Specific features of L-histidine production by Escherichia coli concerned with feedback control of AICAR formation and inorganic phosphate/metal transport

Abstract

Background: In the L-histidine (His) biosynthetic pathway of Escherichia coli, the first key enzyme, ATP-phosphoribosyltransferase (ATP-PRT, HisG), is subject to different types of inhibition. Eliminating the feedback inhibition of HisG by the His end product is an important step that enables the oversynthesis of His in breeding strains. However, the previously reported feedback inhibition-resistant mutant enzyme from E. coli, HisG^E271K, is inhibited by purine nucleotides, particularly ADP and AMP, via competitive inhibition with its ATP substrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is formed not only during His biosynthesis but also during de novo purine biosynthesis, acts as a natural analog of AMP and substitutes for it in some enzymatic reactions. We hypothesized that AICAR could control its own formation, particularly through the His biosynthetic pathway, by negatively influencing HisG enzymatic activity, which would make preventing ATP-PRT transferase inhibition by AICAR crucial for His overproduction.

Results: For the first time, both the native E. coli HisG and the previously described feedback-resistant mutant HisG^E271K enzymes were shown to be sensitive to inhibition by AICAR, a structural analog of AMP. To circumvent the negative effect that AICAR has on His synthesis, we constructed the new His-producing strain EA83 and demonstrated its improved histidine production. This increased production was particularly associated with the improved conversion of AICAR to ATP due to purH and purA gene overexpression; additionally, the PitA-dependent phosphate/metal (Me²⁺-P_i) transport system was modified by a pitA gene deletion. This His-producing strain unexpectedly exhibited decreased alkaline phosphatase activity at low P_i concentrations. AICAR was consequently hypothesized inhibit the two-component PhoBR system, which controls Pho regulon gene expression.

Conclusions: Inhibition of a key enzyme in the His biosynthetic pathway, HisG, by AICAR, which is formed in this pathway, generates a serious bottleneck during His production. The constructed His-producing strain demonstrated the enhanced expression of genes that encode enzymes involved in the metabolism of AICAR to ATP, which is a substrate of HisG, and thus led to improved His accumulation.

Keywords: AICAR (ZMP); ATP-phosphoribosyltransferase; Escherichia coli; Inorganic phosphate/metal transport; L -Histidine; L-Histidine production; Pho regulon; PitA.