【作者】 李莹；

【导师】 唐存多； 王涛；

【作者基本信息】 南阳师范学院 ， 工程硕士（专业学位）， 2023， 硕士

【摘要】 L-2-氨基丁酸（L-2-aminobutyric acid,L-2-ABA）属于有手性性质的非天然的氨基酸,L-2-氨基丁酸既是有着不可替代地位的化工原料,也是重要的医药前体,在化工业和医药行业的应用颇多。它是高需求药物制备流程中的关键中间体,因此L-2-氨基丁酸的需求量也相应提高,由此使得其制备技术成为热门话题。本研究以探索L-2-氨基丁酸的高效绿色合成工艺为主。首先,通过基因工程技术、手段构建了以重组大肠杆菌作为生物催化平台的反应体系。其次,以重组大肠杆菌全细胞为催化剂合成L-2-氨基丁酸。最后,以高活性甲酸脱氢酶构建的辅酶再生系统参与L-2-氨基丁酸的高效全细胞生物合成,并通过单因素实验探索出全细胞催化绿色合成L-2-氨基丁酸的最适反应条件,实现了L-2-氨基丁酸的绿色高效合成。主要研究内容及结果如下:（1）以E.coli BL21（DE3）的基因组序列为模板,利用特异性引物成功获得了苏氨酸脱氨酶的编码基因EcTD。然后将此段编码序列插入表达质粒pACYCDuet-l,构建重组菌株E.coli BL21（DE3）/pACYCDuet-1-EcTD。最后利用特异性引物扩增L-亮氨酸脱氢酶的编码基因EsLeuDH（来源于Exiguobacterium sibiricum）,并将这段基因序列插入表达质粒p ACYCDuet-l另一克隆位点,构建重组菌株E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH。在该重组菌中,苏氨酸脱氨酶与L-亮氨酸脱氢酶成功实现共表达。（2）将重组质粒pACYCDuet-1-EcTD-EsLeuDH和pET28a-CbFDH转入同一个感受态细胞,构建成双质粒共表达重组菌E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH。利用该重组大肠杆菌进行全细胞L-2-氨基丁酸的生物合成。在250 mL三角瓶中构建50 mL反应体系,体系包括:200 mmol/L L-苏氨酸、200 mmol/L甲酸铵、0.75 mmol/L NAD⁺、50 mg冻干的重组大肠杆菌和适量的玻璃珠,并于30℃、220 rpm振荡反应12 h时,测得在上述反应体系中,L-2-氨基丁酸的得率为71%。（3）利用突变体CbFDH^M构建了双质粒共表达重组菌E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH^M。在250 mL三角瓶中构建50mL的反应体系,体系中包括:200 mmol/L L-苏氨酸、200 mmol/L甲酸铵、0.75mmol/L NAD⁺、50 mg冻干的重组大肠杆菌和适量的玻璃珠,并于30℃、220 rpm振荡反应12 h时,测得在上述反应体系中,L-2-氨基丁酸的得率为85%,与以重组菌E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH全细胞催化合成L-2-氨基丁酸的得率相比,L-2-氨基丁酸的得率提高了14%。结果证明通过提高甲酸脱氢酶的表达水平提高辅酶再生能力,的确能够提高L-2-氨基丁酸的生物合成效率。（4）为了探究反应条件对L-2-氨基丁酸合成效率的影响,在本研究中,控制了反应pH和反应温度。最终得到在pH 7.8,温度35℃时,相同反应体系中L-2-氨基丁酸的得率可以达到93.5%,时空产率可以达到3.09 g/(L^-1·h^-1)。更多还原

【Abstract】 L-2-aminobutyric acid（L-2-ABA）is a non-natural amino acid with chirality.L-2-aminobutyric acid is not only an important chemical raw material,but also an important pharmaceutical precursor.It is widely used in the chemical industry and pharmaceutical industry.As an important intermediate in the process of high demand drug synthesis,the demand for L-2-aminobutyric acid also increases,which makes the method of its synthesis become a hot spot.This study focuses on exploring the non-pollution synthesis and efficient technology of L-2-aminobutyric acid.First,recombinant Escherichia coli was constructed through genetic engineering technology as a biocatalytic platform.Secondly,L-2-aminobutyric acid was synthesized using recombinant E.coli whole cell as catalyst.Finally,the coenzyme regeneration system constructed with highly active formate dehydrogenase participated in the efficient whole-cell biosynthesis of L-2-aminobutyric acid,and the optimal reaction conditions for the green synthesis of L-2-aminobutyric acid by whole-cell catalysis were explored through the factorial experiment,realizing the green and highly efficient conpose of L-2-aminobutyric acid.The main research contents and results are following:（1）Using the genome sequence of E.coli BL21（DE3）as a template,the coding gene EcTD of threonine deaminase was successfully obtained using specific primers.Then insert the coding sequence into the expression plasmid pACYCDuet-l to construct the recombinant strain E.coli BL21（DE3）/pACYCDuet-1-EcTD.Finally,the encoding gene EsLeuDH（derived from Exiguobacterium sibiricum）of leucine dehydrogenase was amplified by specific primers,and the gene sequence was inserted into another cloning site of the expression plasmid pACYCDuet-l to construct the recombinant strain E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH.In the recombinant strain,threonine deaminase and leucine dehydrogenase were successfully co-expressed.（2）The recombinant plasmid pACYCDuet-1-EcTD-EsLeuDH and pET28a-CbFDH were transferred into the same E.coli BL21（DE3）cell,and the recombinant strain E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH.The recombinant E.coli was used for the whole cell biosynthesis of L-2-aminobutyric acid.A 50 mL reaction system was constructed in a 250 mL triangular flask,including 200mmol/L L-threonine,200 mmol/L ammonium formate,0.75 mmol/L NAD⁺,50 mg freeze-dried recombinant Escherichia coli and appropriate amount of glass beads,and the reaction was conducted at 30℃,220 rpm for 12 hours.The yield of L-2-aminobutyric acid was 71%in the above reaction system.（3）The recombinant strain E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH^M。A 50 mL reaction system was constructed in a 250 mL triangular flask.The system included 200 mmol/L L-threonine,200 mmol/L ammonium formate,0.75 mmol/L NAD⁺,50 mg of freeze-dried recombinant E.coli and an appropriate amount of glass beads.The reaction was conducted at 30℃and 220 rpm for 12 hours.The yield of L-2-aminobutyric acid in the above reaction system was 85%,compared with that of recombinant E.coli BL21（DE3）/pACYCDuet-1-EcTD-EsLeuDH:pET28a-CbFDH,the yield of L-2-aminobutyric acid catalyzed was 14%higher than that of L-2-aminobutyric acid.The results showed that the biosynthesis efficiency of L-2-aminobutyric acid could be improved by increasing the expression level of formate dehydrogenase to improve the coenzyme regeneration ability.（4）To investigate the effect of reaction conditions on the synthesis efficiency of L-2-aminobutyric acid,the reaction pH and temperature were controlled.Finally,at pH7.8 and temperature 35℃,the yield of L-2-aminobutyric acid in the same reaction system can reach 93.5%,and the space-time yield can reach 3.09 g/(L^-1·h^-1).更多还原