No abstract

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within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process.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.

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 , aroFBL, and serA ), 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 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.

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-Darabino-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 (AroF cg ). 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 AroF cg variants, two yielded > 80% (E154N) and > 50% (P155L) residual activity at 5 mM Tyr and showed > 50% specific activity of the wt AroF cg 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.

We developed plasmid systems pJLIC and pJLRCS that allow the easy cloning of promoter regions upstream of reporter genes (lacZ and gfp) and the subsequent transfer of the resulting reporter cassettes into the chromosome of Escherichia coli by recombineering. Thus, a promoter region can be inserted upstream of lacZ gene in the context of the lactose operon of recipient E. coli strains. Moreover, a promoter–GFP reporter is inserted at a nonessential site of the chromosome. These systems allow for selection and promoter strength analysis at the single copy level. We also provide a collection of E. coli knock‐out mutants in genes for global regulation of C, N, and O metabolism, and combinations of these mutants with the newly constructed chromosomal lacZ and gfp reporter system. This allows assaying transcriptional activities of promoters before the background of different mutants. Both lacZ and gfp reporter cassettes (including appropriate antibiotic resistance markers) and the regulator knock‐outs may be easily transferred by phage P1 mediated transduction to other E. coli strains. As a proof of principle, ß‐galactosidase activity and GFP fluorescence were compared for lacZ and gfp reporter systems placed under the control of the PrssA promoter both in a wild‐type and an isogenic rpoS‐mutant strain.