Increased Lipid Production Research Articles

Regulating the metabolic flux of pyruvate dehydrogenase bypass to enhance lipid production in Saccharomyces cerevisiae

To achieve high efficiency in microbial cell factories, it is crucial to redesign central carbon fluxes to ensure an adequate supply of precursors for producing high-value compounds. In this study, we employed a multi-omics approach to rearrange the central carbon flux of the pyruvate dehydrogenase (PDH) bypass, thereby enhancing the supply of intermediate precursors, specifically acetyl-CoA. This enhancement aimed to improve the biosynthesis of acetyl-CoA-derived compounds, such as terpenoids and fatty acid-derived molecules, in Saccharomyces cerevisiae. Through transcriptomic and lipidomic analyses, we identified ALD4 as a key regulatory gene influencing lipid metabolism. Genetic validation demonstrated that overexpression of the mitochondrial acetaldehyde dehydrogenase (ALDH) gene ALD4 resulted in a 20.1% increase in lipid production. This study provides theoretical support for optimising the performance of S. cerevisiae as a “cell factory” for the production of commercial compounds.

Green integrative large scale treatment of tannery effluent, CO2 sequestration, and biofuel production using oleaginous green microalga Nannochloropsis oculata TSD05: An ecotechnological approach

In this present investigation, the freshwater microalga Nannochloropsis oculata TSD05 was used to demonstrate multiforius environmental applications viz., bioremediation of tannery effluent, carbon sequestration, and green renewable biodiesel production from treated microalgal biomass. The microalga Nannochloropsis oculata TSD05 was exhibited unique heavy metal reduction capabilities and reduced significant amount of heavy metals viz., 96.62% of copper (Cu2+), 99.9% of chromium (Cr3+), 98.36% of zinc (Zn2+), 98.71% of cadmium (Cd2+), 97.96% of lead (Pb2+), 98.88% of Cobalt (Co2+), and 98.76% of nickel (Ni2+). The biosorption capacity rate (qmax) of heavy metals reduction and highest R2 of 97.84% exhibited at 12 days of effluent treatment by Langmuir and Freundlich isotherm models. The phytotoxicity analysis was tested againt treated tannery effluent using Vigna radiate, Sapindus muukorossi seeds and 98.45%, 99.05% seeds were germinated. The Nannochloropsis oculata TSD05 microalga was utilized 98.45% of CO2 by biofixation kinetics, 372 mg g−1 of lipid, 3.65 mg mL−1 of biomass and 4.64 mLg−1 of biodiesel were produced. The produced biodiesel was confirmed and identification of 18 fatty acid methyl ester (biodiesel) compounds using GCMS. The recognition of 17 functional groups was identified using FTIR analysis and the microalga Nannochloropsis oculata TSD05 potential for sustainable and renewable green energy production. The potential future application of Nannochloropsis oculata TSD05 is to increase lipid production and biodiesel yield. Scaling up the bioremediation process can also validate these microalga's feasibility for use in wastewater treament. Enhancing heavy metal biosorption and carbon sequestration efficiency through genetic engineering could also maximize environmental benefits.

Use of plant growth regulators and their combined application to enhance lipid productivity in the filamentous microalga Tribonema sp.

Oleaginous Tribonema sp. has shown great potential in biodiesel production. However, effective strategies are still needed to increase overall lipid productivity for cost-effectiveness. Although the effects of plant growth regulators (PGRs) on enhancing microalgal growth and lipid content have been widely demonstrated, the synergistic effects of their combinations on improving lipid productivity in Tribonema sp. remain unclear. In this study, we investigated the comparative effects of five PGRs (e.g., fulvic acid, melatonin, gibberellin, γ-aminobutyric acid [GABA], and indoleacetic-3-acid [IAA]) at different concentrations (0.1–10 mg/L) on the growth, nitrogen uptake, and lipid content of Tribonema sp. The results showed that 10 mg/L GABA and 0.1 mg/L IAA were optimal conditions, contributing to an approximately 33.00 % increase in lipid productivity compared with the control (without PGRs). Based on these findings, the combination of optimized concentrations of GABA and IAA, especially in equal proportions, resulted in a more desirable fatty acid profile and produced the highest biomass, lipid content, and lipid productivity, with increases of 23.55 %, 25.62 %, and 54.95 %, respectively, over the control. This combination also significantly increased nitrogen removal efficiency in the medium, which was 46.24 % higher that of the control. These results were attributed to the significant upregulation of genes associated with nitrogen metabolism mediated by the synergy of IAA and GABA, promoting nitrate uptake by algal cells in the medium. Genes involved in photosynthesis and lipid synthesis were also upregulated, facilitating simultaneous biomass and lipid accumulation to maximize lipid productivity. Overall, this study is the first to report the combined application of GABA and IAA as an effective strategy to enhance lipid productivity in Tribonema sp., providing insights into the underlying mechanisms. These findings offer a promising solution for economically improving microalgal lipid production.

Effects of Temperature and Nitrogen Limitation on Growth and Lipid Production of Oleaginous Microalgae from Hot Springs

Oleaginous microalgae have gained increasing attention as an alternative feedstock for biodiesel production due to the increasing demand of fuel, climate change, and global warming. This study aimed to isolate and screen robust microalgal strains from hot springs for cultivation in subtropical and tropical areas. The newly isolated oleaginous microalgae were cultivated at 30, 35 and 40 °C. Among mesophilic and thermophilic strains tested, Chlamydomonas sp. HP59 is considered the most robust strain as it showed high cell growth in a broad range of temperatures (30 - 40 °C), with the maximum dried cell weight at 40 °C. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. To increase lipid production, the 2-stage cultivation, in which nitrogen-rich was applied to promote cell growth in the 1st stage and nitrogen-limitation was applied to stimulate lipid accumulation in the 2nd stage, was performed. The high temperature combined with nitrogen-limitation did improve lipid production by all microalgae. With this strategy, Chlamydomonas sp. HP59 showed the highest dried cell weight of 4.33 g/L and lipid production of 1.72 g/L. This study has shown the potential use of the newly isolated oleaginous microalgae from hot springs to be cultivated at high temperatures and under nitrogen-limited conditions for the production of biodiesel feedstocks. HIGHLIGHTS Mesophilic and thermophilic oleaginous microalgae were isolated from hot springs. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. Combined effect of high temperature and nitrogen limitation effectively increased lipid content. Two-stage cultivation gave high dried cell weight and hence overall high lipid production. GRAPHICAL ABSTRACT

Marine algae biomass: A viable and renewable resource for biofuel production: A review

Global energy shortages, as well as rising greenhouse gas emissions, have fueled the search for sustainable renewable energy sources. Recent research has identified microalgae and macroalgae biofuel as one of the major renewable energy sources for environmentally friendly growth, with the potential to replace fossil-based energy sources. The primary disadvantages associated with oil crops and lignocellulose-based biofuels were absent in marine microalgae and macroalgae biofuel. Algae-based renewable energy sources are technically and economically viable, as well as cost competitive; they require no additional land, use little water, and help to reduce atmospheric CO2. Commercial extraction of macroalgae and microalgae biofuel, however, remains impossible due to a small biomass amount and costly downstream procedures. Marine algae-based biofuel production can be made viable by installing sophisticated photobioreactors and developing low-cost biomass collection, drying, and oil recovery technologies. Commercial manufacturing is also achieved through improved genetically modified approaches to stress management, as well as technological metabolic processes to increase lipid production. Furthermore, emerging technologies such as algal-bacterial interactions for improving marine algae growth and lipid production are being investigated. This review focuses on marine algae-based biofuel production and sustainability, economic values, challenges encountered during the commercialization of marine algae biofuels, potential solutions to these challenges, and practicality.

Heterologous glucose-6-phosphate dehydrogenase empowers the biofuel potential of Tetradesmus obliquus via generating lipogenic NADPH

