Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Metabolic evolution and a comparative omics analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for putrescine production

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor l-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To construct a strain of Corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ALA) via the C4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.

Results

The recombinant C. glutamicum strain AP2 harboring a codon-optimized hemA gene from Rhodobacter sphaeroides was used as host strain for 5-ALA production. A plasmid harboring the serine operon, which contained serB, serC and the site-specific mutant serA ^Δ197 , was constructed and introduced into C. glutamicumAP2, leading to an increase of 70% in 5-ALA production. Further overexpression of the glyA gene increased production of 5-ALA by 150% over the control. 5-ALA production was thus significantly enhanced by engineering the glycine biosynthetic pathway. C.glutamicum AG3 produced 3.4 ± 0.2 g 5-ALA/l in shake-flask cultures in CGIIIM medium with the addition of 7.5 g glycine/l.

Conclusion

This is the first report of remodeling the serine and glycine biosynthetic pathway to improve the production of 5-ALA in C. glutamicum.

     

A method for simultaneous gene overexpression and inactivation in the <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> genome

The gene integration method is an important tool to stably express desirable genes in bacteria. To avoid heavy workload and cost, we constructed a rapid and efficient method for genome modification. This method depended on a mobilizable plasmid, which contains a P _tac promoter, an introduced multiple cloning site (iMCS), and rrnBT1T2 terminator. Briefly, the mobilizable plasmid pK18-MBPMT with the P _tac-iMCS-rrnBT1T2 cartridge derived from pK18mobsacB was prepared to directly integrate hetero-/homologous DNA into the Corynebacterium glutamicum genome. Like our previous method, this method was based on insertional inactivation and double-crossover homologous recombination, which simultaneously achieved gene overexpression and inactivation in the genome without the use of genetic markers. Compared to the previous method, this protocol omitted the construction of a recombinant expression plasmid and clone of the target gene(s) cassette, which significantly decreased the workload, cost, and operational time. Using this method, the heterologous gene amy and the homologous gene lysC ^T311I were successfully integrated into the C. glutamicum genome at alaT and avtA loci, respectively. Moreover, the operation time of this method was shorter than that of the previous method, especially for repeated integration. This method, which is based on the mobilizable plasmid pK18-MBPMT, thus represents a potentially attractive protocol for the integration of genes in the course of genetic modification of C. glutamicum.     

Dot1 and Set2 histone methylases control the spontaneous and UV-induced mutagenesis levels in the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeasts

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of histone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-induced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the spontaneous mutagenesis rate in both single and double mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the homologous-recombination-based and the postreplicative DNA repair.     

Overexpression of <Emphasis Type="Italic">ppc</Emphasis> or deletion of <Emphasis Type="Italic">mdh</Emphasis> for improving production of γ-aminobutyric acid in recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced l-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, l-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3?±?3.4, 24.8?±?0.7, and 25.5?±?3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7?±?0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0?±?0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.     

Drug resistance mutations and susceptibility phenotypes of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates in Russia

Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8–36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.     

Two-step production of gamma-aminobutyric acid from cassava powder using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

Production of gamma-aminobutyric acid (GABA) from crop biomass such as cassava in high concentration is desirable, but difficult to achieve. A safe biotechnological route was investigated to produce GABA from cassava powder by C. glutamicum G01 and L. plantarum GB01-21. Liquefied cassava powder was first transformed to glutamic acid by simultaneous saccharification and fermentation with C. glutamicum G01, followed by biotransformation of glutamic acid to GABA with resting cells of L. plantarum GB01-21 in the reaction medium. After optimizing the reaction conditions, the maximum concentration of GABA reached 80.5 g/L with a GABA productivity of 2.68 g/L/h. This is the highest yield ever reported of GABA production from cassava-derived glucose. The bioprocess provides the added advantage of employing nonpathogenic microorganisms, C. glutamicum and L. plantarum, in microbial production of GABA from cassava biomass, which can be used in the food and pharmaceutical industries.     

Mutation Screening of the <Emphasis Type="Italic">CHCHD10</Emphasis> Gene in Chinese Patients with Amyotrophic Lateral Sclerosis

Mutations in the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 gene (CHCHD10), involved in mitochondrial function, have recently been reported as a causative gene of amyotrophic lateral sclerosis (ALS). The aim of this study was to obtain the mutation prevalence of CHCHD10 and the phenotypes with mutations in Chinese ALS patients. A cohort of 499 ALS patients including 487 sporadic ALS (SALS) and 12 familial ALS (FALS), from the Department of Neurology, West China Hospital of Sichuan University, were screened for mutations of all exons of the CHCHD10 gene by Sanger sequencing. Novel candidate mutations or variants were confirmed by polymerase chain reaction-restriction fragment length polymorphism in 466 healthy individuals. All patients identified with mutations of CHCHD10 gene were screened for mutations of the common ALS causative genes including C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1. Three heterozygous variants, including two missense mutations (c.275A?>?G (p.Y92C) and c.306G?>?C (p.Q102H)) and a synonymous change c.306G?>?A (p.Q102Q), were found in exon 3 of CHCHD10 in three alive SALS individuals (with the longest disease duration of 8.6 years), all of which were not detected in healthy controls. No mutation in CHCHD10 was identified in FALS patients. No mutation was found in the aforementioned common ALS causative genes in the patients who carried CHCHD10 mutations. The mutation frequency of CHCHD10 (0.4 %, 2/487) in a Chinese SALS population suggests CHCHD10 gene mutation appears to be an uncommon cause of ALS in Chinese populations. CHCHD10 mutations are associated with a slow progression and long disease duration.     

