【作者】 李宁；

【导师】 陆坚；

【作者基本信息】 广西大学 ， 生物工程（专业学位）， 2020， 硕士

【摘要】 角鲨烯(C₃₀H₅₀)是由六个异戊二烯单元首尾连接形成的三萜化合物,是人参皂苷和类固醇生物合成的前体。其具有多种功能,不仅有抗疲劳、抗感染的作用,更有抗癌、抗氧化的功效。由此被广泛应用于抗癌药物,化妆品,保健品等领域。近年来,市场对角鲨烯的需求越来越大,能够高效生产角鲨烯的方式急待开发。随着代谢工程和合成生物学的快速发展,构建产角鲨烯的细胞工厂已成为一种不错的选择。本研究以大肠杆菌为宿主,通过引入异源的角鲨烯合酶,构建初代产角鲨烯菌株。基于合成生物学的理念,借助代谢工程的手段,对初代菌株从不同角度进行优化,最终获得两株角鲨烯产量可观的菌株,对角鲨烯后续产物及其他萜类化合物的生物合成具有一定的借鉴意义。主要研究内容如下:1,初代产角鲨烯菌株的构建以委托金唯智生物科技有限公司合成的人源角鲨烯合酶为模板,扩增目的基因,构建由L-阿拉伯糖诱导的重组质粒,通过转化的方式得到菌株SQ01。对其进行自诱导培养（Auto-induction medium,ZYM）和全细胞催化,建立从大肠杆菌中提取角鲨烯的方法,建立角鲨烯高效液相色谱仪（i e Uo m Rnce i uid c om Rto R L,HPLC）检测方法。2,多种策略提升菌株产角鲨烯的能力本研究首先通过过表达isp A基因,使更多的IPP和DMAPP流向角鲨烯合成的方向;然后将人源角鲨烯合酶构建在不同拷贝数的载体上,寻求相对最适的蛋白表达水平;因为在研究过程中菌株出现生长不稳定的状况,经过分析对菌株的诱导条件进行优化,使角鲨烯产量在保持原有水平的基础上,菌株的生物量得到提高,且生长稳定;为了使角鲨烯合酶能够更好地表达,也为后续产物提供一个良好的平台,借助C is/CRs9的基因编辑手段,将人源角鲨烯合酶整合至基因组,并将不利基因pox B敲除;为了寻求活力更好的酶,通过构建系统发育树,选定3个新来源的角鲨烯合酶,经过测试,来源于嗜热蓝细菌Thermosynechocoocus elongatus BP-1的角鲨烯合酶产角鲨烯能力相对最强;因为在角鲨烯合成过程中,很多反应需要NADPH作为辅因子,通过敲除基因sth A,使NADPH的含量得到提高,进而使角鲨烯产量再次得到提高。综合以上策略,最终得到菌株SQ13。在24小时内胞内能积累的角鲨烯的量为19.12±0.82 m/,发酵液含量为191.2±8.2 m/L,相比于初代菌株SQ01,细胞干重产量提升3.82倍。3,多拷贝MVA途径菌株的构建因为整合至基因组的单拷贝MVA途径,会存在某些酶表达不足的问题,进而影响途径中酶之间彼此的适配性,降低菌株产角鲨烯的能力。借助位点特异性重组方法（Ni i ecombin Rse medi Rted inte Rtion,NRMI）,在基因组上整合多拷贝MVA途径,弥补单拷贝MVA途径存在的某些酶表达不足的状况,提高彼此之间的适配性,达到更好的催化效果。最终,得到菌株SQ14。在24小时内其胞内能积累的角鲨烯的量为26.51±1.26 m/,发酵液含量为265.1±12.6m/L,相比于初代菌株SQ01,细胞干重产量显著提升5.30倍。本研究以已引入单拷贝MVA途径的菌株为底盘菌,通过引入异源角鲨烯合酶,构建初代产角鲨烯菌株,后续采取多种策略对该菌株进行优化,最终得到菌株SQ13,角鲨烯产量为19.12 m/。考虑到整合至基因组的单拷贝MVA途径存在某些酶表达不足的问题,借助NRMI位点特异性重组方法,在基因组整合多拷贝MVA途径,补充途径中某些酶的表达量,提高彼此之间的适配性,达到更好的催化效果,最终得到菌株SQ14,角鲨烯产量达到26.5 m/。本研究所构菌株及为优化菌株所采取的策略为后续产物和其它萜类化合物的生物合成提供一定的基础和借鉴。更多还原

【Abstract】 Squalene(C30H50)is a triterpene compound formed by the end-to-end connection of six isoprene units.It is the precursor of ginsenoside and steroid biosynthesis.Because its structure is a highly unsaturated olefin,it has anti-oxidation and anti-cancer functions.As a result,it is widely used in anti-cancer drugs,cosmetics,health products and other fields.In recent years,there has been an increasing demand for squalene in the market,and a way to efficiently produce squalene needs to be developed urgently.With the rapid development of metabolic engineering and synthetic biology,the construction of a cell factory producing squalene has become a good choice.IPP and DMAPP are important precursors for the biosynthesis of squalene.There are currently two known pathways for producing IPP and DMAPP,namely 2-C-methyl-D-erythritol 4-phosphate(MEP)pathway and mevalonate pathway(MVA)pathway.In this study,Escherichia coli was used as the research object,and the first squalene-producing strain was constructed by introducing heterologous squalene synthase.Based on the concept of synthetic biology,with the help of metabolic engineering,the primary strains were optimized from different perspectives,and finally two strains with considerable squalene yields were constructed,which has a certain effect on the biosynthesis of the subsequent products of squalene and other terpenoids.The main research contents are as follows:1.Construction of the primary strain producing squaleneUsing the human squalene synthase synthesized by GENEWIZ as a template,the target gene was amplified to construct a recombinant plasmid induced by L-arabinose,and the strain SQ01 was obtained by transformation.Carry out ZYM auto-induction culture and whole-cell catalysis,establish a method for extracting squalene from E.coli,and establish squalene HPLC and GC-MS detection methods.2.Various strategies to improve the ability of strains to produce squaleneIn this study,the ispA gene was overexpressed to make more IPP and DMAPP flow toward the direction of squalene synthesis;then,the human-derived squalene synthase was constructed on vectors with different copy numbers to find the relatively optimal protein expression level.Because of the unstable growth of the strain during the research process,the induction conditions of the strain were optimized after analysis,so that the squalene production was maintained at the original level,the biomass of the strain was increased,and the growth was stable;It enables better expression of squalene synthase and also provides a good platform for subsequent products.With the help of Crispr/Cas9 gene editing methods,human squalene synthase is integrated into the genome and the unfavorable gene poxB is removed;In order to find enzymes with better activity,by constructing a phylogenetic tree,three new sources of squalene synthase were selected.After testing,the squalene synthase derived from Thermosynechocoocus elongatus BP-1 has a relatively high capacity to produce squalene.The strongest;because in the process of squalene synthesis,many reactions require NADPH as a cofactor or substrate.By knocking out the gene sthA,the content of NADPH is increased,and the production of squalene is increased again.Combining the above strategies,the strain SQ13 was finally obtained.The amount of squalene that can accumulate in the cell within 24 hours is 19.12 ± 0.82 mg/g,and the content of the fermentation broth is 191.2±8.2 mg/L.Calculated at the high-density fermentation level of 150 OD,the amplified output on the tank can reach 0.88 g/L,compared with the primary strain SQ01,the cell dry weight yield was significantly increased by 3.82 times.3.Construction of multi-copy MVA pathway strainsBecause of the single-copy MVA pathway integrated into the genome,there is bound to be a problem of insufficient expression of certain enzymes,which in turn affects the adaptability of enzymes in the pathway and reduces the ability of the strain to produce squalene.With the help of NRMI site-specific recombination method,the multi-copy MVA pathway is integrated in the genome to make up for the insufficient expression of certain enzymes in the single-copy MVA pathway,improve mutual adaptability,and achieve better catalytic effects.Finally,the strain SQ14 was obtained.The amount of squalene that can accumulate in the cell within 24 hours is 26.51 ± 1.26 mg/g,and the fermentation broth content is 265.1 ± 12.6 mg/L.Calculated at the high-density fermentation level of 150 OD,the scaled-up output on the tank can reach 1.22 g/L,compared with the original strain SQ01,the cell dry weight yield was significantly increased by 5.30 times.In this study,the strain that has been introduced into the single-copy MVA pathway was used as the chassis bacteria.By introducing heterologous squalene synthase,the primary squalene-producing strain was constructed,and various strategies were used to optimize the strain.Finally,the strain SQ13 was obtained.The yield of squalene was 19.12 mg/g.Considering that the single-copy MVA pathway integrated into the genome has the problem of insufficient expression of certain enzymes,with the help of NRMI site-specific recombination method,the multi-copy MVA pathway is integrated into the genome to supplement the expression of certain enzymes in the pathway and increase the relationship between each other.To achieve better catalytic effect,the strain SQ14 was finally obtained,and the squalene output reached 26.5 mg/g.The constitutive strains in this study and the strategies adopted to optimize the strains provide a certain basis and reference for the biosynthesis of subsequent products and other terpenoids.更多还原