Enhancement of MK-7 synthesis by engineered strains of Bacillus subtilis natto that overexpress men genes and by increasing available dissolved oxygen北大核心CSCD

In recent years, researchers have discovered novel physiological functions of vitamin K2. In addition to promoting blood clotting, it can prevent osteoporosis and cardiovascular disease and is expected to treat some tumors and Parkinson’s disease. Bacillus subtilis natto is a probiotic that has been approved by the U.S. Food and Drug Administration for use as a bioproducer of vitamin K2. Its product’s main form is MK-7, which has a long half-life in the human body and high bioavailability. Bacillus subtilis natto displays great potential for large-scale biological preparation of vitamin K2. In this study, the Sipizizen method of Bacillus subtilis transformation was optimized to make it suitable for molecular transformation of Bacillus subtilis natto. Vectors for overexpression of all 8 genes involved in the menadione synthetic pathway were constructed, and changes in MK-7 fermentation yield after transformation of Bacillus subtilis natto were investigated. Three enzymes were found to exert significant effects on MK-7 synthesis, namely isopentenyltransferase (MenA), 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB), and nornaphthoquinone methyltransferase (MenG). A modified strain (BN-pABG) with higher MK-7 productivity was obtained by concerted overexpression of menA, menB, and menG. In a 5 L bioreactor, MK-7 synthesis was further enhanced by optimizing oxygen supply. The final yield of MK-7 from the modified strain was 62.21 mg/L, 1.26 times higher than that of the original strain. These results show that combined overexpression of menA, menB, and menG strongly promotes MK-7 synthesis by Bacillus subtilis natto, and optimizing the oxygen supply conditions also promotes more robust MK-7 synthesis. © 2022 Authors. All rights reserved.     

抗砷载体的构建及在氧化亚铁硫杆菌中的表达

利用ＤＮＡ体外重组技术，将抗砷质粒ｐＵＭ３经ＨｉｎｄⅢ酶切后得到的４．３ｋｂ抗砷片段克隆到有广泛寄主的ＩｎｃＱ质粒ｐＪＲＤ２１５的ＨｉｎｄⅢ位点上，构建了一个新的抗砷质粒ｐＳＤＸ３。砷抗性研究表明，在大肠杆菌中，ｐＳＤＸ３的抗砷水平与ｐＵＭ３相近。将ｐＳＤＸ３通过接合的方式引入氧化亚铁硫杆菌Ｔｆ－５９中并得到了表达，与对照相比，含质粒ｐＳＤＸ３的Ｔｆ－５９抗砷水平有了很大的提高。     

The influence of introducing soft-shelled turtle on soil nitrogen-fixing microorganisms in paddy field北大核心CSCD

Soil microorganisms are an important part of the soil ecosystem, but studies on the structure of the soil bacterial community, especially the related genes, is lacking. This study aimed to investigate the structure of soil microorganisms and the connection between the soil microorganisms and different farming systems. We used the Illumina Genome Analyzer IIx to compare root soil bacteria (nifH gene) diversity characteristics between the rice-turtle co-culture system (R-T) and rice monoculture system (R-M), aiming to reveal the structural characteristics of soil microorganisms, and the connection between the soil microorganisms and different farming systems. The results of High Throughput Sequencing of nitrogen-fixing bacteria showed that the main nitrogen-fixing bacteria were classified as Bradyrhizobium, Geobacter, Anaeromyxobacter, Desulfovibrio, Sideroxydans, Desulfuromonas, Halorhodospira, Azotobacter, Desulfobulbus, Methylomonas, Pseudacidovorax, and Azospirillum. The diversity index of nitrogen-fixing bacteria in the root soil of R-T was higher than that of R-M, which was 6.85-0.07. The difference of soil nitrogen-fixation bacteria in the root soil of rice paddy was 23.86% among the different farming systems, and the difference in the different rice growth period was 16.97%. In summary, the rice growth period and different farming systems both have an influence on the nitrogen-fixation bacteria in paddy soil; the influence of the growth period on the nitrogen-fixation bacteria in the paddy soil was greater. © 2018 Science Press. All rights reserved.     

Isolation,identification, and the biodegradation characteristics of a benzoylurea insecticides-degrading strain北大核心CSCD

A chlorbenzuron, diflubenzuron, and hexaflumuron-degrading bacterium strain M6, was isolated from the activated sludge of an insecticide factory. The strain was identified as Achromobacter sp. according to an analysis on the 16S rRNA gene sequences, morphological, and physiological characteristics. Strain M6 could degrade more than 91% of 100 mg/L chlorbenzuron, diflubenzuron, and hexaflumuron within 48 hours, which could act as the sole carbon source. Strain M6 showed more chlorbenzuron degradation at a temperature range between 25 and 40 ℃ and a pH range between 6.0 and 8.0. The optimal temperature and the initial pH of medium for chlorbenzuron degradation by strain M6 were 30 ℃ and 7.0, respectively; the maximum chlorbenzuron tolerated concentration of strain M6 was as high as 400 mg/L. Strain M6 hydrolyzed 4-acetaminophenol into a purple-red product. Moreover, an approximately 1.4 kb DNA fragment, which could be expressed into an amidase to degrade amide pesticides, was amplified from the genomic DNA of strain M6. The results preliminarily proved that 3 benzoylurea insecticides could be degraded because of strain M6 hydrolyzing their amide bonds. This study obtained a highly efficient degrading strain and provided new resources and valuable information on benzoylurea insecticide degradation. © 2018 Science Press. All rights reserved.     

东亚飞蝗辅酶Q-细胞色素C还原酶基因克隆和序列分析

辅酶Q-细胞色素C还原酶是组成线粒体呼吸链的4种酶复合体之一,对线粒体呼吸链的电子传递起着重要作用.同时,辅酶Q-细胞色素C还原酶还是杀真菌剂(Fungicide)、抗原虫(Antiprotozoan)、抗癌药物(Anticancer drugs)以及抗疟疾(Antimalarial)药物——阿托喹酮(Atovaquone)、氯胍(Proguanil)、布帕伐醌(Buparvaquone)的作用靶标,可将它作为昆虫的药靶,设计和开发新型的杀虫剂.采用RACE方法,克隆了东亚飞蝗[Locusta migratoria manilensis(Meyen)]辅酶Q-细胞色素C还原酶cDNA全序列(GenBank登录号:GU593056.1).获得的cDNA全长939 bp,其中可读框819 bp,编码272个氨基酸,推测其相对分子质量为29.48 ku,等电点为9.19.通过与其它10个物种的细胞色素C还原酶基因的氨基酸序列进行比对,发现东亚飞蝗与家蚕、赤拟谷盗、黒腹果蝇、埃及伊蚊、致乏库蚊、小家鼠、人、丽蝇蛹集金小蜂、蜜蜂和豌豆蚜的辅酶Q-细胞色素C还原酶氨基酸序列同源性分别为72%、72%、66%、63%、60%、59%、59%、58%、57%和56%.