Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Mechanism of the oxidative stress-mediated increase in lipid accumulation by the bacterium,R. opacus PD630: Experimental analysis and genome-scale metabolic modeling

Appropriate species of oleaginous bacteria, with their high growth rates and lipid accumulation capabilities, can be good contenders for industrial triacylglycerol (TAG) production, compared to microalgae. Further, oxidative stress (OS) can be used to significantly increase TAG yields in oleaginous microbes, but the mechanism is unexplored. In a first, this study explored the mechanism behind OS-mediated increase in TAG accumulation by the bacterium, Rhodococccus opacus PD630, through experimental analysis and metabolic modelling. Two mechanisms that could increase acetyl-CoA (TAG-precursor) levels were hypothesized based on literature information. One was OS-mediated inactivation of the aconitase (TCA cycle), and another was the inactivation of the triosephosphate isomerase (TPI; glycolysis). The results negated the involvement of aconitase in increased acetyl-CoA levels. Analysis of the metabolic model showed that inactivation of TPI, re-routed the flux through the pentose phosphate pathway (PPP), supplying both NADPH and acetyl-CoA for TAG synthesis. Additionally, inactivation of TPI increased TAG flux by 143%, whereas, inactivating both TPI and aconitase, increased it by 152%. We present experimental evidence for OS-mediated decrease in TPI activity and increase in activity of glucose-6-phosphate dehydrogenase (PPP enzyme). The findings indicate that increased flux through PPP can be explored to improve TAG accumulation on a large-scale.     

Indole-3-acetic acid in the culture medium of two axenic green microalgae

In the view of the facts that algal extracts have been used in agriculture asa source of plant growth stimulating agents and IAA has been shown to bepresent in the extracts, a study was planned to establish whether or notaxenic algae can produce IAA. Evidence is provided for extracellular IAAproduction during culture of two axenic green microalgae. IAAidentification was based on co-chromatography with the standard, analysisof UV and fluorescent spectra, and gas chromatography – selectedion-monitoring mass spectrometry. HPLC analyses showed that underthe experimental conditions the amounts of IAA released to the mediumby Scenedesmus armatus and Chlorella pyrenoidosa weregenerally low. IAA tended to occur in Scenedesmus armatus culturemedium at higher concentrations than in that of Chlorellapyrenoidosa. In fast-growing cultures of Scenedesmus armatus,constantly aerated with CO₂/air mixture, the concentration of IAAcalculated per cell was less than in the slow-growing cultures.     

13C Metabolic Flux Analysis of acetate conversion to lipids by Yarrowia lipolytica

Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP.     

The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems

Malic enzyme (ME; NADP⁺-dependent; EC 1.1.40) provides NADPH for lipid biosynthesis in oleaginous microorganisms. Its role in vivo depends on there being an adequate supply of NADH to drive malate dehydrogenase to convert oxaloacetate to malate as a component of a cycle of three reactions: pyruvate → oxaloacetate → malate and, by the action of ME, back to pyruvate. However, the availability of cytosolic NADH is limited and, consequently, ancillary means of producing NADPH are necessary. Stoichiometries are given for the conversion of glucose to triacylglycerols involving ME with and without the reactions of the pentose phosphate pathway (PPP) as an additional source of NADPH. Some oleaginous microorganisms (such as Yarrowia lipolytica), however, lack a cytosolic ME and, if the PPP is the sole provider of NADPH, the theoretical yield of triacylglycerol from glucose falls to 27.6 % (w/w) from 31.6 % when ME is present. An alternative route for NADPH generation via a cytosolic isocitrate dehydrogenase (NADP⁺-dependent) is then discussed.     

Neuroprotection by glucose-6-phosphate dehydrogenase and the pentose phosphate pathway

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme that channels glucose catabolism from glycolysis into the pentose phosphate pathway (PPP), is vital for the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in cells. NADPH is in turn a substrate for glutathione reductase, which reduces oxidized glutathione disulfide to sulfhydryl glutathione. Best known for inherited deficiencies underlying acute hemolytic anemia due to elevated oxidative stress by food or medication, G6PD, and PPP activation have been associated with neuroprotection. Recent works have now provided more definitive evidence for G6PD's protective role in ischemic brain injury and strengthened its links to neurodegeneration. In Drosophila models, improved proteostasis and lifespan extension result from an increased PPP flux due to G6PD induction, which is phenocopied by transgenic overexpression of G6PD in neurons. Moderate transgenic expression of G6PD was also shown to improve healthspan in mouse. Here, the deciphered and implicated roles of G6PD and PPP in protection against brain injury, neurodegenerative diseases, and in healthspan/lifespan extensions are discussed together with an important caveat, namely NADPH oxidase (NOX) activity and the oxidative stress generated by the latter. Activation of G6PD with selective inhibition of NOX activity could be a viable neuroprotective strategy for brain injury, disease, and aging.     

Contribution of hexose-6-phosphate dehydrogenase to NADPH content and redox environment in the endoplasmic reticulum

Abstract

Background: Hexose-6-phosphate dehydrogenase (H6PD) has been considered to be a main source of NADPH in the endoplasmic reticulum. It provides reducing equivalents to 11-hydroxysteroid dehydrogenase type 1 for in situ re-activation of glucocorticoids. H6PD null mice indeed show signs of glucocorticoid deficiency, but also suffer from a skeletal myopathy mainly affecting fast twitch muscles, in which the unfolded protein response (UPR) is activated. Thus, H6PD may have additional functions in muscle.

Materials and methods: To determine the contribution of H6PD to total microsomal NADPH content, we measured NADPH in microsomes from liver and quadriceps, gastrocnemius and soleus muscles. To evaluate the effect of H6PD deficiency on microsomal thiol-disulfide redox environment, we measured reduced and oxidized glutathione and free protein thiols.

Results and conclusions: H6PD deficiency decreased but did not eliminate NADPH content in liver and soleus microsomes. Thus there must be other sources of NADPH within the endoplasmic/sarcoplasmic reticulum. Levels of reduced glutathione and free protein thiols were decreased in gastrocnemius muscle from null mice, indicating a more oxidative environment. Such alterations in redox environment may underlie the myopathy and UPR activation in H6PD null mice.

General significance: H6PD plays a role in maintaining normal NADPH levels and redox environment inside the endoplasmic reticulum. Intrinsic differences in ER metabolism may explain the differing effects of H6PD deficiency in different tissues.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

The mixed culture of microalgae Chlorella pyrenoidosa and yeast Yarrowia lipolytica for microbial biomass production

Microbial biomass which mostly generated from the microbial processes of bacteria, yeasts, and microalgae is an important resource. Recent concerns in microbial biomass production field, especially microbial lipid production for biofuel, have been focused towards the mixed culture of microalgae and yeast. To more comprehensive understanding of the mixed culture for microbial biomass, mono Chlorella pyrenoidosa, mono Yarrowia lipolytica and the mixed culture were investigated in the present work. Results showed that the mixed culture achieved significantly faster cell propagation of microalga and yeast, smaller individual cell size of yeast and higher relative chlorophyll content of microalga. The mixed culture facilitated the assimilation of carbon and nitrogen and drove the carbon flow to carbohydrate. Besides higher lipid yield (0.77 g/L), higher yields of carbohydrates (1.82 g/L), protein (1.99 g/L) and heating value (114.64 kJ/L) indicated the microbial biomass harvested from the mixed culture have more potential utilization in renewable energy, feedstuff, and chemical industry.

     

Efficacy of Chlorella pyrenoidosa and Scenedesmus abundans for Nutrient Removal in Rice Mill Effluent (Paddy Soaked Water)

Microalgae are product of sustainable development owing to its ability to treat variety of wastewater effluents and thus produced biomass can serve as value added product for various commercial applications. This paper deals with the cultivation of microalgae species namely Chlorella pyrenoidosa and Scenedesmus abundans in rice mill effluent (i.e., paddy soaked water) for nutrient removal. In order to investigate the nutrient removal capability, microalgae are subjected to cultivation in both raw and autoclaved samples. The maximum phosphate removal by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 98.3% and 97.6%, respectively, whereas, the removal of ammoniacal nitrogen by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 92% and 90.3%, respectively. The growth (measured in terms of chlorophyll content) of Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 3.88 mg/l and 5.55 mg/l, respectively. The results indicate the suitability of microalgae cultivation in rice mill effluent treatment for nutrient removal.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Constitutive and Chloroplast Targeted Expression of Acetyl-CoA Carboxylase in Oleaginous Microalgae Elevates Fatty Acid Biosynthesis

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.     

