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

Liangsen Liu,Lei Zeng,Lei Chen,Fangzhong Wang,Yali Bi,Weiwen Zhang,Jinjin Diao

doi:10.3389/fmicb.2022.824189

Abstract

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.

Highlights

Docosahexaenoic acid (DHA, 22:6n-3), a long-chain polyunsaturated fatty acid (PUFAs) and a structural constituent of membranes, in components of the nervous system, such as the brain and retina (Ferraro et al, 2013), is essential for human beings, because they are poor DHA synthesizers (Muskiet et al, 2004)
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)
At the end of the fermentation that corresponded to the stationary growth phase, the fermentation broth with the glucose concentration adjusted to 27 g/L was harvested by centrifugation (17696 × g, 20 min, and 4◦C), and the aqueous phase was collected as the fermentation supernatant under ensured sterile operation

Summary

INTRODUCTION

Docosahexaenoic acid (DHA, 22:6n-3), a long-chain polyunsaturated fatty acid (PUFAs) and a structural constituent of membranes, in components of the nervous system, such as the brain and retina (Ferraro et al, 2013), is essential for human beings, because they are poor DHA synthesizers (Muskiet et al, 2004). 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). To improve the DHA productivity of C. cohnii, FS-ALE was developed by gradually increasing the amount of the inhibitory fermentation supernatant collected from fedbatch fermented C. cohnii ATCC 30556 (starting strain) at the stationary growth phase in the regular fermentation medium. This study provides an innovative solution (FS-ALE) to relieve the inhibition of the fermentation supernatant at the late stage of normal fermentation in C. cohnii. This study provides valuable insights for understanding the mechanisms of cell growth and lipid accumulation in C. cohnii

MATERIALS AND METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT