Improvement of Vitamin K2 Production by Bacillus subtilis (natto) withγray irradiation ........................... 82
第一篇 大会报告
小基因组B. amyloliquefaciens菌株的构建以及γ-聚谷氨酸的生物
合成
宋存江 冯俊 谷燕燕 高伟霞 全玉芬 党雨蕾 王淑芳
南开大学分子微生物与技术教育部重点实验室 songcj@，022-23503866
摘要：目前，合成生物学的研究主要集中在以下方面 ① 生物元件标准化及生物模块的设计与构建及其适配性研究；② 最小基因组研究；③ 基因组的设计、合成与组装。
小基因组微生物构建主要步骤包括：① 掌握菌株全基因组数据背景；② 菌株基因组的生物信息学分析（必须基因、基因组岛和复制起始位点等）；③ 建立大片段无痕敲除基因序列的方法；④ 利用所构建的方法进行基因组精简；⑤ 检验所构建的小基因组菌株生长和合成产物状况。 本研究完成了Bacillus amyloliquefaciens LL3的全基因组测序，该菌株拥有一个 3995227 bp染色体组和一个6758bp的内源性质粒。生物信息学分析的结果显示出：原有基因4325个，必须基因273个；具有经过水平转移获得的大片段的基因组岛8个；该菌株复制起始位点位于3994731-3994423 ，另外dnaA 在1753-1973。本研究建立了利用温敏质粒结合反向筛选标记的大片段无痕删除基因序列的方法。利用该方法对基因组进行了精简，共计删除了约10%的基因组序列。小基因组菌株的特征表现为生长速度有所加快。另外，研究组对该菌株进行了改造，构建了γ-聚谷氨酸合成工程菌株。
通过代谢改造γ-PGA合成相关代谢网络包括：副产物合成，γ-PGA降解，谷氨酸前体合成，γ-PGA合成以及自诱导因子合成。构建了一株γ-PGA产量提高的解淀粉芽孢杆菌工程菌。首先在解淀粉芽孢杆菌NK-1出发菌株的基础上敲除了胞外多糖合成基因簇epsA-O，果聚糖合成基因簇sac，脂多糖合成相关基因lps，乙酸合成相关基因pta，得到NK-E7菌株产量较野生型有微小提高从3.8提高到
4.15 g/L。然后在NK-E7菌株基础上敲除γ-PGA降解酶基因pgdS以及细胞壁水解酶基因cwlO，得到NK-E10菌株产量从4.15提高到9.18 g/L。在NK-E10菌株的基础上敲除细胞自诱导因子合成基因luxS，聚谷氨酸产量从9.18提高到9.54 g/L。通过人工设计合成的sRNA对谷氨酸利用途径关键基因进行抑制表达。实验发现当谷氨酸脱氢酶基因rocG抑制时，菌株NK-anti-rocG的γ-PGA产量最高达到11.04 g/L。最后利用5-L发酵罐对NK-anti-rocG菌株进行补料分批发酵实验，以蔗糖为发碳源，菌株在发酵54 h时产量最高达到20.3 g/L。
2014 - 2015发表的论文：
[1] Weitao Geng,Chao Yang,Yanyan Gu,Ruihua,Liu,Wenbin Guo,Xiaomeng Wang,Cunjiang Song* and Shufang Wang* Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans Microbial Biotechnology,2014,7,155–164.
[2] Jun Feng, Weixia Gao, Yanyan Gu, Wei Zhang, Mingfeng Cao, Cunjiang Song*, Peng Zhang, Min Sun, Chao Yang, Shufang Wang*. Functions of poly-gamma -glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance. Appl Microbiol Biotechnol, 2014,98:6397-6407.
[3] Yanyan Gu, Chao Yang, Xiaomeng Wang, Weitao Geng, Yang Sun, Jun Feng, Yuanyuan Wang, Yufen Quan, You Che, Chi Zhang, Ting Gong, Wei Zhang, Weixia Gao, Zhenqiang Zuo, Cunjiang Song*,
Shufang Wang. Genome Sequence of the ε- Poly -L-Lysine-Producing Strain Streptomyces albulus NK660, Isolated from Soil in Gutian, Fujian Province, China. Genome Announcements, 2014, 2(3), e00532-14.
[4] Jun Feng, Yanyan Gu,Yang Sun,Lifang Han,Chao Yang,Wei Zhang,Mingfeng Cao,Cunjiang Song*, Weixia Gao and Shufang Wang*.Metabolic engineering of Bacillus amyloliquefaciens LL3 for
poly-gamma-glutamic acid (γ-PGA) overproduction. Microbial Biotechnology, 2014, 7, 446–455.
[5] Wei Zhang, Cunjiang Song* et al. A markerless gene replacement method for B. Amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production. Appl Microbiol Biotechnol, 2014, 98,8963–8973.
[6] Wei Zhang, Yulian He, Weixia Gao, Jun Feng, Mingfeng Cao, Chao Yang, Cunjiang Song* and Shufang Wang*. Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.Journal of Industrial Microbiology and Biotechnology, 2015, 42,297–305.
[7] Yuanyuan Wang, Chi Zhang, Ting Gong, Zhenqiang Zuo, Fengjie Zhao, Xu Fan, Chao Yang*,
Cunjiang Song*.An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440. Journal of Microbiological Methods, 2015, doi: 10.1016/j.mimet.2015.03.022
[8] Jun Feng#,YanyanGu#,Lifang Han, Kexin Bi, Yufeng Quan, Chao Yang, Wei Zhang, Mingfeng Cao, Shufang Wang,Weixia Gao,Yang Sun and Cunjiang Song?. Construction of a Bacillus amyloliquefaciens strain for high purity levan production. 2015, FEMS Letters, doi: 10.1093/femsle/fnv079
[9] Jun Feng, Yanyan, Yufen Quan, Wei Zhang, Mingfeng Cao, Weixia Gao, Cunjiang Song*, Chao Yang, Shufang Wang* Recruiting a new strategy to improve levan production in
Bacillus amyloliquefaciens. Scientific Reports | 5:13814 | DOi: 10.1038/srep13814.
[10] Jun Feng, Yanyan Gu, Yufen Quan, Mingfeng Cao, Weixia Gao, Wei Zhang, Shufang Wang* , Chao Yang, Cunjiang Song*. Improved poly-γ -glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering. Metabolic Engineering, ( 2015 ) ,