Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

Nucleic acid-based scaffold systems and application in enzyme cascade catalysis.

Enzymes, as elements with catalytic functions, can be rationally designed and multiple assembled to form a composite catalytic system and achieve cascade catalytic functions. Enzyme cascade catalysis could produce various chemical products with high conversion rate in short time. With the development of DNA nanotechnology, assembling enzymes to different nucleic acid-based scaffolds in different spatial organizations could effectively improve the catalytic efficiency of enzymes. Herein, we review the construction and application of nucleic acid-based scaffold systems from the perspective of template assembly in three dimensions. The challenges and future outlooks in the development of enzyme cascades are also discussed. KEY POINTS: • The principles and construction of various nucleic acid scaffolds are summarized • The application of nucleic acid scaffolds in enzyme cascade catalysis is discussed.

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.

Accumulation and conversion of ß-carotene and astaxanthin induced by abiotic stresses in Schizochytrium sp.

Astaxanthin is a kind of ketone carotenoid belonging to tetraterpenoids with an excellent antioxidant activity and it is widely used in nutrition and health-care industries. This study aimed to explore the effect of different abiotic stresses on carotenoid production in Schizochytrium sp. Firstly, the characteristics of carotenoid accumulation were studied in Schizochytrium sp. by monitoring the change of carotenoid yields and gene expressions. Then, different abiotic stresses were systematically studied to regulate the carotenoid accumulation. Results showed that low temperature could advance the astaxanthin accumulation, while ferric ion could stimulate the conversion from carotene to astaxanthin. The glucose and monosodium glutamate ratio of 100:5 was helpful for the accumulation of ß-carotene. In addition, micro-oxygen supply conditions could increase the yield of ß-carotene and astaxanthin by 25.47% and 14.92%, respectively. This study provided the potential regulation strategies for carotenoid production which might be used in different carotenoid-producing strains.

The current status of biotechnological production and the application of a novel antioxidant ergothioneine.

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.

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.

Effect of SDS on release of intracellular pneumocandin B0 in extractive batch fermentation of Glarea lozoyensis.

Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelia of Glarea lozoyensis and inhibits fungal 1,3-ß-glucan synthase. Extractive batch fermentation can promote the release of intracellular secondary metabolites into the fermentation broth and is often used in industry. The addition of extractants has been proven as an effective method to attain higher accumulation of hydrophobic secondary metabolites and circumvent troublesome solvent extraction. Various extractants exerted significant but different influences on the biomass and pneumocandin B0 yields. The maximum pneumocandin B0 yield (2528.67 mg/L) and highest extracellular pneumocandin B0 yield (580.33 mg/L) were achieved when 1.0 g/L SDS was added on the 13th day of extractive batch fermentation, corresponding to significant increases of 37.63 and 154% compared with the conventional batch fermentation, respectively. The mechanism behind this phenomenon is partly attributed to the release of intracellular pneumocandin B0 into the fermentation broth and the enhanced biosynthesis of pneumocandin B0 in the mycelia.

Fermentation performance and metabolomic analysis of an engineered high-yield PUFA-producing strain of Schizochytrium sp.

The ω-3/long-chain polyunsaturated fatty acids (LC-PUFAs) play an important role in human health, but they cannot be synthesized in sufficient amounts by the human body. In a previous study, we obtained an engineered Schizochytrium sp. strain (HX-RS) by exchanging the acyltransferase (AT) gene, and it was able to co-produce docosahexaenoic acid and eicosapentaenoic acid. To investigate the mechanism underlying the increase of PUFA content in HX-RS, the discrepancies of fermentation performance, key enzyme activities and intracellular metabolites between HX-RS and its wild-type parent strain (WTS) were analyzed via fed-batch fermentation in 5-L bioreactors. The results showed that the cell dry weight (CDW) of HX-RS was higher than that of the WTS. Metabolomics combined with multivariate analysis showed that 4-aminobutyric acid, proline and glutamine are potential biomarkers associated with cell growth and lipid accumulation of HX-RS. Additionally, the shift of metabolic flux including a decrease of glyceraldehyde-3-phosphate content, high flux from pyruvate to acetyl-CoA, and a highly active glycolysis pathway were also found to be closely related to the high PUFA yield of the engineered strain. These findings provide new insights into the effects of exogenous AT gene expression on cell proliferation and fatty acid metabolism.

Intracellular response of Bacillus natto in response to different oxygen supply and its influence on menaquinone-7 biosynthesis.

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.

Novel osmotic stress control strategy for improved pneumocandin B0 production in Glarea lozoyensis combined with a mechanistic analysis at the transcriptome level.

Pneumocandin B0, the precursor of the antifungal drug caspofungin, is a secondary metabolite of the fungus Glarea lozoyensis. In this study, we investigated the effects of mannitol as the sole carbon source on pneumocandin B0 production by G. lozoyensis. The osmotic pressure is more important in enhancing pneumocandin B0 production than is the substrate concentration. Based on the kinetic analysis, an osmotic stress control fed-batch strategy was developed. This strategy led to a maximum pneumocandin B0 concentration of 2711 mg/L with a productivity of 9.05 mg/L/h, representing 34.67 and 6.47% improvements, respectively, over the best result achieved by the one-stage fermentation. Furthermore, G. lozoyensis accumulated glutamate and proline as compatible solutes to resist osmotic stress, and these amino acids also provided the precursors for the enhanced pneumocandin B0 production. Osmotic stress also activated ROS (reactive oxygen species)-dependent signal transduction by upregulating the levels of related genes and increasing intracellular ROS levels by 20%. We also provided a possible mechanism for pneumocandin B0 accumulation based on signal transduction. These findings will improve our understanding of the regulatory mechanisms of pneumocandin B0 biosynthesis and may be applied to improve secondary metabolite production.

