Project description:BackgroundTofu whey wastewater (TWW) is the wastewater of tofu processing, which is rich in a variety of nutrients. Rhodotorula glutinis can make full use of TWW to ferment and reproduce yeast cells, produce carotenoids and other nutrients, improve the utilization value of TWW, and reduce environmental pollution and resource waste.MethodsIn this study, the nutrient composition changes of TWW treated by Rhodotorula glutinis were analyzed to reformulate TWW medium, and the optimal composition and proportion of TWW medium that can improve the biomass and carotenoids production of Rhodotorula glutinis were explored. Meanwhile, the Rhodotorula glutinis liquid obtained under these conditions was used to prepare biological feed for laying hens, and the effect of Rhodotorula glutinis growing on TWW as substrate on laying performance and egg quality of laying hens were verified.ResultsThe results showed that the zinc content of TWW after Rhodotorula glutinis fermentation increased by 62.30%, the phosphorus content decreased by 42.31%, and the contents of vitamin B1, B2 and B6 increased to varying degrees. The optimal fermentation conditions of Rhodotorula glutinis in the TWW medium were as follow: the initial pH was 6.40, the amount of soybean oil, glucose and zinc ions was 0.80 ml/L, 16.32 g/L, and 20.52 mg/L, respectively. Under this condition, the biomass of Rhodotorula glutinis reached 2.23 g/L, the carotenoids production was 832.86 μg/g, and the number of effective viable yeast count was 7.08 × 107 cfu/ml. In addition, the laying performance and egg quality of laying hens fed Rhodotorula glutinis biological feed were improved.DiscussionIn this study, we analyzed the composition changes of TWW, optimized the fermentation conditions of Rhodotorula glutinis in TWW medium, explored the influence of Rhodotorula glutinis utilizing TWW on laying layers, and provided a new idea for the efficient utilization of TWW.
Project description:Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the ?-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher ?-carotene (27.13?±?0.66?mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous ?-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.
Project description:The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from Rhodotorula glutinis GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells in vitro, and the strain no longer proliferated in vivo, for safe and controllable polypeptide drug delivery. In vivo, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.
Project description:BackgroundThe regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast.ResultsDifferential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity.ConclusionsThis study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.
Project description:To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of ï¢-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains. Experiment Overall Design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .
Project description:Rhodotorula glutinis ATCC 204091 is an oleaginous oxidative red yeast that can accumulate lipids to >50% of its biomass when grown with appropriate carbon and nitrogen ratios. It produces a red pigment consisting of useful antioxidants, such as carotenoids, torulene, and torularhodin, when cultivated under carbon-deficient conditions.