Adaptive laboratory evolution of Pseudomonas putida and Corynebacterium glutamicum to enhance anthranilate tolerance.

Microbial bioproduction of the aromatic acid anthranilate (ortho-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms Corynebacterium glutamicum and Pseudomonas putida are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for C. glutamicum and P. putida, to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.

Regulation of solvent tolerance in Pseudomonas putida S12 mediated by mobile elements.

Organic solvent-tolerant bacteria are outstanding and versatile hosts for the bio-based production of a broad range of generally toxic aromatic compounds. The energetically costly solvent tolerance mechanisms are subject to multiple levels of regulation, involving among other mobile genetic elements. The genome of the solvent-tolerant Pseudomonas putida S12 contains many such mobile elements that play a major role in the regulation and adaptation to various stress conditions, including the regulation of expression of the solvent efflux pump SrpABC. We recently sequenced the genome of P. putida S12. Detailed annotation identified a threefold higher copy number of the mobile element ISS12 in contrast to earlier observations. In this study, we describe the mobile genetic elements and elaborate on the role of ISS12 in the establishment and maintenance of solvent tolerance in P. putida. We identified three different variants of ISS12 of which a single variant exhibits a high translocation rate. One copy of this variant caused a loss of solvent tolerance in the sequenced strain by disruption of srpA. Solvent tolerance could be restored by applying selective pressure, leading to a clean excision of the mobile element.

Metabolic response of Pseudomonas putida to increased NADH regeneration rates.

Pseudomonas putida efficiently utilizes many different carbon sources without the formation of byproducts even under conditions of stress. This implies a high degree of flexibility to cope with conditions that require a significantly altered distribution of carbon to either biomass or energy in the form of NADH. In the literature, co-feeding of the reduced C1 compound formate to Escherichia coli heterologously expressing the NAD+-dependent formate dehydrogenase of the yeast Candida boidinii was demonstrated to boost various NADH-demanding applications. Pseudomonas putida as emerging biotechnological workhorse is inherently equipped with an NAD+-dependent formate dehydrogenase encouraging us to investigate the use of formate and its effect on P. putida's metabolism. Hence, this study provides a detailed insight into the co-utilization of formate and glucose by P. putida. Our results show that the addition of formate leads to a high increase in the NADH regeneration rate resulting in a very high biomass yield on glucose. Metabolic flux analysis revealed a significant flux rerouting from catabolism to anabolism. These metabolic insights argue further for P. putida as a host for redox cofactor demanding bioprocesses.

A rapid method to estimate NADH regeneration rates in living cells.

We introduce a rapid whole cell assay for the estimation of NADH regeneration rates based on the fluorescent dye resazurin. A co-feed of formate and glucose, known to increase the intrinsic NADH regeneration rate of P. putida KT2440, was chosen as model system for the validation of this assay.

Metabolic Engineering of Pseudomonas putida KT2440 to Produce Anthranilate from Glucose.

The Pseudomonas putida KT2440 strain was engineered in order to produce anthranilate (oAB, ortho-aminobenzoate), a precursor of the aromatic amino acid tryptophan, from glucose as sole carbon source. To enable the production of the metabolic intermediate oAB, the trpDC operon encoding an anthranilate phosphoribosyltransferase (TrpD) and an indole-3-glycerol phosphate synthase (TrpC), were deleted. In addition, the chorismate mutase (pheA) responsible for the conversion of chorismate over prephenate to phenylpyruvate was deleted in the background of the deletion of trpDC to circumvent a potential drain of precursor. To further increase the oAB production, a feedback insensitive version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by the aroG (D146N) gene and an anthranilate synthase (trpE (S40F) G) were overexpressed separately and simultaneously in the deletion mutants. With optimized production conditions in a tryptophan-limited fed-batch process a maximum of 1.54 ± 0.3 g L(-1) (11.23 mM) oAB was obtained with the best performing engineered P. putida KT2440 strain (P. putida ΔtrpDC pSEVA234_aroG (D146N) _trpE (S40F) G).

Nontoxic, Hydrophilic Cationic Polymers-Identified as Class of Antimicrobial Polymers.

Amphiphilic polycations are an alternative to biocides but also toxic to mammalian cells. Antimicrobially active hydrophilic polycations based on 1,4-dibromo-2-butene and tetramethyl-1,3-propanediamine named PBI are not hemotoxic for porcine red blood cells with a hemocytotoxicity (HC50) of more than 40,000 µg · mL(-1). They are quickly killing bacterial cells at their MIC (minimal inhibitory concentration). The highest found selectivity HC50 /MIC is more than 20,000 for S. epidermidis. Investigations on sequentially prepared PBIs with defined molecular weight Mn and tailored end groups revealed that there is a dependence of antimicrobial activity and selectivity on Mn and nature of the end groups.