Harvesting Chlorella species using magnetic iron oxide nanoparticles

Microalgae are very useful organisms as it provides many beneficial products for human use. For large scale cultivation and further applications, its harvesting procedure needs to be enhanced to make the production process of the end product highly affordable. Magnetic nanoparticles have great potential to harvest microalgae as it can easily attract and attach to the algal cell surface forming a layer, which can be harvested quickly under the influence of a magnetic field. Our work on Chlorella pyrenoidosa and Chlorella minutissima shows, 500 mg of the synthesized bare iron oxide nanoparticles, harvests 90% of Chlorella pyrenoidosa (1 g L^?1), in 60 s at pH 3 and 600 mg iron oxide nanoparticles, harvests 85% of Chlorella minutissima (1 g L^?1) in 60 s at pH 5, which can decrease the amount of time and energy consumed in the overall production costs.     

Rewiring of purine metabolism in response to acidosis stress in glioma stem cells

Glioma stem cells (GSCs) contribute to therapy resistance and poor outcomes for glioma patients. A significant feature of GSCs is their ability to grow in an acidic microenvironment. However, the mechanism underlying the rewiring of their metabolism in low pH remains elusive. Here, using metabolomics and metabolic flux approaches, we cultured GSCs at pH 6.8 and pH 7.4 and found that cells cultured in low pH exhibited increased de novo purine nucleotide biosynthesis activity. The overexpression of glucose-6-phosphate dehydrogenase, encoded by G6PD or H6PD, supports the metabolic dependency of GSCs on nucleotides when cultured under acidic conditions, by enhancing the pentose phosphate pathway (PPP). The high level of reduced glutathione (GSH) under acidic conditions also causes demand for the PPP to provide NADPH. Taken together, upregulation of G6PD/H6PD in the PPP plays an important role in acidic-driven purine metabolic reprogramming and confers a predilection toward glioma progression. Our findings indicate that targeting G6PD/H6PD, which are closely related to glioma patient survival, may serve as a promising therapeutic target for improved glioblastoma therapeutics. An integrated metabolomics and metabolic flux analysis, as well as considering microenvironment and cancer stem cells, provide a precise insight into understanding cancer metabolic reprogramming.     

High fructose-containing drinking water-induced steatohepatitis in rats is prevented by the nicotinamide-mediated modulation of redox homeostasis and NADPH-producing enzymes

An imbalance in the redox state, increased levels of lipid precursors and overactivation of de novo lipogenesis determine the development of fibrosis during nonalcoholic steatohepatitis (NASH). We evaluated the modulation of NADPH-producing enzymes associated with the antifibrotic, antioxidant and antilipemic effects of nicotinamide (NAM) in a model of NASH induced by excess fructose consumption. Male rats were provided drinking water containing 40% fructose for 16 weeks. During the last 12 weeks of fructose administration, water containing NAM was provided to some of the rats for 5 h/day. The biochemical profiles and the ghrelin, leptin, lipoperoxidation and TNF-α levels in serum and the glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and NADP⁺-dependent isocitric dehydrogenase (IDP) levels, the reduced/oxidized glutathione (GSH/GSSG) and reduced/oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H/NAD(P)⁺) ratios, and the levels of various lipogenic and fibrotic markers in the liver were evaluated. The results showed that hepatic fibrosis induced by fructose consumption was associated with weight gain, hunger-satiety system dysregulation, hyperinsulinemia, dyslipidemia, lipoperoxidation and inflammation. Moreover, increased levels of hepatic G6PD and ME activity and expression, the NAD(P)H/NAD(P)⁺ ratios, and GSSG concentration and increased expression of lipogenic and fibrotic markers were detected, and these alterations were attenuated by NAM administration. Specifically, NAM diminished the activity and expression of G6PD and ME, and this effect was associated with a decrease in the NADPH/NADP⁺ ratios, increased GSH levels and decreased lipoperoxidation and inflammation, ameliorating fibrosis and NASH development. NAM reduces liver steatosis and fibrosis by regulating redox homeostasis through a G6PD- and ME-dependent mechanism.