Abstract
Backgroundl-Histidine biosynthesis is embedded in an intertwined metabolic network which renders microbial overproduction of this amino acid challenging. This is reflected in the few available examples of histidine producers in literature. Since knowledge about the metabolic interplay is limited, we systematically perturbed the metabolism of Corynebacterium glutamicum to gain a holistic understanding in the metabolic limitations for l-histidine production. We, therefore, constructed C. glutamicum strains in a modularized metabolic engineering approach and analyzed them with LC/MS-QToF-based systems metabolic profiling (SMP) supported by flux balance analysis (FBA).ResultsThe engineered strains produced l-histidine, equimolar amounts of glycine, and possessed heavily decreased intracellular adenylate concentrations, despite a stable adenylate energy charge. FBA identified regeneration of ATP from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as crucial step for l-histidine production and SMP identified strong intracellular accumulation of inosine monophosphate (IMP) in the engineered strains. Energy engineering readjusted the intracellular IMP and ATP levels to wild-type niveau and reinforced the intrinsic low ATP regeneration capacity to maintain a balanced energy state of the cell. SMP further indicated limitations in the C1 supply which was overcome by expression of the glycine cleavage system from C. jeikeium. Finally, we rerouted the carbon flux towards the oxidative pentose phosphate pathway thereby further increasing product yield to 0.093 ± 0.003 mol l-histidine per mol glucose.ConclusionBy applying the modularized metabolic engineering approach combined with SMP and FBA, we identified an intrinsically low ATP regeneration capacity, which prevents to maintain a balanced energy state of the cell in an l-histidine overproduction scenario and an insufficient supply of C1 units. To overcome these limitations, we provide a metabolic engineering strategy which constitutes a general approach to improve the production of ATP and/or C1 intensive products.
Highlights
Background lHistidine was discovered in the late nineteenth century by Kossel and Hedin simultaneously [89] and the l-enantiomer is nowadays categorized as an essential amino acid for human infants and adults, belonging to the 20 standard proteinogenic amino acids [52]
Histidine is a precursor for histamine, which is known to play an important role in regulating human immune response, and is linked to several allergic disorders [67, 69]
Optimizing the histidine biosynthesis In the first step, we chromosomally introduced the nucleotide exchanges ggc to cat and acg to cag in hisG of C. glutamicum ATCC 13032 (Fig. 7) to relieve HisG from feedback inhibition, yielding variant HisGG233H−T235Q [77]
Summary
Background lHistidine (further referred to as histidine) was discovered in the late nineteenth century by Kossel and Hedin simultaneously [89] and the l-enantiomer is nowadays categorized as an essential amino acid for human infants and adults, belonging to the 20 standard proteinogenic amino acids [52]. Histidine has the ability to switch between the protonated and unprotonated states due to the pKa of about 6 of its imidazole group [64]. Histidine is a common ligand of metalloproteins and part of the catalytic triad in several enzymes, underlining its physiologically prominent role [57, 70, 72]. Exceeding physiological levels of histidine in humans has shown to be connected with mutations in histidase and was named histidinemia, a benign inborn error of metabolism [6, 49]. Histidine is a precursor for histamine, which is known to play an important role in regulating human immune response, and is linked to several allergic disorders [67, 69]. Histidine is available as feed supplement and has been reported to have anti-inflammatory and antioxidant properties, which makes it attractive for applications in the medical industry [25, 33, 34, 87, 90, 91]
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