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The effects of propanol and 1, 3-propanediol on fatty acid and biomass accumulation in Schizochytrium ATCC 20888 were explored. Propanol increased the contents of saturated fatty acids and total fatty acids by 55.4 and15.3%, while 1, 3-propanediol elevated the polyunsaturated fatty acids, total fatty acids and biomass contents by 30.7, 17.0, and 6.89%. Although both of them quench ROS to increase fatty acids biosynthesis, the mechanisms are different. The effect of propanol did not reflect on metabolic level while 1, 3-propanediol elevated osmoregulators contents and activated triacylglycerol biosynthetic pathway. The triacylglycerol content and the ratio of polyunsaturated fatty acids to saturated fatty acids were significantly increased by 2.53-fold, which explained the higher PUFA accumulation in Schizochytrium after adding 1, 3- propanediol. At last, the combination of propanol and 1, 3-propanediol further elevated total fatty acids by approximately 1.2-fold without compromising cell growth. These findings are valuable for scale-up production of designed Schizochytrium oil for various application purposes.

Polyunsaturated fatty acids (PUFAs) are important nutrients that play important roles in human health. In eukaryotes, PUFAs can be de novo synthesized through two independent biosynthetic pathways: the desaturase/elongase pathway and the PUFA synthase pathway. Among them, PUFAs synthesized through the PUFA synthase pathway typically have few byproducts and require fewer reduction equivalents. In the past 2 decades, numerous studies have been carried out to identify, analyze and engineer PUFA synthases from eukaryotes. These studies showed both similarities and differences between the eukaryotic PUFA synthase pathways and those well studied in prokaryotes. For example, eukaryotic PUFA synthases contain the same domain types as those in prokaryotic PUFA synthases, but the number and arrangement of several domains are different; the basic functions of same-type domains are similar, but the properties and catalytic activities of these domains are somewhat different. To further utilize the PUFA synthase pathway in microbial cell factories and improve the productivity of PUFAs, many challenges still need to be addressed, such as incompletely elucidated PUFA synthesis mechanisms and the difficult genetic manipulation of eukaryotic hosts. In this review, we provide an updated introduction to the eukaryotic PUFA synthase pathway, summarize the functions of domains and propose the possible mechanisms of the PUFA synthesis process, and then provide future research directions to further elucidate and engineer the eukaryotic PUFA synthase pathway for the maximal benefits of humans.

Docosahexaenoic acid (DHA, 22:6n-3) plays significant roles in enhancing human health and preventing human diseases. The heterotrophic marine dinoflagellate Crypthecodinium cohnii is a good candidate to produce high-quality DHA. To overcome the inhibition caused by the fermentation supernatant in the late fermentation stage of DHA-producing C. cohnii, fermentation supernatant-based adaptive laboratory evolution (FS-ALE) was conducted. The cell growth and DHA productivity of the evolved strain (FS280) obtained after 280 adaptive cycles corresponding to 840 days of evolution were increased by 161.87 and 311.23%, respectively, at 72 h under stress conditions and increased by 19.87 and 51.79% without any stress compared with the starting strain, demonstrating the effectiveness of FS-ALE. In addition, a comparative proteomic analysis identified 11,106 proteins and 910 differentially expressed proteins, including six stress-responsive proteins, as well as the up- and downregulated pathways in FS280 that might contribute to its improved cell growth and DHA accumulation. Our study demonstrated that FS-ALE could be a valuable solution to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation of heterotrophic microalgae.

High DHA production cost caused by low DHA titer and productivity of the current Schizochytrium strains is a bottleneck for its application in competition with traditional fish-oil based approach. In this study, atmospheric and room-temperature plasma with iodoacetic acid and dehydroepiandrosterone screening led to three mutants, 6–8, 6–16 and 6–23 all with increased growth and DHA accumulations. A LC/MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle might be relevant to the increased growth and DHA biosynthesis in the mutants. Finally, the mutant 6–23, which achieved the highest growth and DHA accumulation among all mutants, was evaluated in a 5 L fermentor. The results showed that the DHA concentration and productivity in mutant 6–23 were 41.4 g/L and 430.7 mg/L/h in fermentation for 96 h, respectively, which is the highest reported so far in literature. The study provides a novel strain improvement strategy for DHA-producing Schizochytrium.

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.

Characterizing and engineering microbial communities for lignocellulosic biofuel production has received widespread attention. Previous research has established that Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17 coculture significantly improves overall cellulosic biofuel production efficiency. Here, we investigated this interaction and revealed the mechanism underlying the improved efficiency observed. In contrast to the previously reported mutualistic relationship, a harmful effect toward C. thermocellum JN4 was observed in these microbial consortia. Although T. thermosaccharolyticum GD17 relieves the carbon catabolite repression of C. thermocellum JN4 regarding obtaining more cellobiose or glucose released from lignocellulose, T. thermosaccharolyticum GD17 significantly hampers the growth of C. thermocellum JN4 in coculture. The increased formation of end products is due to the strong competitive metabolic advantage of T. thermosaccharolyticum GD17 over C. thermocellum JN4 in the conversion of glucose or cellobiose into final products. The possibility of controlling and rebalancing these microbial consortia to modulate cellulose degradation was achieved by adding T. thermosaccharolyticum GD17 stimulants into the system. As cellulolytic bacteria are usually at a metabolic disadvantage, these discoveries may apply to a large proportion of cellulosic biofuel-producing microbial consortia. These findings provide a reference for engineering efficient and modular microbial consortia for modulating cellulosic conversion.

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.

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 NADPC- 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. [ABSTRACT FROM AUTHOR]

The lack of selective markers has been a key problem preventing multistep genetic engineering in filamentous fungi, particularly for industrial species such as the lignocellulose degrading Penicillium oxalicum JUA10-1(formerly named as Penicillium decumbens). To resolve this problem, we constructed a genetic manipulation system taking advantage of two established genetic systems: the Cre-loxP system and Tet-on system in P. oxalicum JUA10-1. This system is efficient and convenient. The expression of Cre recombinase was activated by doxycycline since it was controlled by Tet-on system. Using this system, two genes, ligD and bglI, were sequentially disrupted by loxP flanked ptrA. The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi. This system will also play an important role in improving the productivity of interesting products and minimizing by-product when fermented by filamentous fungi. [ABSTRACT FROM AUTHOR]