Subjects: Biology >> Bioengineering submitted time 2018-07-01 Cooperative journals: 《中国生物工程杂志》
Abstract： 铜绿假单胞菌是一种能引起多部位急慢性感染且难以用抗生素控制的机会致病菌，近年来已成为院内感染的主要致病菌之一。大量研究表明，细菌将毒力因子精准输送至宿主细胞是其致病的关键，分泌系统在这一过程中扮演重要作用，其中近期发现的Ⅵ型分泌系统(Type VIsecretion system,T6SS)在铜绿假单胞菌与宿主间的相互作用和促进生物膜的形成等机制中发挥重要作用，已引起国内外学者高度关注。着重对铜绿假单胞菌T6SS的结构组成、效应功能和调节机制等相关研究进行简要综述，旨在为铜绿假单胞菌感染患者的治疗提供新策略。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract：维生素K2是一种人体必需维生素，具有促进凝血酶原产生和骨钙素合成等作用，在损伤细胞修复方面也有明显效果。微生物发酵法制备维生素K2具有环境影响小、生物活性高、生产成本低等优点，是维生素K2规模化制备的发展趋势。利用超声波和表面活性剂提高微生物发酵过程中菌体细胞通透性是一种常见的细胞代谢人工调控方法。低功率超声波的空化作用可以在细胞表面瞬间造成微伤，使细胞膜局部破裂从而改变细胞膜的通透性，有利于胞内物质释放或胞外物质进入细胞内。表面活性剂有助于提高营养物质溶解性，降低培养基表面张力，减小菌体表面和培养基的界面阻力，从而促进营养物质和菌体代谢产物的跨膜传输。 本文对实验室保藏的一株产维生素K2黄杆菌（Flavobacterium.sp）Fla-M进行低功率超声波辐照和表面活性剂处理，考察二者在提高细胞渗漏发酵方面的协同作用。首先在500 mL摇瓶中对Fla-M进行表面活性剂（聚氧乙烯油醚POE）添加时间和添加浓度优化，发现在发酵起始阶段添加1%POE效果最佳，发酵结束时生物量为13.4 g/L，胞外维生素K2产量为36.3 mg/L，相比于未添加POE的对照组（生物量7.32 g/L，胞外维生素K2 0.85 mg/L）分别提高了83.5%和41倍，扫描电镜观察发现在添加POE发酵的菌体表面聚集了大量表面活性剂胶团，由于POE与细胞膜磷脂分子结构相似，二者可能相溶形成混合胶束改变了细胞膜结构，进而改善细胞膜的通透性。其次在500 mL摇瓶中对Fla-M进行了超声方式、超声时机、超声功率以及作用时间研究，发现在菌体生长稳定期（发酵第5 d）、120 W 20 KHz条件下，插入式超声98 S（每次3 S，间隔4 S）效果最佳，发酵结束时生物量为11.1 g/L，胞外维生素K2达到50.1 mg/L, 相比于未超声对照组（生物量7.32 g/L，胞外维生素K2 0.85 mg/L），分别提高了51.6%和58倍。透射电镜观察发现超声波处理后尽管细胞膜完整但磷脂双分子层界限模糊，且细胞膜表面有孔状破损结构，可见疑似内容物外渗现象。在上述最优条件下，在500 mL摇瓶中综合运用POE和超声的处理方法，生物量和胞外维生素K2产量在发酵6 d后达到最大值，分别为生物量11.5 g/L，胞外维生素K2 59.7 mg/L，较单独运用POE或超声的方法发酵周期缩短3 d、胞外维生素K2产量分别提高64.4%和19.1%。运用排斥性染料碘化丙啶(PI)对发酵后细胞进行流式细胞仪检测，设001号为阴性对照，即未加荧光载体的未处理菌体的荧光信号；002号为处理的菌体加荧光载体的荧光信号；003号为未处理菌体加荧光载体的荧光信号；004号为阳性对照，即死细胞加荧光载体的荧光信号，阴性对照的001号菌体自发荧光区域以外的面积M1占总面积的比例预设为0，结果显示004号的M1占总面积的比例17.21%>002号M1占总面积的比例8.89%>003号M1占总面积的比例1.21%，说明死菌体的细胞膜通透性>渗漏培养菌体的细胞膜通透性>无渗漏培养菌体的细胞膜通透性，验证了经超声和表面活性剂处理后，菌体细胞膜通透性大幅提高。本研究对发酵法制备维生素K2的产业化开发具有一定的借鉴意义。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： Vitamin K2 (VK2) is a series of menaquinone compounds with isoprene side chains, which are represented by MK-n depending on the length of the side chains. Highly active VK2 is mainly synthesized by microorganisms and has the physiological function of preventing and treating diseases such as osteoporosis, hemorrhage, liver cirrhosis and Parkinson's disease. Flavobacterium is an important production strain and can synthesize a variety of VK2 homologs including MK4, MK5 and MK6. We found that by regulating the fermentation temperature, the type and yield of VK2 homologs synthesized by Flavobacterium can be controlled. In the range of 20~37℃, Flavobacterium sp. M1-14 grows best at 25℃, the biomass reaches 8.8 g/L, but the fermentation product is completely MK6, the yield is 13.9 mg/L, and the unit cell yield is 1.6 mg/L g. When the fermentation temperature is higher than 30°C, Flavobacterium can synthesize MK4, MK5 and MK6 at the same time. At 37°C, the yields of MK4 and MK5 are the highest, which are 1.6 mg/L and 1.7 mg/L, respectively, and the total amount of VK2 is 12.5 mg/L. At this time, the biomass was only 5.5g/L, and the unit cell yield was 2.3mg/g. In view of the difference in the optimum temperature for the growth of Flavobacterium cells and the synthesis of VK2 homologs, variable temperature fermentation was considered to increase biomass and VK2 production. After optimization of multiple factors, we developed a two-stage temperature change strategy with low temperature first and then high temperature, that is, fermentation at 25°C for 48 hours, and then fermented at 37°C for 96 hours, the VK2 yield reached 20.9 mg/L (among which MK4 was 2.1 mg). /L, MK5 is 2.3 mg/L, MK6 is 16.5 mg/L), the biomass is 8.8 g/L, and the unit cell yield is 2.4 mg/g. Then, on the 30L fermenter, we investigated the oxygen demand of fermentation at different temperatures by controlling the ventilation rate and rotation speed. It was found that at 25℃ and 37℃, the optimum kLa for VK2 synthesis by Flavobacterium fermentation was 360 h-1 and 60 h-1, respectively. A two-stage variable kLa control strategy was developed for the changes in the oxygen demand of bacteria during variable temperature fermentation. After optimization, the kLa was 360 h-1 in the first 24 hours of variable temperature fermentation, and the kLa was 60 h-1 in the next 120 hours. was 22.5 mg/L), which was 107% higher than the initial value, the biomass was 15.5 g/L, and the unit cell yield was 1.9 mg/g. The staged fermentation regulation strategy of changing temperature and kLa can change the type of homologues of Flavobacterium synthesizing VK2, significantly increase the production of VK2, and lay an optimized foundation for realizing the industrialization of VK2 bioproduction.
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