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食品与发酵工业  2022, Vol. 48 Issue (7): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.028234
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
地衣芽胞杆菌发酵合成L-酪氨酸以及莽草酸途径代谢节点的研究
许银彪1,2, 李由然1,2, 石贵阳1,2*
1(粮食发酵工艺与技术国家工程实验室(江南大学),江苏 无锡,214122)
2(江南大学 生物工程学院,江苏 无锡,214122)
L-tyrosine production of Bacillus licheniformis by fermentation and the metabolic nodes of shikimate pathway
XU Yinbiao1,2, LI Youran1,2, SHI Guiyang1,2*
1(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)
2(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
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摘要 L-酪氨酸是多种有价值的次级代谢产物的通用前体。地衣芽胞杆菌中芳香族氨基酸合成是通过莽草酸途径进行,但代谢调控的潜在机制仍不清楚。该研究对地衣芽胞杆菌合成酪氨酸过程中莽草酸途径代谢节点进行挖掘。通过发酵优化、提升细胞摄氧能力、荧光定量PCR检测途径基因转录水平、多个节点基因的不同组合方式过表达以及莽草酸补加等策略,对莽草酸途径代谢节点进行挖掘。在最优发酵条件下,重组菌HGPA合成酪氨酸单位菌体产量可以达到71 mg/L。荧光定量PCR结果表明,vgb基因的过表达后aroCaroD基因转录水平分别是原始菌的2.57和2.91倍,酪氨酸单位菌体产量从71 mg/L提升至100 mg/L,提升了40.8%。在HGPA的基础上分别过表达基因aroC,aroDaroK,其中HGPAD与HGPAK重组菌酪氨酸合成量可以达到1 200 mg/L。发酵过程中补加5 g/L莽草酸时,HGPA与HGPAK的酪氨酸合成分别可以达到1 311和1 490 mg/L。aroDaroK是地衣芽胞杆菌合成酪氨酸过程中莽草酸途径的重要代谢节点。aroDaroK的过表达可以提升L-酪氨酸的合成但是不能彻底解除节点代谢流的限制。该研究为进一步实现地衣芽胞杆菌工业生产L-酪氨酸及其高价值衍生物的应用潜能奠定了基础。
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许银彪
李由然
石贵阳
关键词:  L-酪氨酸  莽草酸途径  地衣芽胞杆菌  发酵优化  荧光定量PCR    
Abstract: L-tyrosine serves as a common precursor for multiple valuable secondary metabolites,such as L-DOPA,melanin-like biomaterials,monolignols,betalain pigments,plant alkaloids and pharmaceuticals.At the same time,it can be used as a food additive in industry.The synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway with D-Erythrose 4-phosphate(E4P) and phosphoenolpyruvate(PEP) as precursors,but the underlying mechanisms involving metabolic regulation on L-tyrosine production remain unclear in this microorganism.In this work,we first optimized the carbon source,such as glucose,sucrose,lactose and xylose,in the fermentation of L-tyrosine.We found that lactose is more conducive to the synthesis of L-tyrosine by B.licheniformis,and the yield of L-tyrosine per OD600 reached 31.05 mg/L which is 54% higher than that with glucose as the carbon source.In addition,the culture conditions,such as temperature,dissolved oxygen(DO) and inoculum size,were optimized in the fermentation process of recombinant strain expressing HGPA.The optimization of cultural conditions obtained a result of inoculum size 3%(by volume),temperature 37 ℃,shaking speed 200 r/min by one-factor-at-a-time method.The production of L-tyrosine per OD600 by recombinant strain HGPA can reach 71 mg/L at the optimal fermentation condition.It has been verified that the overexpression of vgb,a gene encoding Vitreoscilla hemoglobin(VHb),can increase the yield of L-tyrosine per OD600 from 71 to 100 mg/L,increased by 40.8%.To evaluate the effect of gene vgb expression on the transcription of the shikimate pathway,we used quantitative RT-PCR for detecting the expression level of all genes in shikimate pathway of strain CICIM B6902::vgb with wild-type strain as control.The results indicated that the expression of aroC and aroD in strain overexpressing vgb gene was 2.57 and 2.91 times that of the original strain,respectively,which may result in the improvement of L-tyrosine production.Accordingly,we overexpressed genes aroC,aroD,and aroK,respectively,on the basis of HGPA and L-tyrosine production with expressing HGPAD and HGPAK can reach 1 200 mg/L.However,the production of L-tyrosine was constrained by a narrow range relative to recombinant strain expressing HGPA when aroC gene was co-expressed with 3-deoxy-7-phosphoheptulonate(DAHP) synthase(AroGfbr,D146 N) and dual-function chorismate mutase/prephenate dehydrogenase(TyrAfbr,M53I/A354V).It is worth noting that when 5 g/L shikimate was added during the fermentation process,the production of L-tyrosine with expressing HGPA and HGPAK could reach 1 311 and 1 490 mg/L,respectively.This indicated that the overexpression of genes aroD and aroK could not completely lift the restriction of nodal metabolic flux in shikimate pathway of B.licheniformis.This paper evaluated the effect of overexpression of bottleneck genes in shikimate pathway on aromatic amino acids production in B.licheniformis.Furthermore,this research provided the foundation for further realizing the application potential of B. licheniformis,a Generally Recognized As Safe(GRAS) workhorse,in the industrial production of L-tyrosine and its high-value derivatives.
Key words:  L-tyrosine    shikimate pathway    Bacillus licheniformis    fermentation optimization    quantitative RT-PCR
收稿日期:  2021-06-02      修回日期:  2021-09-22           出版日期:  2022-04-15      发布日期:  2022-04-27      期的出版日期:  2022-04-15
基金资助: 国家重点研发计划项目(2020YFA0907700,2018YFA0900300,2018YFA0900504);轻工业技术与工程国家一流学科计划项目(LITE2018-22);国家自然基金项目(31401674);江苏高校拔尖学术项目和江苏普罗旺斯研究生科研与实践创新计划项目(KYCX18_1796)
作者简介:  博士研究生(石贵阳教授为通信作者,E-mail:gyshi@jiangnan.edu.cn)
引用本文:    
许银彪,李由然,石贵阳. 地衣芽胞杆菌发酵合成L-酪氨酸以及莽草酸途径代谢节点的研究[J]. 食品与发酵工业, 2022, 48(7): 1-7.
XU Yinbiao,LI Youran,SHI Guiyang. L-tyrosine production of Bacillus licheniformis by fermentation and the metabolic nodes of shikimate pathway[J]. Food and Fermentation Industries, 2022, 48(7): 1-7.
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