Biology subjects

Marienhagen, J.

Publications and source records attributed to Marienhagen, J..

5 recordsLinked to original sources

bioRxiv2023

Metabolic rewiring compensates for the loss of glutamate and aspartate biosynthesis in Bacillus subtilis

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, with excess nitrogen, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle. It will be interesting to investigate whether the B. subtilis strain using the alternative glutamate biosynthesis route can evolve in such a way that it robustly grows during nitrogen limitation and excess.

microbiology↗

bioRxiv2023

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose

Anthranilate and its derivative aniline are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, aniline is mainly chemically produced from petroleum-derived benzene, but it could be also obtained more sustainably by decarboxylation of the microbially produced shikimate pathway intermediate anthranilate. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy- arabinoheptulosonate-7-phosphate synthase from E. coli, catalyzing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback-resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production, but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates, or any other aromatic compound derived thereof.

bioengineering↗

bioRxiv2021

Aminoglycoside antibiotics inhibit phage infection by blocking an early step of the phage infection cycle

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, hinting at a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect, but retains its antiviral properties. Altogether, this study expands the knowledge of potential aminoglycoside functions in bacterial communities suggesting that aminoglycosides are not only used by their producers as toxic molecules against their bacterial competitors, but could also provide protection against the threat of phage predation at the community level.

microbiology↗

bioRxiv2021

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose with Corynebacterium glutamicum

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of O_SCPLOWDC_SCPLOW-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 {+/-} 12.1 mM (9.6 {+/-} 1.9 g L-1) from O_SCPLOWDC_SCPLOW-xylose as the primary carbon and energy source. The corresponding yield was [Formula], which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on O_SCPLOWDC_SCPLOW-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 {+/-} 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a threechamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

bioengineering↗

bioRxiv2019

Competence of Corynebacterium glutamicum as a host for the production of type I polyketides

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

bioengineering↗