Single-colony Isolates Research Articles

Psychrotrophic Lipase Producers from Arctic Soil and Sediment Samples

Culturable microorganisms were successfully isolated from soil and sediment samples collected in 2011 on the northern coast of Hornsund, West Spitsbergen. A total of 63 single colony isolates from three sampling sites obtained were subjected to temperature dependence study to assess whether they are obligate psychrophilic or psychrotrophic strains. From initial temperature screening, only 53 psychrotrophic isolates were selected that are capable of growing between 4-28 degrees C. The rest that were capable of tolerating higher temperatures up to 37 degrees C were not included in this study. These isolates were chosen for lipase enzyme screening confirmation with the standard plate assay of olive oil and fluorescent dye Rhodamine B. Six lipase positive isolates were also subjected for subsequent lipase enzyme plate screening on tributyrin, triolein, olive oil and palm oil agar. Lipase production by these six isolates was further assayed by using colorimetric method with palm oil and olive oil as the substrate. These isolates with promising lipase activity ranging from 20 U/ml up to 160 U/ml on palm oil and olive oil substrate were successfully identified. Molecular identification by using 16S rRNA revealed that five out of six isolates were Gram-negative Proteobacteria and the other one was a Gram-positive Actinobacteria.

Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N2 -fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2 -fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2 -fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9 × 10(4) and 4.7 × 10(4) nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.

Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

BackgroundAn industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes.Methodology/Principal FindingsIn this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v).Conclusion/SignificanceThe findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially robust strains of consolidated bioprocessing microorganisms.

Automation Highlights from the Literature

Automation Highlights from the Literature

Automation Highlights from the Literature

Automation Highlights from the Literature

Genomic analysis of Saccharomyces cerevisiae isolates that grow optimally with glucose as the sole carbon source

A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants that grew optimally under these conditions. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after aerobic growth in a high glucose medium for approximately 450 generations. After the evolution period, three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was used to evaluate genetic changes. Greater than 100 deletion/insertion changes were found with approximately half of these effecting genes. Additionally, over 180 SNPs were identified with more than one-quarter of these resulting in a nonsynonymous mutation. Affymetrix DNA microarrays and RNseq analysis were used to determine differences in gene expression in the evolved strains compared to the parental strain. It was established that approximately 900 genes demonstrated significantly altered expression in the evolved strains relative to the parental strain. Many of these genes showed similar alterations in their expression in all three evolved strains. Interestingly, genes with altered expression in the three evolved strains included genes with a role in oxidative metabolism. Overall these results are consistent with the physiological observations of optimal growth with glucose as the carbon source. Namely, the decreased ethanol production suggest that the underlying metabolism switched from fermentation to respiration during the selection for optimal growth on glucose.

Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar.

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.

Reply to Frerichs et al.: Chasing the genetic diversity, not source attribution

Frerichs et al. (1) view the prevalence of Vibrio cholerae non-O1/O139 skeptically in the early weeks of the Haitian epidemic (2). Unexpected findings spawn skeptics. Setting aside differences in sampling time frames, differences in recoveries might readily be traced to comprehensive sampling and methodologies. Using a combination of thiosulphate-citrate-bile salt-sucrose and taurocholate tellurite gelatin agar medium to substantiate detection of both sucrose fermenting and nonfermenting V. cholerae reported in this region (3), we picked multiple single-colony isolates (average of 4, depending on availability of well-separated colonies and colony morphologies) on each original plate, which were screened systematically, using a battery of specific biochemical, serological, and molecular methods. Sensitive and discriminating, but more expensive and labor- and time-intensive, our approach is seldom used in settings like Haiti National Public Health Laboratory. Our approach uncovered non-O1/O139 in the numbers reported, which less fastidious conventional screening would underestimate. We advocate for such an approach to become standard.

Isolation and Characterization of Novel Alginate-Degrading Pseudoalteromonas sp. Y-4

To isolate an alginate-degrading bacterium, we conducted a single colony isolation using a solid medium containing alginate as the sole carbon source. A marine bacterium Y-4 capable of degrading alginate was isolated from seawater. The strain was identified to be Pseudoalteromonas sp., based on morphological, biochemical, 16S rDNA homology, and phylogenetic analyses. Moreover, Pseudoalteromonas sp. Y-4 exhibited alginate lyase activity in the presence of 4% alginate even though many known alginate-degrading bacteria degrade in the range of 0.5-1% alginate. The optimum culture conditions for the Y-4 strain were 2% alginate, pH 8.0, and 3% NaCl at <TEX>$30^{\circ}C$</TEX>. The highest alginate lyase activity was also observed under the same conditions. To our knowledge, this is the first reported isolation of a marine bacterium degrading high concentrations of alginate.

