The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite productio

Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

Synopsis In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P . We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12 % Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways

Access to N-alkylated amino acids by microbial fermentation

N-methylated amino acids are found in many pharmaceutically active compounds and have been shown to improve pharmacokinetic properties as constituents of peptide drugs since N-methylation of amino acids may result in conformational changes, improved proteolytic stability and higher lipophilicity of the peptide drug.1 N-methylated amino acids are mainly produced chemically or by biocatalysis, however with low yields or high costs for co-factor regeneration. First, we established a fermentative route for production of N-mehtyl-L-glutamate by Pseudomonas putida from glucose and glycerol. Interception of the C1 assimilation pathway of Methylobacterium extorquence yielded N-methyl-L-glutamate titers of 17.9 g L-1 in fed-batch cultivation.2 Due to high substrate specificity of this C1 assimilation pathway genes, we continued with an independent pathway for extension of the product range. Therefore, we focus on pathway-design for N-methylated amino acids by the industrially relevant production host Corynebacterium glutamicum. Metabolic engineering of C. glutamicum led to an expanded product range of proteinogenic amino acids like L-valine2 but also ω-amino acids like γ-aminobutyrate and diamines like putrescine3. The rare imine reductase DpkA from P. putida KT2440 catalyzes the reductive methylamination of pyruvate as side activity. Implementation of DpkA into the central carbon metabolism of the pyruvate overproducing C. glutamicum strain ELB-P4 yielded N-methyl-L-alanine production. Optimization of carbon- and nitrogen ratios of the minimal medium allowed production of up to 10.5 g L-1 when cultivated in shake flasks. N-methyl-L-alanine titers of 31.7 g L-1 with a yield of 0.71 g per g glucose were achieved in fed-batch cultivation5. Due to the somewhat relaxed substrate scope of DpkA, the product portfolio of N-methylated amino acids produced by fermentation could be successfully extended. Changing the base strain to a glyoxylate producing C. glutamicum strain6 achieved production of 2.6 g L-1 sarcosine, the N-methylated glycine derivative, from glucose. Sarcosine production based on the second generation feedstocks xylose and arabinose led to higher product titers than glucose-based production and optimization of substrate composition led to a titer of 8.7 g L-1 sarcosine. This is the first example in which a C. glutamicum process using lignocellulosic pentoses is superior to glucose-based production. By mutation of the active site of DpkA, a mutant with higher specific activity towards glyoxylate (30.3 ± 2.7 U mg-1; wild type enzyme 25.7 ± 1.8 U mg-1) was identified. Therefore, the mutant DpkAF117L was incorporated into the production strain and enabled faster sarcosine production. Additionally, this mutation led to an increased activity towards reductive ethylamination of glyoxylate (31.2 ± 1.1 U mg-1; wild type enzyme 25.3 ± 3.2 U mg-1). As a result, the fermentative production of N-ethylglycine showed enhanced volumetric productivity compared to the strain harboring the wild type enzyme. Fermentative access to N-methylated amino acids was achieved by two independent pathway designs. First, we enabled N-methyl-L-glutamate production by pathway interception in P. putida. Additionally, introduction of the imine reductase gene dpkA from P. putida into various 2-oxoacid producing C. glutamicum strains extended the product range. Optimization of medium composition, preferred substrate specificity of the strain or the enzyme itself resulted in excellent production yields. 1 Chatterjee J, Rechenmacher F and Kesser H, Angew. Chem. Int. Ed., 2013, 52, 254-269. 2 Mindt M, Walter T, Risser JM and Wendisch VF, Front. Bioeng. Biotechnol., 2018, 6, 159. 3 Wendisch VF, Mindt M and Pérez-García F, Appl. Microbiol. Biotechnol., 2018, 102, 3583-3594. 4 Wieschalka S, Blombach B and Eikmanns BJ, Appl. Microbiol. Biotechnol., 2012, 94, 449-459. 5 Mindt M, Risse JM, Gruß H, Sewald N, Eikmanns BJ and Wendisch VF, Sci. Rep., 2018, 8, 12895. 6 Zahoor A, Otten A and Wendisch VF, J. Biotechnol., 2014, 192, 366-37

One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst

Mindt M, Risse JM, Gruß H, Sewald N, Eikmanns BJ, Wendisch VF. One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst. Scientific Reports. 2018;8(1): 12895.N-methylated amino acids are found in Nature in various biological compounds. N-methylation of amino acids has been shown to improve pharmacokinetic properties of peptide drugs due to conformational changes, improved proteolytic stability and/or higher lipophilicity. Due to these characteristics N-methylated amino acids received increasing interest by the pharmaceutical industry. Syntheses of N-methylated amino acids by chemical and biocatalytic approaches are known, but often show incomplete stereoselectivity, low yields or expensive co-factor regeneration. So far a one-step fermentative process from sugars has not yet been described. Here, a one-step conversion of sugars and methylamine to the N-methylated amino acid N-methyl-l-alanine was developed. A whole-cell biocatalyst was derived from a pyruvate overproducing C. glutamicum strain by heterologous expression of the N-methyl-l-amino acid dehydrogenase gene from Pseudomonas putida. As proof-of-concept, N-methyl-l-alanine titers of 31.7 g L−1 with a yield of 0.71 g per g glucose were achieved in fed-batch cultivation. The C. glutamicum strain producing this imine reductase enzyme was engineered further to extend this green chemistry route to production of N-methyl-l-alanine from alternative feed stocks such as starch or the lignocellulosic sugars xylose and arabinose

The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria

ISSN:0168-6445ISSN:1574-697

Bio-based production of organic acids with Corynebacterium glutamicum

The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG

TCA Cycle and Glyoxylate Shunt of Corynebacterium glutamicum

The enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles of Corynebacterium glutamicum and in particular their regulation have been intensively studied in the past years. Nearly all TCA and glyoxylate cycle genes are subject to growth phase- or carbon source-dependent transcriptional regulation. Seven different regulators were shown to be involved in expression control of TCA and glyoxylate cycle genes, i.e., AcnR, DtxR, GlxR, RamA, RamB, RipA, and SucR. At the level of enzyme activity, the 2-oxoglutarate dehydrogenase (ODH) complex was found to be controlled by the inhibitor protein OdhI in dependency of its phosphorylation state, which is determined by the serine/threonine protein kinases PknG, PknA, PknB, and PknL and by the phospho-serine/threonine protein phosphatase Ppp. OdhI was shown to be crucial for glutamate production. This chapter summarizes new data on TCA cycle enzymes and describes the current knowledge on the regulation of this pathway and of the glyoxylate shunt

Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum▿

Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for oxaloacetate of 1.4 mM and a Vmax of 201 μmol of oxaloacetate converted per min per mg of protein, resulting in a kcat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested

The Alcohol Dehydrogenase Gene adhA in Corynebacterium glutamicum Is Subject to Carbon Catabolite Repression▿

Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene (adhA), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum. Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA, and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose- and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA