Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11?±?0.004 h^?1 and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.     

The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression of ptsG, which encodes the glucose-specific PTS enzyme II. Overexpression of sugR resulted in reduced ptsG mRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lacking sugR, expression of the ptsG'-'cat fusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, including ptsS and the putative NCgl1859-fruK-ptsF operon. Purified SugR bound to DNA regions upstream of ptsG, ptsS, and NCgl1859, and a 75-bp ptsG promoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to the ptsG promoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR represses ptsG, ptsG expression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability.     

Engineering of a xylose metabolic pathway in Corynebacterium glutamicum

The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertès, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.     

Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum

Heterologous expression of a phytase gene (phyC) from Bacillus amyloliquefaciens DSM 7 enabled the growth of Corynebacterium glutamicum with phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) as a new, sole source of phosphorus. Phytate was not used as a carbon source. During growth of the phyC-expressing amino acid (l-lysine)-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) with phytate as the source of phosphorus, merely a small, transient accumulation of inorganic phosphate was observed in the fermentation broth. At the later stages of fermentation, free inorganic phosphate from phytate degradation was no longer detectable. Growth and l-lysine production by the phytase-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) in phytate medium did not differ significantly from control experiments with strain C. glutamicum ATCC 21253 (pWLQ2) conducted with an excess of inorganic phosphate, demonstrating that there was no phosphate limitation when phytate was used as the phosphorus source. Under the expression conditions employed, only part of PhyC was secreted to the culture broth by C. glutamicum, but this did not significantly affect growth or lysine production.     

The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum

In this study, we analyzed the whcA gene from Corynebacterium glutamicum , which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the Δ whcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA -overexpressing cells, whereas its activity in the Δ whcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

Molecular analysis of the Corynebacterium glutamicum transketolase gene

Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.     

Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified. Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998     

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.     

Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum

Despite its key position in central metabolism, L-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, L-serine is consumed at rates up to 19.4 +/- 4.0 nmol min(-1) (mg [dry weight])(-1), resulting in the complete consumption of 100 mM L-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine beta-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent Km, 11 mM) prevents substantial L-serine degradation.     

Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction

AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms.     

Corynebacterium glutamicum whcB, a stationary phase-specific regulatory gene

The function of whcB, one of the four whiB homologues of Corynebacterium glutamicum, was assessed. Cells carrying the P(180)-whcB clone, and thus overexpressing the whcB gene, showed retarded growth, probably due to increased sensitivity to oxidants, whereas cells lacking whcB (ΔwhcB) did not. However, growth retardation was not observed in cells with additionally whcE deleted. Furthermore, the ΔwhcE phenotype, characterized by slow growth and sensitivity to oxidants, was reversed in cells carrying P(180)-whcB. Like the whcE gene, which is also known as a whiB homologue, the whcB gene was preferentially expressed in stationary phase. Determination of the genes under regulation of whcB using two-dimensional polyacrylamide gel electrophoresis identified several genes involved in electron transfer reactions that were regulated in cells carrying P(180)-whcB. Collectively, these findings indicate that whcB function requires whcE. Furthermore, whcB and whcE are paralogues but perform distinct regulatory roles during growth under oxidative stress.     

Impact of transport processes in the osmotic response of Corynebacterium glutamicum

Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum. Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress. For the adaptation to hypoosmotic stress C. glutamicum possesses at least three different mechanosensitive (MS) channels. To overcome hyperosmotic stress C. glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake. Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs. Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine. In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes. BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail. This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation). BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.