Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products.

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction.

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.

Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct.

para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of the shikimate pathway. The resultant strain produced 45mM of PABA in a test-tube scale culture. Under a fermenter-controlled condition, the strain produced up to 314mM (43g/L) of PABA at 48h, with a 20% yield. To our knowledge, this is the highest concentration of PABA produced by a genetically modified microorganism ever reported.

History-Driven Genetic Modification Design Technique Using a Domain-Specific Lexical Model for the Acceleration of DBTL Cycles for Microbial Cell Factories.

The development of microbes for conducting bioprocessing via synthetic biology involves design-build-test-learn (DBTL) cycles. To aid the designing step, we developed a computational technique that suggests next genetic modifications on the basis of relatedness to the user's design history of genetic modifications accumulated through former DBTL cycles conducted by the user. This technique, which comprehensively retrieves well-known designs related to the history, involves searching text for previous literature and then mining genes that frequently co-occur in the literature with those modified genes. We further developed a domain-specific lexical model that weights literature that is more related to the domain of metabolic engineering to emphasize genes modified for bioprocessing. Our technique made a suggestion by using a history of creating a Corynebacterium glutamicum strain producing shikimic acid that had 18 genetic modifications. Inspired by the suggestion, eight genes were considered by biologists for further modification, and modifying four of these genes proved experimentally efficient in increasing the production of shikimic acid. These results indicated that our proposed technique successfully utilized the former cycles to suggest relevant designs that biologists considered worth testing. Comprehensive retrieval of well-tested designs will help less-experienced researchers overcome the entry barrier as well as inspire experienced researchers to formulate design concepts that have been overlooked or suspended. This technique will aid DBTL cycles by feeding histories back to the next genetic design, thereby complementing the designing step.

Homozygous triplicate mutations in three 16S rRNA genes responsible for high-level aminoglycoside resistance in Nocardia farcinica clinical isolates from a Canada-wide bovine mastitis epizootic.

Nocardia farcinica strains showing high-level resistance to amikacin were isolated from clinical cases in a Canada-wide bovine mastitis epizootic. Shotgun cloning of the resistance genes in the amikacin-resistant mastitis isolate N. farcinica IFM 10580 (W6220 [Centers for Disease Control and Prevention]) using a multicopy vector system revealed that the 16S rRNA gene with an A-to-G single-point mutation at position 1408 (in Escherichia coli numbering) conferred "moderate" cross-resistance to amikacin and other aminoglycosides to an originally susceptible N. farcinica strain IFM 10152. Subsequent DNA sequence analyses revealed that, in contrast to the susceptible strain, all three chromosomal 16S rRNA genes of IFM 10580, the epizootic clinical strain, contained the same A1408G point mutations. Mutant colonies showing high-level aminoglycoside resistance were obtained when the susceptible strain N. farcinica IFM 10152 was transformed with a multicopy plasmid carrying the A1408G mutant 16S rRNA gene and was cultured in the presence of aminoglycosides for 3 to 5 days. Of these transformants, at least two of the three chromosomal 16S rRNA genes contained A1408G mutations. A triple mutant was easily obtained from a strain carrying the two chromosomal A1408G mutant genes and one wild-type gene, even in the absence of the plasmid. The triple mutant showed the highest level of resistance to aminoglycosides, even in the absence of the plasmid carrying the mutant 16S rRNA gene. These results suggest that the homozygous mutations in the three 16S rRNA genes are responsible for the high-level aminoglycoside resistance found in N. farcinica isolates of the bovine mastitis epizootic.

Trf4 is a useful gene for discrimination of Candida tropicalis from other medically important Candida species.

Comparative studies of random amplified polymorphic DNA (RAPD) band patterns of Candida tropicalis with those of clinically important Candida species have shown the presence of specific RAPD bands for C. tropicalis. A band specific to C. tropicalis strains (ca. 400 bp) was extracted and sequenced. It was found to belong to a fragment of the Trf4 gene, which is essential for growth of these strains and has a characteristic sequence of C. tropicalis. A PCR primer was designed specifically for C. tropicalis which amplifies the 324 bp band. The PCR primer amplified DNA products for all C. tropicalis strains tested, but did not amplify any PCR bands from C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. kefer, C. krusei, C. parapsilosis, or C. zeylanoides. Usefulness of the PCR primer in differentiating from clinical isolates of other fungal species is discussed.

Enhanced induction of cytochromes P450alk that oxidize methyl-ends of n-alkanes and fatty acids in the long-chain dicarboxylic acid-hyperproducing mutant of Candida maltosa.

