Production of single cell oil by two novel nonconventional yeast strains of Curvibasidium sp. isolated from medicinal lichen.

Oleaginous yeasts utilize renewable resources to produce lipids, which benefits sustainable development, and it is of great interest to screen robust lipid producers. Curvibasidium sp. belongs to nonconventional yeast that are very limitedly studied. Here, two cold-adaptive strains of Curvibasidium sp., namely, Y230 and Y231, isolated from the medicinal lichen Usnea diffracta were investigated for their potential in lipid production. Genome mining of Curvibasidium sp. Y231 was performed, and the special features related to fatty acid biosynthesis were revealed. Glucose, xylose, and glycerol were tested as sole carbon sources for yeast cell growth and lipid production. The total lipid contents of Curvibasidium sp. Y230 and Y231 range from 38.43% to 54.62% of the cell dry cell weight at 20°C, and glucose is the optimal carbon source. These results indicate that the Curvibasidium sp. strains are promising for sustainable lipid production. Our study provides basis for exploration of lichen-derived strains for biotechnological applications, and also benefits utilization of other nonconventional yeasts for sustainable production based on genome-based studies.

Identification dehydratase domains from Schizochytrium sp. and Shewanella sp. and distinct functions in biosynthesis of fatty acids.

Polyunsaturated fatty acid (PUFA) synthase is a special and effective enzyme for PUFA synthesis, and dehydratase (DH) domain played a crucial role in it. In this work, we compared four different DH domains from different strains (Schizochytrium sp. HX-308 and Shewanella sp. BR-2) and different gene clusters. First bioinformatics analysis showed that DH1, 2 and DH3 were similar to FabA and PKS-DH, respectively, and all of them got a hot-dog structure. Second, four DH domains were expressed in Escherichia coli that increased biomass. Especially, Schi-DH1,2 presented the highest dry cell weight of 2.3 g/L which was 1.62 times of that of control. Fatty acids profile analysis showed that DH1,2 could enhance the percentage of unsaturated fatty acids, especially DH1,2 from Schizochytrium sp., while DH3 benefited for the saturated fatty acid biosynthesis. Furthermore, five kinds of fatty acids were added to the medium to study the substrate preferences. Results revealed that DH1,2 domain preferred to acting on C16:0, while DH3 domain trended acting on C14:0 and C15:0, which illustrated DH from different clusters do have specific substrate preference. Besides, DH expression could save the cell growth inhibition by mid-chain fatty acids. This study provided more information about the catalysis mechanism of polyunsaturated fatty acid synthase and might promote the modification study based on this enzyme.

Effects of three antibiotics on growth and antioxidant response of Chlorella pyrenoidosa and Anabaena cylindrica.

Antibiotics are essential for treatments of bacterial infection and play important roles in the fields of aquaculture and animal husbandry. Antibiotics are accumulated in water and soil due to the excessive consumption and incomplete treatment of antibiotic wastewater. The accumulation of antibiotics in ecological systems leads to global environmental risks. The toxic effects of spiramycin (SPI), tigecycline (TGC), and amoxicillin (AMX) on Chlorella pyrenoidesa and Anabaena cylindrica were evaluated based on growth inhibition experiments, and determinations of ROS production and antioxidant enzyme activities (catalase, superoxide dismutase, and malondialdehyde). Half maximal effective concentrations (EC50) of TGC, SPI, and AMX for A. cylindrica were 62.52 µg/L, 38.40 µg/L, and 7.66 mg/L, respectively. Those were 6.20 mg/L, 4.58 mg/L, and > 2 g/L for C. pyrenoidesa, respectively. It was shown that A. cylindrica was much more sensitive to these antibiotics than C. pyrenoidesa. In addition, EC50 values of SPI and TGC were lower than that of AMX. It was indicated that SPI and TGC had higher toxic than AMX to C. pyrenoidesa and A. cylindrica. The current study is helpful to evaluating possible ecological risks of TGC, SPI, and AMX by green microalgae and cyanobacteria.

Kinetic model for effects of simulated flue gas onto growth profiles of Chlorella sp. AE10 and Chlorella sp. Cv.

Microalgae are potential candidate for biofuel production as alternative one for fossil fuels. CO2 in flue gas is available carbon source to support microalgae growth. In this study, the effects of different concentrations of the simulated flue gas onto algal growth and photosynthetic activity were evaluated for both Chlorella sp. AE10 and Chlorella sp. Cv. The growth profiles were correlated by a simple kinetic model. It was indicated that the simulated flue gas led to low pH and the photosynthetic activity was partially destroyed. Chlorella sp. Cv can tolerate full simulated flue gas, 10% CO2  + 200 ppm NOx  + 100 ppm SOx . The pH in medium maintained at 6 and the photosynthetic activity was more than 0.6 at the first 6 days. If the concentration of NOx was more 100 ppm and that of SOx was more than 50 ppm, the pH was declined to 4 at day 2 or 3 for Chlorella sp. AE10. At the same time, the related photosynthetic activities of Chlorella sp. AE10 were less than 0.4, which was not suitable for algal growth. It was shown that Chlorella sp. Cv could be used for CO2 fixation from the simulated flue gas.

Application of the CRISPR/Cas system for genome editing in microalgae.

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.

Enhanced roles of biochar and organic fertilizer in microalgae for soil carbon sink.

Improved soil carbon sink capability is important for the mitigation of carbon dioxide emissions and the enhancement of soil productivity. Biochar and organic fertilizer (OF) showed a significant improving effect on microalgae in soil carbon sink capacity, and the ultimate soil total organic carbons with microalgae-OF, microalgae-biochar, microalgae-OF-biochar were about 16, 67 and 58% higher than that with microalgae alone, respectively, indicating that carbon fixation efficiency of microalgae applied in soil was improved with biochar and OF whilst the soil carbon capacity was promoted, the mechanism of which is illustrated through simulative experiments. Organic fertilizer could spur algal conversion of carbon into cell molecules by increasing intracellular polysaccharide production of microalgae. Biochar could change carbon metabolism pathway of microalgae through altering the yield of intracellular saccharides, and yield and type of extracellular saccharides. There was a superimposition effect on the soil carbon sink when biochar and OF were both present with microalgae.

Enhancing the stability of lutein emulsions with a water-soluble antioxidant and a oil-soluble antioxidant.

Lutein is critical for protecting the eye against light damage. The low solubility and high sensitivity of lutein to environmental stresses prevent its further application. The hypothesis is that the combination of one water-soluble antioxidant and one oil-soluble antioxidant will be beneficial to improve the stability of lutein emulsions. A low-energy method was performed to prepare lutein emulsions. The combination of a lipid-soluble antioxidant (propyl gallate or ethylenediaminetetraacetic acid) and a water-soluble antioxidant (tea polyphenol or ascobic acid) were investigated for improving the lutein retention rates. It was shown that the highest lutein retention rate was achieved by using propyl gallate and tea polyphenol, 92.57%, at Day 7. It was proven that the lutein retention rates of emulsions with propyl gallate and tea polyphenol were 89.8%, 73.5% and 55.2% at 4 °C, 25 °C and 37 °C, respectively, at Day 28. The current study is helpful to prepare for the further application of lutein emulsions for ocular delivery.

Exploring kinetics, removal mechanism and possible transformation products of tigecycline by Chlorella pyrenoidosa.

The accumulation of antibiotics in wastewater leads to broad antibiotic resistance, threating human health. Microalgae have been receiving attention due to their ability to remove antibiotics from wastewater. Tigecycline (TGC) is a broad-spectrum glycylcycline antibiotic. It has not been investigated for removal by microalgae. The removal kinetics of TGC by Chlorella pyrenoidosa were evaluated under different initial dry cell densities, TGC concentrations, temperatures and light intensity conditions. Approximately 90% of TGC could be removed when the TGC concentration was 10 mg∙L-1 and the initial dry cell density was more than 0.2 g∙L-1. A low value of TGC per g dry cell weight ratio led to a high removal efficiency of TGC. The initial dry cell density of microalgae was also critical for the removal of TGC. A high initial dry cell density is better than a low initial dry cell density to remove TGC when the ratio of the TGC concentration to dry cell weight are the same at the beginning of the cultivation. The removal mechanisms were investigated. Photolysis was a slow process that did not lead to removal at the beginning. Adsorption, hydrolysis, photolysis and biodegradation by microalgae were the main contributors to the removal of TGC. TGC was easily hydrolyzed under high -temperature conditions. Three transformation products of TGC by microalgae were identified. The stability of TGC was evaluated in water and salt solutions of citric acid, K2HPO4·3H2O and ferric ammonium citrate. TGC was stable in ultrapure water and citric acid solution. TGC was hydrolyzed in K2HPO4·3H2O and ferric ammonium citrate solutions.

Using nanomaterials to increase the efficiency of chemical production in microbial cell factories: A comprehensive review.

Microbes have proven to be robust workhorses for the large-scale production of many chemicals. Especially, high-value biochemicals (e.g., natural pigments, unsaturated fatty acids) that cannot be derived from fossil fuels, can be produced by engineered microbes. There is a growing interest in both academia and industry to find new technologies that can enhance the efficiencies of microbial cell factories and boost the circular bioeconomy. Rapid technological innovations, such as microbial genome editing and synthetic biology, have greatly advanced the production of chemicals in engineered microbes. Nanomaterial-based technologies that exploit the unique physiochemical properties of nano-scale materials (e.g., large surface area, excellent catalytic activity, tunable optical and electrical performance) have demonstrated great potential and attracted increasing attention. There are many studies showing that nanomaterials can assist microbes in the synthesis of chemicals by providing micronutrients, inducing anti-ROS responses, promoting gas-liquid mass transfer, immobilizing microbial cells and promoting electron transfer in electrosynthesis. Furthermore, the latest studies demonstrate that nanomaterials can be used to construct photocatalyst-microbe hybrids and achieve solar driven chemical production. In this review, we comprehensively summarize these advances and discuss the current gaps as well as future perspectives. With the rapid development of synthetic biology and nanotechnology, we believe more nanomaterial-based technologies will be developed and used to improve the productivity of microbial cell factories.

Engineering the xylose metabolism in Schizochytrium sp. to improve the utilization of lignocellulose.

BACKGROUND: Schizochytrium sp. is a heterotrophic, oil-producing microorganism that can efficiently produce lipids. However, the industrial production of bulk chemicals using Schizochytrium sp. is still not economically viable due to high-cost culture medium. Replacing glucose with cheap and renewable lignocellulose is a highly promising approach to reduce production costs, but Schizochytrium sp. cannot efficiently metabolize xylose, a major pentose in lignocellulosic biomass. RESULTS: In order to improve the utilization of lignocellulose by Schizochytrium sp., we cloned and functionally characterized the genes encoding enzymes involved in the xylose metabolism. The results showed that the endogenous xylose reductase and xylulose kinase genes possess corresponding functional activities. Additionally, attempts were made to construct a strain of Schizochytrium sp. that can effectively use xylose by using genetic engineering techniques to introduce exogenous xylitol dehydrogenase/xylose isomerase; however, the introduction of heterologous xylitol dehydrogenase did not produce a xylose-utilizing engineered strain, whereas the introduction of xylose isomerase did. The results showed that the engineered strain 308-XI with an exogenous xylose isomerase could consume 8.2 g/L xylose over 60 h of cultivation. Xylose consumption was further elevated to 11.1 g/L when heterologous xylose isomerase and xylulose kinase were overexpressed simultaneously. Furthermore, cultivation of 308-XI-XK(S) using lignocellulosic hydrolysates, which contained glucose and xylose, yielded a 22.4 g/L of dry cell weight and 5.3 g/L of total lipid titer, respectively, representing 42.7 and 30.4% increases compared to the wild type. CONCLUSION: This study shows that engineering of Schizochytrium sp. to efficiently utilize xylose is conducive to improve its utilization of lignocellulose, which can reduce the costs of industrial lipid production.

Adaptive Evolution Improves Algal Strains for Environmental Remediation.

