 15 degrees, 1 7 degrees 19 degrees and 23 degrees for the 800 nm starch nanoparticles and native rice starch, while the characteristic diffraction peaks of other starch nanoparticles disappeared in varying degrees due to the changed crystal structure. (C) 2020 Elsevier B.V. All rights reserved.
摘要：
Enzyme production as well as rice straw saccharification and fermentation were integrated to produce bioethanol in this study. Submerged fermentation of Na2CO3 pretreated rice straw to produce a complex enzyme for saccharification by Aspergillus fumigatus was performed. The major component of rice straw, that is cellulose, hemicellulose and lignin was almost completely degraded in 24 h. Using whole pretreated rice straw slurry as the substrate, the maximum concentrations of reducing sugar and ethanol were obtained with values of 63.6 g/L and 30.9 g/L, respectively, in a total hydrolysis and fermentation time at a substrate concentration of 8% (based on the original amount of rice straw). Fed-batch fermentation was employed for enzymolysis and fermentation of rice straw residue. Under a 32% total substrate concentration, 108.6 g/L ethanol was obtained in a total enzymolysis and fermentation time of 40 h. The results from different fermentation methods showed that the laccase produced by A. fumigatus in situ could effectively promote the enzymatic hydrolysis and fermentation through detoxifying the phenols produced during pretreatment and by the enzymatic hydrolysis processes. (C) 2020 Elsevier Ltd. All rights reserved.
作者机构：
[Liu, Yong-Nan; Liu, Bi-Yang; Liu, Gao-Qiang; Ma, You-Chu] Cent South Univ Forestry & Technol, Hunan Prov Key Lab Forestry Biotechnol, Changsha, Peoples R China.;[Liu, Yong-Nan; Liu, Bi-Yang; Liu, Gao-Qiang; Ma, You-Chu] Cent South Univ Forestry & Technol, Int Cooperat Base Sci & Technol Innovat Forest Re, Changsha, Peoples R China.;[Yang, Hai-Long] Wenzhou Univ, Coll Environm & Life Sci, Wenzhou, Peoples R China.
通讯机构：
[Liu, Gao-Qiang] C;Cent South Univ Forestry & Technol, Hunan Prov Key Lab Forestry Biotechnol, Changsha, Peoples R China.;Cent South Univ Forestry & Technol, Int Cooperat Base Sci & Technol Innovat Forest Re, Changsha, Peoples R China.
摘要：
The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.
