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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.

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.

The heterotrophic microalga Crypthecodinium cohnii accumulates lipids with a high fraction of docosahexaenoic acid (DHA). However, lack of genomic information limits the understanding of its physiological metabolism for better DHA production. In this study, de novo assembly of the C. cohnii transcriptome from three growth stages (i.e., fast growth, fatty acid accumulation and DHA conversion stages) was conducted, leading to identification of a total of 82,106 unigenes with an N50 of 1822 bp, among which 64.7% were annotated based on sequence similarity to known genes in the database. In addition, pathway enrichment analysis showed that transcripts related to fatty acid biosynthesis, starch and sucrose metabolism as well as biosynthesis of unsaturated fatty acids were significantly up-regulated during late-stage fermentation. Interestingly, several polyketide synthases (PKSs) and multiple fatty acid desaturases potentially involved in DHA biosynthesis were identified in the C. cohnii transcriptome, most of which were significantly up-regulated during lipid and DHA accumulation, implying that C. cohnii might utilize a combination of PKS systems and desaturase steps for DHA biosynthesis. The results were further confirmed by qRT-PCR and GC–MS-based metabolomic analyses. Overall, integrative analysis of de novo transcriptomic and metabolomic data provided important functional genomic information necessary for not only a better understanding of C. cohnii growth and DHA biosynthesis but also future genetic engineering of industry-important C. cohnii.

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. Electronic supplementary material The online version of this article (10.1186/s13068-019-1484-x) contains supplementary material, which is available to authorized users.

Docosahexaenoic acid (DHA, C22: 6), which can be synthesized by the heterotrophic microalga Crypthecodinium cohnii, has received significant interest recently, due to its important roles in human health. However, the high production cost caused by low productivity and yield is still a major hurdle for industrial production of DHA using C. cohnii. The reduction of starch content has been found to be a promising mean to drive more carbon source to lipid biosynthesis in C. cohnii; however, it usually affects cell growth negatively, which limits its practical application. In this study, atmospheric and room temperature plasma mutagenesis associated with high-throughput screening based on iodine vapour was developed to identify a starch-deficient C. cohnii mutant 16D. The mutant 16D accumulated more biomass, lipid and DHA, with less starch and extracellular polysaccharide. The productivity and the yield of DHA were elevated 1.7 and 1.3 fold, reaching levels of 57.7 mg/L-h and 52.0 mg/g, respectively, in fed-batch fermentation, which are the highest reported so far for DHA production using C. cohnii. LC- and GC–MS based metabolomic analyses revealed multiple possible mechanisms relevant to increased growth and lipid content in mutant 16D, including strengthened Embden-Meyerhof, Krebs cycle and pentose phosphate pathways to provide more CoA, ATP and NADPH for supporting growth and lipid accumulation; and a significant increase in the intracellular abundance of tagatose, which was demonstrated to be involved in lipid content enhancement possibly by activating the triacylglycerol biosynthesis pathway in mutant 16D. The study presents a new strain improvement strategy for DHA-producing C. cohnii and new insights for switching metabolism from starch to growth and lipid in C. cohnii. In addition, the study also demonstrates the inductive effects of tagatose on lipogenesis in heterotrophic microalgae.

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.