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.

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.

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.

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.

Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation.

Microalgae have drawn great attention as promising sustainable source of lipids and carotenoids. Their lipid and carotenoids accumulation machinery can be trigged by the stress conditions such as nutrient limitation or exposure to the damaging physical factors. However, stressful conditions often adversely affect microalgal growth and cause oxidative damage to the cells, which can eventually reduce the yield of the desired products. To overcome these limitations, two-stage cultivation strategies and supplementation of growth-promoting agents have traditionally been utilized, but developing new highly adapted strains is theoretically the simplest strategy. In addition to genetic engineering, adaptive laboratory evolution (ALE) is frequently used to develop beneficial phenotypes in industrial microorganisms during long-term selection under specific stress conditions. In recent years, many studies have gradually introduced ALE as a powerful tool to improve the biological properties of microalgae, especially for improving the production of lipid and carotenoids. In this review, strategies for the manipulation of stress in microalgal lipids and carotenoids production are summarized and discussed. Furthermore, this review summarizes the overall state of ALE technology, including available selection pressures, methods, and their applications in microalgae for the improved production of lipids and carotenoids.

Adaptive evolution of microalgae Schizochytrium sp. under high salinity stress to alleviate oxidative damage and improve lipid biosynthesis.

Lipid accumulation of Schizochytrium sp. can be induced by stress condition, but this stress-induction usually reduce cell growth and cause oxidative damage, which can eventually lower the lipid yield. Here, adaptive laboratory evolution (ALE) combined high salinity was performed to enhance the antioxidant system and lipid accumulation. The final strain ALE150, which was obtained after 150 days, showed a maximal cell dry weight (CDW) of 134.5 g/L and lipid yield of 80.14 g/L, representing a 32.7 and 53.31% increase over the starting strain, respectively. Moreover, ALE150 exhibited an overall higher total antioxidant capacity (T-AOC) and lower reactive oxygen species (ROS) levels than the starting strain. Furthermore, the regulatory mechanisms responsible for the improved performance of ALE150 were analyzed by transcriptomic analysis. Genes related to the antioxidant enzymes and central carbon metabolism were up-regulation. Moreover, the metabolic fluxes towards the fatty acid synthase (FAS) and polyketide synthase (PKS) pathways were also changed.

Development of a cooperative two-factor adaptive-evolution method to enhance lipid production and prevent lipid peroxidation in Schizochytrium sp.

BACKGROUND: Schizochytrium sp. is a marine microalga with great potential as a promising sustainable source of lipids rich in docosahexaenoic acid (DHA). This organism's lipid accumulation machinery can be induced by various stress conditions, but this stress induction usually comes at the expense of lower biomass in industrial fermentations. Moreover, oxidative damage induced by various environmental stresses can result in the peroxidation of lipids, and especially polyunsaturated fatty acids, which causes unstable DHA production, but is often ignored in fermentation processes. Therefore, it is urgent to develop new production strains that not only have a high DHA production capacity, but also possess strong antioxidant defenses. RESULTS: Adaptive laboratory evolution (ALE) is an effective method for the development of beneficial phenotypes in industrial microorganisms. Here, a novel cooperative two-factor ALE strategy based on concomitant low temperature and high salinity was applied to improve the production capacity of Schizochytrium sp. Low-temperature conditions were used to improve the DHA content, and high salinity was applied to stimulate lipid accumulation and enhance the antioxidative defense systems of Schizochytrium sp. After 30 adaptation cycles, a maximal cell dry weight of 126.4 g/L and DHA yield of 38.12 g/L were obtained in the endpoint strain ALE-TF30, which was 27.42 and 57.52% higher than parental strain, respectively. Moreover, the fact that ALE-TF30 had the lowest concentrations of reactive oxygen species and malondialdehyde among all strains indicated that lipid peroxidation was greatly suppressed by the evolutionary process. Accordingly, the ALE-TF30 strain exhibited an overall increase of gene expression levels of antioxidant enzymes and polyketide synthases compared to the parental strain. CONCLUSION: This study provides important clues on how to overcome the negative effects of lipid peroxidation on DHA production in Schizochytrium sp. Taken together, the cooperative two-factor ALE process can not only increase the accumulation of lipids rich in DHA, but also prevent the loss of produced lipid caused by lipid peroxidation. The strategy proposed here may provide a new and alternative direction for the industrial cultivation of oil-producing microalgae.

[Bioactive compounds from marine sponges and cell culture of marine sponges].

Presented a survey of bioactive compounds discovered from marine sponges in the recent five years, including the classes, distribution and their potential pharmaceutical uses. In particular, the compounds with antitumor, antivirus and antibacteria activity were discussed with their originating marine sponge species. Whereas the "Supply Problems" were identified to hinder the clinical tests and commercial applications of most of the sponge bioactive compounds. In vitro cell culture of marine sponges is one of the most promising approaches to solve this problem. The state-of-the art of marine sponge cell culture and the challenging areas were discussed. A brief summary of the R&D status was also given on the bioactive compounds from marine sponges in Chinese oceans. It is crucial to invest more efforts on studying marine sponges and their bioactive compounds in our country in order to develop new marine drugs of independent intellectual property.