Metabolomics profiling reveals the mechanism of increased pneumocandin B0 production by comparing mutant and parent strains.

Metabolic profiling was used to discover mechanisms of increased pneumocandin B0 production in a high-yield strain by comparing it with its parent strain. Initially, 79 intracellular metabolites were identified, and the levels of 15 metabolites involved in six pathways were found to be directly correlated with pneumocandin B0 biosynthesis. Then by combining the analysis of key enzymes, acetyl-CoA and NADPH were identified as the main factors limiting pneumocandin B0 biosynthesis. Other metabolites, such as pyruvate, α-ketoglutaric acid, lactate, unsaturated fatty acids and previously unreported metabolite γ-aminobutyric acid were shown to play important roles in pneumocandin B0 biosynthesis and cell growth. Finally, the overall metabolic mechanism hypothesis was formulated and a rational feeding strategy was implemented that increased the pneumocandin B0 yield from 1821 to 2768 mg/L. These results provide practical and theoretical guidance for strain selection, medium optimization, and genetic engineering for pneumocandin B0 production.

CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art.

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.

Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up.

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B0 production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B0 yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B0 production to 1134 mg/L when compared with the parent strain (810 mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B0 concentration to 1873 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B0 production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.

CFD investigation of Schizochytrium sp. impeller configurations on cell growth and docosahexaenoic acid synthesis.

Effects of impeller configurations on docosahexaenoic acid production and flow characteristics were investigated by Schizochytrium sp. in a 15 L bioreactor. 6-straight blade disc turbine (6-SBDT), 6-arrowy-blade disc turbine (6-ABDT) and down-pumping propeller (DPP) were combined to form different impeller configurations. Simulated results showed that configuration SSA consisting of upper two 6-SBDT and one bottom 6-ABDT possessed the worst oxygen supply capacity. But it obtained the highest DHA percentage of 48.17 % and DHA yield of 21.42 g/L, indicating that it was beneficial for DHA synthesis and converting glucose to biomass and lipids. Configuration SAS consisting of one middle 6-ABDT and two 6-SBDT provided better mixing capacity, which resulted in the maximum glucose consumption rate of 2.86 g/L h and the highest biomass of 108.09 g/L. This study would improve insight into understanding the relationship between flow field and the physiology of Schizochytrium sp. for the scale-up of industrial DHA production.

The roles of different salts and a novel osmotic pressure control strategy for improvement of DHA production by Schizochytrium sp.

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.

[Establishment of genetic transformation system of Schizochytrium sp. by homologous recombination].

OBJECTIVE: Schizochytrium sp. is a marine fungus that can produce DHA efficiently. Genetic engineering has been successfully used in industrial strain improvement and metabolic studies. In order to use genetic engineering to modified Schizochytrium sp., we established an genetic transformation system of Schizochytrium sp. METHODS: A genetic transformation system of Schizochytrium sp. was established by 18S rDNA-targeted homologous recombination. The targeting vector contained a part of 18S rDNA from Schizochytrium sp. and the ble gene. This targeting vector was transformed into Schizochytrium sp. by electroporation and then selected by Zeocin-containing plates. The incorporation of exogenous ble gene into the genome of Schizochytrium was inspected by PCR amplification. RESULTS: Fermentation results show that the transformants had similar cell dry weight, lipid yield, DHA content, and composition of other fatty acids to the wild type strain. CONCLUSION: Our results show that the introduction of resistance gene did not affect the cell growth and lipid metabolism. This system could be used to introduce new functional genes into Schizochytrium sp.

Fungal arachidonic acid-rich oil: research, development and industrialization.

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.

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors.

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.

Regulation of docosahexaenoic acid production by Schizochytrium sp.: effect of nitrogen addition.

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.

Improvement of egg yolk powder properties through ultrasound coupled sodium sulfite pretreatment assisted enzymatic hydrolysis and underlying mechanism.

Egg yolk is an excellent protein source for the production of bioactive peptides. However, the recent method need to remove lipid first which involves wastage and pollution of organic reagents. Therefore, the process of directly using oily yolk powder to prepare egg yolk peptides has attracted much attention. This study developed a one-step process to simultaneously extract oil and hydrolyze proteins based on an ultrasound coupled sodium sulfite pretreatment (UCSSP) assisted enzymatic hydrolysis for egg yolk powder. Results showed that UCSSP increased the oil extraction rate from zero to 75 % with 59.35 g/L of soluble protein and 33.99 g/L of peptide. Further analysis of the underlying mechanism demonstrated that ultrasound pre-treatment could change the secondary structure of EYP while sodium sulfite pre-treatment softened the protein and induced more hydrophobic groups exposed, thus inducing more lipoprotein released for hydrolysis. In addition, the proportion of peptides ranging from 180 Da to 3000 Da in the UCSSP group increased from 31.19 % before to 79.74 %, which was 31.27 % and 6.16 % higher than that of UP and SP. Furthermore, the obtained peptides showed obvious activities in uric acid-lowering, anti-obesity and antioxidant with 56.24 % inhibition in XOD activity and close antioxidant activity to vitamin C, implying it a potential health product.