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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (4): 60-68    DOI: 10.13523/j.cb.20190408
技术与方法     
应用合成生物学策略构建全细胞生物催化剂合成(S)-乙偶姻 *
李检秀1,2**,陈先锐1**,陈小玲1,黄艳燕1,莫棋文1,谢能中1,2***(),黄日波1,2***()
1 广西科学院非粮生物质酶解国家重点实验室 国家非粮生物质能源工程技术研究中心 广西生物炼制重点实验室 南宁 530007
2 广西大学生命科学与技术学院 亚热带农业生物资源保护与利用国家重点实验室 南宁 530004
Construct Whole-cell Biocatalyst and Produce (S)-Acetoin via Synthetic Biology Strategy
Jian-xiu LI1,2**,Xian-rui CHEN1**,Xiao-ling CHEN1,Yan-yan HUANG1,Qi-wen MO1,Neng-zhong XIE1,2***(),Ri-bo HUANG1,2***()
1 National Engineering Research Center for Non-food Biorefinery, State Key Laboratory of Non-food Biomass Energy and Enzyme Technology, and Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China
2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science andTechnology, Guangxi University, Nanning 530004, China
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摘要：

目的:在大肠杆菌宿主中过量表达丁二酮还原酶(DAR),同时构建辅酶NADH原位再生系统,利用全细胞高效催化丁二酮不对称还原合成(S)-乙偶姻。方法:PCR克隆多黏芽孢杆菌(Paenibacillus polymyxa)dar基因连到质粒pETDuet-1,转化至大肠杆菌(Escherichia coli)BL21(DE3),构建重组菌E.coli BL21(DE3)-DAR;通过HiTrap TALON柱亲和层析纯化表达产物DAR酶蛋白,测定DAR的比酶活和分子动力学参数。在重组菌E.coli BL21(DE3)-DAR中构建辅酶NADH原位再生系统,协同表达枯草芽孢杆菌(Bacillus subtilis)的葡萄糖脱氢酶(GDH),构建重组菌E.coli BL21(DE3)-DAR/GDH,并以此重组菌为全细胞生物催化剂,优化催化条件,提高(S)-乙偶姻的产量和产率。结果:获得重组工程菌E.coli BL21(DE3)-DAR和E.coli BL21(DE3)-DAR/GDH。DAR以NADH为辅酶还原丁二酮的米氏常数Km、最大催化速率Vmax、催化常数Kcat分别为2.59mmol/L、1.64μmol/(L·min·mg)、12.3/s,还原丁二酮生成(S)-乙偶姻光学的纯度为95.86%,具有较好的催化效率和立体异构体选择性。构建辅酶NADH原位再生系统后,重组菌E.coli BL21(DE3)-DAR/GDH可高效催化丁二酮合成乙偶姻。在最优催化条件下分批补料,乙偶姻产量达51.26g/L,转化率为81.37%,生产速率为5.13g/(L·h)。结论:使用非手性化合物原料丁二酮生产高附加值的手性化合物(S)-乙偶姻,以重组菌为全细胞生物催化剂合成(S)-乙偶姻,不需额外添加昂贵的辅酶,具有较高的生产应用价值。
关键词： 全细胞生物催化剂;  辅酶再生系统;  丁二酮还原酶;  乙偶姻    
Abstract:

