Metabolic control analysis enables rational improvement of E. coli L-tryptophan producers but methylglyoxal formation limits glycerol-based production.

BACKGROUND: Although efficient L-tryptophan production using engineered Escherichia coli is established from glucose, the use of alternative carbon sources is still very limited. Through the application of glycerol as an alternate, a more sustainable substrate (by-product of biodiesel preparation), the well-studied intracellular glycolytic pathways are rerouted, resulting in the activity of different intracellular control sites and regulations, which are not fully understood in detail. Metabolic analysis was applied to well-known engineered E. coli cells with 10 genetic modifications. Cells were withdrawn from a fed-batch production process with glycerol as a carbon source, followed by metabolic control analysis (MCA). This resulted in the identification of several additional enzymes controlling the carbon flux to L-tryptophan. RESULTS: These controlling enzyme activities were addressed stepwise by the targeted overexpression of 4 additional enzymes (trpC, trpB, serB, aroB). Their efficacy regarding L-tryptophan productivity was evaluated under consistent fed-batch cultivation conditions. Although process comparability was impeded by process variances related to a temporal, unpredictable break-off in L-tryptophan production, process improvements of up to 28% with respect to the L-tryptophan produced were observed using the new producer strains. The intracellular effects of these targeted genetic modifications were revealed by metabolic analysis in combination with MCA and expression analysis. Furthermore, it was discovered that the E. coli cells produced the highly toxic metabolite methylglyoxal (MGO) during the fed-batch process. A closer look at the MGO production and detoxification on the metabolome, fluxome, and transcriptome level of the engineered E. coli indicated that the highly toxic metabolite plays a critical role in the production of aromatic amino acids with glycerol as a carbon source. CONCLUSIONS: A detailed process analysis of a new L-tryptophan producer strain revealed that several of the 4 targeted genetic modifications of the E. coli L-tryptophan producer strain proved to be effective, and, for others, new engineering approaches could be derived from the results. As a starting point for further strain and process optimization, the up-regulation of MGO detoxifying enzymes and a lowering of the feeding rate during the last third of the cultivation seems reasonable.

Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition. Corynebacterium glutamicum, the industrial workhorse for amino acid production, has two isoenzymes of DAHPS, AroF (Tyr sensitive) and AroG (Phe and Tyr sensitive). Here, we introduce feedback resistance against Tyr in the class I DAHPS AroF (AroFcg). We pursued a consensus approach by drawing on structural modeling, sequence and structural comparisons, knowledge of feedback-resistant variants in E. coli homologs, and computed folding free energy changes. Two types of variants were predicted: Those where substitutions putatively either destabilize the inhibitor binding site or directly interfere with inhibitor binding. The recombinant variants were purified and assessed in enzyme activity assays in the presence or absence of Tyr. Of eight AroFcg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroFcg in the absence of Tyr. Evaluation of two and four further variants at positions 154 and 155 yielded E154S, completely resistant to 5 mM Tyr, and P155I, which behaves similarly to P155L. Hence, feedback-resistant variants were found that are unlikely to evolve by point mutations from the parental gene and, thus, would be missed by classical strain engineering. KEY POINTS: • We introduce feedback resistance against Tyr in the class I DAHPS AroF • Variants at position 154 (155) yield > 80% (> 50%) residual activity at 5 mM Tyr • The variants found are unlikely to evolve by point mutations from the parental gene.

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate.

L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H+-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error. Metabolic analyses and subsequent extensive data evaluation were performed based on the data of the parallel analysis reactors and the production process. Extracellular rates and intracellular metabolite concentrations displayed evident deflections in cell metabolism and particularly in chorismate biosynthesis due to the perturbations with shikimate. Intracellular flux distributions were estimated using a thermodynamics-based flux analysis method, which integrates thermodynamic constraints and intracellular metabolite concentrations to restrain the solution space. Feasible flux distributions, Gibbs reaction energies and concentration ranges were computed simultaneously for the genome-wide metabolic model, with minimum bias in relation to the direction of metabolic reactions. Metabolic control analysis was applied to estimate elasticities and flux control coefficients, predicting controlling sites for L-tryptophan biosynthesis. The addition of shikimate led to enhanced deviations in chorismate biosynthesis, revealing a so far not observed control of 3-dehydroquinate synthase on L-tryptophan formation. The relative expression of the identified target genes was analysed with RT-qPCR. Transcriptome analysis revealed disparities in gene expression and the localisation of target genes to further improve the microbial L-tryptophan producer by metabolic engineering.

Metabolic control analysis of L-tryptophan production with Escherichia coli based on data from short-term perturbation experiments.

E. coli strain NT1259 /pF112aroFBLkan was able to produce 14.3 g L-1 L-tryptophan within 68 h in a fed-batch process from glycerol on a 15 L scale. To gain detailed insight into metabolism of this E. coli strain in the fed-batch process, a sample of L-tryptophan producing cells was withdrawn after 47 h, was separated rapidly and then resuspended in four parallel stirred-tank bioreactors with fresh media. Four different carbon sources (glucose, glycerol, succinate, pyruvate) were supplied individually with varying feeding rates within 19 min and the metabolic reactions of the cells in the four parallel reactors were analyzed by quantification of extracellular and intracellular substrate, product and metabolite concentrations. Data analysis allowed the estimation of intracellular carbon fluxes and of thermodynamic limitations concerning intracellular concentrations and reaction energies. Carbon fluxes and intracellular metabolite concentrations enabled the estimation of elasticities and flux control coefficients by applying metabolic control analysis making use of a metabolic model considering 48 enzymatic reactions and 56 metabolites. As the flux control coefficients describe connections between enzyme activities and metabolic fluxes, they reveal genetic targets for strain improvement. Metabolic control analysis of the recombinant E. coli cells withdrawn from the fed-batch production process clearly indicated that (i) the supply of two precursors for L-tryptophan biosynthesis, L-serine and phosphoribosyl-pyrophosphate, as well as (ii) the formation of aromatic byproducts and (iii) the enzymatic steps of igps and trps2 within the L-tryptophan biosynthesis pathway have major impact on fed-batch production of L-tryptophan from glycerol and should be the targets for further strain improvements.

2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity.

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.

Fed-batch production of l-tryptophan from glycerol using recombinant Escherichia coli.

l-tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of the recombinant l-tryptophan producer Escherichia coli NT367 was studied in controlled fed-batch processes. The chromosome of the recombinant l-tryptophan producer was equipped with additional genes coding for enzymes of the aromatic amino acids biosynthetic pathway and l-serine biosynthesis, including genes for feedback-resistant enzyme variants ( trpE fbr , aroFBL, and serA fbr ), deletions of enzymatic steps for the degradation of precursors or the product l-tryptophan ( sdaB and tnaA), and alterations in the regulation of l-tryptophan metabolism (deletion of trpL and trpR). The impact of glycerol supply rates as well as the application of a multicopy plasmid (pF112- aroFBL -kan) were investigated in fully controlled stirred-tank bioreactors on a 15 L scale. The combination of E. coli NT367 carrying pF112- aroFBL -kan and an appropriate biomass-specific glycerol supply-rate resulted in the highest final product concentration of 12.5 g L -1 l-tryptophan with the lowest concentrations of other aromatic amino acids. Fed-batch production of l-tryptophan from glycerol was shown for the first time with recombinant E. coli.