Reliability of the search for 19 common mutations in the <Emphasis Type="Italic">CFTR</Emphasis> gene in Russian cystic fibrosis patients and the calculated frequency of the disease in Russian Federation

A study of Russian cystic fibrosis (CF) patient DNA was conducted to assess the incidence frequency of 19 mutations, namely CFTRdele2,3(21kb), F508del, I507del, 1677delTA, 2143delT, 2184insA, 394delTT, 3821delT, L138ins, 604insA, 3944delGT, G542X, W1282X, N1303K, R334W, and 3849 + 10kbC > T, S1196X, 621 + 1g > t, and E92K of the CFTR gene. We also sought to determine the estimated CF frequency in Russian Federation. In addition, we determined the total information content of the approach for 19 common mutations registration in the CFTR gene, 84.6%, and the allelic frequencies of the examined mutations: three mutations were observed with a frequency exceeding 5% (F508del, 53.98%, E92K, 6.47%, CFTRdele2,3(21kb), 5.35%); other mutations were observed with frequencies ranging from 0.13 to 3.0%. The CF population carrier frequency was 1 in 38 subjects, while the predicted CF frequency was 1 in 5776 newborns.     

Analysis of endophytic actinobacteria species diversity in the stem of <Emphasis Type="Italic">Gynura cusimbua</Emphasis> by 16S rRNA gene clone library

Endophytic actinobacteria that lived in any associations with plant tissues represented a rather unexplored area of actinobacteria compared with soils. Gynura cusimbua was a kind of medicinal plant which had prevention effects for high blood pressure, coronary heart disease, Alzheimer’s disease, atherosclerosis, etc. Endophytic actinobacteria of G. cusimbua might produce some secondary metabolites which had the same function as their host. Stem samples of G. cusimbua collected from Hainan Province were used to study their endophytic actinobacteria to find some new compounds. In order to avoid vast proportions of the host plant DNA in the metagenomic library, the strategies of enrichment of the microorganism cells after tissue digestion and exclusion of 16S rRNA gene derived from the plastid by digested with PvuII were used. Two sets of actinobacteria specific primers were used for targeting endophytic actinobacteria from metagenomic library. 63 positive clones of actinobacteria were selected for sequencing and constructing the phylogenetic tree of 16S rRNA gene, and the 16S rRNA gene sequence of 59 strains among them had higher similar to the closest type strain and belonged to Microbacterium, Arthrobacter, Micrococcus, Curtobacterium, Okibacterium, Quadrisphaera and Kineococcus, respectively. Others were in low similarity and belonged to unclassified Micrococcineae, unclassified Intrasporangiaceae and unclassified Microbacteriaceae.     

Native promoters of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application in <Emphasis Type="SmallCaps">l</Emphasis>-lysine production

Objective

To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering.

Results

Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using β-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R ² = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction.

Conclusions

The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.

     

Molecular characterization of methicillin-resistant Panton-valentine leukocidin positive <Emphasis Type="Italic">staphylococcus aureus</Emphasis> clones disseminating in Tunisian hospitals and in the community

Background

The spread of MRSA strains at hospitals as well as in the community are of great concern worldwide. We characterized the MRSA clones isolated at Tunisian hospitals and in the community by comparing them to those isolated in other countries.

Results

We characterized 69 MRSA strains isolated from two Tunisian university hospitals between the years 2004-2008. Twenty-two of 28 (79%) community-associated MRSA (CA-MRSA) strains and 21 of 41 (51%) healthcare-associated MRSA (HA-MRSA) strains were PVL-positive. The PVL-positive strains belonged to predicted founder group (FG) 80 in MLST and carried either type IVc SCCmec or nontypeable SCCmec that harbours the class B mec gene complex. In contrast, very diverse clones were identified in PVL-negative strains: three FGs (5, 15, and 22) for HA-MRSA strains and four FGs (5, 15, 45, and 80) for CA-MRSA strains; and these strains carried the SCCmec element of either type I, III, IVc or was nontypeable. The nucleotide sequencing of phi7401PVL lysogenized in a CA-MRSA strain JCSC7401, revealed that the phage was highly homologous to phiSA2mw, with nucleotide identities of more than 95%. Furthermore, all PVL positive strains were found to carry the same PVL phage, since these strains were positive in two PCR studies, identifying gene linkage between lukS and mtp (major tail protein) and the lysogeny region, both of which are in common with phi7401PVL and phiSa2mw.

Conclusions

Our experiments suggest that FG80 S. aureus strains have changed to be more virulent by acquiring phi7401PVL, and to be resistant to β-lactams by acquiring SCCmec elements. These novel clones might have disseminated in the Tunisian community as well as at the Tunisian hospitals by taking over existing MRSA clones.

     

Bipolar distribution of the cyst-forming dinoflagellate <Emphasis Type="Italic"> Polarella glacialis</Emphasis>

Morphological investigations of motile cells and cysts of a small dinoflagellate (strain CCMP 2088) isolated from Canadian Arctic waters were carried out under both light and scanning electron microscopy. This species strongly resembled Polarella glacialis (strain CCMP 1383), which up to now was known only from Antarctic sea ice. The photosynthetic pigment composition of strain CCMP 2088 is typical of dinoflagellates, with peridinin as a major accessory pigment. Phylogenetic relationships between the two strains and other dinoflagellate species were inferred from SSU nrDNA using Neighbour Joining and weighted parsimony analyses. Our results showed that strain CCMP 2088 and P. glacialis (strain CCMP 1383) grouped in the same clade (Suessiales clade), showing high similarity values (0.99%). Morphological and molecular data support the assignment of the Arctic strain to P. glacialis. The free-living Gymnodinium simplex and the two P. glacialis strains have a basal position in the Suessiales clade, as compared to Symbiodinium spp.