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

Abstract

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 fedbatch 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.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66

“…There are some similar studies on oleagineous model systems including thraustochytrids [29][30][31][32]44,45] but none covering with quantitative methods the whole central carbon metabolism, redox-cofactors and lipid composition as completely as presented here. Those studies have mostly applied untargeted LC-MS and GC-MS profiling, few internal standards and usually only normalized to the total peak area.…”

“…Yang and coworkers studied Schizochytrium strains with different DHA content and reported downregulated glycolytic and upregulated TCA pools in the high DHA strain [31]. Liu and coworkers applied GC-MS and LC-MS metabolite profiling to maintain high DHA productivity during repeated fed-batch cultivation of Crypthecodinium [32]. All these studies provide interesting information on the relative cellular adjustments to changes in cultivation conditions and growth stages which can be used to further improve yields and productivities of the DHA-bioprocess.…”

“…Establishment of genetic tools for thraustochytrid strain engineering has been challenging, but several successful attempts have been reported [24][25][26][27][28]. Most thraustochytrid studies are reported with cultivation data, total lipids and DHA-production, while only scarce information is available about intracellular metabolite levels [13,[29][30][31][32]. Such information is requested in ME projects, both to generate biological knowledge as basis for selection of targets to manipulate, but, equally important, to characterize the phenotype of the resulting mutants.…”

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

“…All cultivations were carried out with the strain E. coli BL21(DE3), transformed with a pET30a + plasmid carrying the gene for the cytoplasmic protein (CP) and periplasmic Crypthecodinium cohnii docosahexaenoic acid 4 cycles, 80% medium replacement Liu et al, 2020 Aspergillus terreus Lovastatin 3 cycles, 37% yield in crease Novak et al, 1997;Kumar et al, 2000 Gluconobacter oxydans Dihydroxyacetone 4 cycles, repeated fed-batch process using two spatially separated vessels Bauer et al, 2005 Kluyveromyces marxianus Ethanol 5 cycles, product yield constant Ozmihci and Kargi, 2007 Kitasatospora ε-Poly-L-lysine 5 cycles Zhang et al, 2010 Yarrowia lipolytica Citric acid 10 cycles, productivity decrease over cultivation time Moeller et al, 2011 Pichia pastoris human serum albumin (rHSA) 4 cycles, 47% yield increase Ohya et al, 2005 Pichia pastoris Malaria vaccine candidates stable productivity for 2-8 cycles, methanol induction Martens et al, 2011;Fricke et al, 2013 Escherichia coli Pyruvate 5 cycles, q p increased fivefold Zelić et al, 2004 protein (PP), respectively. The cytoplasmic protein contained no disulfide bonds, had a isoelectric point (PI) of 5.62 and a protein size of 26.9 kDa.…”

Rewiring the Metabolic Network to Increase Docosahexaenoic Acid Productivity in Crypthecodinium cohnii by Fermentation Supernatant-Based Adaptive Laboratory Evolution

“…In recent years, significant efforts have been made to improve DHA productivity in C. cohnii using approaches such as optimizing fermentation conditions (Liu et al, 2018), employing different carbon sources (De Swaaf et al, 2003a,b), and enhancing strain improvement using the acetyl-CoA carboxylase inhibitor sethoxydim (Liu et al, 2017). However, certain bottlenecks remain, such as low tolerance to the inhibitory fermentation supernatant at the late stage of fermentation, which significantly limits further improvements of DHA productivity (Liu et al, 2020). It has been previously reported that the fermentation supernatant, in which the accumulation of unspent medium components or metabolites exported by C. cohnii at the late stage of fermentation occurs, could inhibit the growth and DHA synthesis in C. cohnii (Liu et al, 2020).…”