Although L-serine proceeds in just three steps from the glycolytic intermediate 3-phosphoglycerate, and as much as 8% of the carbon assimilated from glucose is directed via L-serine formation, previous attempts to obtain a strain producing L-serine from glucose have not been successful. We functionally identified the genes serC and serB from Corynebacterium glutamicum, coding for phosphoserine aminotransferase and phosphoserine phosphatase, respectively. The overexpression of these genes, together with the third biosynthetic serA gene, serA ⌬197 , encoding an L-serine-insensitive 3-phosphoglycerate dehydrogenase, yielded only traces of L-serine, as did the overexpression of these genes in a strain with the L-serine dehydratase gene sdaA deleted. However, reduced expression of the serine hydroxymethyltransferase gene glyA, in combination with the overexpression of serA ⌬197 , serC, and serB, resulted in a transient accumulation of up to 16 mM L-serine in the culture medium. When sdaA was also deleted, the resulting strain, C. glutamicum ⌬sdaA::pK18mobglyA (pEC-T18mob2serA ⌬197 CB), accumulated up to 86 mM L-serine with a maximal specific productivity of 1.2 mmol h ؊1 g (dry weight) ؊1 . This illustrates a high rate of L-serine formation and also utilization in the C. glutamicum wild type. Therefore, metabolic engineering of L-serine production from glucose can be achieved only by addressing the apparent key position of this amino acid in the central metabolism.The demand of L-serine is about 300 tons per year, and this amino acid is required for the pharmaceutical and the cosmetic industries, in addition to being a building block for chemical and biochemical purposes (6). The current production relies mainly on its enzymatic or cellular conversion from the precursor glycine plus a C 1 compound. Utilizing the condensing activity of serine hydroxymethyltransferase, an enzymatic system has been elaborated to convert glycine plus formaldehyde to L-serine (15). The cellular systems employed, among others, resting cells of methanol-utilizing bacteria such as Hyphomicrobium methylovorum where L-serine accumulation from glycine plus methanol was achieved (16). Also, a fermentative production of L-serine from glycine alone by Corynebacterium glycinophilum was described (19). However, there is not much information on the direct fermentative production of L-serine from glucose. Attempts to isolate L-serine-producing strains using different bacteria by applying undirected mutagenesis yielded mutants accumulating only traces of L-serine (38). Apparently, the direct conversion of glucose is a demanding challenge, probably due to the role of L-serine as a central intermediate for a number of cellular reactions (Fig. 1).We are interested in the amino acid-synthesizing capabilities of Corynebacterium glutamicum, which is traditionally used for the large-scale production of L-glutamate and L-lysine (9). In general, the efforts to engineer producing strains were focused on the enzymes of the biosynthesis pathways. For instance, con...

The amino acid L-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of L-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on P tac and lacI Q are unstable, acquiring mutations in lacI Q , for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum ⌬sdaA strain (pserACB) accumulates only traces of L-serine, with the C. glutamicum ⌬pabABC⌬sdaA strain (pserACB), L-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM L-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM L-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial L-serine production is available.L-Serine is a nonessential amino acid but plays an important role in stabilizing the blood sugar concentration in the liver (16). It relates, furthermore, to many other substances, including sphingosine and the phosphatides, which are part of the myelin covering of the nerves, as well as the formation of activated C 1 units used for a number of anabolic processes (20). Therefore, L-serine is present in selected infusion solutions and also has other applications. For instance, it is an ingredient of skin lotions to ensure a proper hydration status. The total annual demand for L-serine is estimated to be 300 tons (5).The production processes currently used still rely on the extraction of L-serine from protein hydrolysates or from molasses, as well as on the enzymological conversion of glycine plus a C 1 compound, like methanol, to L-serine. The latter uses the reverse reaction of the serine hydroxymethyltransferase (SHMT) (6). Thus, an enzymatic system has been designed to convert glycine plus formaldehyde to L-serine (4). The cellular systems assayed employed, among other things, resting cells of methanol-utilizing bacteria, such as Hyphomicrobium methylovorum, where L-serine formation from glycine plus methanol was achieved (6). In such a system, up to 45 g liter Ϫ1 L-serine accumulation was possible, but only at a glycine yield of 50%, thus making the system less attractive. Also, alginate-entrapped cells of Corynebacterium glycinophilum were used for L-serine formation from glycine (21). It is self-evident that it would be most profitable to directly convert cheap sugar into L-serine. Although microbial processes for amino acid production are in general advancing quickly, attempts to develop L-serin...

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A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactanpeptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of basestable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.A common feature of members of the Corynebacterineae is that they possess an unusual cell wall dominated by a heteropolysaccharide termed an arabinogalactan (AG), which is linked to both mycolic acids and peptidoglycan, forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex (5,10,12,15,24,25,34). The formation of the arabinan domain in the mAGP complex, consisting mainly of ␣135, ␣133, and ␤132 glycosyl linkages, results from the subsequent addition of arabinofuranose (Araf) from the lipid-linked sugar donor ␤-D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) by a set of unique membrane-bound arabinofuranosyltransferases (5,7,12,18,34).The deletion of Corynebacterium glutamicum emb (emb Cg ) (4) and a chemical analysis of the cell wall revealed a novel truncated AG structure possessing only terminal Araf residues with a corresponding loss of cell wall-bound mycolic acids (4). The presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb Cg proteins led to the identification of AftA, which belongs to the glycosyltransferase C (GT-C) superfamily (5). Recently, additional GT-C enzymes have been identified, termed AftB, which is responsible for the attachment of terminal ␤(132) Araf residues (34), and AftC, which is involved in AG branching (12) before decoration with mycolic acids, both of which are conserved within the Corynebacterineae (12, 34). It is clear that additional glycosyltransferases involved in both AG and lipoarabinomannan biosynthesis still remain to be identified. Indeed, Liu and Mushegian (22) identified 15 members of the GT-C superfamily residing in the Corynebacterineae, representing candidates involved in the biosynthesis of cell wall-related glycans and lipoglycans (22). We have continued our earlier studies (5, 12, 34) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex by studying mutants of C. glutamicum and the orthologous genes and enzymes of Mycobacterium tuberculosis.A particularly interesting feature of...

Gene expression in the obligately aerobic acetic acid bacterium Gluconobacter oxydans responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the FNR family in this species. Evidence was obtained that GoxR contains an iron-sulfur cluster, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches including a transcriptome comparison of a ΔgoxR mutant with the wild type and detection of in vivo GoxR binding sites by ChAP-Seq. Prominent targets were the cioAB genes encoding a cytochrome bd oxidase with low O2 affinity, which were repressed by GoxR, and the pnt operon, which was activated by GoxR. The pnt operon encodes a transhydrogenase (pntA1A2B), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of G. oxydans due to the periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to an inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the pnt operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by G. oxydans under oxygen-restricted conditions in a GoxR-dependent manner. IMPORTANCE Gluconobacter oxydans serves as cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of G. oxydans has been studied experimentally up to now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because G. oxydans has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and NADH reoxidation by reduction of acetaldehyde to ethanol.