Search

Phytoene synthase (PSY) as the key rate-limiting enzyme in carotenoid metabolism can be regulated by various regulators and factors. We found that DsPSY1 played a dominant role in carotenogenesis in the β-carotene-accumulating Dunaliella salina , and two amino acid residues critical in the substrate binding were associated with the functional variance between DsPSY1 and DsPSY2.

Previous studies have found that probiotic supplements could comfort mental behavioral disorders. This study investigated the effects of Lactiplantibacillus plantarum DMDL 9010 (LP9010) intake on the depression-like behavior induced by dextran sodium sulfate (DSS) and its possible mechanism. Male C57BL/6N mice were fed with DSS to establish the model of ulcerative colitis. LP9010 intake reduced DSS-induced inflammatory response, and repaired intestinal barrier damage, as well as lightened depression-like behavior. LP9010 supplementation also inhibited neuroinflammation by up-regulating the levels of neurotransmitters especially 5-HT, NE, DA, and 5-HIAA. Moreover, intake of LP9010 reorganized the gut microbiome by increasing the relative abundance of Bacteroidetes and Firmicutes, and decreasing the relative abundance of Proteobacteria and Verrucomicrobia. Furthermore, treatment with LP9010 also increased the levels of short-chain fatty acids such as butyric acid and propionic acid. In conclusion, LP9010 intake was a promising probiotic intervention strategy for the prevention of colitis-induced behavioral disorders through the microbiota-gut-brain axis.

Polymyxin A1 was a rarely investigated member in the polymyxins family produced by Bacillus aerosporus. As a cyclic non-ribosomal lipopeptide, it was purified from Paenibacillus thiaminolyticus for the first time. The producing strain SY20 was screened from Chinese natural fermented bamboo shoots and identified as P. thiaminolyticus SY20 using 16S rRNA homology along with whole genome sequencing. The optimum incubation time was 32 h by the growth kinetics of antimicrobial agent production. The proteinaceous nature of antimicrobial agents was characterized according to the physicochemical properties of the cell-free supernatant. Subsequently, the active antimicrobial agent was purified from the supernatant using ammonium sulfate–graded precipitation, ion-exchange chromatography, and C18-H chromatography. The active agent was identified as polymyxin A1 with a molecular weight 1156.7 Da and antimicrobial activity mainly against Gram-negative bacteria. The molecular structure, a cyclic heptapeptide and a tripeptide side chain acylated by a fatty acid at the amino terminus, was elucidated using the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), amino acid analysis, and whole genome mining tool. Meanwhile, the biosynthetic gene cluster of polymyxin A1 including five open reading frames (ORFs) was demonstrated in the genome. The compound should be further explored for its efficacy and toxicity in vivo to develop its application.

As one of the sources of biodiesel, microalgae are expected to solve petroleum shortage. In this study, different concentrations of piperonyl butoxide were added to the culture medium to investigate their effects on the growth, pigment content, lipid accumulation, and content of carotenoids in

As one of the core equipment components of oil and gas drilling rig, derrick is also the main heavy-duty component. Working environment of derrick is quite bad, the material for which should have not only high strength, but also good plasticity, toughness and weldability. In this paper, the chemical composition, microstructure, tensile properties, impact properties and hardness of three typical Q690D high strength low alloy steel manufactured from different methods were studied. The application feasibility of Q690 for drilling derrick manufacture was analyzed. The experimental results show that the carbon equivalent, tensile properties and impact absorption energy of Q690D steel with different thickness obtained by TMCP and Q-T process were obviously higher than the requirement of corresponding standard. TMCP Q690D steel had better toughness, lower carbon content and carbon equivalent, and its micro-structure was granular bainite. Carbon content of Q-T Q690D steel plate was relatively higher than that of TMCP Q690D, and the toughness was slightly lower than that of TMCP Q690D. The microstructure Q-T Q690D was tempered sorbite, and there was certain zonal segregation in center part. The thickness of steel plate used in derrick was usually less than 40 mm, whose manufacturing technology of Q690D was mature. There is broad application prospect in the field of drilling equipment. The application of Q690D for drilling equipment not only improve the strength grade of equipment materials, but also help to optimize the design of components and ensure service safety.

The pull out and well drop accident caused by serious thread galling damage of low alloy steel tubing connections were investigated in this paper. Representative samples were selected from five failure accidents, and the test and verification program were designed. Based on the experiment and analysis it is considered that the material used for tubing and the quality of thread processing meet the requirements of the standard. The field end of tubing connection pull out may be due to the serious thread galling damage, which reduced the joint strength. Using the torque-position control method to make-up the thread connections in oil field may ensure the connection quality and the connection strength, meanwhile, and hence to avoid the occurrence of tubing pulling out effectively.

Drilling equipment is the key of oil and gas drilling development. Its manufacturing quality and service performance have important influence on oil and gas drilling development safety. The application of high strength grade steel plays an important role in improving drilling equipment manufacturing level and service performance. In this paper, the chemical composition, microstructure, tensile properties, impact properties and hardness of TMCP Q550D and Q-T Q550D high-strength low-alloy steel were tested and compared, and the application feasibility for drilling equipment manufacture was analyzed comprehensively. The experimental results show that the mechanical properties of Q550D by two different methods were obviously higher than the requirement of national standard. Q550D steel had excellent plasticity and toughness, which meets the requirement of drilling equipment manufacture. The main difference between different steel lied in their chemical composition and micro-structure. Carbon content of TMCP Q550D steel plate was lower than that of QT Q550D, and TMCP Q550 was mainly depend on TMCP technology and micro-alloy elements, whose micro-structure was mainly granular bainite. Q-T Q550D was mainly depend on Q-T technology, and its microstructure was tempered sorbite with obvious banded structure and slightly low toughness.

Microalgae are considered to be a very promising class of raw material for carotenoid production. In this study, melatonin (MLT), a widely used plant growth regulator, was added to the autotrophic medium of Dunaliella bardawil to explore its effects on the growth and pigment accumulation of Dunaliella bardawil. The results showed that the induction of exogenous MLT alone was not beneficial to the growth and pigment accumulation of Dunaliella bardawil, and the higher the concentration, the more obvious the inhibitory effect on the algal cells. Therefore, a strategy to promote carotenoid accumulation in Dunaliella bardawil by combining exogenous MLT and light induction was carried out. Under 4500 LUX light intensity, the content of zeaxanthin was significantly increased under exogenous MLT induction. In the 200 μg/mL, 300 μg/mL, and 400 μg/mL MLT-treated groups, the zeaxanthin single-cell content in the 300 μg/mL-treated group was as high as 0.38 ng/mL (0.17 ng/mL in the control group), which was 1.24-fold higher compared to the control. Under 9500 LUX light intensity, all carotenoids showed an increasing trend in all experimental groups, except for zeaxanthin, which showed a decreasing trend. The effect of 300 μg/mL showed the most obvious in the 200 μg/mL,300 μg/mL, and 400 μg/mL MLT treatment groups, where the lutein, α-carotene and β-carotene contents were 1.24, 1.14 and 1.31 times higher than those of the control group, respectively. Overall, exogenous MLT at high light intensities had a significant effect on pigment accumulation in Dunaliella bardawil.

Polymyxin A1 was a rarely investigated member in the polymyxins family produced by

The salt-tolerant unicellular alga Dunaliella bardawil FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, i.e., β-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from D. bardawil. Their substrate specificities and enzyme activities were tested through functional complementation assays in Escherichia coli. It was showed that DbBCH could catalyze the hydroxylation of the β-rings of both β- and α-carotene, and displayed a low level of e-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate β-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the β-ring and e-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the β-ring of α-carotene. The high accumulation of lutein in D. bardawil may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga D. bardawil FACHB-847.

Dunaliella salina can accumulate a large amount of β-carotene which is generally considered to be its terminal product of carotenoid metabolism. In this study, it was proved that D. salina has the ketolase (DsBKT) of catalyzing the synthesis of astaxanthin, the downstream products of β-carotene. Therefore, the reason why D. salina does not synthesize astaxanthin is the purpose of this study. The enzymatic activity of DsBKT was detected by functional complementation assays in Escherichia coli, results showed that DsBKT had efficient ketolase activity toward β-carotene and zeaxanthin to produce astaxanthin, indicating that there were complete astaxanthin-producing genes in Dunaliella. Unlike the induced expression of Lycopene cyclase (catalyzing β-carotene synthesis) under salt stress, the expression of DsBKT was very low under both normal and stress conditions, which may be the main reason why D. salina cannot accumulate astaxanthin. On the contrary, with the astaxanthin-rich Haematococcus pluvialis as a control, its BKT gene was significantly upregulated under salt stress. Further study showed that DsBKT promoter had strong promoter ability and could stably drive the expression of ble-egfp in D. salina. Obviously, DsBKT promoter is not the reason of DsBKT not being expressed which may be caused by Noncoding RNA.

Lactarius volemus Fr. has been regarded as a great edible medicinal fungal resource in China. In this study, L. volemus Fr. polysaccharide (LVP) with an average molecular weight of 16.842 kDa was obtained by water extraction. The structure of LVP was characterized to be mannan, and the linkages in the mannan were found to comprise the Manp, (1→4)-α-Man and (1→4,6)-α-Man. Furthermore, intraperitoneal administration of LVP increased the thymus, spleen and liver indices, dose-dependently. Additionally, LVP enhanced the immune response and the phagocytic activities. Pathological evaluations showed that LVP in mice increased the proliferation of red medullary lymphocytes (60–70%). Collectively, these results indicated that LVP might be a potential resource of raw material for further investigations of functional foods.

Biological remediation of toxic oxygen-containing anions such as nitrate that are common in the environment is of great significance. Therefore, it is necessary to understand the specific role of nitrate and nitrite reductase in the bioremediation process. Bacillus cereus LJ01, which was isolated from traditional Chinese soybean paste, effectively degraded nitrite (such as NaNO2) at 0–15 mmol L−1 in LB medium. Moreover, the nitrite-degrading active substance (ASDN) was isolated and purified from B. cereus LJ01. The nitrite-degrading activity of nitrite reductase (named LJ01-NiR) was 4004.89 U mg−1. The gene encoding the assimilation of nitrite reductase in B. cereus LJ01 was cloned and overexpressed in E. coli. The purified recombinant LJ01-NiR has a wide range of activities under temperature (20–60 °C), pH (6.5–8.0) and metal ions (Fe3+, Fe2+, Cu2+, Mn2+, and Al3+). Kinetic parameters of LJ01-NiR, including the values of Km and Vmax were 1.38 mM and 2.00 μmol g−1 min−1, respectively. The results showed that LJ01-NiR could degrade nitrite with or without an electron donor. In addition, sequence analysis revealed that LJ01-NiR was a ferredoxin-dependent nitrite reductase given the presence of conserved [Fe4–S4] cluster and heme-binding domain. The nitrite ion binds to the LJ01-NiR active site by forming three hydrogen bonds with the residues ASN72, ALA133 and ASN140. Due to its high nitrite-degrading activity, LJ01-NiR could potentially be used for environmental pollution treatment.

Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.

A two-step strategy was employed to culture Dunaliella tertiolecta, an oleaginous unicellular green alga, combined by the salt stress and sodium azide intervention, to observe their effects on its lipid accumulation. When the algae cultured at different salt concentrations reached the logarithmic growth phase, sodium azide was added. The results showed that the addition of sodium azide significantly increased the lipid content and had no significant effect on cell biomass. The lipid yield and single cell lipid content under 50 μM sodium azide increased by 10.4% and 21.7%. Under the two-step culture condition, combining of the treatment of 50 μM sodium azide and 2.5 M salt stress, the total lipid productivity and single-cell lipid content were 10% and 70.5% higher than that of the control. It seemed that sodium azide and salinity might have a synergistic effect on the lipid accumulation of D. tertiolecta. It can be concluded that sodium azide is an effective inducer of lipid accumulation in D. tertiolecta, and two-stage cultivation is a feasible way to improve lipid accumulation in microalgae.

Dunaliella bardawil, a unicellular green alga, can accumulate a large amount of lutein and β-carotene under stresses. Using chemical inducers combined with abiotic stress to promote the accumulation of high value-added products such as lipids and carotenoids in microalgae has attracted more and more attention. In this study, creatinine was added into autotrophic medium to investigate its effects on the growth, chlorophyll content, and the ingredients and content of carotenoids in D. bardawil. The results showed that creatinine alone could significantly increase the biomass, chlorophyll and carotenoid contents of D. bardawil, among which the contents of lutein and β-carotene were further increased, while the content of zeaxanthin was decreased. In order to further improve the content of the two carotenoids, different light intensities combined with creatinine have been adopted. Under 6.589 W/m2 light intensity, creatinine could effectively increase the production of lutein, zeaxanthin, α-carotene and β-carotene. Compared with the control, the content of lutein increased by 46 % and the content of β-carotene increased by 77 % when the concentration of creatinine was 500 μg/mL. In conclusion, creatinine can effectively improve the production lutein and β-carotene in D. bardawil, which is more conducive under lower light intensity.

The salt-tolerant unicellular alga

Microalgae have been considered as alternative sustainable resources for high-value bioproducts such as lipids (especially triacylglycerides [TAGs]), polyunsaturated fatty acids (PUFAs), and carotenoids, due to their relatively high photosynthetic efficiency, no arable land requirement, and ease of scale-up. It is of great significance to exploit microalgae for the production of high-value bioproducts. How to improve the content or productivity of specific bioproducts has become one of the most urgent challenges. In this review, we will describe high-value bioproducts from microalgae and their biosynthetic pathways (mainly for lipids, PUFAs, and carotenoids). Recent progress and strategies for the enhanced production of bioproducts from microalgae are also described in detail, and these strategies take advantages of optimized cultivation conditions with abiotic stress, chemical stress (addition of metabolic precursors, phytohormones, chemical inhibitors, and chemicals inducing oxidative stress response), and molecular approaches such as metabolic engineering, transcriptional engineering, and gene disruption strategies (mainly RNAi, antisense RNA, miRNA-based knockdown, and CRISPR/Cas9).

The nitrite that normally exists in fermented foods is a threat to public health. Nitrite degradation by lactic acid bacteria (LAB) has the advantages of safety and low cost. This study investigated the effect of Lactobacillus plantarum DMDL 9010 (LP9010) on growth phenotype under nitrite stress from the perspective of whole genome sequencing. The results showed that LP9010 has certain tolerance to nitrite. However, high nitrite concentration (5–12.5 mmol/L) inhibited the growth and acid production. Nitrite could not only affect the morphology of LP9010 cells with longer phenotype, shrink and dent on the surface (even crack), but also decreased cell surface hydrophobicity. Correspondingly, 19 genes encoding stress proteins have been found in the genome. In addition, genes related to osmotic regulation proteins and peptidoglycans synthesis were found in the genome of LP9010, which may affect the cell wall structure under nitrite stress. Low pH lactic acid could degrade nitrite, whereas nitrite was hardly degraded when pH > 4. In addition, we found the putative nitrite reductase gene in LP9010, the nitrite ion bound to the Cd1NiR (pgl) active site by forming two hydrogen bonds with the residues PRO87 and PRO88. This study could provide theoretical reference to reduce nitrite in food products.

Carotenoids are essential for photosynthesis and photoprotection in photosynthetic organisms and beneficial for human health. Apocarotenoids derived from carotenoid degradation can serve critical functions including hormones, volatiles, and signals. They have been used commercially as food colorants, animal feed supplements, and nutraceuticals for cosmetic and pharmaceutical purposes. This review focuses on the molecular evolution of carotenogenic enzymes and carotenoid cleavage oxygenases (CCOs) from bacteria, fungi, cyanobacteria, algae, and plants. The diversity of carotenoids and apocarotenoids as well as their complicated biosynthetic pathway in different species can shed light on the history of early molecular evolution. Some carotenogenic genes (such as phytoene synthases) have high protein sequence similarity from bacteria to land plants, but some (such as phytoene desaturases, lycopene cyclases, carotenoid hydroxylases, and CCOs) have low similarity. The broad diversity of apocarotenoid volatile compounds can be attributed to large numbers of carotenoid precursors and the various cleavage sites catalyzed by CCOs enzymes. A variety of carotenogenic enzymes and CCOs indicate the functional diversification of carotenoids and apocrotenoids in different species. New carotenoids, new apocarotenoids, new carotenogenic enzymes, new CCOs, and new pathways still need to be explored.

HBscFv-IFNγ, a fusion protein constructed by fusing γ-interferon (IFNγ) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNγ, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNγ were connected into HBscFv-IFNγ by the linker (Gly4Ser)3, and then the multicopy recombinant plasmids pPICZαA/(HBscFv-IFNγ)1,2,4 were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNγ, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNγ displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNγ in a purity of 95-98%. The HBscFv-IFNγ was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNγ has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNγ in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.

Causes were analyzed on the thread-tripping failure of the Φ88.90 mm × 6.45 mm L80 EU tubing used in a certain gas well western China. The physical and chemical properties, macro/micro morphology were used to analyze the failure samples. The thread parameters inspection and the full scale test were carried out by using the same batch of unused tubing. It is showed that the physical and chemical properties, thread parameters and the full scale test results were all meet the requirements of the corresponding standard. The failure is a low-load thread stripping case and the main cause for such a failure is thread gluing which result in the reduction of the connection strength. The reason caused the thread gluing is thread alternating which occurred during the making-up process.

Microalgae are unicellular organisms that act as the crucial primary producers all over the world, typically found in marine and freshwater environments. Most of them can live photo-autotrophically, reproduce rapidly, and accumulate biomass in a short period efficiently. To adapt to the uninterrupted change of the environment, they evolve and differentiate continuously. As a result, some of them evolve special abilities such as toleration of extreme environment, generation of sophisticated structure to adapt to the environment, and avoid predators. Microalgae are believed to be promising bioreactors because of their high lipid and pigment contents. Genetic engineering technologies have given revolutions in the microalgal industry, which decoded the secrets of microalgal genes, express recombinant genes in microalgal genomes, and largely soar the accumulation of interested components in transgenic microalgae. However, owing to several obstructions, the industry of transgenic microalgae is still immature. Here, we provide an overview to emphasize the advantage and imperfection of the existing transgenic microalgal bioreactors.

Paocai is one of the most popular traditional fermented products in China. Different lactic acid bacteria (LAB) have an important influence on the microbial changes, the dynamic changes of metabolites, and the sensory qualities of paocai. In this study, five different paocai groups were designed which were fermented by Lactobacillus plantarum DMDL 9010 (LP), Lactobacillus brevis GIM 1.773 (LB), Leuconostoc mesenteroides GIM 1.774 (LM), mixed three kinds of LAB (LP + LB + LM), pasteurization (PST) and natural fermentation (NF) without LAB. The nitrite, nitrate, pH, titratable acidity, viable count (LAB and yeast) and metabolites (organic acids, sugars and volatile substances) of these paocai groups were measured. Compared with each single bacterial fermentation (LP, LB and LM), NF and PST, the mixed three kinds of LAB (LP + LB + LM) had a lower nitrite peak, which could be attributed to the significant accumulation of organic acids, and positively affected sensory quality of paocai. Flavor substances produced in paocai samples confirmed as alcohols, ketones and olefins. Sensory evaluation showed that multi-bacteria mixed fermentation of paocai had a better flavor than that of NF, PST and single bacterial fermentation. Therefore, the use of multiple LAB as starter cultures contributes to enhance paocai qualities and control the levels of nitrite.

ATP-citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is responsible for the biosynthesis of fatty acids, lipids, and flavonoid. Here, cDNAs of the small and large subunits of ACL (DtACLA and DtACLB) and their genomic sequences were isolated from Dunaliella tertiolecta using RT-PCR, RACEs and genomic walking techniques. It was found that there were 12 exons and 11 introns in DtACLA, and 20 exons and 19 introns in DtACLB. PlantPAN and PlantCARE revealed a number of putative transcription factor binding sites in the 5′-flanking regions of DtACLA and DtACLB. Conserved domain analysis and phylogenetic analysis showed that ACLA and SCSβ may share the common evolutionary origin, and ACLB may have a common origin from the fusion and divergence of SCSα and CS. The recombinant DtACLA and DtACLB in E. coli BL21 (DE3) seemed to be expressed mainly in the form of insoluble fraction. The transcription levels of DtACLA and DtACLB were fairly consistent with lipid accumulation in response to nitrogen deficiency, suggesting that both subunits of ACL from D. tertiolecta may be coordinate to function in the catalysis, and their activity was related to the lipid accumulation in D. tertiolecta.

In the carotenoid biosynthesis, lycopene β-cyclase (LCYb) is a key regulatory enzyme involved in the conversion of lycopene into β-carotene. Under stress conditions, such as high salinity, high light and nutrient deprivation, large amounts of β-carotene can be accumulated in Dunaliella bardawil. To study on the molecular responses of salt stress in D. bardawil is of great significance to reveal the mechanisms of salt tolerance and engineer crop plants to be salt-tolerant. In this study, the full-length coding sequence of lcyb from D. bardawil (Dblcyb, GenBank: KX218392) was isolated by transcriptome sequencing. Then, the genomic sequence, promoter and terminator regions of Dblcyb were isolated by genome walking. The Dblcyb promoter (GenBank: KX218393) contained several typical transcription boxes, multiple light response elements and a salt-regulated element (SRE, GT1GMSCAM4). Dbpsy and Dblcyb responsible for β-carotene biosynthesis in D. bardawil was shown to be up-regulated under salt stress and their promoters contained the common SRE. By element deletion analysis and using Ble-EGFP as the reporter, the salt-inducible SRE was confirmed to confer salt-induced expression of Dblcyb promoter. It was indicated that the salt-regulated expression of Dblcyb may be attributed to the salt-responsive element (GT1GMSCAM4) and the GT-rich region in its genomic sequence.

Dunaliella bardawil is characterized as a mass accumulator of β-carotene through lycopene cyclization by lycopene cyclase catalysis. The present research tried to inhibit the formation of β-carotene in D. bardawil to accumulate lycopene using triethylamine, a lycopene cyclase inhibitor. Results showed that 50 ∼ 100 ppm triethylamine could steadily trigger lycopene production, and 150 ppm triethylamine treatment for 3 days led to maximum proportion of lycopene with lowest proportion of β-carotene. The upstream genes (GGPS, PSY, PDS, and ZDS) involved in lycopene biosynthesis of D. bardawil were upregulated after triethylamine treatment for 3 days. While the transcription levels of the downstream genes (LycB, LycE, and ChyB) were decreased at high triethylamine concentration (50 ∼ 150 ppm). Triethylamine seemed to increase lycopene metabolic flow by inhibiting the expressions of LycB and LycE, which stimulated the expression levels of the upstream genes. The increased expression of upstream genes resulting from the inhibition of lycopene cyclases in D. bardawil provides a new insight into the desired pathway of carotenoid synthesis in algae and plants.

Yogurt-flavored bases are widely used in food industry for enhancing flavors. In this study, yogurt-flavored bases by mixed lactic acid bacteria with or without lipase were investigated, which included Streptococcus lactis ACCC 11093 (SL) and Lactobacillus casei subsp. rhamnosus 6013 (LCR), SL and Lactobacillus acidophilus 1.1878 (LAP), and SL and Lactobacillus plantarum DMDL 9010 (LP), respectively. Compared with the combination of SL and LCR and the combination of SL and LAP, the combination of SL and LP had higher production of total amino acids, volatile acids and esters, suggesting that the combination of SL and LP was more efficient to produce yogurt-flavored bases. In addition, lipase significantly increased the production of amino acids associated with sweet and bitter flavors, and resulted in rougher milk globule surface and tighter network structure compared with fermentations without the addition of lipase. Furthermore, a total of 54 volatile compounds were detected in yogurt-flavored bases. The production of volatile compounds in fermentations with and without the addition of lipase, was mainly affected by acids and esters, respectively. Therefore, the lipase is a favorable supplement for producing yogurt-flavored bases.

Dunaliella tertiolecta is a highly oil-producing microalgae. Here, we report its de novo transcriptome assembly and quantitative gene expression analysis of the intervention of triethylamine, with a focus on the complex interactions of pathways involved in the production of triacylglycerol (TAG) biofuel precursors. After growing under 100 ppm triethylamine, we quantified the cellular content of the major biomolecules, including total lipids, single cell oil content, and lipid components. The transcriptome was assembled de novo and the main functional classes, related pathways, and expression of important genes were quantified by the mapping of reads to the transcriptome. Almost 65,925 high-quality readings were produced on the Illumina HiSeq sequencing platform. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during triethylamine was not through the Kennedy pathway, but the bypass pathway by high expression of phosphate: diacylglycerol acyltransferase. The glycolytic pathway has also been down-regulated, not only in the cytoplasm but also in the chloroplast. Triosephosphate translocator was downregulated, and the DNA binding with One Finger protein gene was upregulated. The quantitative transcriptome study reveals a broad overview of how triethylamine stress leads to excessive TAG production in Dunaliella, and provides various genetic engineering goals and strategies to improve biofuel precursors in oleaginous microalgae.

Methyl Acetate (MA), Ethyl Acetate (EA), n-Propyl Acetate (PA), n-Butyl Acetate (BA), Ethyl Propionate (EP) and Ethyl Butyrate (EB) are common ester components for Nitrocellulose (NC) lacquer thinner. They exhibit good solubility for NC lacquer, but they are flammable and explosive and could also bring negative effect on environment. In this paper, their dissolution rates, lower explosive limits (LELs) and the half maximal inhibitory concentrations (IC50) (on Dunaliella bardawil) were studied. Their dissolution rates for NC lacquer are about 39.64, 32.81, 23.56, 19.76, 26.50 and 19.36 mg/min, respectively. Their LELs are about 3.145%, 2.060%, 1.845%, 1.540%, 1.740% and 1.425%, respectively. And their IC50 are about 20.47, 11.35, 3.18, 1.00, 3.37 and 1.98 g/L, respectively. Considering from the three aspects, MA is a better ester component for NC lacquer thinner than other five esters, while BA and EB are relative worse components for NC lacquer thinner.

Microalgae are promising alternatives for sustainable biodiesel production. Previously, it was found that 100 ppm triethylamine greatly enhanced lipid production and lipid content per cell of Dunaliella tertiolecta by 20% and 80%, respectively. However, triethylamine notably reduced biomass production and pigment contents. In this study, a two-stage cultivation with glycerol and triethylamine was attempted to improve cell biomass and lipid accumulation. At the first stage with 1.0 g/liter glycerol addition, D. tertiolecta cells reached the late log phase in a shorter time due to rapid cell growth, leading to the highest cell biomass (1.296 g/liter) for 16 days. However, the increased glycerol concentrations with glycerol addition decreased the lipid content. At the second-stage cultivation with 100 ppm triethylamine, the highest lipid concentration and lipid weight content were 383.60 mg/liter and 37.7% of dry cell weight (DCW), respectively, in the presence of 1.0 g/liter glycerol, which were 27.36% and 72.51% higher than those of the control group, respectively. Besides, the addition of glycerol alleviated the inhibitory effect of triethylamine on cell morphology, algal growth, and pigment accumulation in D. tertiolecta. The results indicated that two-stage cultivation is a viable way to improve lipid yield in microalgae. IMPORTANCE Microalgae are promising alternatives for sustainable biodiesel production. Two-stage cultivation with glycerol and triethylamine enhanced the lipid productivity of Dunaliella tertiolecta, indicating that two-stage cultivation is an efficient strategy for biodiesel production from microalgae. It was found that glycerol significantly enhanced cell biomass of D. tertiolecta, and the presence of glycerol alleviated the inhibitory effect of triethylamine on algal growth. Glycerol, the major byproduct from biodiesel production, was used for the biomass accumulation of D. tertiolecta at the first stage of cultivation. Triethylamine, as a lipid inducer, was used for lipid accumulation at the second stage of cultivation. Two-stage cultivation with glycerol and triethylamine enhanced lipid productivity and alleviated the inhibitory effect of triethylamine on the algal growth of D. tertiolecta, which is an efficient strategy for lipid production from D. tertiolecta.

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil (DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of DbGGPS was 1814 bp, containing a 1074 bp ORF encoding 357 amino acids with a calculated mass of 38.88 kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that DbGGPS possessed a 48 amino acid transit peptide at its N terminus. Bioinformatic analysis revealed that DbGGPS was a member of a group of polyprenyltransferases with five conserved domains and two highly conserved aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay, and gene deletion analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene sources for carotenoid genetic engineering.

Dunaliella tertiolecta, a halotolerant alga, can accumulate large amounts of neutral lipid, which makes it a potential biodiesel feedstock. In this study, neutral lipids of D. tertiolecta induced by different salinities or N or P starvation were analyzed by thin-layer chromatography (TLC), flow cytometry (FCM), and confocal laser scanning microscopy (CLSM). High salinities or N or P starvation resulted in a decrease in cell growth and chlorophyll contents of D. tertiolecta. Neutral lipid contents increased markedly after 3-7 days of N starvation or at low NaCl concentrations (0.5-2.0 M). N starvation had a more dramatic effect on the neutral lipid contents of D. tertiolecta than P starvation. Four putative ME isozymes in different conditions can be detected by using isozyme electrophoresis. Two alternative acetyl-CoA producers, ACL and ACS genes, were up-regulated under low salinities and N starvation. It was suggested that low salinities and N starvation are considered efficient ways to stimulate lipid accumulation in D. tertiolecta.

The halophilic green alga Dunaliella bardawil has been used for commercial production of natural β-carotene by large-scale outdoor cultivation, which often suffers from heat stress especially at noon in hot summers. In this study, the effects of heat stress on cell growth, pigment contents, and activities of antioxidant system in D. bardawil were studied, and RNA-seq experiment was conducted to analyze the transcriptional response to heat stress (42 °C for 2 h) in D. bardawil. High temperature (42 °C) for short time treatment (≤3 h) did not severely affect the cell growth and pigment accumulation of D. bardawil. Multiple genes encoding heat shock proteins for protein folding and antioxidant enzymes against toxic reactive oxygen species were substantially up-regulated significantly under heat stress. D. bardawil cells tended to shift from aerobic to glycolytic metabolism for energy production to increase survival chances under heat stress. Furthermore, the enrichment of ascorbate-glutathione cycle, up-regulation of genes responsible for chloroplast membranes, and changes in lipid characteristics like carbon chain length and unsaturation degree could play a vital role in achieving thermotolerance of D. bardawil. Taken together, this study improved our understanding of the molecular mechanisms of heat stress responses in D. bardawil.

BACKGROUND To decrease the methanol content of the sugar cane sprits, mutagenesis of ultraviolet (UV) coupled with diethyl sulfate (DES) was used to generate a mutant of Saccharomyces cerevisiae with lower methanol content. Meanwhile, the effects of the additions of pectinase, cellulase and glycine on the production of methanol in sugar cane spirits were evaluated. RESULTS After mutagenesis of UV coupled with DES, a mutant S. cerevisiae DU9 with low production of methanol (97.3 ± 1.7 mg/L) was selected, with a 12.3% decrease of that of S. cerevisiae D4 only with DES treatment, and with a 27.8% reduction of that of the strain without any treatment. Pectinase and cellulase significantly increased the methanol levels of the sugar cane spirits. The results showed that there was linear relationship between glycine (concentration within 0∼0.9 g/L) and methanol in sugar cane sprits and the linear equation was y = 104.7 × −4.79 with the conversion rate of glycine conversion to methanol as 24.56%. CONCLUSION Mutagenesis of UV coupled with DES is an efficient way to generate a mutant of S. cerevisiae with lower methanol content. Also, it is necessary to control the additions of pectinase, cellulase and glycine in the fermentation medium, and other unknown ways to generate methanol metabolic pathway in yeasts may need further study. © 2014 Society of Chemical Industry

