Opening a novel biosynthetic pathway to dihydroxyacetone and glycerol in Escherichia coli mutants through expression of a gene variant (fsaAA129S) for fructose 6-phosphate aldolase

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.Ministerium für Wissenschaft und Kunst of Baden-Württember

Complementation of an Escherichia coli K-12 mutant strain deficient in KDO synthesis by forming D-arabinose 5-phosphate from glycolaldehyde with fructose 6-phosphate aldolase (FSA)

KDO (2-keto-3-deoxy-D-manno-octulosonate) is a landmark molecule of the Gram-negative outer membrane. Mutants without KDO formation are known to be barely viable. Arabinose 5-phosphate (A5P) is a precursor of KDO biosynthesis and is normally derived from ribulose 5-phosphate by A5P isomerases, encoded by kdsD and gutQ genes in E. coli K-12. We created a kdsD gutQ-deficient double mutant of strain BW25113 and confirmed that these cells are A5P auxotrophs. Fructose 6-phosphate aldolase (FSA) is known to utilize (among other donors such as dihydroxyacetone or hydroxyacetone) glycolaldehyde (GoA) as a donor compound and to provide A5P in vitro when glyceraldehyde 3-phosphate is the acceptor. We show here that this FSA function in vivo fully reverses the growth defect and the A5P deficiency in kdsD gutQ double mutants. Expression of both plasmid-encoded fsaA, fsaAA129S, or fsaB genes as well as a chromosomally integrated form of fsaAA129S led to maximal OD600 values of >2.2 when GoA was added exogenously (together with glucose as a C source) at a concentration of 100 µM (Ks values in the range of 4-10 µM). Thus, a novel bio-orthogonal bypass to overcome an A5P deficiency was opened. Lower GoA concentrations led to lower growth yields. Interestingly, mutant strains with recombinant fsa genes showed considerable growth yields even without exogenous GoA addition, pointing to yet unknown endogenous GoA sources in E. coli metabolism. This is a further example of the usefulness of FSA in rewiring central metabolic pathways in E. coli

Escherichia coli reporter strains allow for the in vivo evaluation of recombinant elongation factor protein (EF-P)

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats.Deutsche Akademische Austauschdienst (German Academic Exchange Service DAAD

Stability of a mutualistic Escherichia coli co‐culture during violacein production depends on the kind of carbon source

The L‐tryptophan-derived purple pigment violacein (VIO) is produced in recombinant bacteria and studied for its versatile applications. Microbial synthetic co‐cultures are gaining more importance as efficient factories for synthesizing high‐value compounds. In this work, a mutualistic and cross‐feeding Escherichia coli co‐culture is metabolically engineered to produce VIO. The strains are genetically modified by auxotrophies in the tryptophan (TRP) pathway to enable a metabolic division of labor. Therein, one strain produces anthranilate (ANT) and the other transforms it into TRP and further to VIO. Population dynamics and stability depend on the choice of carbon source, impacting the presence and thus exchange of metabolites as well as overall VIO productivity. Four carbon sources (D‐glucose, glycerol, D‐galactose, and D‐xylose) were compared. D‐Xylose led to co‐cultures which showed stable growth and VIO production, ANT‐TRP exchange, and enhanced VIO production. Best titers were ∼126 mg L -1 in shake flasks. The study demonstrates the importance and advantages of a mutualistic approach in VIO synthesis and highlights the carbon source's role in co‐culture stability and productivity. Transferring this knowledge into an up‐scaled bioreactor system has great potential in improving the overall VIO production.German Research Foundation (DFG

Metabolic Engineering of Escherichia coli for para-Amino-Phenylethanol and para-Amino-Phenylacetic Acid Biosynthesis

Aromatic amines are an important class of chemicals which are used as building blocks for the synthesis of polymers and pharmaceuticals. In this study we establish a de novo pathway for the biosynthesis of the aromatic amines para-amino-phenylethanol (PAPE) and para-amino-phenylacetic acid (4-APA) in Escherichia coli. We combined a synthetic para-amino-l-phenylalanine pathway with the fungal Ehrlich pathway. Therefore, we overexpressed the heterologous genes encoding 4-amino-4-deoxychorismate synthase (pabAB from Corynebacterium glutamicum), 4-amino-4-deoxychorismate mutase and 4-amino-4-deoxyprephenate dehydrogenase (papB and papC from Streptomyces venezuelae) and ThDP-dependent keto-acid decarboxylase (aro10 from Saccharomyces cerevisiae) in E. coli. The resulting para-amino-phenylacetaldehyde either was reduced to PAPE or oxidized to 4-APA. The wild type strain E. coli LJ110 with a plasmid carrying these four genes produced (in shake flask cultures) 11 ± 1.5 mg l−1 of PAPE from glucose (4.5 g l−1). By the additional cloning and expression of feaB (phenylacetaldehyde dehydrogenase from E. coli) 36 ± 5 mg l−1 of 4-APA were obtained from 4.5 g l−1 glucose. Competing reactions, such as the genes for aminotransferases (aspC and tyrB) or for biosynthesis of L-phenylalanine and L-tyrosine (pheA, tyrA) and for the regulator TyrR were removed. Additionally, the E. coli genes aroFBL were cloned and expressed from a second plasmid. The best producer strains of E. coli showed improved formation of PAPE and 4-APA, respectively. Plasmid-borne expression of an aldehyde reductase (yahK from E. coli) gave best values for PAPE production, whereas feaB-overexpression led to best values for 4-APA. In fed-batch cultivation, the best producer strains achieved 2.5 ± 0.15 g l−1 of PAPE from glucose (11% C mol mol-1 glucose) and 3.4 ± 0.3 g l−1 of 4-APA (17% C mol mol−1 glucose), respectively which are the highest values for recombinant strains reported so far

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.Projekt DEALDeutsche Forschungsgemeinschaf

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L−1). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.Fil: Fries, Alexander Erich. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Mazzaferro, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina. Albert Ludwigs University of Freiburg; AlemaniaFil: Grüning, Björn. Albert Ludwigs University of Freiburg; AlemaniaFil: Bisel, Philippe. Albert Ludwigs University of Freiburg; AlemaniaFil: Stibal, Karin. Albert Ludwigs University of Freiburg; AlemaniaFil: Buchholz, Patrick C. F.. University of Stuttgart; AlemaniaFil: Pleiss, Jürgen. Universität Stuttgart;Fil: Sprenger, Georg A.. Universität Stuttgart;Fil: Müller, Michael. Albert Ludwigs University of Freiburg; Alemani

Genetic engineering approaches for the fermentative production of phenylglycines

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic β-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs.Deutsche ForschungsgemeinschaftBaden-Württemberg-StiftungDeutsches Zentrum für InfektionsforschungProjekt DEA

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach.

Funder: The Russian Government Program of Competitive Growth of Kazan Federal UniversityFor the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections