Study on the mechanism of efficient extracellular expression of toxic streptomyces phospholipase D in Brevibacillus choshinensis under Mg2+ stress.

BACKGROUND: Phospholipase D (PLD) has significant advantages in the food and medicine industries due to its unique transphosphatidylation. However, the high heterologous expression of PLD is limited by its cytotoxicity. The present study sought to develop an efficient and extracellular expression system of PLD in the non-pathogenic Brevibacillus choshinensis (B. choshinensis). RESULTS: The extracellular PLD was effectively expressed by the strong promoter (P2) under Mg2+ stress, with the highest activity of 10 U/mL. The inductively coupled plasma-mass spectrometry (ICP-MS) results elucidated that the over-expression of PLD by P2 promoter without Mg2+ stress induced the ionic homeostasis perturbation caused by the highly enhanced Ca2+ influx, leading to cell injury or death. Under Mg2+ stress, Ca2+ influx was significantly inhibited, and the strengths of P2 promoter and HWP gene expression were weakened. The study results revealed that the mechanism of Mg2+ induced cell growth protection and PLD expression might be related to the lowered strength of PLD expression by P2 promoter repression to meet with the secretion efficiency of B. choshinensis, and the redistribution of intracellular ions accompanied by decreased Ca2+ influx. CONCLUSIONS: The PLD production was highly improved under Mg2+ stress. By ICP-MS and qPCR analysis combined with other results, the mechanism of the efficient extracellular PLD expression under Mg2+ stress was demonstrated. The relatively low-speed PLD expression during cell growth alleviated cell growth inhibition and profoundly improved PLD production. These results provided a potential approach for the large-scale production of extracellular PLD and novel insights into PLD function.

Genetic regulation and fermentation strategy for squalene production in Schizochytrium sp.

Squalene, as an important terpenoid, is extensively used in the medicine and health care fields owing to its functions of anti-oxidation, blood lipid regulation and cancer prevention. The marine microalgae, Schizochytrium sp., which acts as an excellent strain with potential of high squalene production was selected as the starting strain. The overexpressed strain with sqs gene got the reduced biomass and lipid, while the squalene titer was increased by 79.6% ± 4.7% to 12.8 ± 0.2 mg/L. In order to further increase squalene production, the recombinant strain (HS strain) with sqs and hmgr gene co-overexpression was further constructed. The biomass and squalene titer of the HS strain were increased by 13.6% ± 1.2% and 88.8% ± 5.3%, respectively, which indicated the carbon flux of the mevalonate pathway was enhanced for squalene accumulation. Regarding the squalene synthesis is completely coupled with cell growth, fermentation strategy to prolong the logarithmic growth phase was conducive to improve squalene production. Under the condition of optimal composition and concentrated medium, the squalene titer of HS strain was 27.0 ± 1.3 mg/L, which was 2.0 times that of the basal medium condition (13.5 ± 0.4 mg/L). This study which combined the metabolic engineering and fermentation strategy provides a new strategy for squalene production in Schizochytrium sp. KEY POINTS: •The overexpression of sqs and hmgr genes promoted carbon metabolism for squalene. •The optimal and concentrated media can increase squalene yield.

Enhancing astaxanthin accumulation in Xanthophyllomyces dendrorhous by a phytohormone: metabolomic and gene expression profiles.

Xanthophyllomyces dendrorhous is a promising source of natural astaxanthin due to its ability to accumulate high amounts of astaxanthin. This study showed that 6-benzylaminopurine (6-BAP) is an effective substrate that enhances cell biomass and astaxanthin accumulation in X. dendrorhous. In the current study, the biomass and astaxanthin content in X. dendrorhous were determined to be improved by 21.98% and 24.20%, respectively, induced by 6-BAP treatments. To further understand the metabolic responses of X. dendrorhous to 6-BAP, time-course metabolomics and gene expression levels of X. dendrorhous cultures with and without 6-BAP feeding were investigated. Metabolome analysis revealed that 6-BAP facilitated glucose consumption, promoted the glycolysis, suppressed the TCA cycle, drove carbon flux of acetyl-CoA into fatty acid and mevalonate biosynthesis, and finally facilitated the formation of astaxanthin. ROS analysis suggested that the antioxidant mechanism in X. dendrorhous can be induced by 6-BAP. Additionally, the process of 6-BAP significantly upregulated the expression of six key genes involved in pathways related to astaxanthin biosynthesis. This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production iNn X. dendrorhous and presents a new strategy to improve astaxanthin production to prevent the dilemma of choosing between accumulation of astaxanthin and cell biomass.

Functions of Enyolreductase (ER) Domains of PKS Cluster in Lipid Synthesis and Enhancement of PUFAs Accumulation in Schizochytrium limacinum SR21 Using Triclosan as a Regulator of ER.

The polyketide synthase (PKS) cluster genes are supposed to synthesize polyunsaturated fatty acids (PUFAs) in S. limacinum. In this study, two enyolreductase (ER) genes located on PKS cluster were knocked out through homologous recombination to explore their functions. The knock-out of OrfB-ER (located on OrfB subunit) decreased lipid content and had obvious decrease on PUFAs content, indicating OrfB-ER domain played a vital role on PUFAs synthesis; the knock-out of OrfC-ER (located on OrfC subunit) decreased SFAs content and increased total lipid content, indicating OrfC-ER domain was likely to be related with SFAs synthesis, and lipid production could be improved by down-regulating OrfC-ER domain expression. Therefore, the addition of triclosan as a reported regulator of ER domain induced the increase of PUFAs production by 51.74% and lipids yield by 47.63%. Metabolic analysis indicated triclosan played its role through inhibiting the expression of OrfC-ER to reduce the feedback inhibition of SFAs and further to enhance NADPH synthesis for lipid production, and by weakening mevalonate pathway and tricarboxylic acid (TCA) cycle to shift precursors for lipid and PUFAs synthesis. This research illuminates functions of two ER domains in S. limacinum and provides a potential targets for improving lipid production.

The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381.

BACKGROUND: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and ß-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. RESULTS: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the ß-carotene production was increased by 53% at 96 h. The increase of squalene and ß-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. CONCLUSION: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

Mn-doped CoSe2 nanosheets as high-efficiency catalysts for the oxygen evolution reaction.

In this work, we introduce for the first time an aqueous solution method followed by a selenization step to prepare Mn-doped CoSe2 nanosheets supported on nickel foam for the oxygen evolution reaction. These findings provide us highly efficient electrocatalysts instead of noble metal catalysts for the oxygen evolution reaction.

The critical utilization of active heterotrophic microalgae for bioremoval of Cr(VI) in organics co-contaminated wastewater.

Considering the importance of removing toxic Cr(VI) from practical wastewater containing complex pollutants, this study presented for the first time the utilization of the live heterotrophic microalgae (Botryocossuss sp. NJD-1) to achieve a concurrent abatement of Cr(VI), TOC, NO3-N and PO4-P, through a comprehensive biochemical process. The experimental results showed that the Cr(VI) removal efficiencies in the culture with different types of organic descended in the order of sodium acetate, ethanol and methanol. The highest removal efficiencies were achieved as 94.2%, 98.2%, 66.9% and 99.2% for Cr(VI), TOC, NO3-N and PO4-P, respectively, in the culture with 5 mg L-1 Cr(VI) and sodium acetate of equivalent to 2.92 g C L-1. Through mass balance calculation and characterization, the fate of Cr(VI) and Cr(III) was tracked and quantified in the culture system, which indicates that 87.17% of initial Cr(VI) were reduced to Cr(III) and then adsorbed in algal biomass for the optimal removal case. Consequently, the mechanism demonstrating the correlation among the removal process of Cr(VI), the biological activity of microalgae and the effect of organic compounds was proposed.

Assessment upon heterotrophic microalgae screened from wastewater microbiota for concurrent pollutants removal and biofuel production.

Heterotrophic microalgae, capable of converting organic carbons to biofuel, as well as assimilating nutrients, have a great prospective in wastewater treatment. Meanwhile, the knowledge about heterotrophic microalgae is still far less than the autotrophic conterpart. Hence, in this study, 20 heterotrophic microalgal strains were isolated from a domestic wastewater treatment plant, and identified according to morphology and partial 18S and 23S rRNA gene sequences. Further, their biological traits were assessed in terms of N, P, TOC removal efficiencies, growth parameters, self-settleability and lipids production, expressed through a comprehensive selection index. By such, the optimal strains were chosen and applied back to treat the real wastewater, with or without pretreatment of sterilization. An organic-adaptable strain, i.e., Botryococcus sp. NJD-1, was ultimately recommended to achieve the concurrent biofuel production (up to 61.7% lipid content) and pollutants removal (up to 64.5%, 89.8% and 67.9% for N, P and TOC) in pristine wastewater.

Synergistic effect of Trichoderma reesei cellulases on agricultural tea waste for adsorption of heavy metal Cr(VI).

This is the first attempt to study the synergistic effect between Trichoderma reesei cellulases and the abundant agricultural tea waste in absorption of heavy metal Cr(VI) as well as its kinetic model development. The properties of tea waste were first analyzed by near infrared spectroscopy (NIR), particle size distribution (PSD), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) examination with EDX for comparison between its original (UN-TW) and cellulase-hydrolyzed (TRCEL-TW) conditions. Then, an advanced kinetic model in the form of -d[Cr(VI)]/dt = A[H+](n)e(-Ea/RT) [Cr(VI)](m)(0), which can successfully predict the time-dependent Cr(VI) concentration of various pHs, initial Cr(VI) concentrations and temperatures was developed. The demonstrated synergistic effects of T. reesei cellulases on tea waste suggested that cellulosic material provides more accessibility area for absorption of heavy metal. This study also provides an alternative approach to remove toxic Cr(VI) from aqueous solutions and extend the utilization of agricultural tea waste.

Metal and metalloid contaminant availability in Yundang Lagoon sediments, Xiamen Bay, China, after 20 years continuous rehabilitation.

This study is focused on the pollutant level of sediments after 20 years continuous rehabilitation in Yundang Lagoon, Xiamen Bay, one of the most developed harbors in China. The modified BCR fractionation scheme was employed in sediments for the partition of particulating metals and metalloids. Moreover, the potential environment risks of these pollutants were evaluated by using Håkanson indexes. The results showed that the sediments were slightly contaminated by Cu, Pb, Zn, Cr, Ni, Mn, Ag, As and Se. A major portion (30-46%) of Mn was strongly associated with the acid soluble fraction. Copper (Cu), Cr and Ni exhibit significant oxidisable fraction (from 22 to 32%), Pb, Cr, As, Zn, Se and Ag showed considerable concentration (>50%) in the residual phase. Environment risk evaluation showed that the pollution in Yundang Lagoon is moderate and the ranking of the contaminants followed the order: As>Ni>Pb>Cu>Cr>Zn. Importantly, though these results indicate that the concentrations of heavy metals in sediments here are also higher than the sediment quality guidelines and may pose a potential threat to these areas and so the efforts in remediation should be continued.