Microalgae have potential for environmental remediation, but they must better tolerate stress. Adaptive laboratory evolution (ALE) is effective to construct evolved strains, but its efficiency is low. Highly efficient ALE relies on selecting suitable environmental stress, original strain selection, and optimizing initial cell density and stress strategy.

Growth Performance and Antioxidative Response of Chlorella pyrenoidesa, Dunaliella salina, and Anabaena cylindrica to Four Kinds of Ionic Liquids.

Ionic liquids are widely used for lipid and pigment extractions from microalgae. It is possible that ionic liquids are discharged into environments. The evaluation of growth performance and antioxidative response of ionic liquids to microalgae is helpful to explore the stress regulation mechanism and investigate possible environmental risk. Ionic liquids induce production of reactive oxygen species (ROS) to microalgae. These oxidative stresses are possible from cations, anions, and salinity. In this study, the growth inhibitions of [BMIM]Br, [BMIM]Cl, [EMIM]Cl, and [EMIM]EtOSO3 to Anabaena cylindrica, Chlorella pyrenoidesa, and Dunaliella salina were evaluated. It was interesting that Br- and two kinds of cations, [BMIM] and [EMIM], had significant effects on growth inhibitions of these microalgae. IC50 values of these ionic liquids for A. cylindrica, C. pyrenoidesa, and D. salina were also estimated based on the results of growth inhibitions. It was proved that [EMIM]Cl is relatively harmless to C. pyrenoidesa and D. salina, and [EMIM]EtOSO3 is relatively or practically harmless to C. pyrenoidesa. [BMIM]Br and [BMIM]Cl are practically harmless to A. cylindrica and C. pyrenoidesa, and relatively harmless to D. salina. More than 0.8 g/L [EMIM]EtOSO3 led to bleaching of both A. cylindrica and D. salina at 48 h which was shown that the anion, EtOSO3-, had higher inhibition to A. cylindrica and D. salina than Cl-. In addition, high concentration of ionic liquids led to reductions of chlorophyll content in these three kinds of microalgae, increase of ROS levels and malondialdehyde contents for most of the cases. High concentration of ionic liquids also increased the activities of superoxide dismutase in three kinds of microalgae. There were positive correlations between ROS levels or MDA content, and inhibitions ratios of these ionic liquids to microalgae except [EMIM]Cl to A. cylindrica. These antioxidant enzymes were beneficial for reducing the ROS induced by ionic liquids.

Genetic modification of flux for flux prediction of mutants.

MOTIVATION: Gene deletion and overexpression are critical technologies for designing or improving the metabolic flux distribution of microbes. Some algorithms including flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) predict a flux distribution from a stoichiometric matrix in the mutants in which some metabolic genes are deleted or non-functional, but there are few algorithms that predict how a broad range of genetic modifications, such as over- and underexpression of metabolic genes, alters the phenotypes of the mutants at the metabolic flux level. RESULTS: To overcome such existing limitations, we develop a novel algorithm that predicts the flux distribution of the mutants with a broad range of genetic modification, based on elementary mode analysis. It is denoted as genetic modification of flux (GMF), which couples two algorithms that we have developed: modified control effective flux (mCEF) and enzyme control flux (ECF). mCEF is proposed based on CEF to estimate the gene expression patterns in genetically modified mutants in terms of specific biological functions. GMF is demonstrated to predict the flux distribution of not only gene deletion mutants, but also the mutants with underexpressed and overexpressed genes in Escherichia coli and Corynebacterium glutamicum. This achieves breakthrough in the a priori flux prediction of a broad range of genetically modified mutants. SUPPLEMENTARY INFORMATION: Supplementary file and programs are available at Bioinformatics online or http://www.cadlive.jp.

Exergy analysis for docosahexaenoic acid production by fermentation and strain improvement by adaptive laboratory evolution for Schizochytrium sp.

Exergy analysis is powerful tool for process optimization and mechanism analysis. In this study, exergy analysis was performed for docosahexaenoic acid (DHA) fermentation process. More than 86% of input exergy was contributed by glucose. The exergy of biomass was about 64.66% of the total output exergy when the phosphate concentration was 4 g L-1. The exergy efficiencies of DHA (ηDHA) for the starting strains and the evolved strains under high oxygen concentration, low temperature, and two-factor conditions were also investigated. The ηDHA in the collected experimental data was not more than 20.9%. It was proved that there was a positive correlation between ηDHA and the biomass yield. It was indicated that adaptive laboratory evolution (ALE) improved biomass yield which had the most important effect on enhancing ηDHA and DHA yield (or DHA productivity). It is necessary to improve ηDHA through process optimization and ALE in future.

Extended CADLIVE: a novel graphical notation for design of biochemical network maps and computational pathway analysis.

Biochemical network maps are helpful for understanding the mechanism of how a collection of biochemical reactions generate particular functions within a cell. We developed a new and computationally feasible notation that enables drawing a wide resolution map from the domain-level reactions to phenomenological events and implemented it as the extended GUI network constructor of CADLIVE (Computer-Aided Design of LIVing systEms). The new notation presents 'Domain expansion' for proteins and RNAs, 'Virtual reaction and nodes' that are responsible for illustrating domain-based interaction and 'InnerLink' that links real complex nodes to virtual nodes to illustrate the exact components of the real complex. A modular box is also presented that packs related reactions as a module or a subnetwork, which gives CADLIVE a capability to draw biochemical maps in a hierarchical modular architecture. Furthermore, we developed a pathway search module for virtual knockout mutants as a built-in application of CADLIVE. This module analyzes gene function in the same way as molecular genetics, which simulates a change in mutant phenotypes or confirms the validity of the network map. The extended CADLIVE with the newly proposed notation is demonstrated to be feasible for computational simulation and analysis.

Current states and challenges of salt-affected soil remediation by cyanobacteria.

Natural and human activities lead to soil degradation and soil salinization. The decrease of farmlands threatens food security. There are approximately 1 billion ha salt-affected soils all over of world, which can be made available resources after chemical, physical and biological remediation. Nostoc, Anabaena and other cyanobacterial species have outstanding capabilities, such as the ability to fix nitrogen from the air, produce an extracellular matrix and produce compatible solutes. The remediation of salt-affected soil is a complex and difficult task. During the past years, much new research has been conducted that shows that cyanobacteria are effective for salt-affected soil remediation in laboratory studies and field trials. The related mechanisms for both salt tolerance and salt-affected soil remediation were also evaluated from the perspective of biochemistry, molecular biology and systems biology. The effect of cyanobacteria on salt-affected soil is related to nitrogen fixation and other mechanisms. There are complicated interactions among cyanobacteria, bacteria, fungi and the soil. The interaction between cyanobacteria and salt-tolerant plants should be considered if the cyanobacterium is utilized to improve the soil fertility in addition to performing soil remediation. It is critical to re-establish the micro-ecology in salt-affected soils and improve the salt affected soil remediation efficiency. The first challenge is the selection of suitable cyanobacterial strain. The co-culture of cyanobacteria and bacteria is also potential approach. The cultivation of cyanobacteria on a large scale should be optimized to improve productivity and decrease cost. The development of bio-remediating agents for salt-affected soil remediation also relies on other technical problems, such as harvesting and contamination control. The application of cyanobacteria in salt-affected soil remediation will reconstruct green agriculture and promote the sustainable development of human society.

Effect of light quality on growth rate, carbohydrate accumulation, fatty acid profile and lutein biosynthesis of Chlorella sp. AE10.

Microalgae are feedstocks for multiple product development based on algal biorefinery concept. The effects of light quality (white, red and blue light emitting diodes) and macro-element starvations on Chlorella sp. AE10 were investigated under 20% CO2 and 850 µmol m-2 d-1. Nitrogen and phosphorus starvations had negative effects on its growth rate. The biomass productivities were decreased from day 1 and the highest one was 1.90 g L-1 d-1 under white light conditions. Phosphorus starvation promoted carbohydrate accumulation under three LED light sources conditions and the highest carbohydrate content was 75.9% using red light. Blue light increased lutein content to 9.58 mg g-1. The content of saturated fatty acids was significantly increased from 37.51% under blue light and full culture medium conditions to 77.44% under blue light and nitrogen starvation conditions. Chlorella sp. AE10 was a good candidate for carbohydrate and lutein productions.