Trace Amounts of Furan-2-Carboxylic Acids Determine the Quality of Solid Agar Plates for Bacterial Culture

BackgroundMany investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed.Methodology/Principal FindingsAccording to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L−1 (13 and 21 nmol L−1), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium.Conclusions/SignificanceAgar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.

Pathogenicity and Characterization of Geographically Distributed Isolates of Erysiphe polygoni

The incidence of powdery mildew disease on pea (Pisum sativum L.) is a major limiting factor to maximizing yields in different ecological and geographical zones due to environmental factors and the presence of pathogenic variation in Erysiphe polygoni DC. In order to develop a widely adopted powdery mildew–resistant cultivar it is necessary to challenge genotypes with all available pathogenic variants. In order to examine pathogenic variation, 23 naturally occurring single colony isolates of E. polygoni, collected from geographically diverse locations, were subjected to pathogenicity testing and characterization using a detached leaf assay. Isolates demonstrated small differences in pathogenicity against pea. Isolates MUZ-1 and MUZ-2 were the most virulent and produced disease symptoms even on the resistant cultivars. Isolates TTS-2 and TTS-6 were less virulent. Significant variation occurred among isolates for quantitative and qualitative parameters regardless of the degree of pathogenicity. There was variation for conidium length, conidium width, cleistothecia number, number of hyphae, ascus number, ascus length, ascospore number, and ascospore width. An unweighted pair group method with arithmetic mean phenogram, constructed on the basis of quantitative parameters, indicated that isolates were morphologically different but pathogenicity was not controlled by morphological features of the isolates, though morphological features can be used to distinguish between isolates.

Activation of the Dormant Secondary Metabolite Production by Introducing Gentamicin-Resistance in a Marine-Derived Penicillium purpurogenum G59

A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1–4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 μg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites.

Passage of Campylobacter jejuni through the chicken reservoir or mice promotes phase variation in contingency genes Cj0045 and Cj0170 that strongly associates with colonization and disease in a mouse model

Human illness due to Camplyobacter jejuni infection is closely associated with consumption of poultry products. We previously demonstrated a 50 % shift in allele frequency (phase variation) in contingency gene Cj1139 (wlaN) during passage of C. jejuni NCTC11168 populations through Ross 308 broiler chickens. We hypothesized that phase variation in contingency genes during chicken passage could promote subsequent colonization and disease in humans. To test this hypothesis, we passaged C. jejuni strains NCTC11168, 33292, 81-176, KanR4 and CamR2 through broiler chickens and analysed the ability of passaged and non-passaged populations to colonize C57BL6 IL-10-deficient mice, our model for human colonization and disease. We utilized fragment analysis and nucleotide sequence analysis to measure phase variation in contingency genes. Passage through the chicken reservoir promoted phase variation in five specific contingency genes, and these 'successful' populations colonized mice. When phase variation did not occur in these same five contingency genes during chicken passage, these 'unsuccessful' populations failed to colonize mice. Phase variation during chicken passage generated small insertions or deletions (indels) in the homopolymeric tract (HT) in contingency genes. Single-colony isolates of C. jejuni strain KanR4 carrying an allele of contingency gene Cj0170 with a10G HT colonized mice at high frequency and caused disease symptoms, whereas single-colony isolates carrying the 9G allele failed to colonize mice. Supporting results were observed for the successful 9G allele of Cj0045 in strain 33292. These data suggest that phase variation in Cj0170 and Cj0045 is strongly associated with mouse colonization and disease, and that the chicken reservoir can play an active role in natural selection, phase variation and disease.

Short communication: Identification and differentiation of bifidobacteria obtained from Ukraine

Ten freeze-dried bifidobacterial strains used as probiotics in Ukrainian dairy foods, identified by the supplier as Bifidobacterium adolescentis (2), Bifidobacterium bifidum (2), Bifidobacterium longum (4), Bifidobacterium animalis (1), and Bifidobacterium infantis (1), were characterized. Following rehydration and anaerobic growth on de Man, Rogosa, and Sharpe-cysteine medium at 37°C for 72h, single-colony isolates were picked and evaluated using PCR primers specific for the Bifidobacterium genus, for the supplier-identified species, and for B. animalis ssp. lactis. All isolates were identified as members of the genus Bifidobacterium; however, species-specific PCR revealed all 10 isolates were actually strains of B. animalis ssp. lactis. Further evaluation using pulsed-field gel electrophoresis was only able to separate a single strain (RT 09) from the other 9 strains evaluated. Application of genome-wide allelic profiling to the Ukrainian bifidobacterial strains revealed 4 distinct groups. Interestingly, 6 (60%) of the isolates fell into the same cluster as that containing the common commercial probiotic strain BB-12. Two of the strains (RT 02 and RT 09) were found to be in the same group as ATCC 27536 and one strain (RT 08) was in the same group as the RB 7239 (a previously evaluated commercial strain). One strain, RT 04, was placed on a unique branch. These results highlight the importance of employing routine typing of bifidobacterial isolates, demonstrate the utility of single nucleotide polymorphism/insertion-deletion polymorphism-based allelic typing in B. animalis ssp. lactis strain differentiation and further point to the limited genetic variability of B. animalis ssp. lactis strains and the worldwide distribution of a small number of commercial strains.

