Arthroconidia-mediated Repeated Batch Fermentation Strategy For Antrodia Camphorata And Underlying Mechanism Of Asexual Sporulation

Antrodia camphorata (sys. Antrodia cinnamomea, A. camphorata ) is a precious edible and medicinal mushroom that grows only on cinnamomun kanehirae in Taiwan, China, and exhibits various biological functions, such as liver protection, anti-tumor, anti-cancer.anti-oxidation, anti-inflammatory, vaso-relaxation, etc. However, the wild fruiting bodies of A.camphorata are difficult to satisfy the consumption demand due to its host-specificity, rarity in nature, and slow growth rate. Submerged fermentation was considered as the most efficient artificial cultivation way to produce A. camphorata because of short fermentation period and high efficiency. However, large-scale submerged fermentation of A. camphorata still encounters some engineering problems, such as tedious and time-consuming for inoculum preparation, the production period need to shorten further, the production efficiency need to improve further. In our previous studies, we first reported that A. camphorata produced lots of arthroconidia at the end of submerged fermentation under appropriate environmental conditions, and found that the used the arthroconidia as inoculum for submerged fermentation can shorten fermentation period and improve the triterpenoid production compared with the mycelial inoculum. The repeated batch fermentation (RBF) integrated seed culture and fermentation processes together, saved lots of time and cost, and improved the production efficiency and benefit. Thus, in this study, we established an efficient arthroconidia-mediated RBF process to produce bioactive metabolites of A. camphorata. This RBF process for A.camphorata was established based on asexual sporulation and easy to separate the arthroconidia from mycelium pellets. However, produced enough arthroconidia in time is the basic requirement to ensure the RBF process works sustainedly and stably. Thus, in order to control the conidiation of A. camphorata in submerged fermentation, we used the proteomics and transcriptomics methods to reveal the molecular mechanism of conidiation of A.camphorata, then verified the mechanism via inducing conidiation by nutrition starvation and Ca2+. The main results of this study are as follows:(1) An efficient arthroconidia-mediated RBF process to produce bioactive metabolites production of A. camphorata was established, which shortened the production period, saved the production cost, and enhanced the production efficiency. The RBF process can stably produce 8 cycles, sustained for 2 monthes. Compared with traditional batch fermentation, the RBF process shortened the overall production time of eight cycles to 58 d from 80 d,shortened the average production period to 7 d from 10 d. Accordingly, the crude intercellular polysaccharide productivity was improved by 40.3%, crude triterpenoids productivity was improved by 43.2%, antrodin A productivity was improved by 42.5%, and antrodin B productivity was improved by 58.6%.(2) The FluG-mediated signal pathway of submerged conidiation of A. camphotara was proposed, which help to control the conidiation of A. camphorate in submerged fermentation.The 2DE, RNA-seq, and RT-qPCR methods were used to reveal the molecular mechanism of conidiation of A. camphorata in submerged fermentation, and then a FluG-mediated signal pathway of submerged conidiation was proposed. That is, the SfgA proteins inhibited the expression of flbA,flbB, and flbC through binding to their promoters. At the same time, the FluG protein participated in synthesizing an extracellular sporulation inducing factor (ESIF)and accumulation with incubation time. When FluG proteins were accumulated to some level or induced by some conidiation signal, the ESIF activated flbA, flbB, and flbC through removing the sfgA. Then, the FlbA promote conidiation through suppressing the G-protein-mediated signalling pathway, which inhibit conidiation but promote growth via pkaA. The flbD was activated by FlbB and then form an FlbB-FlbD hetero-dimer, which activates brlA together with FlbC through removing the NsdD protein that binding to the promoter of brlA; the BrlA activate abaA, then wetA was activated by AbaA. Finally, the wetA directly result in conidiation and conidial maturation. In addition, the AbaA promote the expression of vosA and velB, then produce a VosA-VelB hetero-dimer, which help to conidial maturation and inhibit the expression of brlA. In addition, the VeA inhibit the expression of brlA, but stuA promote he expression of brlA.(3) The molecular mechanism of conidiation response to nutrition starvation for A.camphotara was explored, which verified the FluG-mediated signal pathway of submerged conidiation. The RNA-seq and RT-qPCR methods were used to analyze the differential expression genes of mycelia incubated with different nutrition, then a signalling pathway of submerged conidiation induced by nutrition starvation for A camphotara was proposed. That is, when nitrogen starvation, the relevant sensors transfer the signal to genes of areA and tmpA,then the AreA promotes the expression of flbD directly or indirectly, then promotes the expression of brlA further, the TmpA promotes the expression of brlA directly or indirectly;When carbon starvation, the sensor of Rco-3 transfer the signal to brlA directly or indirectly and promotes its expression; BrlA promotes the expression of abaA and wetA, and finally promotes conidiation; When nutrition (nitrogen or carbon) starvation, the relevant sensors transfer the signals to genes of guIC and chsD directly or indirectly, which contribute to the cell wall integrity, then promote conidial mature together. In addition, the gulC also promotes the autolysis of cells, which provide energy and building blocks to conidiation.(4) The molecular mechanism of conidiation response to Ca2+ induceding for A.camphotara were explored, which further verified the FluG-mediated signal pathway of submerged conidiation. The 2DE and RT-qPCR methods were used to analyze the differential expression genes of mycelia incubated with different concentrations of Ca2+, then a Ca2+/calmodulin- and FluG-mediated signaling pathway of submerged conidiation was proposed. That is, the exocellular Ca2+ entry the cytoplasm via the calcium channel proteins(Cchl and Mid1) and bind to the calmodulin (CaM), then activated the calcineurin as Ca2+-CaM complex, at the same time, lots of heat shock proteins are produced. The crzl is activated by calcineurin, and Crzl directly acts on abaA to promotes its expression as a transcription factor. Simultaneously, the calcineurin or HSP90 promotes the expressions of flbB and flbC directly or indirectly, while the FlbB and FlbC promotes the expressions of brlA,and then begin the central regulatory pathway to improve conidiation. The HSP90 and WetA contribute to the cell wall integrity, then promote the conidial mature together.