The association of plasma peroxiredoxin 3 with insulin in pregnant women.

Peroxiredoxin 3 (PRX3) is predominantly located in mitochondria and plays a major role in scavenging hydrogen peroxide of mitochondria. In the present study, we detected plasma PRX3 in pregnant women receiving oral glucose tolerance test at 24-28 gestational weeks. Plasma PRX3 was significantly increased about 1 h later than insulin secretion. In vitro detection of PRX3 in mouse islet cells showed up-regulation by more than 2-fold at 1 h and reached its top at 2 h of glucose stimulation, and the PRX3 level in cultured mediums was concomitantly elevated in a glucose concentration-dependent manner. In addition, both fasting plasma insulin and PRX3 were significantly higher in the subjects of term pregnancy as compared to that at 24-28 gestational weeks, and there was a positive correlation between plasma PRX3 and insulin. Our results indicate that PRX3 plays an active role in the response to insulin release. The positive association of plasma PRX3 and insulin suggest PRX3 to be a potential indicator of high insulin resistance.

Plasma level of peroxiredoxin 3 in patients with polycystic ovarian syndrome.

BACKGROUND: As a member of peroxiredoxin (PRX) family, PRX3 is predominantly located in mitochondria and plays an important role of free radical scavenging. Since a body of evidence demonstrated the involvement of PRX3 in insulin secretion, insulin sensitivity, and glucose metabolism, the present study was conducted to investigate the role of PRX3 in the pathogenesis of polycystic ovarian syndrome (PCOS) featured in insulin resistance. METHODS: Enzyme-linked immunosorbent assay was performed to detect plasma PRX3 in PCOS patients and control subjects. Levels of reactive oxygen species (ROS) and oxidized PRXs were detected in mouse islet cells treated with gradient glucose. RESULTS: We did not find significant difference of fasting plasma PRX3 between PCOS patients and controls. No association was noticed between fasting plasma PRX3 and fasting plasma glucose or insulin. After oral glucose tolerance test (OGTT), PCOS patients showed higher levels of both glucose and insulin as compared to controls. The plasma level of PRX3 was significantly increased at 2 h and began to fall back at 3 h of OGTT. There was a one-hour time lag of peak values between plasma PRX3 and insulin, and the plasma PRX3 at 2 h was positively correlated with the insulin level at 1 h of OGTT of PCOS patients. In addition, the level of ROS was significantly elevated at 1 h and oxidized PRX3 was increased dramatically at 2 h of 16.7mM glucose stimulation in mouse islet cells. CONCLUSION: It seems that PRX3 does not show its antioxidant function under baseline conditions. Instead, PRX3 responds to oxidative stress induced by rapid increase of insulin and glucose in patients with PCOS.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

BACKGROUND: Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. RESULTS: The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. CONCLUSIONS: These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

The Potential Role of Probiotics in Cancer Prevention and Treatment.

The human gut microbiota has a significant effect on many aspects of human physiology such as metabolism, nutrient absorption, and immune function. Imbalance of the microbiota has been implicated in many disorders including inflammatory bowel disease, obesity, asthma, psychiatric illnesses, and cancers. As a kind of functional foods, probiotics have been shown to play a protective role against cancer development in animal models. Clinical application of probiotics indicated that some probiotic strains could diminish the incidence of postoperative inflammation in cancer patients. Chemotherapy or radiotherapy-related diarrhea was relieved in patients who were administered oral probiotics. The present review summarizes the up-to-date studies on probiotic effects and the underlying mechanisms related to cancer. At present, it is commonly accepted that most commercial probiotic products are generally safe and can improve the health of the host. By modulating intestinal microbiota and immune response, some strains of probiotics can be used as an adjuvant for cancer prevention or/and treatment.

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences.

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆(12)-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆(15)-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆(9)-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Effects of different carbon sources on the growth, fatty acids production, and expression of three desaturase genes of Mortierella alpina ATCC 16266.

On the molecular and biochemical levels, the effects of different carbon sources on biomass production, fatty acid biosynthesis, and gene expression of three desaturases were investigated in Mortierella alpina ATCC 16266, at a stationary phase, which is an important filamentous fungus capable of producing various polyunsaturated fatty acids (PUFAs). The maximum mycelial biomass was achieved using sucrose as carbon source. However, the highest productivity of total lipids was shown to be no biomass associated. In addition, glucose was the preferred carbon source for the expression of three desaturase genes compared to others, but the change at the corresponding fatty acid product's level of these desaturase genes was not in accordance with the change measured at the mRNA level among those carbon sources that we utilized. Significant discrepancies between the mRNA expression and the product abundance may indicate post-transcriptional regulatory mechanisms of these desaturases.

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil.

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y. lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y. lipolytica. These findings provide valuable insights into the engineering of Y. lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70Δ. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

The functional role of peroxiredoxin 3 in reactive oxygen species, apoptosis, and chemoresistance of cancer cells.