Large-scale production of biofuels from oleaginous microalgal feedstocks in a commercially feasible manner is hampered by their relatively slow growth rates and overall lipid yield than the rapidly proliferating algal species. Therefore, it is pivotal to obviate this bottleneck by augmenting lipid overproduction in fast-growing microalgal species by heterologous overexpression lipogenic genes from model oleaginous species. In this regard, the pentose phosphate pathway of Tetradesmus obliquus was perturbed by heterologous overexpression of glucose-6-phosphate dehydrogenase (PtG6PD) from the model pennate diatom Phaeodactylum tricornutum, known for its oleaginicity. The role of PtG6PD in increasing lipid production was reported in the heterologous T. obliquus. Total lipid content was increased by 2.09- and 2.31-fold in transgenic lines than WT by elevating the lipogenic NADPH, without any negative impact on their growth rates. Interestingly, lipid yields in the transgenic strains reached up to 93 mg/L and 103 mg/L which showed a further 2-fold enhancement under nitrogen-deficient conditions. Furthermore, it was demonstrated that PtG6PD overexpression also altered fatty acid composition, with a specific increment in total monounsaturated fatty acids. Our results uncovered the pivotal role of the pentose phosphate pathway in supplying lipogenic NADPH in T. obliquus and identified key genetic targets for lipid biosynthesis enhancement in alternative hosts. Altogether, these findings open avenues for metabolic engineering in non-model and fast-growing non-oleaginous microalgal species, thereby presenting a promising strategy for empowering commercially viable photosynthetic cell factories for biofuel production.

The phycosphere and its role in algal biofuel production

Oleaginous microalgae have become a focus for large-scale biofuel production due to their ability to accumulate large quantities of lipids. However, production is currently limited by cost and predation. At present, algal biofuel cultivation is optimized through starvation, supplementing media with nutrients, or genetic engineering; these methods can often be costly with little to no increase in lipid production or the culture’s defense. Investigating the phycosphere of algal-bacterial interactions may overcome these current barriers to large-scale production. The phycosphere of algal-bacterial interactions have formed over millions of years through mutualistic and symbiotic relationships and can provide a more direct source of nutrients compared to adding the nutrients in bulk. The most promising of these interactions include the production of phytohormones and quorum signaling compounds that alter the behaviors of the consortia. Phytohormones can improve algal growth rates, lipid production, and stress resistance. Quorum signaling could create consortia capable of warding off invaders—such as rotifers—while self-regulating and altering behavior based on population density. Mechanisms within the algal phycosphere present many opportunities for the development of novel engineering strategies to further improve algal lipid production and operational costs. This review outlines previous preliminary phycosphere research as well as posing possible opportunities to be pursued in future biofuel production.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids

BackgroundShort-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth.ResultsIn this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain.ConclusionsA more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Enhancing lipid production in Chlorella under successive stresses of periodic micro-current and salinity: Performance and contribution

Two-stage cultivation of Chlorella with periodic micro-current coupled with NaCl stress was investigated to analyze the performance and contribution of increased lipid production. In the growth stage, periodic micro-current of 3 mA resulted in an escalation of 290.06 mg/L of Chlorella’s biomass by 61.25 % in comparison to the control. During the induction stage, under a NaCl concentration of 8 g/L, the highest lipid content and yield attained were 42.11 % and 199.32 mg/L, revealing an increment of 53.13 % and 39.11 % respectively when compared to the control. Additionally, the polyunsaturated fatty acids content (PUFA) decreased from 30.11 % to 23.09 %, while the saturated fatty acids content (SFA) increased from 31.86 % to 42.39 %. Chlorella demonstrated adaptability to the alterations in the cultivation environment by an initial upsurge and later a reduction in SOD and CAT enzyme activities, and concurrently facilitated lipid synthesis by similar changes in the activities of ACCase and DGAT. The above cultivation enabled a synergistic effect of swift growth and efficient lipid accumulation in Chlorella, providing groundwork for large-scale biofuel production.

Optimization of stage conversion time and modification of cell metabolism to enhance lipid production of Auxenochlorella pyrenoidosa in two-stage cultivation

Traditionally, the time of maximum biomass concentration in stage I is the widely adopted stage conversion time in two-stage microalgae culture. This study challenges this conventional approach, demonstrating that the optimal stage conversion time in stage I is 72 h rather than 120 h for achieving maximum biomass concentration. A comparison of cell characteristics revealed that algal cells at 72 h exhibited better growth potential, leading to a higher biomass concentration after transfer to stage II and, consequently, increased lipid productivity. Moreover, the use of phosphorus repletion (5-fold) in stage II directed carbon flux toward biomass growth and lipid accumulation, thereby enhancing lipid productivity. By optimizing the stage conversion time and implementing phosphorus repletion, the mean lipid productivity of Auxenochlorella pyrenoidosa cultured under autotrophy-nitrogen starvation and autotrophy-high light conditions increased by 31 % and 60 %, respectively. This study underscores the importance of reevaluating the currently widely used stage conversion time.

