Library selection with a randomized repertoire of (βα)8-barrel enzymes results in unexpected induction of gene expression.
Library selection with a randomized repertoire of (βα)8-barrel enzymes results in unexpected induction of gene expression.
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使用 (βα)8 桶酶的随机库选择库会导致基因表达的意外诱导
DOI：
10.1021/acs.biochem.9b00579
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Sterner
中科院分区：
文献类型：
--
作者：
Rohweder;Lehmann;Eichner;Rajendran;Ruperti;Treiber;Dettmer;Meister;Gronwald;Sterner
通过随机诱变和体内选择相结合的方法，检测了常见的（βα）8桶折叠获得新功能的潜力。为此，将52种不同的磷酸结合（βα）8桶蛋白编码基因进行易出错PCR，并克隆到表达质粒中。所得到的混合库用于转化不同的营养不良大肠杆菌，每一种都缺乏一种含磷酸盐底物的酶。不同的转化体在最小培养基上孵育后，只有两株菌株生长，缺乏丝氨酸磷酸酶塞尔维亚或丝氨酸转氨酶SerC基因。同样的突变体。coligenesnanE（编码一种推定的en -乙酰甘油三胺-6-磷酸2- epimase）和pdxj（编码吡哆醇5 ' -磷酸合成酶）负责挽救ΔserB和ΔserC。出乎意料的是，互补的NanE和PdxJ变体在体外没有催化塞尔维亚或SerC反应。相反，RT-qPCR、RNAseq和转录组分析表明，它们通过内源性ouse的帮助来挽救这些缺失。通过组氨酸操纵子转录的特异性上调来表达HisB和HisC。虽然HisB的混杂性塞尔维亚活性是公认的，但我们的数据表明，HisC对SerC反应也是混杂的。NanE和PdxJ通过点突变和额外氨基酸的募集成功挽救ΔserB和ΔserC，为（βα）8桶折叠的适应性提供了另一个例子。
The potential of the frequently encountered (βα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selectionin vivo. For this purpose, the genes encoding 52 different phosphate-binding (βα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophicEscherichia colistrains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of theE. coligenesnanE (encoding a putativeN-acetylmannosamine-6-phosphate 2-epimerase) andpdxJ (encoding the pyridoxine 5′-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactionsin vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenousE. colienzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βα)8-barrel fold.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
对酶混杂性的全系统预测揭示了大肠杆菌中吡哆醛 5-磷酸盐生产的新地下替代路线
DOI：
--
发表时间：
2016
期刊：
PLoS Comput. Biol.
影响因子：
--
作者：
M. Oberhardt;Raphy Zarecki;L. Reshef;Fangfang Xia;Miquel Duran;Rachel Schreiber;C. Henry;N. Ben;D. Dwyer;U. Gophna;E. Ruppin
通讯作者：
E. Ruppin
影响因子：
4.7
作者：
Digianantonio, Katherine M.;Korolev, Maria;Hecht, Michael H.
通讯作者：
Hecht, Michael H.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL