Riboflavin Production Research Articles

Adaptive Evolution-Assisted Riboflavin Production of Ashbya gossypii from Cane Molasses

In this study, adaptive evolution was used to improve the adaptation of Ashbya gossypii ATCC 10895 to the substrate (cane molasses) to ensure enhanced substrate utilization and increased riboflavin yield. During the adaptation process, antioxidant activity and riboflavin production ability were evaluated using shake flask fermentation to evaluate the strain response to environmental conditions. After the seventh day of fermentation, results showed that compared with the yield of the parent strain, that of A. gossypii A-24 increased by 97.5%, and the dry cell weight increased by 125%. The A-24 strain was used to further study the cofermentation process of cane molasses and glucose under different ratios. When the ratio of glucose to cane molasses was 1 : 4, the yield of riboflavin was 978 ± 5.37 mg/L, which was 3.7 times that of cane molasses monofermentation.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Riboflavin integrates cellular energetics and cell cycle to regulate maize seed development.

SummaryRiboflavin is the precursor of essential cofactors for diverse metabolic processes. Unlike animals, plants can de novo produce riboflavin through an ancestrally conserved pathway, like bacteria and fungi. However, the mechanism by which riboflavin regulates seed development is poorly understood. Here, we report a novel maize (Zea mays L.) opaque mutant o18, which displays an increase in lysine accumulation, but impaired endosperm filling and embryo development. O18 encodes a rate‐limiting bifunctional enzyme ZmRIBA1, targeted to plastid where to initiate riboflavin biosynthesis. Loss of function of O18 specifically disrupts respiratory complexes I and II, but also decreases SDH1 flavinylation, and in turn shifts the mitochondrial tricarboxylic acid (TCA) cycle to glycolysis. The deprivation of cellular energy leads to cell‐cycle arrest at G1 and S phases in both mitosis and endoreduplication during endosperm development. The unexpected up‐regulation of cell‐cycle genes in o18 correlates with the increase of H3K4me3 levels, revealing a possible H3K4me‐mediated epigenetic back‐up mechanism for cell‐cycle progression under unfavourable circumstances. Overexpression of O18 increases riboflavin production and confers osmotic tolerance. Altogether, our results substantiate a key role of riboflavin in coordinating cellular energy and cell cycle to modulate maize endosperm development.

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis.

Riboflavin is an essential vitamin widely used in the food, pharmaceutical, and feed industries. However, the insufficient supply of precursors caused by the imbalance of intracellular metabolic flow limits the riboflavin synthesis by industrial strains. Here, we increase riboflavin production by tuning multiple gene expression to balance intracellular metabolic flow. First, we tuned the expression of mCherry and egfp genes within operons by generating libraries of tunable intergenic regions (TIGRs) and confirmed the relative expression of the two reporter genes. The TIGR library can coordinate the expression ratio of reporter genes more than 180 times in Escherichia coli and more than 70 times in Bacillus subtilis. Next, we used this strategy to tune the expression of zwf, ribBA, and ywlf genes within operons through the TIGR library to increase the intracellular precursor pool for riboflavin biosynthesis. Based on the fluorescence characteristics of riboflavin, 96-well plates were used to screen the optimal combination mutants quickly. The best-engineered strain was selected from the library, which produced 2.7 g/L riboflavin, increasing by 64.35% in the shake flask. Finally, the riboflavin titer increased by 59.27% to 11.77 g/L in fed-batch fermentation. The strategy described here will contribute to the industrial production of riboflavin and related products by B. subtilis.

From Brown Seaweed to a Sustainable Microbial Feedstock for the Production of Riboflavin.

The increasing global demand for food and energy production encourages the development of new production strategies focused on sustainability. Often, microbial bioprocesses rely on food or feed competitive feedstocks; hence, there is a trending need for green substrates. Here, we have proven the potential of brown seaweed biomass as microbial feedstock on account of its content of mannitol and the glucose polymer laminarin. Our host, Corynebacterium glutamicum, was engineered to enable access to mannitol as a carbon source through the heterologous expression of the mannitol-specific phosphotransferase system and the mannitol-1-phosphate-5-dehydrogenase from Bacillus subtilis. Overproduction of riboflavin was coupled with mannitol and glucose consumption via constitutive overexpression of the biosynthetic riboflavin operon ribGCAH from C. glutamicum. Brown seaweed extract and brown seaweed hydrolysate from Laminaria hyperborea, containing mannitol and glucose, were used as a carbon source for flask and bioreactor fermentations. In a seaweed-based fed-batch fermentation, the riboflavin final titer, yield, and volumetric productivity values of 1,291.2 mg L−1, 66.1 mg g−1, and 17.2 mg L−1 h−1, respectively, were achieved.

Co-Overexpression of RIB1 and RIB6 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin or vitamin B2 is a water-soluble vitamin and a precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which play a key role as enzyme cofactors in energy metabolism. Candida famata yeast is a promising producer of riboflavin, as it belongs to the group of so-called flavinogenic yeasts, capable of riboflavin oversynthesis under conditions of iron starvation. The role of the particular structural genes in the limitation of riboflavin oversynthesis is not known. To study the impact of overexpression of the structural genes of riboflavin synthesis on riboflavin production, a set of plasmids containing genes RIB1, RIB6, and RIB7 in different combinations was constructed. The transformants of the wild-type strain of C. famata, as well as riboflavin overproducer, were obtained, and the synthesis of riboflavin was studied. It was found that overexpression of RIB1 and RIB6 genes coding for enzymes GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase, which catalase the initial steps of riboflavin synthesis, elevated riboflavin production by 13–28% relative to the parental riboflavin-overproducing strains.

Enhancing the biomass and riboflavin production of Ashbya gossypii by using low-intensity ultrasound stimulation

The riboflavin (vitamin B2) produced by the fermentation of Ashbya gossypii is an essential nutrient for humans and animals. In this study, favorable low-intensity ultrasound (US) with a power of 210 W was applied to stimulate A. gossypii for 45 min at 33 kHz during the logarithmic phase of 48 h. Compared with those of the untreated A. gossypii, the extracellular riboflavin, total riboflavin, and biomass of the US-stimulated A. gossypii increased by 23.5%, 26.95%, and 71.99%, at the 168th h, respectively. The optimal US stimulation increased the mycelial membrane permeability, decreased the mycelial diameter, and changed mycelial morphology to promote mass transfer, causing the enhancement of biomass and riboflavin production. Furthermore, US stimulation also increased the reactive oxygen species generation of A. gossypii, which may be attributed to this fungus’ US-induced stress defense that enhanced riboflavin production. Therefore, combining US and fermentation is a feasible and efficient method for enhancing the production of riboflavin and other compounds.

Using fermentation waste of ethanol-producing yeast for bacterial riboflavin production and recycling of spent bacterial mass for enhancing the growth of oily plants

This study aims to use fermentation waste of ethanol production (solid and liquid) for riboflavin and recycling of bacterial biomass as biofertilizers to enhance the growth of some oily crop plants. Out of 10 yeast isolates from fresh milk, Clavispora lusitaniae ASU 33 (MN583181) was able to ferment different concentrations of glucose (2.5%, 5%, 7.5%, 10%, 15% and 20%) into ethanol with high efficiency at 10%. Among seven non-Lactobacillus bacterial isolates recovered from cheese samples, two bacterial isolates Bacillus subtlis-SR2 (MT002768) and Novosphingobium panipatense-SR3 (MT002778) were selected for their high riboflavin production. Different media (control medium, fermentation waste medium and a mixture of the fermentation waste medium and control medium [1:1]) were used for riboflavin production. These media were inoculated by a single or mixture of B. subtlis-SR2, N. panipatense-SR3. The addition of the waste medium of ethanol production to the control medium (1:1) had a stimulatory effect on riboflavin production whether inoculated with either a single strain or a mixture of B. subtlis-SR2 and N. panipatense-SR3. A mixture of fermentation waste and control media inoculated with N. panipatense produced a high riboflavin yield in comparison with other media. Inoculation of Zea mays and Ocimum basilicum plants with either the bacterial biomass waste of riboflavin production (B. subtlis or N. panipatense) or a mixture of B. subtlis and N. panipatense) shows a stimulatory effect on the plant growth in comparison with control (uninoculated plants). These results demonstrate the possibility of minimizing the cost of riboflavin and biofertilizer manufacturing via interlinking ethanol and riboflavin with the biofertilizer production technology. This study outlines the methods of evaluating the strength of spent media by applying procedures developed in the vitamin production industries. Furthermore, bacterial biomass waste can act as an environmentally friendly alternative for agrochemicals.

Genome Sequence and Analysis of the Flavinogenic Yeast Candida membranifaciens IST 626.

The ascomycetous yeast Candida membranifaciens has been isolated from diverse habitats, including humans, insects, and environmental sources, exhibiting a remarkable ability to use different carbon sources that include pentoses, melibiose, and inulin. In this study, we isolated four C. membranifaciens strains from soil and investigated their potential to overproduce riboflavin. C. membranifaciens IST 626 was found to produce the highest concentrations of riboflavin. The volumetric production of this vitamin was higher when C. membranifaciens IST 626 cells were cultured in a commercial medium without iron and when xylose was the available carbon source compared to the same basal medium with glucose. Supplementation of the growth medium with 2 g/L glycine favored the metabolization of xylose, leading to biomass increase and consequent enhancement of riboflavin volumetric production that reached 120 mg/L after 216 h of cultivation. To gain new insights into the molecular basis of riboflavin production and carbon source utilization in this species, the first annotated genome sequence of C. membranifaciens is reported in this article, as well as the result of a comparative genomic analysis with other relevant yeast species. A total of 5619 genes were predicted to be present in C. membranifaciens IST 626 genome sequence (11.5 Mbp). Among them are genes involved in riboflavin biosynthesis, iron homeostasis, and sugar uptake and metabolism. This work put forward C. membranifaciens IST 626 as a riboflavin overproducer and provides valuable molecular data for future development of superior producing strains capable of using the wide range of carbon sources, which is a characteristic trait of the species.

Neuroprotective Effect of Riboflavin Producing Lactic Acid Bacteria in Parkinsonian Models.

Oxidative stress and inflammatory processes might contribute to the cascade of events leading Parkinson disease (PD); and vitamins such as riboflavin can exert protection on vulnerable neurons in neurodegenerative conditions. Previously, it was demonstrated that a mixture of lactic acid bacteria (including a riboflavin-producing strain) improved motor skills in a parkinsonian model. The aim of the present work was to investigate the neuroprotective potential of Lactiplantibacillus (L.) plantarum CRL2130, a riboflavin-producing strain in PD models. In vitro, N2a differentiated neurons were exposed the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and treated with intracellular bacterial extracts or commercial riboflavin. In vivo, adult male C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid, and received orally L. plantarum CRL2130, L. plantarum CRL725 (parent strain that produces low levels of riboflavin) or commercial vitamin. Results showed that when N2a cells were incubated with intracellular extract from L. plantarum CRL2130 maintained the viability, and significantly decreased the release of IL-6 and the formation of reactive oxygen species (ROS), all affected by MPP+. In vivo, the administration of L. plantarum CRL2130 attenuated motor deficits and prevented dopaminergic neuronal death. Decrease of pro-inflammatory cytokines and increase of IL-10 in both serum and brain were observed in samples from mice that received L. plantarum CRL2130 compared to MPTP control group (without treatment). In addition, these beneficial effects were similar or improved when compared with animals that received commercial riboflavin. In conclusion, L. plantarum CRL2130 showed a neuroprotective effect in both PD models through anti-oxidant/anti-inflammatory mechanisms.

High-dimensional linear mixed model selection by partial correlation

We wish to perform variable selection in high-dimensional linear mixed models where the number of the potential covariates is much larger than the sample size and where the random effects are utilized to describe correlated observations. We propose a variable selection procedure based on the Thresholded Partial Correlation (TPC) algorithm (Li, Liu, and Lou 2017) to conduct variable selection using the partial correlation between the covariates and the response variable conditional on the random effects, and this procedure is called the conditional Thresholded Partial Correlation, denoted by TPCc. This TPCc approach is able to select the fixed effects in high-dimensional data when the covariates are highly correlated. We investigate the performance of the proposed method (TPCc) in a variety of simulated high-dimensional data sets. The simulation results show that the TPCc outperforms the TPC in selecting the most appropriate model among the candidate pool in the mixed modeling setting. We also apply the proposed method to a real high-dimensional data set in the production of riboflavin.

Production of vitamin B2 (riboflavin) by Bacillus subtilis

AbstractRiboflavin is one of the main micronutrients required for the human body for cellular respiration. It is the precursor of flavin mononucleotide and flavin adenine dinucleotide coenzymes crucial for the production of energy. The development of biotechnology has given rise to microbial procedures that strongly compete with the chemical synthesis approach and hence the fermentation process of riboflavin by microorganisms was initiated. Therefore, the main aim of this review is to investigate the production of riboflavin by Bacillus subtilis fermentation. The introduction of the microbial synthesis method has found that regulatory and structural genes involved in the synthesis of vitamin B2 are overexpressed, which has eventually enhanced the strain productivity and production of the vitamin via industrial fermentation. Using B. subtilis to synthesize riboflavin can reduce the production cost and increase the yield of riboflavin. The emergence of microbial synthesis methods has effectively increased the yield of riboflavin, and the study of the process of riboflavin synthesis by B. subtilis is of great significance for improving production, reducing costs and using renewable raw materials. © 2021 Society of Chemical Industry (SCI).

In silico evaluation of genomic characteristics of Streptococcus infantarius subsp. infantarius for application in fermentations.

This study aims to evaluate the in silico genomic characteristics of Streptococcus infantarius subsp. infantarius, isolated from Coalho cheese from Paraíba, Brazil, with a view to application in lactic fermentations. rRNA sequences from the 16S ribosomal region were used as input to GenBank, in the search for patterns that could reveal a non-pathogenic behavior of S. infantarius subsp. infantarius, comparing mobile genetic elements, antibiotic resistance genes, pan-genome analysis and multi-genome alignment among related species. S. infantarius subsp. infantarius CJ18 was the only complete genome reported by BLAST/NCBI with high similarity and after comparative genetics with complete genomes of Streptococcus agalactiae (SAG153, NJ1606) and Streptococcus thermophilus (ST106, CS18, IDCC2201, APC151) revealed that CJ18 showed a low number of transposases and integrases, infection by phage bacteria of the Streptococcus genus, absence of antibiotic resistance genes and presence of bacteriocin, folate and riboflavin producing genes. The genome alignment revealed that the collinear blocks of S. thermophilus ST106 and S. agalactiae SAG153 have inverted blocks when compared to the CJ18 genome due to gene positioning, insertions and deletions. Therefore, the strains of S. infantarius subsp. infantarius isolated from Coalho cheese from Paraíba showed genomic similarity with CJ18 and the mobility of genes analyzed in silico showed absence of pathogenicity throughout the genome of CJ18, indicating the potential of these strains for the dairy industry.

Elucidation of the biosynthetic pathway of B-group vitamins via genome mining of food-derived \(\textit{Bacillus velezensis}\) VTX20

B-vitamins are micronutrients that play an important role in various cellular processes of organisms, which are only synthesized by plants, yeasts, and bacteria. Since animals and humans lack the ability to synthesize B-vitamins, supplements of vitamins from dietary and the B-vitamin producing bacteria are required. In this study, we, for the first time, shed some light on biosynthetic pathways involved in folate (vitamin B9), riboflavin (vitamin B2), and biotin (vitamin B7) production in Bacillus velezensis VTX20. The genome-wide comparison revealed that B. velezensis VTX20 shared high similarities with B. tequilensis KCTC 13622, B. subtilis 168, B. amyloliquefaciens DSM 7. Genomic analysis revealed the presence of a complete folate biosynthesis pathway in which some core components were not found in most Bacillus species. Moreover, strain VTX20 also had the metabolic pathways for riboflavin and biotin that are important probiotic traits. These results highlighted that B. velezensis VTX20 is a producer of B-vitamins, which can be applied further in the agricultural biotechnology industry.

Development of nutrient medium for riboflavin biosynthesis by Eremothecium ashbyi ascomycetes

The object of the study is the riboflavin producer Eremothecium ashbyi Guilliermond 1935 VKPM F-340, the subject of the study is the regularities of riboflavin biosynthesis by the E. ashbyi F-340 strain under different cultivation conditions. Riboflavin is an important micronutrient that is a precursor of the coenzymes flavin mononucleotide and flavinadine dinucleotide, it is necessary for biochemical reactions in all living cells. Population growth and an increase in human needs for vitamin-fortified food and agricultural products is the reason for an increase in demand for riboflavin preparations. Considering this, it is important and economically beneficial to improve the technology for the production of vitamin B2. An important factor that affects the yield of the product is the nutrient medium. At present, the influence of agricultural waste on the biosynthesis of riboflavin is being actively studied in the world. However, not all of the studied types of raw materials are typical for the agriculture of this or that country. Therefore, in order to determine whether this direction of research is promising, it is important to check the effect on the biosynthetic activity of the riboflavin producer of the most common wastes of the domestic industry. In this work, this is done on the example of Ukraine. In the course of the study, microbiological (surface and deep cultivation of E. ashbyi F-340), physicochemical (determination of the amount of biomass by the gravimetric method, determination of the concentration of riboflavin by the spectrophotometric method) and mathematical methods were used. The proposed media with the addition of agricultural waste, providing a higher yield of riboflavin compared to conventional media. The influence of different types of agricultural waste on the biosynthesis of riboflavin by the producer E. ashbyi F-340 was evaluated. The efficiency of using sunflower cake as a component of the nutrient medium is shown. The optimal sources of carbon for the nutrient medium with oil cake have been determined, which increases the yield of riboflavin. Due to the large amount of sunflower cake obtained in Ukraine, its use for modifying the nutrient medium in order to increase the yield of riboflavin in the future will lead to a decrease in the cost of the target product due to the use of cheap and ecological raw materials.

Effects of L. plantarum HY7715 on the Gut Microbial Community and Riboflavin Production in a Three-Stage Semi-Continuous Simulated Gut System.

Probiotics should be well established in the gut, passing through the digestive tract with a high degree of viability, and produce metabolites that improve the gut environment by interacting with the gut microbiome. Our previous study revealed that the Lactiplantibacillus plantarum HY7715 strain shows good bile acid resistance and a riboflavin production capacity. To confirm the interaction between HY7715 and gut microbiome, we performed a metabolite and microbiome study using a simulated gut system (SGS) that mimics the intestinal environment. Changes in the microbiome were confirmed and compared with L. plantarum NCDO1752 as the control. After 14 days, the HY7715 treatment group showed a relatively high butyrate content compared to the control group, which showed increased acetate and propionate concentrations. Moreover, the riboflavin content was higher in the HY7715 treatment group, whereas the NCDO1752 treatment group produced only small amounts of riboflavin during the treatment period and showed a tendency to decrease during the washout stage; however, the HY7715 group produced riboflavin continuously in the ascending colon during the washout period. A correlation analysis of the genus that increased as the content of riboflavin increased revealed butyrate-producing microorganisms, such as Blautia and Flavonifractor. In conclusion, treatment with L. plantarum HY7715 induced the production and maintenance of riboflavin and the enrichment of the intestinal microbiome

New Promoters for Metabolic Engineering of Ashbya gossypii.

Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. In addition, metabolically engineered strains of A. gossypii have also been described as valuable biocatalysts for the production of different metabolites such as folic acid, nucleosides, and biolipids. Hence, bioproduction in A. gossypii relies on the availability of well-performing gene expression systems both for endogenous and heterologous genes. In this regard, the identification of novel promoters, which are critical elements for gene expression, decisively helps to expand the A. gossypii molecular toolbox. In this work, we present an adaptation of the Dual Luciferase Reporter (DLR) Assay for promoter analysis in A. gossypii using integrative cassettes. We demonstrate the efficiency of the analysis through the identification of 10 new promoters with different features, including carbon source-regulatable abilities, that will highly improve the gene expression platforms used in A. gossypii. Three novel strong promoters (PCCW12, PSED1, and PTSA1) and seven medium/weak promoters (PHSP26, PAGL366C, PTMA10, PCWP1, PAFR038W, PPFS1, and PCDA2) are presented. The functionality of the promoters was further evaluated both for the overexpression and for the underexpression of the A. gossypii MSN2 gene, which induced significant changes in the sporulation ability of the mutant strains.

Lactose Inducible Expression of Transcription Factor Gene SEF1 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin (vitamin B2) is required for synthesis of the flavin coenzymes: riboflavin-5’-phosphate (flavin mononucleotide) and flavin adenine dinucleotide. Riboflavin is important biotechnological commodity with annual market around 250 million US dollars. It is mostly used as component of feed premixes for animals (80%), in food industry as food colorant, in medicine and component of multivitamin mixtures and as drug for treatment of some diseases. Over the past two decades, the microbial production of riboflavin by fermentation completely replaces the chemical synthetic route. The main producers of riboflavin in industry are engineered strains of the bacterium Bacillus subtilis and of the mycelial fungus Ashbya gossypii. Flavinogenic yeast Candida famata has great biosynthetic potential. Using combination of classical selection and metabolic engineering (overexpression of SEF1, RIB1 and RIB7 genes coding the positive regulator, the first and the last structural enzymes of riboflavin synthesis) resulted in the construction of genetically stable strain of C. famata that produces 16 gram of riboflavin per liter in bioreactor. However, the productivity of riboflavin biosynthesis remains still insufficient for industrial production of this vitamin. Studies of transcriptional regulation of genes involved in riboflavin synthesis and using of strong promoters of C. famata for construction of efficient producers of vitamin B2 are areas of both scientific and industrial interest. Aim. The aim of the current work was to improve riboflavin oversynthesis by the available C. famata strains in synthetic and natural lactose-containing media. Methods. The plasmid DNA isolation, restriction, ligation, electrophoresis in agarose gel, electrotransformation, and PCR were carried out by the standard methods. Riboflavin was assayed fluorometrically using solution of synthetic riboflavin as a standard. The cultivation of yeasts was carried out in YNB or YPD media containing different source of carbon and on whey. Results. The strains of C. famata expressed additional copy of central regulatory gene SEF1 under control of the promoter of LAC4 gene (coding for β–galactosidase) C. famata were constructed. The influence of SEF1 gene expression under control of lactose inducible promoter of CfLAC4 gene on riboflavin production was studied. It was shown that the C. famata strains containing “pLAC4_cf-SEF1_cf” expression cassette revealed 1.6-2.1-fold increase in riboflavin yield on lactose when compared to the parental strain. The riboflavin production constructed strains on whey reached 1.69 gram per liter in flask batch culture. Conclusions. The constructed strains containing additional copy of SEF1 gene under the control of LAC4 promoter is a perfect platform for development of industrial riboflavin production on by-product of dairy industry, whey.

Rational Engineering of Escherichia coli for High-Level Production of Riboflavin.

Riboflavin is widely used as a food additive. Here, multiple strategies were used to increase riboflavin production in Escherichia coli LS31T. First, purR deletion and co-overexpression of fbp, purF, prs, gmk, and ndk genes resulted in an increase of 18.6% in riboflavin titer (reaching 729.7 mg/L). Second, optimization of reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide ratio and respiratory chain activity in LS31T increased the titer up to 1020.2 mg/L. Third, the expression level of the guaC gene in LS31T was downregulated by ribosome binding site replacement, and the riboflavin production was increased by 10.6% to 658.5 mg/L. Then, all the favorable modifications were integrated together, and the resulting strain LS72T produced 1339 mg/L of riboflavin. Moreover, the riboflavin titer of LS72T reached 21 g/L in fed-batch cultivation, with a yield of 110 mg riboflavin/g glucose. To our knowledge, both the riboflavin titer and yield obtained in fed-batch fermentation are the highest ones among all the rationally engineered strains.

A nonlinear mixed–integer programming approach for variable selection in linear regression model

Modern statistical studies often encounter regression models with high dimensions in which the number of features p is greater than the sample size n. Although the theory of linear models is well–established for the traditional assumption p < n, making valid statistical inference in high dimensional cases is a considerable challenge. With recent advances in technologies, the problem appears in many biological, medical, social, industrial, and economic studies. As known, the LASSO method is a popular technique for variable selection/estimation in high dimensional sparse linear models. Here, we show that the prediction performance of the LASSO method can be improved by eliminating the structured noises through a mixed–integer programming approach. As a result of our analysis, a modified variable selection/estimation scheme is proposed for a high dimensional regression model which can be considered as an alternative of the LASSO method. Some numerical experiments are made on the classical riboflavin production and some simulated data sets to shed light on the practical performance of the suggested method.