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Tyrosine Biosynthesis

Tyrosine Biosynthesis. Yao 05/12/12. Pathway design. glk. P trc. P trc. P lac-UV5. P lac-UV5. ptsG -. p15A ori. pMB1 ori. 6-P-G. 6-P-GA. aroG fbr. tyrB. aroA. ppsA. pckA. tyrA fbr. aroB. aroE. T1. T1. T2. T2. tktA. aroL. evgA. glk. Phe. csrA53:FRT. tyrR -. tktA.

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Tyrosine Biosynthesis

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  1. Tyrosine Biosynthesis Yao 05/12/12

  2. Pathway design glk Ptrc Ptrc Plac-UV5 Plac-UV5 ptsG- p15A ori pMB1 ori 6-P-G 6-P-GA aroGfbr tyrB aroA ppsA pckA tyrAfbr aroB aroE T1 T1 T2 T2 tktA aroL evgA glk Phe csrA53:FRT tyrR- tktA E4P pykF- pheA- pykA- aroL aroGfbr aroE aroA aroB PEP CA DAHP tyrAfbr ppsA pyruvate tyrB pckA ppc- Tyr oxaloacetate Gene regulation Gene overexpression Gene deletion Original gene fbr:feedback-inhibition-resistant

  3. Plasmid construction 10~15 copy 15~20 copy

  4. peasyT1-aroGfbr-tyrAfbr-aroE Colony PCR verification Primer up： M13R Primer down：aroG down 1200bp

  5. peasyT1-aroGfbr-tyrAfbr-aroE Enzyme digest HindIII +speI 3600bp+3300bp

  6. pACYC-ppsA-tktA-glk Enzyme digest verification EcoRI +BamHI 4700bp+5400bp Colony PCR verification Primer up： pACYC up Primer down：glk down Theoretical size: 1862 bp

  7. pYMB1-aroGfbr-tyrAfbr-aroE Enzyme digest HindIII +speI 2700bp+3300bp Colony PCR verification Primer up： tyrB down Primer Rev：pYMB down Theoreticalsize: 1400 bp

  8. pYMB1-aroGfbr-tyrAfbr-aroE-glk-tktA-ppsA Colony PCR verification Primer up： pYMB up Primer Rev：ppsA down Theoretical size: 2862 bp

  9. peasyT1-tyrB-pckA Enzyme digest HindIII +speHI 3170bp+2700bp Colony PCR verification Primer up： pckA down Primer Rev：tyrB up Theoretical size: 2870 bp

  10. Summary glk Ptrc Ptrc Plac-UV5 Plac-UV5 ptsG- p15A ori pMB1 ori 6-P-G 6-P-GA aroGfbr tyrB aroA ppsA pckA tyrAfbr aroB aroE T1 T1 T2 T2 tktA aroL evgA glk Phe csrA53:FRT tyrR- tktA E4P pykF- pheA- pykA- aroL aroGfbr aroE aroA aroB PEP CA DAHP tyrAfbr ppsA pyruvate tyrB pckA ppc- Tyr oxaloacetate Gene regulation Gene overexpression Gene deletion Original gene fbr:feedback-inhibition-resistant

  11. Work plan Ptrc Plac-UV5 p15A ori tyrB aroA pckA aroB T1 T2 aroL evgA 做一下pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA的qRT-PCR，看一下各个基因的转录情况。 转化pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA至已经敲除好的菌株中，发酵，测一下产量。 测量转化pYMB1-aroGfbr-tyrAfbr-aroE -glk-tktA-ppsA的菌株中的胞内产物，主要是E4P、PEP、Shikimicacid的胞内含量。

  12. LC-MSmethods 样品淬灭 指数生长后期发酵液 5 mL，20 mL，－40℃ 甘油 /0. 9% NaCl( 60∶ 40，v / v) 溶液混合，12 000 r/min，－19 ℃3 min 去上清液，用 5 mL，4 ℃ ，0. 9% NaCl溶液洗涤菌体，再于12 000 r/min，4 ℃，3 min，弃去上清液，得到新鲜干净的菌体于离心管中，供提取使用。 样品提取 在含有菌体的离心管中加入5 mL 50%甲醇水溶液，漩涡混合混匀，液氮冰冻3 min，冰上解冻5 min，漩涡混合，重复上述液氮冻融过程2 次，15 000 r / min，4 ℃ 离心 5 min，上清液于干净的离心管中，冷冻干燥后，－80 ℃保存，待进样作 LC-MS/MS 分析。 分析条件 色谱柱: Waters Atlantis HILIC silica柱( 150 mm ×2. 1 mm，3 μm，Ireland) ; （AgelaVenusilHILIC 4000） 柱温: 35 ℃; 进样体积: 5 μL; 流速: 250 μL/min; 流动相 A: 10mmol / L 乙酸铵溶液，流动相 B: 乙腈 梯度洗脱程序: 10% A 保持 3 min; 3 ～ 15 min，10% A 线性变化至 50%A 并保持至 25 min; 25 ～27 min 50% A 线性 变化至 10%A 并平衡至 35 min。 MS / MS 条件。电喷雾离子 ( ESI) 源; 负离子扫描; 喷雾电压 －4. 2 kV; 离子源温度 500 ℃; 气帘气压25 psi; 喷雾气压 45 psi; 加热辅助气压 40 psi。多反应监测扫描模式( MRM) : 入口电压 － 5 V，出口电压－ 3 V

  13. Thanks Yuanfeng Yao +86 15900318354 yuanfengyao@gmail.com

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