Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Substrate inhibition in the tricarboxylic acid cycle

It has been shown in the experiments on rat liver mitochondria under glucose hexo-kinase load that excess of substrates of (1-20 mM) pyruvate, acetate, propionate, pent-4-enoate and malate may induce oxidation of NAD(P)H and inhibition of mitochondrial respiration (by 20-50% and more) due to a decreased rate of hydrogen production by tricarboxylic acid cycle. It has been concluded from the analysis of mathematical models and metabolite-testings which remove this inhibition that for pyruvate and acetate this inhibition is an autocatalytic one. It is related to a decreased level of CoA and oxaloacetate due to the formation of "traps" such as acetyl-CoA and alpha-kotoglutarate. For propionate and pent-4-enoate in the bicarbonate-free medium suppression of the flux in the cycle is concerned with a decreased level of CoA, acetyl-CoA and succionoyl CoA due to the accumulation of propionyl-CoA. It seems to be also concerned with the inhibition of citrate-synthetase and alpha-ketoglutarate-dehydrogenase by propionyl-CoA. Malate (in the presence of malonate) can inhibit respiration at the expense of direct inhibition of citrate-synthetase.     

Supramolecular organization of tricarboxylic acid cycle enzymes

We propose a spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon). The structure is based on an analysis of data on the interaction between tricarboxylic acid cycle enzymes and the mitochondrial inner membrane, as well as on data on enzyme-enzyme interactions. The alpha-ketoglutarate dehydrogenase complex, adsorbed along one of the 3-fold symmetry axes of the mitochondrial inner membrane, plays a key role in formation of the metabolon. In the interaction with the membrane, two association sites of the alpha-ketoglutarate dehydrogenase complex participate, placed on opposite sides of the complex. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleoside-diphosphate kinase. Succinate dehydrogenase, which is the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of the metabolon on the membrane. The molecular mass of the complex (without regard to succinate dehydrogenase) is 8 x 10(6) Da. The metabolon symmetry corresponds to the D3 point symmetry group.     

Organization of Krebs tricarboxylic acid cycle enzymes

Binding of enzymes of the Krebs TCA cycle to biological membranes was characterized with respect to intracellular location, susceptibility to various chemical and physical treatments, and extractability as a macromolecular component of the mitochondrial inner membrane. It was shown that citrate synthase and malate dehydrogenase bind to the inner membrane in an ionic strength-sensitive, saturable, and specific manner to a relatively thermostabile component manifested on the inner (matrix) surface of the inner membrane of the mitochondrion. From these data several arguments in support of the physiological applicability of these processes were deduced, and the question of whether these two enzymes bind to the same or different membrane components was considered. Also, experiments preliminary to purification of the citrate synthase binding component were presented.     

Incomplete tricarboxylic acid cycle in a type I methylotroph, Methylococcus capsulatus.

Alpha-Ketoglutaratedehydrogenase was undetectable in extracts of Methylococcus capsulatus. Cells incorporated [1-14-C] acetate into only four protein amino acids (glutamate, proline, arginine, and leucine) and the C5, but not C1, of glutamate.