Methanol, often generated in brewed wine, is highly toxic for human health. To decrease the methanol content of the brewed wine, atmospheric and room-temperature plasma (ARTP) was used as a new mutagenesis tool to generate a mutant of Saccharomyces cerevisiae with lower methanol content. Headspace gas chromatography was used to determine the identity and concentration of methanol with butyl acetate as internal standard in brewed wine. With 47.4% higher and 26.3% positive mutation rates were obtained, the ARTP jet exhibited a strong effect on mutation breeding of S. cerevisiae. The mutant S. cerevisiae S12 exhibited the lowest methanol content, which was decreased by 72.54% compared with that of the wild-type strain. Subsequently, the mutant S. cerevisiae S12 was used to ferment different combinations of malt and adjuncts for lower methanol content and higher alcoholic content. It was shown that the culture 6#, which was 60% malt, 20% wheat, and 20% corn, was the best combinations of malt and adjuncts, with the lowest methanol content (104.8 mg/L), and a relatively higher alcoholic content (15.3%, v/v). The optimal malt-adjunct culture 6#, treated with the glucoamylase dose of 0.04 U/mg of grain released the highest reducing sugars (201.6 mg/mL). It was indicated that the variation in reducing sugars among the combinations of malt and different adjuncts could be due to the dose of exogenous enzymes.

The X80 girth welds were produced by solid-wire gas metal arc welding (GMAW) and shield metal arc welding (SMAW) using two welding consumables respectively, which contained different mass fraction of C, Mo and Ni. The tensile properties, notch toughness, hardness, and microstructures of welded joints were evaluated. The results indicate that high strength and good toughness of welded joints can be achieved. But the tensile properties of all weld metal of GMAW and SMAW process were evidently different because of the difference of mass fraction of C, Mo, Ni. The strength reduced slightly in softening zone of HAZ. Using welding consumable which contain higher Mo additions, the microstructure in filler weld and fusion zones were IAF+GB and GB+M respectively. Furthermore, the mechanical properties of X80 pipeline welded by single wire welding and double wire welding respectively have been compared. The double wire welds exhibited lower yield strength but higher toughness compared to the corresponding single wire welds.

The halophilic green alga Dunaliella bardawil FACHB-847 is rich in lutein and α-carotene, which has great potential for carotenoid production in open ponds. In this study, genes encoding lycopene β- and ε-cyclases (DbLcyB and DbLcyE) from D. bardawil FACHB-847 were functionally identified by genetic complementation in E. coli. The bifunctional DbLcyB not only catalyzed the formation of both mono- and bi-cyclic β-rings with a major β-cyclase activity, but also possessed a weak ε-cyclase activity. In contrast, DbLcyE preferred to convert lycopene into monocyclic δ-carotene, and possessed a weak β-monocyclase activity. Lutein and α-carotene were the prominent carotenoids in D. bardawil FACHB-847, which was in agreement with the result of genetic complementation of co-expression of DbLcyB and DbLcyE in E. coli with α-carotene as the prominent product. The bifunctional DbLcyB and DbLcyE may contribute to the high accumulation of α-carotene in D. bardawil FACHB-847. Interestingly, the accumulation of lutein in D. bardawil FACHB-847 was more sensitive to salt stress, while the accumulation of β-carotene in D. salina CCAP 19/18 was induced by salt stress. In brief, the production of different carotenoid compositions from these two Dunaliella species can be induced by different growth conditions.

Drop Weight Tear Testing (DWTT) method is widely used for determining a materials ability to arrest a propagating crack. The influence factors of DWTT are discussed for X70 thick wall linepipe steel. The effects of nonuniformity of the material, ductile-brittle transition temperature, notch type, evaluation method of the fracture surface, machining method and the test equipments are discussed. Some solutions and recommendations to the problems are proposed.

The influence of Cr on corrosion behavior of P110 steel was studied in the simulated CO2 environment. The result demonstrated that the Cr cant change the effect of temperature on P110 steel. The corrosion rates of the two kinds of materials reached the peak at 90°C. The temperature has dual effects on corrosion, which caused the adhesive and productivity of corrosion product formed up 90°C are better than that formed below 90°C; The adding of Cr in the P110 steel can prohibit the local corrosion to occur.