Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved ß-carotene production in Saccharomyces cerevisiae.

BACKGROUND: The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve ß-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. RESULTS: The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of ß-carotene in S. cerevisiae. However, due to the competition for precursor between ß-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on ß-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in ß-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to ß-carotene pathway and achieved additional 31.7% increase in ß-carotene content without adversely affecting cell growth. By inducing an extra constitutive ß-carotene synthesis pathway for further conversion precursor farnesol to ß-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of ß-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. CONCLUSIONS: This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.

The Protective Effect of Cx43 Protein-Mediated Phosphocreatine on Myocardial Ischemia/Reperfusion Injury.

OBJECTIVES: To verify the protective effect of phosphocreatine on myocardium in an ischemic model and the possible mechanism of action. METHODS: The model of myocardial ischemia/reperfusion (I/R) was established by the ligation balloon method. 30 SD rats were randomly divided into three groups, n = 10 in each group. Sham operation group: the coronary artery was not blocked and observed for 120 minutes. The ischemia/reperfusion (I/R) group was given ischemia for 30 minutes and ischemia reperfusion for 90 minutes. Phosphocreatine (PCr) group: after 30 minutes of ischemia, the rats were intraperitoneally injected with PCr (200 mg/kg) for 90 minutes. The animal groups of myocardial ischemia/reperfusion model in vitro were the same as those in vivo. The heart was removed by thoracotomy and washed immediately in H-K buffer solution. Then, the heart was installed on the Langendorff instrument. The concentration of PCr perfusion fluid in the PCr group was 10 mmol/L. The changes in coronary blood flow in isolated myocardium were recorded. The heart rate and electrocardiogram were recorded by RM6240BT. At the end of the experiment, myocardial pathological sections and Cx43 immunofluorescence staining were made, and the contents of malondialdehyde (MDA) in myocardial tissue were detected. RESULTS: Phosphocreatinine treatment improved the myocardial ischemia model, performance in electrocardiogram (ECG) changes (ST segment apparent), and histological changes (decrease in necrotic myocardial cells, inflammatory cell infiltration, and a reduction in myocardial edema). At the same time, MDA decreased, while coronary blood flow and Cx43 expression significantly improved. CONCLUSIONS: Phosphocreatine can improve the electrocardiogram and restore histologic changes in ischemic myocardium and coronary blood flow. The postulated mechanism is by inhibiting the generation of free oxygen radicals and restoring the expression of Cx43 protein.

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of ß-carotene in Saccharomyces cerevisiae.

BACKGROUND: Product toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (ß-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae. RESULTS: Substrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for ß-carotene efflux. The accumulation of ß-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of ß-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of ß-carotene secretion and a 1.71-fold increase of the intracellular ß-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01. CONCLUSIONS: Overall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

Characterization of two Vitis vinifera carotenoid cleavage dioxygenases by heterologous expression in Saccharomyces cerevisiae.

Norisoprenoids are produced from carotenoids under oxidative degradation mediated by carotenoid cleavage dioxygenases (CCDs) and contribute to floral and fruity notes in grape berries and wine. The diversity of CCD substrates and products has been demonstrated by in vitro recombinant proteins extracted from Escherichia coli expressing CCD genes and of in vivo proteins in an E. coli system co-expressing genes for carotenoid synthesis and cleavage. In the current study, VvCCD1 and VvCCD4b were isolated from the cDNA library of Vitis vinifera L. cv. Cabernet Sauvignon and then transformed into carotenoid-accumulating recombinant Saccharomyces cerevisiae strains. The expression of the target genes was monitored during the yeast growth period, and the accumulation of carotenoids and norisoprenoids in the recombinant strains was measured. The results indicated that both of the VvCCDs cleaved ß-carotene at the 7, 8 (7', 8') position into ß-cyclocitral for the first time. Additionally, the two enzymes also degraded ß-carotene at the 9, 10 (9', 10') position to generate ß-ionone and cleaved lycopene at the 5, 6 (5', 6') position into 6-methyl-5-hepten-2-one. These findings suggested that the VvCCDs may possess more cleavage characteristics under the eukaryotic expression system in S. cerevisiae than the prokaryotic system in E. coli, which could better explain the biochemical functions of VvCCDs in grape berries.

Transcriptional Comparison Investigating the Influence of the Addition of Unsaturated Fatty Acids on Aroma Compounds During Alcoholic Fermentation.

The levels of unsaturated fatty acids (UFAs) in grape must significantly influence yeast metabolism and the production of aroma compounds. In this work, cDNA microarray technology was applied to analyze the transcriptional discrepancies of wine yeast (commercial wine yeast Lalvin EC1118) fermenting in synthetic grape must supplemented with different concentrations of a mixture of UFAs (including linoleic acid, oleic acid, and α-linolenic acid). The results showed that the initial addition of a high level of UFAs can significantly enrich the intracellular UFAs when compared to a low addition of UFAs and further increase the cell population and most volatiles, including higher alcohols and esters, except for several fatty acids. Microarray analyses identified that 63 genes were upregulated, and 91 genes were downregulated during the different fermentation stages. The up-regulated genes were involved in yeast growth and proliferation, stress responses and amino acid transportation, while the repressed genes were associated with lipid and sterol biosynthesis, amino acid metabolism, TCA cycle regulation, mitochondrial respiration, and stress responses. Unexpectedly, the genes directly related to the biosynthesis of volatile compounds did not vary substantially between the fermentations with the high and low UFA additions. The beneficial aromatic function of the UFAs was ascribed to the increased biomass and amino acid transportation, considering that the incorporation of the additional UFAs in yeast cells maintains high membrane fluidity and increases the ability of the cells to resist deleterious conditions. Our results highlighted the importance of UFAs in the regulation of aroma biosynthesis during wine fermentation and suggested that the improvement of the resistance of yeast to extreme stresses during alcoholic fermentation is essential to effectively modulate and improve the production of aroma compounds. A potential way to achieve this goal could be the rational increase of the UFA contents in grape must.

Comparing the Effects of Different Unsaturated Fatty Acids on Fermentation Performance of Saccharomyces cerevisiae and Aroma Compounds during Red Wine Fermentation.

To understand the individual enological function of different unsaturated fatty acids (UFAs), the separated effects of three different UFAs, linoleic acid (LA), oleic acid (OA), and α-linolenic acid (ALA), on yeast fermentation and aroma compounds were investigated in the alcoholic fermentation of Cabernet Sauvignon wine. The results showed that, besides concentration, UFAs types could also influence fermentation process and volatiles in final wine. Low concentrations of UFAs (12 and 60 mg/L), especially LA and OA, significantly promoted fermentation activity and most volatiles when compared to the control, however, the effect became the inhibition with increasing concentrations of UFAs (120 and 240 mg/L). It was interesting to find that OA addition (12 and 60 mg/L) could generate more acetate esters (especially isoamyl acetate) in wine, while 12 mg/L LA facilitated more fatty acids formation (octanoic acid and decanoic acid). In comparison, 120 and 240 mg/L ALA produced more amount of C6 alcohols (1-hexanol) and higher alcohols (isobutyl alcohol and 2,3-butanediol). UFAs additions were unfavorable for ethyl esters formation, except for an increment of ethyl hexanoate in 12 mg/L OA wine. As a result, different aromatic profiles of wines were generated by variations of UFAs types and levels, as shown by PCA. The transcriptional data revealed that the expressions of aroma-related genes, such as BAT1, BAT2, PDC1, PDC5, PDC6, ACC1, FAS1, ATF1, EEB1, and EHT1 were correlated with aroma compounds productions in different treatments. Our data suggested that the three UFAs have different enological functions and they could generate different aromatic profiles. Thus, besides concentrations, it is essential to consider the types of UFAs when applying the strategy to adjust UFAs contents to modulate the aromatic quality of wines.

Use of Indigenous Hanseniaspora vineae and Metschnikowia pulcherrima Co-fermentation With Saccharomyces cerevisiae to Improve the Aroma Diversity of Vidal Blanc Icewine.

Using novel non-Saccharomyces strains is regarded as an effective way to improve the aroma diversity of wines to meet the expectations of consumers. The non-Saccharomyces Hanseniaspora vineae and Metschnikowia pulcherrima have good aromatic properties useful for the production of table wine. However, no detailed information is available on their performances in icewine fermentation. In this study, simultaneous and sequential fermentation trials of indigenous M. pulcherrima CVE-MP20 or H. vineae CVE-HV11 with S. cerevisiae (SC45) were performed in 50-L fermenters of Vidal icewine, respectively. The results showed that SC45 cofermented with different non-Saccharomyces strains could generate a distinct aroma quality of icewine compared with four S. cerevisiae strain monocultures as evidenced by principal component analysis (PCA). Mixed fermentation of MP20/SC45 produced higher contents of acetate esters and ß-damascenone with lower C6 alcohols relative to SC45 monoculture. Interestingly, HV11/SC45 generated the highest amounts of C6 alcohols [(Z)-3-hexen-1-ol and (E)-3-hexen-1-ol], higher alcohols (isobutanol, isopentanol, and 2-phenylethanol), acetate esters (2-phenethyl acetate and isoamyl acetate), cis-rose oxide, ß-damascenone, and phenylacetaldehyde. Compared with simultaneous inoculation, sequential inoculation could achieve higher aroma diversity and produce higher intensity of fruity, flowery, and sweet attributes of icewine as assessed by calculating the odor activity values (OAVs). Our results verified the desired enological characteristics of H. vineae strain in icewine fermentation and also demonstrated that using indigenous non-Saccharomyces and Saccharomyces strains is a feasible way to improve aroma diversity of icewine products, which could provide an alternative way to meet the requirement of wine consumers for diversified aromatic quality.