PURPOSE: The mammalian peroxiredoxin (PRX) family contains six members that provide antioxidant defense in different cell types by removing reactive oxygen species (ROS) through conserved active cysteines. Different from other members, PRX3 is predominantly located in mitochondria, a major apoptosis mediator. The purpose of this review is to summarize the findings on PRX3 concerning its role in ROS removal, apoptosis, and chemoresistance of cancer cells. METHODS: The relevant literature from PubMed and Medline databases is reviewed in this article (1994-2014). RESULTS: Because of fast growth and relatively low supply of oxygen in cancer cells, ROS production from mitochondria is exaggerated to an extent that overwhelms cellular antioxidant defenses resulting in oxidative stress. As an active responder to oxidative stress, PRX3 is accordingly up-regulated in cancer cells to remove cellular ROS and inhibit apoptosis, which provides a favorable microenvironment for cell proliferation. CONCLUSION: Since most of chemotherapy or radiotherapy for cancers is through ROS increase and apoptotic induction, PRX3 might be involved in the chemotherapeutic resistance of cancers.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel.

BACKGROUND: Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical. RESULTS: We engineered Saccharomyces cerevisiae to produce fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences. To boost the ability of S. cerevisiae to produce the aforementioned esters, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted to increase flux towards fatty acyl-CoAs. In addition, five isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, and Adh7) targeted into the mitochondria were overexpressed to enhance production of alcohol precursors. By combining these engineering strategies with high-cell-density fermentation, over 230 mg/L fatty acid short- and branched-chain alkyl esters were produced, which is the highest titer reported in yeast to date. CONCLUSIONS: In this work, we engineered the metabolism of S. cerevisiae to produce biodiesels in the form of fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters. To our knowledge, this is the first report of the production of fatty acid isobutyl and active amyl esters in S. cerevisiae. Our findings will be useful for engineering S. cerevisiae strains toward high-level and sustainable biodiesel production.

Production of Fatty Acid-derived valuable chemicals in synthetic microbes.

Fatty acid derivatives, such as hydroxy fatty acids, fatty alcohols, fatty acid methyl/ethyl esters, and fatty alka(e)nes, have a wide range of industrial applications including plastics, lubricants, and fuels. Currently, these chemicals are obtained mainly through chemical synthesis, which is complex and costly, and their availability from natural biological sources is extremely limited. Metabolic engineering of microorganisms has provided a platform for effective production of these valuable biochemicals. Notably, synthetic biology-based metabolic engineering strategies have been extensively applied to refactor microorganisms for improved biochemical production. Here, we reviewed: (i) the current status of metabolic engineering of microbes that produce fatty acid-derived valuable chemicals, and (ii) the recent progress of synthetic biology approaches that assist metabolic engineering, such as mRNA secondary structure engineering, sensor-regulator system, regulatable expression system, ultrasensitive input/output control system, and computer science-based design of complex gene circuits. Furthermore, key challenges and strategies were discussed. Finally, we concluded that synthetic biology provides useful metabolic engineering strategies for economically viable production of fatty acid-derived valuable chemicals in engineered microbes.

Transcriptional regulation of desaturase genes in Pichia pastoris GS115.

Here we investigated the regulation of Pichia pastoris desaturase genes by low temperature and exogenous fatty acids in the late-exponential phase at the transcriptional level. Time-course studies of gene expression showed that mRNA levels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous oleic acid. Stearic acid showed no obvious repression of mRNA levels of Fad12 and Fad15 and a slight increase in Fad9A and Fad9B mRNA levels. Using a promoter-reporter gene construct, we demonstrated that the pFAD15 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by unsaturated fatty acids. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. Disruption of Spt23 resulted in a decrease in transcript levels of Fad9A and Fad9B, but had little effect on the other desaturase genes. Consistent with these observations, a decrease in the relative amount of oleic acid (OLA) and an increase in the relative content of linoleic acid and ALA with different degrees were clearly observed in the stationary phase cells of ΔSpt23 mutant. Further analysis showed that the effect of low-temperature activation and OLA inhibition on expression of Fad9A and Fad9B seemed to disappear after disruption of the Spt23 gene, which indicated that Spt23p is essential for the expression of two Δ9-desaturase genes internally and probably involved in the regulation of Δ9-desaturase genes transcription in response to external stimuli, and thereby plays a role in the synthesis of OLA.

Cloning and functional identification of delta5 fatty acid desaturase gene and its 5'-upstream region from marine fungus Thraustochytrium sp. FJN-10.

A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-γ-linolenic acid (20:3 Δ(8,11,14)), resulting in arachidonic acid (20:4 Δ(5,8,11,14)) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5'-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5'-upstream region of other delta5 desaturases showed that the 5'-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5'-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5'-upstream region of fad5 could drive the expression of green fluorescent protein.