Objective: The whole-cell biocatalyst, overexpressing diacetyl reductase (DAR) and introduced in situ-NADH regeneration systems was applied to improve (S)-acetoin production from prochiral diacetyl.Methods: The gene encoding DAR from Paenibacillus polymyxa was cloned and expressed in Escherichia coli. Recombine DAR was purified by HiTrap TALON affinity chromatography, then enzyme activities and molecular kinetic parameters of purified DAR were measured. NADH in situ regeneration system based on glucose dehydrogenase (GDH) from Bacillus subtilis was introduced. The whole-cell biocatalyst, overexpressing DAR and GDH was applied to (S)-acetoin produce and the reaction conditions were optimized.Results: DAR showed a high catalytic efficiency and enantioselective (enantiomeric purity 95.86%). The Km, Vmax and Kcat values of DAR for diacetyl were 2.59mmol/L, 1.64μmol/(L·min·mg) and 12.3/s, respectively. The whole-cell biocatalyst, introduced in situ-NADH regeneration systems resulted in higher (S)-acetoin concentration, productivity and yield form diacetyl. Under optimal conditions in fed-batch bioconversion, 51.26g/L (S)-acetoin was produced from 63g/L diacetyl with a productivity of 5.13g/(L·h).Conclusion: The compound of prochiral diacetyl was used as substrate for asymmetric synthesis of high value chiral (S)-acetoin. The results demonstrated that whole-cell biocatalyst, introduced in situ-NADH regeneration systems, can effectively improve the production of (S)-acetoin with good applicability and economic performance.
Key words: Whole-cell biocatalyst    Cofactor regeneration    Diacetyl reductase    (S)-Acetoin
收稿日期: 2018-10-08 出版日期: 2019-05-08
ZTFLH:  Q819  
基金资助: * 广西科技计划(桂科合14125008-2-22);国家自然科学基金资助项目(21466007);国家自然科学基金资助项目(31400079)
通讯作者: 谢能中,黄日波     E-mail: xienengzhong@gxas.cn;guruace@gxas.cn
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引用本文:

李检秀,陈先锐,陈小玲,黄艳燕,莫棋文,谢能中,黄日波. 应用合成生物学策略构建全细胞生物催化剂合成(S)-乙偶姻 *[J]. 中国生物工程杂志, 2019, 39(4): 60-68.

Jian-xiu LI,Xian-rui CHEN,Xiao-ling CHEN,Yan-yan HUANG,Qi-wen MO,Neng-zhong XIE,Ri-bo HUANG. Construct Whole-cell Biocatalyst and Produce (S)-Acetoin via Synthetic Biology Strategy. China Biotechnology, 2019, 39(4): 60-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190408    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/60

Strains, plasmids and primers Feature or sequence Source
Strains Feature
P. polymyxa DSM 365 Mutant 本实验室选育
B. subtilis 168 ATCC 27370 本实验室保存
E. coli DH5α supE44 △lacU169 (φ80 lacZ△M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Novagen
E. coli BL21(DE3) F- ompT hsdSB (rB-mB-) gal (λ c I 857 ind1 Sam7 nin5 lacUV5 T7gene1) dcm (DE3) Novagen
E. coli BL21(DE3)-pETDuet E. coli BL21(DE3)carrying pETDuet-1 This study
E. coli BL21(DE3)-DAR E. coli BL21(DE3) carryingpETDuet-dar This study
E. coli BL21(DE3)-DAR/GDH E. coli BL21(DE3) carryingpETDuet-dar/gdh This study
Plasmids Feature
pETDuet-1 Overexpression vector; Ampr Novagen
pETDuet-dar dar in pETDuet-1 This study
pETDuet-dar/gdh dar and gdh in pETDuet-1 This study
Primers Sequence
P1 (dar- BamH I-F) 5'- CGCGGATCCGGAACTTAAGAATAAAACAGC -3'
P2 (dar- Hind III-R) 5'- CCCAAGCTTCTACTGTGGGTTGGTTGT -3'
P3 (gdh-Nde I-F) 5'-GGAATTC CATATG TATCCGGATTTAAAAGG-3'
P4 (gdh-Aat II-R) 5'-TATCCA GACGTC TTAACCGCGGCCTGC-3'
表1  菌株、质粒与引物
图1  重组质粒的酶切验证
图2  DAR和GDH在E. coli BL21(DE3)中的表达及纯化
菌株 丁二酮(g/L) 乙偶姻(g/L) 生产速率 [g/(L·h)]
E. coli BL21(DE3)-pETDuet 14.35 ± 0.28 ND* ND
E. coli BL21(DE3)-DAR 12.72 ± 0.05 2.21 ± 0.33 0.74 ± 0.11
E. coli BL21(DE3)-DAR/GDH 9.76 ± 0.39 5.03 ± 0.48 1.68 ± 0.16
表2  全细胞生物催化剂的生物转化
图3  全细胞生物催化剂催化条件的优化
图4  优化催化条件下的补料生物合成
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