Tunable expression rate control of a growth-decoupled T7 expression system by L-arabinose only.
Tunable expression rate control of a growth-decoupled T7 expression system by L-arabinose only.
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DOI：
10.1186/s12934-021-01512-7
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发表时间：
2021-02-01
影响因子：
6.4
通讯作者：
Mairhofer J
中科院分区：
文献类型：
--
作者：
Stargardt P;Striedner G;Mairhofer J
基因表达的精确调控对于复杂膜蛋白（MP）、酶或其他对宿主细胞有毒的蛋白质的产生至关重要。在本文中，我们展示了在正常异丙基β-d-1-硫代半乳糖苷（IPTG）诱导的PT7-lacO启动子控制下的基因可以在新构建的大肠杆菌表达宿主BL21-AI<gp2>中被l-arabinose单独诱导，BL21-AI<gp2>是基于最近发表的噬菌体激发生长解偶重组蛋白生产方法的菌株。在这里，我们发现BL21-AI<gp2>能够在细胞水平上以l-阿拉伯糖浓度依赖的方式精确调节蛋白质的产生速率，同时允许代谢资源的重新分配，这是由于l-阿拉伯糖诱导的生长解耦被噬菌体来源的抑制剂肽gp2。我们成功地在类似补料批的条件下在微规模培养（800µL）下对该系统进行了表征，并通过在线gfp信号、FACS分析、SDS-PAGE和western blotting生成了数据，证明6种不同的大肠杆菌衍生的MP-GFP融合蛋白的特定产量相关增加。在所有测试情况下，BL21-AI<gp2>优于亲本菌株BL21-AI，在生长相关生产模式下运行。特异性MP-GFP融合蛋白的产量提高了2.7倍。因此，这种方法允许微调MP生产或多酶途径的表达，例如，必须满足特定的化学计量来优化产品通量。
Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. Here, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. In all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.
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影响因子：
16
作者：
James, Ellen;Liu, Minhao;Sheppard, Carol;Mekler, Vladimir;Camara, Beatriz;Liu, Bing;Simpson, Pete;Cota, Ernesto;Severinov, Konstantin;Matthews, Steve;Wigneshweraraj, Sivaramesh
通讯作者：
Wigneshweraraj, Sivaramesh
影响因子：
3
作者：
Acosta, MBR;Ferreira, RCC;Costa, SOP
通讯作者：
Costa, SOP
影响因子：
56.9
作者：
Daley, DO;Rapp, M;von Heijne, G
通讯作者：
von Heijne, G
影响因子：
2.8
作者：
Khlebnikov, A;Datsenko, KA;Keasling, JD
通讯作者：
Keasling, JD
影响因子：
4.8
作者：
Breazeale, SD;Ribeiro, AA;Raetz, CRH
通讯作者：
Raetz, CRH