Ultraconserved elements from transcriptome and genome data provide insight into the phylogenomics of Sternorrhyncha (Insecta: Hemiptera).

Sternorrhyncha, one of the four major suborders of Hemiptera, is a phytophagous taxon inclusive of nearly 18 000 described species. The phylogenetic relationships within the taxon and the earliest-branching lineage of its infraorders remain incompletely understood. This study attempted to illuminate the phylogenetic relationships within Sternorrhyncha through the use of maximum likelihood, Bayesian inference and maximum parsimony analyses, employing ultraconserved element (UCE) data from 39 genomic and 62 transcriptomic datasets and thereby representing most families within the taxon. The probe set Hemiptera 2.7Kv1 was used to recover a total of 2731 UCE loci: from 547 to 1699 (with an average of 1084) across all genomic datasets and from 108 to 849 (with an average of 329) across all transcriptomic datasets. All three types of phylogenetic analyses employed in this study produced robust statistical support for Sternorrhyncha being a monophyletic group. The different methods of phylogenetic analysis produced inconsistent descriptions of topological structure at the infraorder level: while maximum likelihood and Bayesian inference analyses produced strong statistical evidence (100%) indicating the clade Psylloidea + Aleyrodoidea to be a sister of the clade Aphidoidea (Aphidomorpha) + Coccoidea (Coccomorpha), the maximum parsimony analysis failed to recover a similar result. Our results also provide detail on the phylogenetic relationships within each infraorder. This study presents the first use of UCE data to investigate the phylogeny of Sternorrhyncha. It also shows the viability of amalgamating genomic and transcriptomic data in studies of phylogenetic relationships, potentially highlighting a resource-efficient approach for future inquiries into diverse taxa through the integration of varied data sources.

Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing.

By directly converting solar energy and carbon dioxide into biobased products, cyanobacteria are promising chassis for photosynthetic biosynthesis. To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales, exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance. To simplify and accelerate the screening for fast-growing cyanobacteria strains, a method called Individual Cyanobacteria Vitality Tests and Screening (iCyanVS) was established. We show that the 13C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations, thus greatly accelerating the process screening for fastest-growing cells. The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing. Therefore, this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.

Patterns in the Incidence of Scarlet Fever Among Children Aged 0-9 Years - China, 2010-2019.

Introduction: This study investigates the patterns of scarlet fever among Chinese children aged 0-9 years from 2010 to 2019. The objective is to provide insights that may inform potential adjustments to China's current prevention and control tactics for this illness. Methods: The present study utilized data on the occurrence of scarlet fever in children from 2010 to 2019, sourced from the National Notifiable Disease Reporting System database, managed by the Chinese Center for Disease Control and Prevention. This research implemented SAS9.4 software to construct trajectory models representing the temporal incidence of scarlet fever, accounting for key variables such as sex, geographic region, urban versus rural dwellings, and various age brackets. Results: From 2010 to 2019, a total of 554,695 scarlet fever cases were reported among children aged 0-9 years in the 31 mainland Chinese provincial-level administrative divisions, signifying a rate of 35.36 per 100,000 individuals. An inconsistent yet generally rising trend was observed, evidenced by a 3.17-fold increase in reported cases and a 3.02-fold escalation in incidence rate over this period. Examination of these trends revealed three distinctive developmental patterns for both males and females, with the lowest prevalence in the first trajectory and the highest in the third. The incidence was consistently higher among males than females in all trajectories. The urban and northern regions displayed equal or greater trajectory rates than their rural and southern counterparts, respectively. In terms of age groups, the lowest incidence was observed in the 0-1-year age group, while the highest was recorded in the 4-5 and 6-7-year age groups. Conclusions: Between 2010 and 2019, there was a marked increase in the incidence of scarlet fever among children in China. The disease predominantly impacts urban-dwelling children, ranging from 4 to 7 years old, in the northern regions of the country. The incidence is reported to be higher among boys compared to girls.

Unlocking the potentials of cyanobacterial photosynthesis for directly converting carbon dioxide into glucose.

Glucose is the most abundant monosaccharide, serving as an essential energy source for cells in all domains of life and as an important feedstock for the biorefinery industry. The plant-biomass-sugar route dominates the current glucose supply, while the direct conversion of carbon dioxide into glucose through photosynthesis is not well studied. Here, we show that the potential of Synechococcus elongatus PCC 7942 for photosynthetic glucose production can be unlocked by preventing native glucokinase activity. Knocking out two glucokinase genes causes intracellular accumulation of glucose and promotes the formation of a spontaneous mutation in the genome, which eventually leads to glucose secretion. Without heterologous catalysis or transportation genes, glucokinase deficiency and spontaneous genomic mutation lead to a glucose secretion of 1.5 g/L, which is further increased to 5 g/L through metabolic and cultivation engineering. These findings underline the cyanobacterial metabolism plasticities and demonstrate their applications for supporting the direct photosynthetic production of glucose.

Degradation of sulfamethoxazole and antibiotic resistance genes from surface water in the photocatalyst-loading bionic ecosystems.

The behavior and removal of sulfamethoxazole (SMX) and 3 typical corresponding antibiotic resistance genes (ARGs) including sul1, sul2, sul3, and 16S rDNA in surface water were investigated in the photocatalyst-loading bionic ecosystems (PCBEs). Synthesized composite photocatalyst g-C3N4/TiO2 showing higher catalytic activity than Fe/g-C3N4/TiO2 was selected in the PCBEs. Five PCBEs, i.e., A-the control (without bionic grass or photocatalyst), B-bionic grass loaded with 4.12 g/m2 g-C3N4/TiO2, C-bionic grass loaded with 8.25 g/m2 g-C3N4/TiO2, D-bionic grass loaded with 12.37 g/m2 g-C3N4/TiO2, and E-bionic grass loaded with 16.5 g/m2 g-C3N4/TiO2 were constructed and operated in a medium-scale running cyclical flume. SMX could be photolyzed efficiently by g-C3N4/TiO2 with an optimal unit load on the bionic grass of 12.37 g/m2. 3-amino-5-methylisooxazole and p-aminobenzene sulfonamide were selected as main intermediates through the analyses of SMX degradation mechanisms and pathways, and detected in the aqueous phase and bionic grass. The intermediates were higher in the underwater part of the bionic grass than the above-water part. The overall removal of SMX ranged from 31.7 % to 82.3 % in 5 PCBEs, and the removal of sul1and sul2 were 0.2 %- 62.9 % in the aqueous phase and 8.4 %-63.2 % in the sediment. PCBE D might be the best construction when SMX and ARGs' removal was considered comprehensively. Moreover, the microbial structures showed Proteobacteria as the most dominant bacterial species had a relative abundance of 22.2 %-26.6 % and 33.4 %-68.2 % in the aquatic phase and sediment respectively, illustrating that the removal of the antibiotic and ARGs was bound up with the variations of dominant bacteria in the ecosystems. The findings illustrated that ecosystems with bionic grass and photocatalysts could be a promising technology for the removal of typical antibiotics and ARGs from surface water.

Antibiotic fate in an artificial-constructed urban river planted with the algae Microcystis aeruginosa and emergent hydrophyte.

The behavior and removal of six antibiotics, that is, azithromycin, clarithromycin, sulfathiazole, sulfamethoxazole, ciprofloxacin, and tetracycline, in an artificial-controllable urban river (ACUR) were investigated. The ACUR was constructed to form five artificial eco-systems by planting three emergent hydrophytes and Microcystis aeruginosa: (1) Control; (2) MA: M. aeruginosa only; (3) MA-J-C: M. aeruginosa combined with Juncus effusus and Cyperus alternifolius; (4) MA-C-A: M. aeruginosa combined with C. alternifolius and Acorus calamus L.; (5) MA-A-J: M. aeruginosa combined with A. calamus L. and J. effusus. The MA-C-A system achieved the best removal of azithromycin and clarithromycin after 15-day test with the final concentrations 0.92 and 0.83 µg/L. The contents of ciprofloxacin and tetracycline in sediment were highest, up to 1453 and 1745 ng/g. The antibiotic plant bioaccumulation was higher in roots rather than the shoots (stem and leaves). No target antibiotics were detected in algae cells. The combination of hybrid hydrophytes had a certain effect on the removal of antibiotics, and thus selecting appropriate hydrophytes in urban rivers could greatly improve water quality. The overall removal of six antibiotics was greatly improved by the ACUR containing the hybrid hydrophytes and the algae, indicating a synergistic effect on antibiotic removal. PRACTITIONER POINTS: Controllable-mobile artificial eco-systems were developed with emergent hydrophytes and M. aeruginosa. The M. aeruginosa + Cyperus alternifolius + Acorus calamus L. system removed azithromycin and clarithromycin most at the end of tests. Emergent hydrophytes and M. aeruginosa have a synergistic effect on the removal of antibiotics. The combination of emergent hydrophytes did play an important role in the removal of antibiotics. The artificial eco-systems containing the hybrid hydrophytes and the algae could greatly improve the overall removal of antibiotics.

Salt-Tolerant Synechococcus elongatus UTEX 2973 Obtained via Engineering of Heterologous Synthesis of Compatible Solute Glucosylglycerol.

The recently isolated cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) is characterized by a faster growth rate and greater tolerance to high temperature and high light, making it a good candidate chassis for autotrophic photosynthetic microbial cell factories. However, Syn2973 is sensitive to salt stress, making it urgently important to improve the salt tolerance of Syn2973 for future biotechnological applications. Glucosylglycerol, a compatible solute, plays an important role in resisting salt stress in moderate and marine halotolerant cyanobacteria. In this study, the salt tolerance of Syn2973 was successfully improved by introducing the glucosylglycerol (GG) biosynthetic pathway (OD750 improved by 24% at 60 h). In addition, the salt tolerance of Syn2973 was further enhanced by overexpressing the rate-limiting step of glycerol-3-phosphate dehydrogenase and downregulating the gene rfbA, which encodes UDP glucose pyrophosphorylase. Taken together, these results indicate that the growth of the end-point strain M-2522-GgpPS-drfbA was improved by 62% compared with the control strain M-pSI-pSII at 60 h under treatment with 0.5 M NaCl. Finally, a comparative metabolomic analysis between strains M-pSI-pSII and M-2522-GgpPS-drfbA was performed to characterize the carbon flux in the engineered M-2522-GgpPS-drfbA strain, and the results showed that more carbon flux was redirected from ADP-GLC to GG synthesis. This study provides important engineering strategies to improve salt tolerance and GG production in Syn2973 in the future.

Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions.

In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.

[Removal of Antibiotics and Antibiotic Resistance Genes from Urban Rivers Using Artificial Ecosystems].

Four antibiotics[azithromycin (AZM), sulfamethoxazole (SMZ), ciprofloxacin (CIP), and tetracycline (TCY)], and the antibiotic resistance genes (ARGs)[sulfonamides (sul1 and sul2), tetracyclines (tetX and tetM), quinolones (qnrS and qnrD), macrolides (ermB), and 16S rDNA] were selected as target compounds. Artificial ecosystems were constructed with combinations of two emergent plants and Microcystis aeruginosa (Acorus calamus+Cordyceps, algae+Cordyceps, algae+Acorus calamus, and algae+Acorus calamus+Cordyceps) in an indoor-simulated river system. Throughout the artificial ecosystems, changes in antibiotic concentrations and other pollution indicators (i.e., COD, NH4+-N, TP, and TN) were monitored in different media (the aqueous phase, sediment phase, and in plants), and the distribution and removal of ARGs in aqueous and sediment phases were explored. Removal of the target compounds was calculated based on mass balance, and the correlation between ARG abundance and environmental factors in the aqueous and sediment phases was analyzed. The results showed that the constructed artificial ecosystem achieved removal rates of COD, NH4+-N, TP, and TN ranging from 60.2% to 74.8%, 63.4% to 77.4%, 64.0% to 73.2%, and 46.8% to 54.8%, respectively. The antibiotics in the aqueous phase were notably removed and the artificial ecosystem 'algae+Acorus calamus+Cordyceps' achieved the best removal efficiency for the four antibiotics. Removal rates of the antibiotics in the sediment phase were ranked in the order TCY>CIP>AZM>SMZ; the removal efficiency of TCY in the 'algae+Acorus calamus+Cordyceps' system reached up to 53.5%. The total removal rates of antibiotics obtained by the ecosystems were ranked in the following order:algae+Acorus calamus+Cordyceps > algae+Cordyceps > algae+Acorus calamus > Acorus calamus+Cordyceps. Removal of the four ARGs was very efficient and was higher in the aqueous phase than in the sediment phase. Correlations between the ARGs, the other pollution indicators, and the antibiotics were variable; tetX and environmental factors were correlated in the aqueous phase, while AZM and its corresponding ARGs were not significantly correlated in the sediment phase. The results showed that ARGs can be targeted under corresponding antibiotic pressure and other types of environmental pressure. In the study system, the concentrations of antibiotics did not directly affect the transmission of ARGs. Overall, this study shows that artificial ecosystems constructed with emergent plants and Microcystis aeruginosa can be effective at purifying water and reducing the environmental risks of antibiotics in urban rivers.

Deciphering and engineering photosynthetic cyanobacteria for heavy metal bioremediation.

Environmental pollution caused by heavy metals has received worldwide attentions due to their ubiquity, poor degradability and easy bioaccumulation in host cells. As one potential solution, photosynthetic cyanobacteria have been considered as promising remediation chassis and widely applied in various bioremediation processes of heavy-metals. Meanwhile, deciphering resistant mechanisms and constructing tolerant chassis towards heavy metals could greatly contribute to the successful application of the cyanobacteria-based bioremediation in the future. In this review, first we summarized recent application of cyanobacteria in heavy metals bioremediation using either live or dead cells. Second, resistant mechanisms and strategies for enhancing cyanobacterial bioremediation of heavy metals were discussed. Finally, potential challenges and perspectives for improving bioremediation of heavy metals by cyanobacteria were presented.

A transporter Slr1512 involved in bicarbonate and pH-dependent acclimation mechanism to high light stress in Synechocystis sp. PCC 6803.

High light (HL) exposure leads to photoinhibition and excess accumulation of toxic reactive oxygen species (ROS) in photosynthetic organisms, negatively impacting the global primary production. In this study, by screening a mutant library, a gene related with bicarbonate transport, slr1512, was found involved in HL acclimation in model cyanobacterium Synechocystis sp. PCC 6803. Comparative growth analysis showed that the slr1512 knockout mutant dramatically enhanced the tolerance of Synechocystis towards long-term HL stress (200 µmol photons m-2 s-1) than the wild type, achieving an enhanced growth by ~1.95-folds after 10 d. The phenotype differences between Δslr1512 and the wild type were analyzed via absorption spectrum and chlorophyll a content measurement. In addition, the accessible bicarbonate controlled by slr1512 and decreased PSII activity were demonstrated, and they were found to be the key factors affecting the tolerance of Synechocystis against HL stress. Further analysis confirmed that intracellular bicarbonate can significantly affect the activity of photosystem II, leading to the altered accumulation of toxic ROS under HL. Finally, a comparative transcriptomics was applied to determine the differential responses to HL between Δslr1512 and the wild type. This work provides useful insights to long-term acclimation mechanisms towards HL and valuable information to guide the future tolerance engineering of cyanobacteria against HL.

Tailoring cyanobacteria as a new platform for highly efficient synthesis of astaxanthin.

With the ability to recycle CO2 into value-added chemicals, cyanobacteria have been considered as renewable microbial cell factories. Astaxanthin, a highly valued carotenoid with potent antioxidant activity, could be beneficial to human health. Astaxanthin biosynthesis in engineered chassis has been achieved previously, but it generated a relatively low yield. Here, we successfully constructed a highly efficient astaxanthin biosynthetic pathway in cyanobacterium Synechocystis sp. PCC 6803, and achieved more than a 500-fold increase in astaxanthin production via stepwise reconstruction of the biosynthetic pathway and rational rewiring of the endogenous metabolism. The engineered strain produced up to 29.6 mg/g of astaxanthin (dry cell weight), which is the highest yield reported in the engineered chassis to date. Moreover, multi-omics analyses revealed that establishing a high astaxanthin flux may enhance photosynthesis and central metabolism in the engineered strain to compensate for the depleted pigments, which could be valuable for astaxanthin overproduction. This study presents a novel alternative for high-efficiency biosynthesis of astaxanthin directly from CO2.

Improved Salt Tolerance and Metabolomics Analysis of Synechococcus elongatus UTEX 2973 by Overexpressing Mrp Antiporters.

The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) is a promising candidate for photosynthetic microbial factory. Seawater utilization is necessary for large-scale cultivation of Syn2973 in the future. However, Syn2973 is sensitive to salt stress, making it necessary to improve its salt tolerance. In this study, 21 exogenous putative transporters were individually overexpressed in Syn2973 to evaluate their effects on salt tolerance. The results showed the overexpression of three Mrp antiporters significantly improved the salt tolerance of Syn2973. Notably, overexpressing the Mrp antiporter from Synechococcus sp. PCC 7002 improved cell growth by 57.7% under 0.4 M NaCl condition. In addition, the metabolomics and biomass composition analyses revealed the possible mechanisms against salt stress in both Syn2973 and the genetically engineered strain. The study provides important engineering strategies to improve salt tolerance of Syn2973 and is valuable for understanding mechanisms of salt tolerance in cyanobacteria.

Engineering salt tolerance of photosynthetic cyanobacteria for seawater utilization.

Photosynthetic cyanobacteria are capable of utilizing sunlight and CO2 as sole energy and carbon sources, respectively. With genetically modified cyanobacteria being used as a promising chassis to produce various biofuels and chemicals in recent years, future large-scale cultivation of cyanobacteria would have to be performed in seawater, since freshwater supplies of the earth are very limiting. However, high concentration of salt is known to inhibit the growth of cyanobacteria. This review aims at comparing the mechanisms that different cyanobacteria respond to salt stress, and then summarizing various strategies of developing salt-tolerant cyanobacteria for seawater cultivation, including the utilization of halotolerant cyanobacteria and the engineering of salt-tolerant freshwater cyanobacteria. In addition, the challenges and potential strategies related to further improving salt tolerance in cyanobacteria are also discussed.

Repeated fed-batch strategy and metabolomic analysis to achieve high docosahexaenoic acid productivity in Crypthecodinium cohnii.

BACKGROUND: Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC- and GC-MS metabolomic analyses. RESULTS: It was discovered that glucose concentration at 15-27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC- and GC-MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, L-tryptophan, L-threonine, L-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. CONCLUSIONS: The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31.

BACKGROUND: Docosahexaenoic acid (DHA, C22:6) and odd-chain fatty acids (OCFAs, C15:0 and C17:0) have attracted great interest, since they have been widely used in food and therapeutic industries, as well as chemical industry, such as biodiesel production and improvement. The oil-producing heterotrophic microalgae Schizochytrium sp. 31 is one of main DHA-producing strains. Recently, it was found that Schizochytrium can also synthesize OCFAs; however, contents and titers of DHA and OCFAs in Schizochytrium are still low, which limit its practical application. RESULTS: In this study, we found that acetyl-CoA carboxylase suffered from a feedback inhibition by C16-CoA in Schizochytrium, and relief of the inhibition resulted in improved both lipid content and the ratio of OCFAs in total fatty acids. Based on this finding, a novel strategy for elevating both DHA and OCFAs contents was established. First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from Mortierella alpina, which encodes an elongase enzyme responsible for converting C16 into C18 fatty acids. After the above two-step engineering, contents of DHA and OCFAs were increased by 1.39- and 3.30-fold, reaching a level of 26.70 and 25.08% of dry cell weight, respectively, which are the highest contents reported so far for Schizochytrium. The titers of DHA and OCFAs were elevated by 1.08- and 2.57-fold, reaching a level of 3.54 and 3.32 g/L, respectively. Notably, the OCFAs titer achieved was 2.66-fold higher than the highest reported in Escherichia coli (1.25 g/L), implying potential value for industry application. To reveal the potential metabolic mechanism for the enhanced biosynthesis of both DHA and OCFAs, LC-MS metabolomic analysis was employed and the results showed that the pentose phosphate pathway and the glycolysis pathway were strengthened and intracellular propionyl-CoA concentration were also significantly increased in the engineered Schizochytrium, suggesting an increased supply of NADPH, acetyl-CoA, and propionyl-CoA for DHA and OCFAs accumulation. CONCLUSIONS: The discovery provides a new source of OCFAs production, and proposes a new strategy to improve contents and titers of both DHA and OCFAs in Schizochytrium. These will be valuable for improving commercial potential of Schizochytrium and guiding the engineering strategy in other fatty acids producing heterotrophic microalga.

Rewiring metabolic network by chemical modulator based laboratory evolution doubles lipid production in Crypthecodinium cohnii.

Dietary omega-3 long-chain polyunsaturated fatty acids docosahexaenoic acid (DHA, C22:6) can be synthesized in microalgae Crypthecodinium cohnii; however, its productivity is still low. Here, we established a new protocol termed as "chemical modulator based adaptive laboratory evolution" (CM-ALE) to enhance lipid and DHA productivity in C. cohnii. First, ACCase inhibitor sethoxydim based CM-ALE was applied to redirect carbon equivalents from starch to lipid. Second, CM-ALE using growth modulator sesamol as selection pressure was conducted to relive negative effects of sesamol on lipid biosynthesis in C. cohnii, which allows enhancement of biomass productivity by 30% without decreasing lipid content when sesamol was added. After two-step CM-ALE, the lipid and DHA productivity in C. cohnii was respectively doubled to a level of 0.046 g/L/h and 0.025 g/L/h in culture with addition of 1 mM sesamol, demonstrating that this two-step CM-ALE could be a valuable approach to maximize the properties of microalgae.

Nitrogen Feeding Strategies and Metabolomic Analysis To Alleviate High-Nitrogen Inhibition on Docosahexaenoic Acid Production in Crypthecodinium cohnii.

It is well-known that high-nitrogen content inhibits cell growth and docosahexaenoic acid (DHA) biosynthesis in heterotrophic microalgae Crypthecodinium cohnii. In this study, two nitrogen feeding strategies, pulse-feeding and continuous-feeding, were evaluated to alleviate high-nitrogen inhibition effects on C. cohnii. The results showed that continuous-feeding with a medium solution containing 50% ( w/v) yeast extract at 2.1 mL/h during 12-96 h was the optimal nitrogen feeding strategy for the fermentation process, when glucose concentration was maintained at 15-27 g/L during the same period. With the optimized strategy, 71.2 g/L of dry cell weight and DHA productivity of 57.1 mg/L/h were achieved. In addition, metabolomic analysis was applied to determine the metabolic changes during different nitrogen feeding conditions, and the changes in amino acids, polysaccharides, purines, and pentose phosphate pathway were observed, providing valuable metabolite-level information for exploring the mechanism of the high-nitrogen inhibition effect and further improving DHA productivity in C. cohnii.

13C Metabolic Flux Analysis of Enhanced Lipid Accumulation Modulated by Ethanolamine in Crypthecodinium cohnii.

The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.

Screening of chemical modulators for lipid accumulation in Schizochytrium sp. S31.

Schizochytrium sp. is a promising candidate for docosahexaenoic acid (DHA) production due to its high content of lipid and DHA proportions. To further enhance the lipid accumulation, seven chemical modulators were screened to evaluate their roles on lipid accumulation. Notably, among the seven tested chemical modulators, the addition of naphthoxyacetic acid (BNOA) or jasmonic acid (JA) was able to increase the lipid accumulation of Schizochytrium sp. S31. In addition, the effects of BNOA and JA were demonstrated dose-dependent and time-dependent, achieving a highest increase in lipid content by 11.16% and 12.71% when 2.0 mg/L of BNOA or 20 mg/L of JA was added into culture at 48 h after inoculation, respectively. In addition, the combination of 2 mg/L BNOA and 20 mg/L JA further increased lipid accumulation up to 16.79%. These results provided valuable strategy on promoting the lipid accumulation and DHA production by chemical modulators in Schizochytrium sp. S31.