Simultaneously maximizing microalgal biomass and lipid productivities by machine learning driven modeling, global sensitivity analysis and multi-objective optimization for sustainable biodiesel production

Simultaneous enhancement in the productivities of microalgal biomass and lipids that are inversely correlated to each other has been a long-standing challenge before the scientists engaged in algal biodiesel research and innovation. This study, develops an Artificial Neural Network (ANN) model and uses multi-objective optimization approach to determine the process parameters, namely, light intensity, CO2%, air flow rate, and C/N ratio to maximize biomass and lipid productivities of microalga, Chlorella sorokiniana, simultaneously. Experimental data based on central composite design (CCD) were used to train a feed-forward multilayer ANN with four critical parameters. The global sensitivity analysis was performed on the trained ANN model using Sobol's method to assess the relative significance of the four process variables. Since the objectives of maximizing biomass and lipid productivities conflict with each other, multi-objective optimization approach was used for deriving the optimal process parameters, experimental validation of which resulted in approximately 3 times increase in biomass productivity and 7 times increase in lipid productivity. On transesterification of the lipid, the biodiesel product with saturated to unsaturated fatty acids ratio of 48:52 conformed very well to the international standards, ASTM D6751 and EN14214, thereby making it an environment friendly green biofuel. Thus, the present study showcases the successful application of machine learning tool in analysing the global sensitivity analysis of process variables, and employ it for multi-objective optimization towards achieving maximum microalgal biomass and lipid productivities concomitantly for potentially sustainable production of biodiesel.

Optimization of Bioenergy Production from Algae Using Bioreactor Design

Bioenergy production from algae has gained significant attention due to its potential as a sustainable and renewable energy source. This research investigates the optimization of bioenergy production by focusing on bioreactor design to enhance the efficiency of algal biomass growth and lipid extraction. The study involves analyzing key bioreactor parameters such as light intensity, nutrient concentration, pH levels, and carbon dioxide (CO₂) supply. Experimental results demonstrated a 25% increase in lipid production through optimized conditions, showing promising improvements in biofuel yield. The findings of this research provide valuable insights into the potential scalability of algae-based bioenergy systems.

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Differences in phytoplankton population vulnerability in response to chemical activity of mixtures.

Hydrophobic organic contaminants (HOCs) affect phytoplankton at cellular to population levels, ultimately impacting communities and ecosystems. Baseline toxicants, such as some HOCs, predominantly partition to biological membranes and storage lipids. Predicting their toxic effects on phytoplankton populations therefore requires consideration beyond cell uptake and diffusion. Functional traits like lipid content and profile can offer insights into the diverse responses of phytoplankton populations exposed to HOCs. Our study investigated the vulnerability of five phytoplankton species populations to varying chemical activities of a mixture of polycyclic aromatic hydrocarbons (PAHs). Population vulnerability was assessed based on intrinsic sensitivities (toxicokinetic and toxicodynamic), and demography. Despite similar chemical activities in biota within the exposed algae, effects varied significantly. According to the chemical activity causing 50% of the growth inhibition (Ea50), we found that the diatom Phaeodactylum tricornutum (Ea50 = 0.203) was the least affected by the chemical exposure and was also a species with low lipid content. In contrast, Prymnesium parvum (Ea50 = 0.072) and Rhodomonas salina (Ea50 = 0.08), both with high lipid content and high diversity of fatty acids in non-exposed samples, were more vulnerable to the chemical mixture. Moreover, the species P. parvum, P. tricornutum, and Nannochloris sp., displayed increased lipid production, evidenced as 5-10% increase in lipid fluorescence, after exposure to the chemical mixture. This lipid increase has the potential to alter the intrinsic sensitivity of the populations because storage lipids facilitate membrane repair, reconstitution and may, in the short-term, dilute contaminants within cells. Our study integrated principles of thermodynamics through the assessment of membrane saturation (i.e. chemical activity), and a lipid trait-based assessment to elucidate the differences in population vulnerability among phytoplankton species exposed to HOC mixtures.