Frequency of antibiotic resistance in Helicobacter pylori strains isolated from the northern population of Iran

The purpose of this study was to evaluate the primary resistance rates of recent clinical Helicobacter pylori isolates to the most commonly used antibiotics in Iran. Two hundreds and ten patients presenting with gastric maladies between January and July of 2009 were enrolled in this study. Endoscopy was performed, and biopsy specimens were collected from each patient for subsequent bacterial culture of H. pylori. Single colony isolates from each patient were then used for antimicrobial susceptibility testing. The disk diffusion method was used to determine susceptibility patterns. One hundred and ninety-seven of the patients were H. pylori positive (93.8%). The rates of resistance to tetracycline, amoxicillin, ciprofloxacin, metronidazole, clarithromycin, and furizoladone were 37.1%, 23.9%, 34.5%, 65.5%, 45.2%, and 61.4%, respectively. A significant association between amoxicillin resistance and disease state (P<0.05) was identified. Furthermore, some double, triple, quadruple, and quintuple combinations of antibiotic resistance were found to be associated with disease state. This study evaluated the prevalence of H. pylori resistance to the most commonly prescribed antibiotics used in Iran and showed that resistance rates were generally higher than previously reported. This data adds to the growing body of evidence that suggests there is increasing antibiotic resistance among H. pylori isolates, which likely is responsible for the decreasing efficacy of anti-H. pylori therapy at the local and global level. Hence, there is a need for continued monitoring of resistance patterns, especially at the local level, and for incorporation of that information into treatment regimens for H. pylori infections.

Biochemical and Molecular Characterization of Secreted α-Xylosidase from Aspergillus niger

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.

Nonylphenol 분해 미생물 컨소시엄 균주 개발

Nonylphenol (NP), which is well known as an endocrine disrupter, has been detected widely in untreated sewage or waste water streams. Given the necessity of discovering an eco-friendly method of degrading this toxic organic compound, this study was conducted to isolate NP-degrading microorganisms from the aqueous environment. NP-degrading microbes were isolated through NP-containing enrichment culture. Finally, a microbial consortium, SW-3, capable of degrading NP with high efficiency, was selected from the mixture sample. The microbial consortium SW-3 was able to degrade over 99% of 100 ppm NP in the culture medium for 40 days at <TEX>$25^{\circ}C$</TEX>. The microbial consortium SW-3 seemed to utilize NP as a carbon source, since NP was the sole carbon source in the culture medium. In order to isolate the NP-degrading bacterium, we further conducted single colony isolation using the microbial consortium SW-3. Four strains isolated from SW-3 exhibited lower NP-degradation efficiency than that of SW-3, suggesting that NP was degraded by the co-metabolism of the microbial consortium. We suggest that the microbial consortium obtained in this study would be useful in developing an eco-friendly bioremediation technology for NP degradation.

Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches

The microbiota of two traditional Iranian cheeses (Lighvan and Koozeh) made of raw ewe’s milk or mixtures of ewe’s and goat’s milk without starter addition was explored by culture-independent and culture-dependent approaches. Three batches of Lighvan and one of Koozeh were subjected to culture-independent polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through manufacturing and ripening. In addition, culturing in elective media for lactic acid bacteria (M17, MRS and KAA), isolation of single colonies (n = 130), molecular identification by PCR-amplified ribosomal DNA restriction analysis and sequencing, and differentiation at the strain level by repetitive extragenic palindromic PCR was also performed. DGGE analysis showed that the dominant amplicons in all four cheese batches belonged to Lactococcus lactis and Streptococcus parauberis. In addition, Escherichia coli and Lactococcus garvieae were frequently identified in both Lighvan and Koozeh, while Streptococcus thermophilus was found occasionally. In contrast, Enterococcus faecium and Enterococcus faecalis were found to be dominant among the isolates in all batches. These species showed a high genetic diversity. The discrepancy between culturing and DGGE results suggested that dominant populations were in a nonrecoverable state in the used media. This reinforces the idea that culture-dependent and culture-independent techniques provide complementary data, ultimately affording a better description of cheese ecosystems. These data could be of help in the selection of commercial starters for industrial-scale manufacture of Lighvan and Koozeh cheeses using pasteurised milk. Alternatively, microbial analysis would allow the selection of appropriate strains for designing of specific starters for traditional cheese manufacture.

The improvement of riboflavin production in Ashbya gossypii via disparity mutagenesis and DNA microarray analysis

We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin. By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites. One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon flux from β-oxidation to the riboflavin biosynthetic pathway.