Karin Krumbach

A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level

We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes.

Linking Central Metabolism with Increased Pathway Flux: l-Valine Accumulation by Corynebacterium glutamicum

ABSTRACT Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize l-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for d-pantothenate. When the resulting strain, C. glutamicum 13032ΔilvAΔpanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM l-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the l-valine biosynthesis pathway.

MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria

Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development.

The two carboxylases of Corynebacterium glutamicum essential for fatty acid and mycolic acid synthesis

The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique alpha-branched beta-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the alpha-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated alpha-subunit AccBC, the beta-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar beta-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two beta-subunits present serve to lend specificity to this unique large multienzyme complex.

Characterization of myo-Inositol Utilization by Corynebacterium glutamicum: the Stimulon, Identification of Transporters, and Influence on l-Lysine Formation

Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.

Inactivation of Corynebacterium glutamicum NCgl0452 and the role of MgtA in the biosynthesis of a novel mannosylated glycolipid involved in lipomannan biosynthesis.

Mycobacterium tuberculosis PimB has been demonstrated to catalyze the addition of a mannose residue from GDP-mannose to a monoacylated phosphatidyl-myo-inositol mannoside (Ac1PIM1) to generate Ac1PIM2. Herein, we describe the disruption of its probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type Corynebacterium glutamicum and the C. glutamicum::pimB mutant. Following a careful analysis, two related glycolipids, Gl-A and Gl-X, were found in the parent strain, but Gl-X was absent from the mutant. The biosynthesis of Gl-X was restored in the mutant by complementation with either Cg-pimB or Mt-pimB. Subsequent chemical analyses established Gl-X as 1,2-di-O-C16/C18:1-(α-d-mannopyranosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol (ManGlcAGroAc2) and Gl-A as the precursor, GlcAGroAc2. In addition, C. glutamicum::pimB was still able to produce Ac1PIM2, suggesting that Cg-PimB catalyzes the synthesis of ManGlcAGroAc2 from GlcAGroAc2. Isolation of lipoglycans from C. glutamicum led to the identification of two related lipoglycans. The larger lipoglycan possessed a lipoarabinomannan-like structure, whereas the smaller lipoglycan was similar to lipomannan (LM). The absence of ManGlcA-GroAc2 in C. glutamicum::pimB led to a severe reduction in LM. These results suggested that ManGlcAGroAc2 was further extended to an LM-like molecule. Complementation of C. glutamicum::pimB with Cg-pimB and Mt-pimB led to the restoration of LM biosynthesis. As a result, Cg-PimB, which we have assigned as MgtA, is now clearly defined as a GDP-mannose-dependent α-mannosyltransferase from our in vitro analyses and is involved in the biosynthesis of ManGlcAGroAc2.

Biosynthesis of mycobacterial arabinogalactan: identification of a novel α(1→3) arabinofuranosyltransferase

The cell wall mycolyl‐arabinogalactan–peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt‐EmbA and Mt‐EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT‐C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG α(1→3)‐linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild‐type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor β‐d‐arabinofuranosyl‐1‐monophosphoryl‐decaprenol (DPA) and a newly synthesized linear α(1→5)‐linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching α(1→3) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target.

Disruption of Cg-Ppm1, a Polyprenyl Monophosphomannose Synthase, and the Generation of Lipoglycan-less Mutants in Corynebacterium glutamicum

The glycosyl donor, polyprenyl monophosphomannose (PPM), has been shown to be involved in the biosynthesis of the mycobacterial lipoglycans: lipomannan and lipoarabinomannan. The mycobacterial PPM synthase (Mt-ppm1) catalyzes the transfer of mannose from GDP-mannose to polyprenyl phosphates. Based on sequence homology to Mt-ppm1, we have identified the PPM synthase from Corynebacterium glutamicum. In the present study, we demonstrate that the corynebacterial synthase is composed of two distinct domains; a catalytic domain (Cg-ppm1) and a membrane domain (Cg-ppm2). Through the inactivation of Cg-ppm1, we observed a complex phenotype that included altered cell growth rate and inability to synthesize PPM molecules and lipoglycans. When Cg-ppm2 was deleted, no observable phenotype was noted, indicating the clear organization of the two domains. The complementation of the inactivated Cg-ppm1 strain with the corresponding mycobacterial enzyme (Mt-Ppm1/D2) led to the restoration of a wild type phenotype. The present study illustrates, for the first time, the generation of a lipoglycan-less mutant based on a molecular strategy in a member of the Corynebacterianeae family. Lipoglycans are important immunomodulatory molecules involved in determining the outcome of infection, and so the generation of defined mutants and their subsequent immunological characterization is timely.

Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates.

Biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. The industrially important platform organism Corynebacterium glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of α-ketoglutarate and its derivatives (e.g. l-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum. This five-step pathway, encoded by the xylXABCD-operon, enabled a recombinant C. glutamicum strain to utilize d-xylose in d-xylose/d-glucose mixtures. Interestingly, this strain exhibited a tri-phasic growth behavior and transiently accumulated d-xylonate during d-xylose utilization in the second growth phase. However, this intermediate of the implemented oxidative pathway was re-consumed in the third growth phase leading to more biomass formation. Furthermore, C. glutamicum pEKEx3-xylXABCDCc was also able to grow on d-xylose as sole carbon and energy source with a maximum growth rate of μmax=0.07±0.01h(-1). These results render C. glutamicum pEKEx3-xylXABCDCc a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on d-xylose containing substrates.

Identification and transcriptional analysis of the dapB-ORF2- dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes involved in L-lysine synthesis

The dapA gene encoding dihydrodipicolinate synthase of Corynebacterium glutamicum is pivotal for high-level L-lysine production. The entire dapB-ORF2- dapA-ORF4 gene cluster was cloned, its sequence of 5907 bp completed, and a Northern analysis was performed. Whereas deletion of ORF2 had no detectable consequences for the cell, the interruption of ORF4 resulted (i) in a decreased growth rate, (ii) an increased specific activity of the diaminopimelate decarboxylase, and (iii) a decreased specific activity of the diaminopimelate dehydrogenase. Based on these physiological features, and structural features of the ORF4 encoded protein shared with the initiation factor IF2 of Streptococcus faecium, we hypothesize that ORF4 is part of a transcriptional control element in C. glutamicum.