The content of linoleic acid in grape must influences the aromatic effect of branched-chain amino acids addition on red wine.

The effect of adding amino acids on wine aroma is largely influenced by nutritional status of grape must. In this study, the effects of linoleic acid (LA) content on the aromatic function of branched-chain amino acids (BCAAs) addition were investigated in alcoholic fermentation of Cabernet Sauvignon wine. The results showed that initial LA content in must significantly influenced the effect of BCAAs addition on volatiles in final wine. Adding BCAAs (140 mg/L of l-leucine, 117 mg/L of l-isoleucine and 118 mg/L of l-valine) in must with low LA content (12 mg/L) promoted the production of most volatiles, including higher alcohols (isobutanol, 2-phenylethanol), fatty acids (hexanoic acid, octanoic acid, decanoic acid) and esters (ethyl acetate, isoamyl acetate, 2-phenethyl acetate and ethyl octanoate), which were well consistent with previous literatures. However, this function disappeared or even became inhibition with increasing LA content in must, especially in 120 mg/L LA must, the total contents of higher alcohol, acetate esters and ethyl esters were 33.9%, 18.1% and 54.2% lower than those in the control without BCAAs addition, respectively. The transcriptional data revealed that several major genes including GAP1, ADH1, ATF1, ACC1, FAS1 and OLE1 were marked repressed by high LA content. Our data indicated that LA can regulate the expressions of related functional genes to efficiently influence the formations of volatiles in BCAAs supplemented wines. Therefore, it is essential to consider initial content of unsaturated fatty acids (LA) in must when using the strategy that supplying amino acids (BCAAs) to modulate aromatic quality of wines.

Use of Torulaspora delbrueckii Co-fermentation With Two Saccharomyces cerevisiae Strains With Different Aromatic Characteristic to Improve the Diversity of Red Wine Aroma Profile.

The use of selected Saccharomyces and non-Saccharomyces strains as mixed starters has advantages over pure fermentation due to achieving wine products with distinctive and diversified aroma expected by consumers. To obtain a way to improve the aroma diversity and increase the differentiation of wine product, in this study, the aromatic effect of multi-culture of indigenous Torulaspora delbrueckii (TD12), simultaneous and sequential inoculation with two Saccharomyces strains (indigenous icewine yeast SC45 and commercial yeast BDX) with different enological characteristics were investigated in laboratory-scale 20 L fermenter, respectively. The results showed that T. delbrueckii co-fermented with different S. cerevisiae strain could generate diversified physicochemical and aromatic quality of wine as evidenced by PCA. Mixed fermentation of SC45/TD12 produced higher contents of higher alcohol (3-methyl-1-pentanol and phenylethyl alcohol), ethyl esters (ethyl decanoate and ethyl butanoate), terpenes and phenylacetaldehyde with less fatty acids (hexanoic acid, octanoic acid) and acetic acid, while BDX/TD12 generated more C6 alcohol (1-hexanol) and acetate esters (ethyl acetate and isoamyl acetate). Compared to simultaneous inoculation, sequential inoculation could achieve higher aroma diversity, and generate higher intensity of fruity, flowery and sweet attributes of wine as assessed by calculating the odor activity values. The different S. cerevisiae strain and inoculation method in alcoholic fermentation could further influence the formations of aromatic compounds in malolactic fermentation. Our results highlighted the importance of S. cerevisiae strain in shaping the aromatic quality of wine in mixed fermentation, and also suggested that using different S. cerevisiae strains with distinct aromatic characteristics co-fermentation with specific non-Saccharomyces strain is a potential way to increase the aromatic diversity and quality of wine product, which could provide an alternative way to meet the requirement of wine consumers for diversified aromatic quality.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

Enhancement of ß-carotene production by over-expression of HMG-CoA reductase coupled with addition of ergosterol biosynthesis inhibitors in recombinant Saccharomyces cerevisiae.

In this study, the synergistic effect of overexpressing the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene and adding ergosterol synthesis inhibitor, ketoconazole, on ß-carotene production in the recombinant Saccharomyces cerevisiae was investigated. The results showed that the over-expression of HMG-CoA reductase gene and adding 100 mg/l ketoconazole alone can result in 135.1 and 15.6% increment of ß-carotene concentration compared with that of the control (2.05 mg/g dry weight of cells), respectively. However, the combination of overexpressing HMG-CoA reductase gene and adding ketoconazole can achieve a 206.8% increment of pigment content (6.29 mg/g dry weight of cells) compared with that of the control. Due to the fact that over-expression of the HMG-CoA reductase gene can simultaneously improve the flux of the sterol and carotenoid biosynthetic pathway, it can be concluded that under the circumstances of blocking sterol biosynthesis, increasing the activity of HMG-CoA reductase can result in more precursors FPP fluxing into carotenoid branch and obtain a high increment of ß-carotene production. The results of this study collectively suggest that the combination of overexpressing HMG-CoA reductase gene and supplying ergosterol synthesis inhibitor is an effective strategy to improve the production of desirable isoprenoid compounds such as carotenoids.

Enhanced production of ß-carotene by recombinant industrial wine yeast using grape juice as substrate.

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their ß-carotene production in real grape juice. The results showed that highest ß-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous ß-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.

Important role of catalase in the production of ß-carotene by recombinant Saccharomyces cerevisiae under H2O2 stress.

The effect of H(2)O(2) supplement on cell growth and ß-carotene productions in recombinant Saccharomyces cerevisiae CFW-01 and CFW-01 ctt1 deficiency in cytosolic catalase were investigated in shaking flasks. The results showed that supplement of H(2)O(2) (0.5 and 1.0 mM) can significantly stimulate the ß-carotene production. However, ß-carotene levels of CFW-01 ctt1Δ under 0.5 and 1 mM H(2)O(2) were 16.7 and 36.7% lower than those of CFW-01, respectively. Although lacking cytosolic catalase, no significant differences in cell growth were observed between CFW-01 ctt1Δ and CFW-01 under the same level of H(2)O(2) stress. These results suggest that ß-carotene can act as an antioxidant to protect the recombinant yeast from H(2)O(2) oxidative damage in the absence of cytosolic catalase. However, catalase still plays an important role in the production of ß-carotene under H(2)O(2) stress. If catalase can not timely decompose H(2)O(2), the free radicals such as OH· derived from H(2)O(2) can result in decrease of ß-carotene concentration. Therefore, in the production of ß-carotene by H(2)O(2) stress, not only the level of oxidative stress, but also the activities of catalase in cells should be considered.

Biosynthesis of anthocyanins and their regulation in colored grapes.

Anthocyanins, synthesized via the flavonoid pathway, are a class of crucial phenolic compounds which are fundamentally responsible for the red color of grapes and wines. As the most important natural colorants in grapes and their products, anthocyanins are also widely studied for their numerous beneficial effects on human health. In recent years, the biosynthetic pathway of anthocyanins in grapes has been thoroughly investigated. Their intracellular transportation and accumulation have also been further clarified. Additionally, the genetic mechanism regulating their biosynthesis and the phytohormone influences on them are better understood. Furthermore, due to their importance in the quality of wine grapes, the effects of the environmental factors and viticulture practices on anthocyanin accumulation are being investigated increasingly. The present paper summarizes both the basic information and the most recent advances in the study of the anthocyanin biosynthesis in red grapes, emphasizing their gene structure, the transcriptional factors and the diverse exterior regulation factors.

Changes of flavan-3-ols with different degrees of polymerization in seeds of 'Shiraz', 'Cabernet Sauvignon' and 'Marselan' grapes after veraison.

Flavan-3-ols consist of flavan-3-ol monomers and polymers with different degrees of polymerization (DP). In this study, flavan-3-ol extracts from grape seeds were well separated into three fractions including monomers, oligomers (2 < DP < 10) and polymers (DP > 10), by means of normal-phase HPLC-MS. The different patterns of these three fractions were analyzed in three Vitis vinifera cultivars ('Shiraz', 'Cabernet Sauvignon' and 'Marselan') seeds from veraison to harvest. The results showed: (1) polymers were the main form of flavan-3-ols in grape seeds and monomers accounted for only a small proportion; (2) the contents of flavan-3-ol monomers in the seeds of three grape cultivars all exhibited a gradually decreasing trend with a little fluctuation, whereas the patterns of the change of contents of oligomers and polymers were extremely different among grape cultivars; the contents of flavan-3-ol oligomers were enhanced in the seeds of 'Cabernet Sauvignon', but were reduced in the other two cultivars; (3) with regard to the proportion of flavan-3-ols with a certain DP to total flavan-3-ols, both flavan-3-ol monomers and flavan-3-ols with low DP fell in proportion, while the flavan-3-ols with high DP increased correspondingly. These findings indicate that flavan-3-ol polymerization in developing seeds is variety-dependent and may be genetically regulated.