Metabolic equilibrium and reproductive resilience: Freshwater gastropods under nanoplastics exposure

Nanoplastics (NPs) have gained increasing attention due to their widespread presence in aquatic environments and potential adverse effects on organisms. The interaction between NPs and freshwater gastropods can lead to a range of physiological and reproductive disturbances. In this study, we investigated the adverse effects of NPs (two size: 20 nm and 100 nm; three concentrations: 0.5, 50 and 100 ppm) on energy metabolism and reproductive fitness in freshwater gastropods Lymnean stagnalis after 21 days exposure. Briefly, the condition index negatively correlated with increasing NPs concentrations for both sizes. Bioaccumulation revealed a concentration-dependent trend in the 100 nm group, and the highest accumulation appeared in the 100 ppm group, compared to all the rest groups. This phenomenon could be attributed to the larger surface area which facilitates stronger attachment to tissues, while smaller particles could be cleared more readily from body. Carbohydrate and protein reserves remained largely unaffected at all concentrations. However, 100 nm NPs triggered stress responses, increasing lipid production, and 20 nm NPs potentially interfered with mitochondrial function, affecting electron transport system activity. Despite the variations observed in lipid levels and energy cost, the ratio of available energy to energy cost remained stable across for both NPs sizes, and this resilience suggests that cellular energy allocation endured undisturbed, hinting at mechanisms that enable gastropods to maintain their metabolic equilibrium. Reproductively, NPL-exposed groups had fewer clutches, with clutches per collection time decreasing over time for both sizes. In terms of egg development, shell growth and hatching rates remained unaffected, suggesting resilience in aquatic ecosystems.In conclusion, this study underscores the substantial impact of NPs on freshwater gastropods, raising ecological and reproductive concerns. The intricate interplay between nanoparticle size, concentration, and physiological responses highlights the complexity of NPs interactions in aquatic ecosystems, necessitating further research and regulatory measures.

Theoretical Models Constructed by Artificial Intelligence Algorithms for Enhanced Lipid Production: Decision Support Tools

Theoretical models that predict the lipid content of microalgae are an important tool for increasing lipid productivity. In this study, response surface methodology (RSM), RSM combined with artificial neural network (ANN), and RSM combined with ensemble learning algorithms (ELA) for regression were used to calculate the maximum lipid percentage (%) from Chlorella minutissima (C. minutissima). We defined one set of rules to achieve the highest lipid content and used trees.RandomTree (tRT) to simulate the process parameters under various conditions. Among the various models, results showed the optimum values of the root mean squared error (0.2156), mean absolute error (0.1167), and correlation coefficient (0.9961) in the tRT model. RSM combined with tRT estimated that the lipid percentage was 30.3% in wastewater (< 35%), lysozyme (≥ 3.5 U/mL), and chitinase (< 15 U/mL) concentrations, achieving the best model based on experimental data. The optimal values of wastewater concentration, chitinase, and lysozyme were 20% (v/v), 5 U/mL, and 10 U/mL, respectively. Also, the if-then rules obtained from tRT were also used to test the process parameters. The tRT model served as a powerful tool to obtain maximum lipid content. The final rankings of the performance of various algorithms were determined. Furthermore, the models developed can be used by the fuel industry to achieve cost-effective, large-scale production of lipid content and biodiesel.

Progress of Algaculture technology as Biofuel

As the problem of climate change becomes more and more serious, improving the carbon emission situation will be an effective way to solve various climate problems. Algal biofuels can build a complete carbon cycle and reduce carbon emissions. In this paper, the conditions required for microalgae cultivation and how diverse factors play a role in the growth rate and yield of microalgae were reviewed. And show how different factors like pH value, illumination, temperature, medium of culture, and nutrients in the medium affect the content of different substances in the final microalgae to help breed the matching microalgae. Macroscopically, this paper introduces both open raceway ponds and photobioreactors as microalgae cultivation systems and their respective characteristics. Microalgae will produce higher lipid content and hydrogen production capacity in different acid-base environments, respectively, and provide a nitrogen-deficient environment to further increase lipid production. The two-stage hybrid system combining an open raceway pond and photobioreactor has more obvious advantages in terms of growth rate and yield.

