| Pneumocandin B0 is the precursor of an antifungal drug caspofungin.However,the industrial production of pneumocandin B0 still faces problems due to low yield,long fermentation period,and the resulting high cost has seriously restricted its medical applications.In this work,Glarea lozoyensis,an industrial production strain of pneumocandin B0,was used as a study object.Systems-biological methods,such as metabolomics,transcriptomics,real-time quantitative PCR,and key enzymes activity analysis were used.Based on a deep analysis of the mechanisms responsible for high yields of pneumocandin B0,an accumulation mechanism of pneumocandin B0 based on reactive oxygen species?ROS?balance was put forward.Therefore,three strategies for improving fermentation and strains were also promulgated.The main results are as follows:?1?In order to explore the key metabolites and pathways in the biosynthesis of pneumocandin B0,the metabolic profiles of the mutated strain G.lozoyensis Q1 and the parent strain G.lozoyensis ATCC 74030 were analyzed.A total of 79 metabolites were detected,15 of which were identified as key metabolites.It was found that a continuous supply of NADPH and sufficient acetyl-CoA was a prerequisite for highly efficient synthesis of pneumocandin B0.In addition,the abundance of?-aminobutyric acid and unsaturated fatty acids was directly proportional to the synthesis of pneumocandin B0 in the mutated strain.Gamma-aminobutyric acid and unsaturated fatty acids are involved in the elimination of intracellular ROS,which implies that the synthesis of pneumocandin B0 might be related to the oxidative stress reaction.?2?In order to verify the effect of ROS on the synthesis of pneumocandin B0,the formation of intracellular ROS was induced in G.lozoyensis Q1 by increasing the osmotic pressure.The lower osmotic stress was helpful for increase the biomass,while the higher osmotic stress was helpful for increase the pneumocandin B0 per biomass.Consequently,a novel osmotic stress control strategy for improved pneumocandin B0 production in G.lozoyensis was developed.The fermentation period was shortened 72 h and the productivity increased by 44%.The transcription factors YAP1,which is related to the stress reaction,is involved in the production of secondary metabolites.?3?To further explore the relationship between yap1-mediated ROS signaling and the biosynthesis of pneumocandin B0 at the molecular level,a yap1 gene knockout strain?Glyap1 was constructed by homologous recombination.?Glyap1did not have the ability to restore the cellular redox balance upon disturbance,which led to higher ROS levels.The ROS increased the yield of pneumocandin B0 per cell during the early fermentation stage.But the excessive ROS damage to cells during the late fermentation stage.Finally,a hypothesis of the regulation of pneumocandin B0synthesis mechanism based on the cellular oxidation-reduction balance was proposed.?4?The endogenous ROS elimination system was enhanced using low-temperature adaptive laboratory evolution?ALE?,in order to resist the excessive ROS in the late fermentation stage.G.lozoyensis Q1 was used as the starting strain,and after 50 runs of low-temperature ALE,the final evolved strain ALE50 was obtained.The low-temperature ALE strains had higher permeability of the cell membrane,higher acetyl coenzyme A concentration,antioxidant enzyme activity and proline concentration.These four functions promoted the synthesis of pneumocandin B0.Ultimately,combining the ALE50 strain and osmotic stress control strategy,the final yield of pneumocandin B0 reached 3120 mg/L at 384 h.Furthermore,the pneumocandin B0 per biomass was 49.52 mg/g DCW.Compared with the fermentation of G.lozoyensis Q1 without the regulation strategy,the yield and pneumocandin B0 per biomass was increased by 71 and 90%,respectively.Thus,the ALE50 strain showed a significantly increased production capacity of pneumocandin B0,bringing us closer to cost-competitive industrial production of this important next-generation antifungal agent. |
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