gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation
gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation
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短乳杆菌NCL912的gadA基因位点及其在补料分批发酵过程中的表达
DOI：
10.1111/1574-6968.12301
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发表时间：
2013-12-01
影响因子：
2.1
通讯作者：
Cao, Yusheng
中科院分区：
文献类型：
--
作者：
Li, Haixing;Li, Wenming;Cao, Yusheng
正常情况下，短乳杆菌有两个谷氨酸脱羧酶(GAD)基因：GADA和GADB。利用聚合酶链式反应技术，从短链乳杆菌高产菌株NCL912中克隆了GABA高产菌株的GADA基因。然而，尽管使用了61对不同的引物，包括来自保守区的简并引物，我们仍然无法使用PCR从NCL912菌株中克隆gadB。此外，我们不能通过基因组行走在Aldo-keto还原酶基因下游3000bp以上的方式来克隆它，Aldo-keto还原酶基因是一个单拷贝基因，位于L.brevis ATCC367中gadB上游1003bp。总之，这些数据表明，短链乳杆菌NCL912不包含gadB基因。通过基因组步行的方法，克隆了GADA基因的上下游区域，获得了包含完整GADA基因的4615bp的DNA片段。该基因座包含GAD基因(GADA)和谷氨酸：GABA逆向转运蛋白基因(GADC)，它们似乎在操纵子(GadCA)中转录，以及gadCA的转录调节基因(GadR)。在整个补料分批发酵过程中，GadR、GADC和GADA的表达是同步的，并且与GABA的产量有很好的相关性。我们从NCL912中克隆的GADA基因座与其他短链球菌GADA基因座的同源性较低，这些差异可能解释了NCL912在培养过程中产生更高水平GABA的能力。
Normally, Lactobacillus brevis has two glutamate decarboxylase (GAD) genes; gadA and gadB. Using PCR, we cloned the gadA gene from L.brevis strain NCL912, a high yield strain for the production of gamma-aminobutyric acid (GABA). However, despite using 61 different primer pairs, including degenerate primers from conserved regions, we were unable to use PCR to clone gadB from the NCL912 strain. Furthermore, we could not clone it by genomic walking over 3000bp downstream of the aldo-keto reductase gene, a single-copy gene that is located 1003bp upstream of gadB in L.brevis ATCC367. Altogether, the data suggest that L.brevis NCL912 does not contain a gadB gene. By genomic walking, we cloned regions upstream and downstream of the gadA gene to obtain a 4615bp DNA fragment that included the complete gadA locus. The locus contained the GAD gene (gadA) and the glutamate:GABA antiporter gene (gadC), which appear to be transcribed in an operon (gadCA), and a transcriptional regulator (gadR) of gadCA. During whole fed-batch fermentation, the expression of gadR,gadC and gadA was synchronized and correlated well with GABA production. The gadA locus we cloned from NCL912 has reduced homology compared with gadA loci of other L.brevis strains, and these differences might explain the ability of NCL912 to produce higher levels of GABA in culture.