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform

ABSTRACT Cutaneotrichosporon curvatum and Yarrowia lipolytica can accumulate microbial oils using short-chain fatty acids (SCFA) as carbon sources. SCFAs-rich media often contain significant amounts of nitrogen that prevent high carbon:nitrogen (C:N) ratios necessary to boost lipid production. This work assessed the intrinsic ability of C. curvatum and Y. lipolytica to produce high amounts of microbial oils from these unusual carbon sources. Results demonstrated that minor differences in SCFA concentration (only 2 g/L) had a significant effect on yeast growth and lipid production. A C:N of 80 promoted yeast growth at all SCFA concentrations and favored SCFA consumption at 19 g/L SCFAs. The different SCFA uptake preferences in C. curvatum and Y. lipolytica highlighted the importance of considering the SCFA profile to select a suitable yeast strain for microbial oils production. At the most challenging SCFA concentration (19 g/L), 57.2% ±1.6% (w/w) and 78.4 ± 0.6% (w/w) lipid content were obtained in C. curvatum and Y. lipolytica, respectively. These values are among the highest reported for wild-type strains. To circumvent the challenges associated with media with high nitrogen content, this report also proved struvite precipitation as an effective method for increasing lipid production (from 17.9 ± 3.9% (w/w) to 41.9 ± 2.6% (w/w)) after nitrogen removal in food waste-derived media.

Age related changes in hyaluronan expression leads to Meibomian gland dysfunction

The prevalence of dry eye disease (DED) ranges from ∼5 to 50 % and its associated symptoms decrease productivity and reduce the quality of life. Approximately 85 % of all DED cases are caused by Meibomian gland dysfunction (MGD). As humans and mice age, their Meibomian glands (MGs) undergo age-related changes resulting in age related-MGD (ARMGD). The precise cause of ARMGD remains elusive, which makes developing therapies extremely challenging. We previously demonstrated that a hyaluronan (HA)-rich matrix exists surrounding the MG, regulating MG morphogenesis and homeostasis. Herein, we investigated whether changes to the HA matrix in the MG throughout life contributes towards ARMGD, and whether altering this HA matrix can prevent ARMGD. For such, HA synthase (Has) knockout mice were aged and compared to age matched wild type (wt) mice. MG morphology, lipid production, PPARγ expression, basal cell proliferation, stem cells, presence of atrophic glands and MG dropout were analyzed at 8 weeks, 6 months, 1 year and 2 years of age and correlated with the composition of the HA matrix. We found that as mice age, there is a loss of HA expression in and surrounding the MGs of wt mice, while, in contrast, Has1−/−Has3−/− mice present a significant increase in HA expression through Has2 upregulation. At 1 year, Has1−/−Has3−/− mice present significantly enlarged MGs, compared to age-matched wt mice and compared to all adult mice. Thus, Has1−/−Has3−/− mice continue to develop new glandular tissue as they age, instead of suffering MG atrophy. At 2 years, Has1−/−Has3−/− mice continue to present significantly larger MGs compared to age-matched wt mice. Has1−/−Has3−/− mice present increased lipid production, increased PPARγ expression and an increase in the number of proliferating cells when compared to wt mice at all-time points analyzed. Taken together, our data shows that a loss of the HA matrix surrounding the MG as mice age contributes towards ARMGD, and increasing Has2 expression, and consequently HA levels, prevents ARMGD in mice.

High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering

Nervonic acid benefits the treatment of neurological diseases and the health of brain. In this study, we employed the oleaginous yeast Yarrowia lipolytica to overproduce nervonic acid oil by systematic metabolic engineering. First, the production of nervonic acid was dramatically improved by iterative expression of the genes ecoding β-ketoacyl-CoA synthase CgKCS, fatty acid elongase gELOVL6 and desaturase MaOLE2. Second, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of glycerol-3-phosphate acyltransferases and diacylglycerol acyltransferases from Malania oleifera in endoplasmic reticulum (ER). Third, overexpression of a newly identified ER structure regulator gene YlINO2 led to a 39.3% increase in lipid production. Fourth, disruption of the AMP-activated S/T protein kinase gene SNF1 increased the ratio of nervonic acid to lignoceric acid by 61.6%. Next, pilot-scale fermentation using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).