RESUMO
Ergothioneine is a sulfur-containing histidine derivative, that possessesexcellent antioxidant activity and has been used in the food and cosmetics industries. It plays a significant role in anti-aging and the prevention of various diseases. This review will briefly introduce the functions and applications of ergothioneine, elaborate the biosynthetic pathways of ergothioneine and describe several strategies to increase the production of ergothioneine. Then the efficient extraction and detection methods of ergothioneine will be presented. Finally, several proposals are put forward to increase the yield of ergothioneine, and the development prospects of ergothioneine will be discussed.
Assuntos
Ergotioneína , Antioxidantes , Vias Biossintéticas , Biotecnologia , Ergotioneína/metabolismo , Histidina/metabolismoRESUMO
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.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Microalgas/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica , Marcação de Genes , Engenharia Genética , RNA Guia de Cinetoplastídeos/genética , Transformação GenéticaRESUMO
Menaquinone-7 (MK-7) plays an important role in blood clotting, cardiovascular disease and anti-osteoporosis, and has been wildly used in the food additives and pharmaceutical industries. The aim of this study was to investigate the mechanism of menaquinone-7 biosynthesis in response to different oxygen supplies in Bacillus natto. The differences of fermentation performance, intracellular metabolites, oxidative stress reaction and enzyme activities of Bacillus natto R127 were analyzed under different KLa. Glycerol consumption rate and MK-7 yield at 24.76 min- 1 was 2.1 and 7.02 times of that at 18.23 min- 1. Oxidative stress analysis showed the cell generated more active oxygen and possessed higher antioxidant capacity at high oxygen supply condition. Meanwhile, high pyruvate kinase and high cytochrome c oxidase activities were also observed at 24.76 min- 1. Furthermore, comparative metabolomics analyses concluded series of biomarkers for high MK-7 biosynthesis and cell rapid growth. Besides, several metabolic responses including low glyceraldehyde-3-phosphate accumulation, low flux from pyruvate to lactic acid, high active TCA pathway, were also found to be associated with high MK-7 accumulation at high oxygen supply conditions. These findings provided the information for better understanding of oxygen effect on MK-7 biosynthesis and lay a foundation for further improvement of MK-7 production as well.
Assuntos
Bacillus subtilis/metabolismo , Glicerol/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , Oxigênio/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/metabolismoRESUMO
In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.
Assuntos
Sistemas CRISPR-Cas , Fungos/genética , Edição de Genes/métodos , Genoma Fúngico , Microbiologia Industrial/métodos , Agricultura/métodos , Indústria Alimentícia/métodosRESUMO
The effects of different osmotic pressure, changed by six salts (NaCl, Na2SO4, (NH4)2SO4, KH2PO4 and MSG), on cell growth and DHA synthesis by Schizochytrium sp. were investigated. Six optimal mediums were obtained to study different osmotic pressure combinations at cell growth stage and DHA synthesis stage. Results showed that cultivated cell in higher osmotic pressure condition and fermented in lower osmotic pressure condition was benefit to enhance DHA synthesis. Combination 17-6 could get the maximum cell dry weight of 56.95 g/L and the highest DHA percentage in total fatty acids of 55.21%, while combination 17-B could get the highest lipid yield of 33.47 g/L with 42.10% DHA in total fatty acids. This was the first report about the enhancement of DHA production by osmotic regulation and this work provided two novel osmotic control processes for high lipid yield and high DHA percentage in total fatty acids.
Assuntos
Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/biossíntese , Pressão Osmótica , Estramenópilas/metabolismoRESUMO
Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.
Assuntos
Ácido Araquidônico/metabolismo , Fungos/metabolismo , Óleos/metabolismo , Indústrias , PesquisaRESUMO
Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.
Assuntos
Ácido Araquidônico/biossíntese , Reatores Biológicos , Mortierella/metabolismo , Consumo de Oxigênio/fisiologia , Oxigênio/metabolismoRESUMO
Docosahexaenoic acid (DHA) percentage in total fatty acids (TFAs) is an important index in DHA microbial production. In this study, the change of DHA percentage in response to fermentation stages and the strategies to increase DHA percentage were investigated. Two kinds of conventional nitrogen sources, monosodium glutamate (MSG) and ammonium sulfate (AS), were tested to regulate DHA synthesis. Results showed that MSG addition could accelerate the substrate consumption rate but inhibit lipid accumulation, while AS addition could increase DHA percentage in TFAs effectively but extend fermentation period slightly. Finally, the AS addition strategy was successfully applied in 7,000-L fermentor and DHA percentage in TFAs and DHA yield reached 46.06 % and 18.48 g/L, which was 19.54 and 17.41 % higher than that of no-addition strategy. This would provide guidance for the large-scale production of the other similar polyunsaturated fatty acid, and give insight into the nitrogen metabolism in oil-producing microorganisms.
