Transcriptional regulation of histidine biosynthesis genes in Corynebacterium glutamicum.
ABSTRACT Corynebacterium glutamicum, a gram-positive bacterium, has been widely used for industrial amino acid production. Corynebacterium glutamicum his genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. The latter his operon starts the transcription at the C residue localized 196 bp upstream of the hisD ATG start codon. Our computer-based sequence analysis showed that the region corresponding to the untranslated 5' end of the transcript, named the hisD leader region, displays the typical features of the T-box transcriptional attenuation mechanism. Therefore, expression of the cat reporter gene under the control of the wild-type or mutated hisD leader regions was tested in multi-copy (pProm and pTer series) and in single-copy (pInt series) systems under conditions of sufficient or limited histidine. Our mutational studies led to the conclusion that the CAU histidine specifier and 5'-UGGA-3' sequence in the hisD leader region are required for the hisDCB-orf1-orf2-hisHA-impA-hisFI gene regulation. The cat gene expression from the wild-type leader region was negatively regulated by histidine. However, the cat gene expression from mutated leader regions was irresponsive to the level of histidine in the growth medium. Taken together, we propose that a T-box mediated attenuation mechanism is responsible for the gene expression of the hisDCB-orf1-orf2-hisHA-impA-hisFI operon in C. glutamicum.
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ABSTRACT: l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.Microbial Biotechnology 04/2013; · 3.21 Impact Factor
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ABSTRACT: Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18hisDPtac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The L-histidine titer at 0.85 g l(-1) was 80 % greater in the transformed bacterium and production of byproducts, L-alanine and L-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l(-1)). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production.Biotechnology Letters 01/2013; · 1.85 Impact Factor
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ABSTRACT: Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole L-Tryptophan biosynthetic operon, while in certain closely related actinobacteria, e.g. Mycobacterium, the trpF gene is missing. In Mycobacterium the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in L-Histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs a novel (βα)8 isomerase sub-family with a specialized function in L-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism.Molecular Biology and Evolution 06/2013; · 10.35 Impact Factor