Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

Modification of histidine biosynthesis pathway genes and the impact on production of L-histidine in Corynebacterium glutamicum.

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.

Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli.

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of transport system for aromatic amino acids in L-tryptophan producing strains has not been fully explored. In this study, the fact was revealed that L-tryptophan production and cell growth were affected by the modification of transport systems based on YddG functioning as aromatic amino acid excretion and AroP functioning as general aromatic amino acid permease. Through comparing glucose conversion rates of recombinant strains such as Escherichia coli TRTH ΔaroP, E. coli TRTH-Y, and E. coli TRTH ΔaroP-Y, the moderate modification of transport system resulted in the metabolic flux redistribution of L-tryptophan biosynthesis pathway. In the fed-batch fermentation by E. coli TRTH and E. coli TRTH-Y in 30-liter fermentor, the final production of L-tryptophan fermented by E. coli TRTH-Y was 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH.