1,3-丁二醇合成细胞的构建及优化  

Improvement of 1,3-Butanediol Production by Engineered Escherichia coli

作　　者：林海英[1,2] 于艺洁 陈怡平 张曼 LIN Haiying;YU Yijie;CHEN Yiping;ZHANG Man(College of Biological Science and Technology,Fuzhou University,Fuzhou 350108,China;Key Laboratory of Marine Enzyme Engineering of Fujian Province,Fuzhou 350116,China)

机构地区：[1]福州大学生物科学与工程学院,福州350108 [2]福建省海洋酶工程重点实验室,福州350116

出　　处：《中国生物工程杂志》2024年第11期30-38,共9页China Biotechnology

摘　　要：1,3-丁二醇(1,3-butanediol)是应用于化工合成、食品药品、日化生产等领域的重要二元醇,以大肠杆菌BL21(DE3)为宿主菌,利用乙酰辅酶A乙酰基转移酶PhaA、NAD(P)H依赖型乙酰乙酰辅酶A还原酶PhaB1、丁醛脱氢酶Bld构建1,3-丁二醇代谢合成途径,在此基础上探究醛酮还原酶PA1127及NAD激酶(NAD kinase, NADK)对重组菌株代谢生产1,3-丁二醇的影响,并对产1,3-丁二醇重组菌株进行培养条件优化,以期提高重组菌株的1,3-丁二醇产量。采用多质粒共转化系统和多顺反子系统两种形式构建重组质粒,转化获得多酶共表达重组菌株。在转化试验或发酵结束后,利用丹磺酰法(DNS法)检测葡萄糖消耗量、通过吸光值检测比较菌体量和NADPH水平、采用GC和HPLC对1,3-丁二醇和副产物进行定量分析。成功构建由PhaA、PhaB1、Bld组成的1,3-丁二醇代谢合成途径,并实现葡萄糖向1,3-丁二醇的转化;NADK过表达使重组菌株合成1,3-丁二醇的能力提高,同时使乙酸、乳酸、乙醇等副产物产量增加,发酵过程培养条件及培养基组分的优化可提高重组菌株1,3-丁二醇产量达0.531 g/L。此结论可为1,3-丁二醇生物合成研究提供理论依据。1,3-butanediol is an important diol used in chemical synthesis,food and medicine,daily chemical production,and more.In this study,Escherichia coli BL21(DE3)was used as the host bacterium.The 1,3-butanediol metabolic synthesis pathway was constructed using acetyl-CoA acetyltransferase PhaA,NAD(P)H-dependent acetyl-CoA reductase PhaB1 and butaldehyde dehydrogenase Bld.On this basis,the effects of aldehyde-ketone reductase PA1127 and NAD kinase were explored.The effects of NADK on the metabolic production of 1,3-butanediol in recombinant strains were studied,and the culture conditions of 1,3-butanediol producing recombinant strains were optimized in order to improve the production of 1,3-butanediol in the recombinant strains.The recombinant plasmid was constructed in the form of multi-plasmid co-transformation system and multi-cis-trans subsystem,and the recombinant strain was obtained by transformation.After the conversion experiment or fermentation,glucose consumption was determined by the DNS method,bacterial volume and NADPH levels were compared by light absorption value,and 1,3-butanediol and by-products were quantitatively analyzed by GC and HPLC.The 1,3-butanediol metabolic synthesis pathway consisting of PhaA,PhaB1 and Bld was successfully constructed,and the conversion of glucose to 1,3-butanediol was realized.Overexpression of NADK improved the ability of recombinant strains to synthesize 1,3-butanediol,but increased the yield of acetic acid,lactic acid,ethanol and other by-products.By optimizing culture conditions and medium components during fermentation,the yield of 1,3-butanediol was increased to 0.531 g/L.This conclusion provides a theoretical basis for the study of 1,3-butanediol biosynthesis.

关 键 词：1 3-丁二醇 代谢途径 醛/酮还原酶PA1127 NAD激酶 

分 类 号：Q819[生物学—生物工程]