In the long-chain dicarboxylic acids (DCA)-hyperproducing mutant Candida maltosa strains, methyl-ends of n-alkanes and fatty acids are hydroxylated by n-alkane inducible cytochromes P450 (P450alk), presumably as an essential step in DCA production. A significantly higher production of P450alks was observed in response to n-alkane in the DCA-hyperproducing mutant strain M2030 than in the wild-type strain 1098. Northern analysis demonstrated that n-tetradecane induction levels of mRNAs of all four ALK genes encoding major P450alk isoforms involved in n-alkane assimilation were significantly higher in the DCA-hyperproducing mutant than in the wild-type strain. Among these four ALK genes, enhancement of the transcriptional induction level of ALK5, which prefers fatty acids as substrates, was prominent in the mutant. In agreement with Northern analysis, promoters of ALK genes, especially that of ALK5, more strongly responded to n-alkanes in the DCA-hyperproducing mutant than in the wild-type strain. These results suggest that the transcriptional control of ALK genes in the DCA-hyperproducing mutant strains was altered preferably to accelerate DCA production.

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli.

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.

Construction of a pair of practical Nocardia-Escherichia coli shuttle vectors.

We constructed a pair of Nocardia-Escherichia coli shuttle vectors, pNV18 and pNV19, by combining the mycobacterial plasmid pAL5000 with the E. coli vector pK18 or pK19. These vectors have a number of useful features, including small size (4.4 kb), a multiple cloning site, and blue/white selection. To our knowledge, pNV18 and pNV19 are the first cloning vectors for practical use in Nocardia spp.

Identification of Nocardia farcinica by a PCR primer amplifying a specific DNA band for the bacterium.

A PCR primer specific to Nocardia farcinica was prepared based on sequence information of random amplified polymorphic DNA (RAPD) analysis. The PCR primer amplifies N. farcinica species only; no amplification was observed in 25 other Nocardia strains that we tested. Specificity of the primer for N. farcinica was also confirmed using other fungal and bacterial strains that are frequently isolated from clinical samples such as sputa and broncho alveolar lavage (VAL).

Multilocus microsatellite typing for Cryptococcus neoformans var. grubii.

Fifteen randomly selected microsatellites (simple sequence repeats; SSRs), from the H99 Cryptococcus neoformans var. grubii (serotype A) genome, were sequenced, characterized and applied to sequence 87 clinical and environmental C. neoformans var. grubii isolates from 12 different countries based on Multilocus Microsatellite Typing (MLMT). Among the 15 SSR loci, three (designated CNG1, CNG2 and CNG3) were polymorphic, while the remaining 12 SSR loci showed no variations. The specific PCR primers of the polymorphic microsatellites, i.e., CNG1, CNG2 and CNG3, amplified those loci only from strains of C. neoformans (C. neoformans var. grubii, C. neoformans var. neoformans and the AD hybrid) but not from Cryptococcus gattii, suggesting a species-specific association. The three polymorphic microsatellites are useful markers for strain genotyping, population genetic analysis, epidemiological studies, and may be helpful for the diagnosis of cryptococcosis due to C. neoformans.

Nocardia terpenica sp. nov., isolated from Japanese patients with nocardiosis.

Two bacterial strains isolated from different hospitals in Japan were subjected to a polyphasic analysis. Strains IFM 0406 and IFM 0706(T), producers of novel terpenoid antibiotics, were found to have morphological, biochemical, physiological and chemotaxonomic properties consistent with their classification in the genus Nocardia, except for the presence of MK-8(H(4)) as one of the predominant menaquinones in addition to the major menaquinone MK-8(H(4 omega-cyc)). The 16S rRNA gene sequence similarity of strains IFM 0406 and IFM 0706(T) was 99.9 %, and the closest members of Nocardia to these strains were the type strains of Nocardia nova and Nocardia mexicana, showing similarity of 97.5 and 97.1 %, respectively. Based on their characteristic phenotypic and phylogenetic properties, a novel species of the genus Nocardia, Nocardia terpenica sp. nov. is proposed for the two strains. The type strain is IFM 0706(T) (=JCM 13033(T)=DSM 44935(T)=NBRC 100888(T)).

Majority of Actinomadura clinical isolates from sputa or bronchoalveolar lavage fluid in Japan belongs to the cluster of Actinomadura cremea and Actinomadura nitritigenes, and the description of Actinomadura chibensis sp. nov.

In Japan during 1996-2004, 21 actinomycete strains that have madurose as the diagnostic cell-wall sugar and show true branching in their substrate and aerial mycelia were isolated from sputa or bronchoalveolar lavage (BAL) fluid of patients with pulmonary infections or who were suspected of having related infections. Chemotaxonomic studies showed that all the isolates belong to the genus Actinomadura. Among them, six and seven strains were classified respectively into clusters of Actinomadura nitritigenes and Actinomadura cremea based on 16S rDNA analyses because their 16S rDNA similarities to those respective species were greater than 99.5%. To our knowledge, this is first report that strains of above two species were isolated from clinical specimens. Neither Actinomadura madurae nor Actinomadura pelletieri strain was isolated, and one new species, Actinomadura chibensis, was proposed; the remaining seven strains were not assigned into any known species, suggesting the presence of another new Actinomadura species.

n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa.

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.