Assuntos
Sulfato de Amônio/farmacologia , Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Glutâmico/farmacologia , Estramenópilas/crescimento & desenvolvimentoRESUMO
Docosahexaenoic acid (DHA) is an important and widely used infant food additive. In this study, the effects of phosphate concentration on lipid and especially DHA synthesis in the oleaginous fungi Schizochytrium sp. HX-308 have been investigated in batch cultures. The maximum DHA yield (8.9 g/L) and DHA productivity (148.3 mg/L h) in 0.1 g/L KH2PO4 concentration were higher than the DHA yield (6.2 g/L) and DHA productivity (86.1 mg/L h) in 4 g/L KH2PO4 concentration. Furthermore, differences in related enzyme activities (malic enzyme, glucose-6-phosphate dehydrogenase and NAD(+)-isocitrate dehydrogenase) between phosphate-sufficient and phosphate-limitation conditions were assayed. The results showed that the phosphate-limitation condition could maintain higher activities of malic enzyme and glucose-6-phosphate dehydrogenase in addition to lower activity of NAD(+)-isocitrate dehydrogenase. In addition, glucose-6-phosphate dehydrogenase might be the main supplier of NADPH at the early stage of fermentation while malic enzyme might be the provider at the late stage. This information might explain the regulation mechanism of phosphate limitation for lipid production and be useful for further DHA production enhancement.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Fosfatos/metabolismo , Compostos de Potássio/metabolismo , Estramenópilas/crescimento & desenvolvimento , Soluções Tampão , Oxirredutases/metabolismo , Fosfatos/farmacologia , Compostos de Potássio/farmacologia , Estramenópilas/citologia , Estramenópilas/enzimologiaRESUMO
Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l(-1) and 138.8 mg l(-1) h(-1), respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Fermentação , Biologia Marinha , Estramenópilas/metabolismo , Meios de CulturaRESUMO
Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.
Assuntos
Butileno Glicóis/metabolismo , Repressão Catabólica , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Klebsiella oxytoca/metabolismo , Xilose/metabolismo , Proteína Receptora de AMP Cíclico/biossíntese , Proteína Receptora de AMP Cíclico/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Expressão Gênica , Klebsiella oxytoca/genética , Dados de Sequência Molecular , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , Análise de Sequência de DNARESUMO
The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Eucariotos/crescimento & desenvolvimento , Eucariotos/metabolismo , Biomassa , Reatores Biológicos/microbiologia , Meios de Cultura/química , Glucose/metabolismo , Metabolismo dos Lipídeos , Oxigênio/metabolismoRESUMO
Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Butileno Glicóis/metabolismo , Klebsiella oxytoca/metabolismo , Fermentação , Técnicas de Silenciamento de Genes , Engenharia Genética , Klebsiella oxytoca/genética , Klebsiella oxytoca/crescimento & desenvolvimentoRESUMO
A stable culture environment is the key for optimal growth and metabolic activity of microorganisms, especially in marine species, and intermittent feeding during DHA production using Schizochytrium sp. generates an unstable culture environment. To investigate the effect of unstable culture environment on the cells' physiological status and DHA synthesis, fermentations with different feeding strategies were performed on the lab scale. The intermittent feeding strategy caused fluctuations of substrate concentration and osmotic pressure, which had a negative effect on cell division and product synthesis. The physiological status and metabolic level of Schizochytrium sp. were relatively stable under a continuous feeding strategy with a relatively stable substrate concentration of 20-25 g/L, which was beneficial for the efficient transformation of substrate, leading to an improvement of DHA productivity. This strategy was further applied to pilot scale, whereby the DHA content, DHA productivity, convert ratio of glucose to lipid and DHA reached 55.02%, 320.17 mg/(L·h), 24.35%, and 13.40%, respectively. This study therefore provides an efficient strategy for ensuring a stable culture environment for the production of DHA and similar metabolites. Graphical Abstract.
Assuntos
Técnicas de Cultura/métodos , Ácidos Docosa-Hexaenoicos/biossíntese , Fermentação , Estramenópilas/crescimento & desenvolvimento , Estramenópilas/metabolismo , Reatores Biológicos , Biotecnologia , BiotransformaçãoRESUMO
Docosahexaenoic acid (DHA) production in Schizochytrium sp. HX-308 was evaluated by detecting enzymatic activities of ATP:citrate lyase (EC 4.1.3.8), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at different fermentation stages. According to the analysis, a regulation strategy was proposed which reinforced acetyl-CoA and NADPH supply at a specific fermentation stage. DHA content of total fatty acids was increased from 35 to 60% by the addition of 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid content also showed an apparent increase of 35% and reached 19 g/L when 40 mL ethanol/L was added at the late lipid accumulation stage.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A/metabolismo , Reatores Biológicos/microbiologia , Biotecnologia , Meios de Cultura , Eucariotos/crescimento & desenvolvimento , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Cinética , Malato Desidrogenase/metabolismo , NADP/metabolismoRESUMO
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.
Assuntos
Ácidos Graxos Insaturados/biossíntese , Engenharia Metabólica/métodos , Microalgas/crescimento & desenvolvimento , Engenharia Genética , Metabolismo dos Lipídeos , Microalgas/genética , Microalgas/metabolismo , Fotossíntese , Policetídeo SintasesRESUMO
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.
Assuntos
Microalgas , Biocombustíveis , Engenharia Genética , LipídeosRESUMO
The aim of this work was to reduce the algae-residue emission and make use of cane molasses as fermentation materials for docosahexaenoic acid (DHA) fermentaion by Schizochytrium sp., which further could cut the cost of DHA production. Algae-residue and cane molasses were respectively used as nitrogen and carbon sources to replace yeast extract and glucose. A significant DHA yield of 18.58â¯g/L was obtained using algae-residue, while cane molasses could not be used well as sole carbon source due to the presence of undesirable substance. A two-stage culture strategy with glucose followed by pretreated cane molasses as carbon source was developed, resulting in a final DHA yield of 15.22â¯g/L. This study therefore offers an economical and green strategy for DHA production by Schizochytrium sp.
Assuntos
Bengala , Ácidos Docosa-Hexaenoicos/biossíntese , Estramenópilas/metabolismo , Carbono/metabolismo , Fermentação , Glucose/metabolismo , Melaço , Nitrogênio/metabolismoRESUMO
In this study, the effects of salt stress on the physiological, lipidomic and transcriptomic profiles of halophilic microalga Schizochytrium sp. were investigated. In general, Schizochytrium sp. could survive under high osmotic fermentation medium containing 30â¯g/L NaCl, and showed a significant increase in C14:0 percentage in total fatty acids. In lipidomic analysis, C14:0 was specifically enriched in phosphatidylcholine (PC), and membrane phospholipids participated in the salt stress response mostly. Specially, one novel signal lipid N-acylphosphatidylethanolamine (NAPE) (18:0/20:3/14:0) was upregulated significantly. Transcriptomic analysis revealed glycerol-3-phosphate acyltransferase (GPAT) and phospholipase ABHD3 (PLABDH3) were involved in C14:0 metabolism and NAPE biosynthesis. Signalling pathways they mediated were activated as evident by high expression level of Myristoyl-CoA: protein N-myristoyltransferase (NMT) and NAPE-hydrolyzing PLD (NAPE-PLD). This study gives us an insight in specific responses to salt stress in Schizochytrium sp. and provides a considerable proportion of novel genes that could commendably be used for engineering modification.
Assuntos
Estresse Salino , Transcriptoma , Ácidos Graxos , Fosfolipídeos , Transdução de SinaisRESUMO
Enhancing lipid productivity and reducing oxidative damage is essential for lipid overproduction in microalgae. In this study, addition of 20 mg/L fulvic acid (FA) resulted a 34.4% increase of lipid yield in Schizochytrium sp. Furthermore, the cooperative effect of FA and EDTA on cell growth and lipid production was investigated. The combined addition of 20 mg/L FA and 1.0 g/L EDTA yielded a maximal cell dry weight of 130.7 g/L and lipid productivity of 1.16 g/L/h, representing 36.4% and threefold increase over the non-supplemented group, respectively. Moreover, compared with the non-supplemented group, the combined addition strategy exhibited overall lower levels of reactive oxygen species and malondialdehyde, which accompanied with 66.7% and 81.9% higher superoxide dismutase and catalase activity, respectively. Furthermore, a 24.1-37.1% increase of malic enzyme and 19.4-25.2% decrease of phosphoenolpyruvate carboxylase activity was observed during the entire fermentation stage (0-108 h). Results suggested that the combined addition strategy not only enhanced lipid accumulation, but also prevented the lipid peroxidation.