Application of chemicals for enhancing lipid production in microalgae-a short review.

Microalgae have attracted great attention as a promising sustainable resource for biofuel production. In studies aiming to improve lipid accumulation, many key enzymes involved in lipid biosynthesis were identified and confirmed, but genetic engineering remains a challenge in most species of microalgae. In an alternative approach, various chemical modulators can be used to directly regulate the lipid biosynthesis pathway, with similar effects to gene overexpression and interference approaches, including improving the precursor supply and blocking competing pathways. The produced lipid can be protected from being converted into other metabolites by the chemicals such as lipase inhibitors. In addition, a few chemicals were also demonstrated to greatly influence cell growth and lipid accumulation by indirect regulation of the lipid biosynthesis pathway, such as increasing cell permeability or regulating oxidative stress. Thus, adding chemical modulators can be a useful alternative strategy for improving lipid accumulation in large-scale cultivation of microalgae.

Enhancement of lipid accumulation in microalgae by metabolic engineering.

Microalgal lipids have drawn great attention as a promising sustainable resource for biodiesel or food supplement production. The development of high-performance strains of microalgae by metabolic engineering is invaluable for increasing the quantity or quality of desired lipids. The synthesis routes of lipids used as biodiesel in microalgae are based on fatty acid synthase (FAS) and triacylglycerols (TAG) biosynthesis pathway. Polyunsaturated fatty acids (PUFAs), including ω-6 and ω-3 fatty acids, are essential nutrients for humans. Notably, microalgae possess two distinct pathways for polyunsaturated fatty acids (PUFAs) biosynthesis, including the desaturase/elongase pathway and the polyketide synthase (PKS) pathway. Thus, it is necessary to identify which biosynthetic pathways are responsible for PUFA synthesis in particular microalgae species. In recent years, various key enzymes and functional domains involved in fatty acid and TAG biosynthesis pathway were identified and potentially regulated by genetic engineering approaches to elevate specific lipids content. In addition, other studies have reported the implementation of strategies to increase lipid accumulation based on increasing acetyl-CoA/NADPH supply, enhancing photosynthetic efficiency, or blocking competing pathways. Furthermore, other efforts have used transcription factor engineering to simultaneously regulate multiple genes related to lipid accumulation. This review summarizes recent research about a variety of microalgae lipid biosynthesis pathways, and discusses multiple gene manipulation strategies that have been employed for specific lipid overproduction in industrial microalgae.

Adaptive evolution and carbon dioxide fixation of Chlorella sp. in simulated flue gas.

Carbon dioxide and other greenhouse gas emissions leads to global warming. Biological capture through microalgae is a potential approach for solving this environmental problem. It is still a technical challenge to enhance the tolerance of microalgae to flue gas if CO2 is fixed from flue gas directly. A new strain, Chlorella sp. Cv was obtained through adaptive evolution (46 cycles) against simulated flue gas (10% CO2, 200 ppm NOx and 100 ppm SOx). It was confirmed that Chlorella sp. Cv could tolerate simulated flue gas conditions and the maximum CO2 fixation rate was 1.2 g L-1 d-1. In a two-stage process, the biomass concentration was 2.7 g L-1 and the carbohydrate content was 68.4%. Comparative transcriptomic analysis was performed for Chlorella sp. Cv under simulated flue gas and control conditions (10% CO2). These responses against simulated flue gas uncovered the significant difference between the evolved strain and the original strain. The metabolic responses to flue gas were explored with focus on various specific genes. Upregulation of several genes related to photosynthesis, oxidative phosphorylation, CO2 fixation, sulfur metabolism and nitrogen metabolism was beneficial for the evolved strain to tolerate the simulated flue gas. The upregulation of genes related to extracellular sulfur transport and nitrate reductase was essential to utilize the sulfate and nitrate from dissolved SOx and NOx. The results in this study are helpful to establish a new process for CO2 capture directly from industrial flue gas.