Fermentation Of Marine Fungus Aspergillus Glaucus For Aspergiolide A Production And Heterologous Expression Of Fungal Polyketide Synthase Gene Tspks1

Aspergiolide A, a structurally novel anthraquinone derivative isolated from cultures of a marine-derived fungus Aspergillus glaucus HB1-19 by Ocean University of China, was proven to hold striking anti-tumor activities and be synthesized via polyketide pathway. However, aspergiolide A production from the original cultures was quite low (< 4 mg/L). In present work, we aim to enhance aspergiolide A production using metabolic regulation, process optimization and fermentation scale-up to provide sufficient quantity of compound for further pharmacy studies of animal experiments, clinic research and structure activity relationship. We are also dedicated to introduce some useful information to marine microorganism fermentations owing to the lack of systematic culture techniques of marine microbes. In addition, cloning and function analysis of fungal polyketide synthase (PKS) gene have developed rapidly in recent years. By contrast, heterologous expression of fungal PKS gene is still in its infancy. Here we attempted to heterologously express Tspks1 from Talaromyces stipitatus ATCC 10500.Aspergiolide A production enhancement by citrate and its effects on growth and sexual development of marine-derived fungus Aspergillus glaucus HB1-19 were first investigated. In agar plate culture,15 mmol/L citric acid decreased colony radial growth and aspergiolide A production by 31.5% and 23.0%, respectively. It also improved sexual cleistothecium formation by 360% but depressed asexual conidiospore generation by 84.8%. In submerged culture, adding 40 mmol/L citric acid finally promoted aspergiolide A production by 80.0%, which accompanied with 16.7% increase of biomass and 10.0% enhancement of sugar utilization. Citrate and low pH could significantly improve pyruvate accumulation but inhibit succinate and fumarate production. Moreover, low pH was favorable to citrate utilization.Medium development is important for the whole fermentation optimization. Statistical methodologies including orthogonal design, Plackett-Burman design and response surface methodology were used to develop new medium to facilitate aspergiolide A production. Under the proposed optimized conditions, the experimental aspergiolide A production reached 71.2 mg/L, which increased 4.22 times compared to that in original medium. Elemental analysis was finally conducted, and carbon-nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. High carbon-nitrogen ratio facilitates acetyl-CoA accumulation and further improves aspergiolide A production.Even with metabolic regulation and high yield medium, aspergiolide A production in 5 L stirred-tank bioreactor is still too low (<6.0 mg/L). Study about shear stress and dissolved oxygen tension in shaking flask cultures revealed that the marine-derived fungus Aspergillus glaucus HB1-19 is typically shear sensitive and mechanical shear stress severely damaged mycelia and diminished aspergiolide A production. Moreover, aspergiolide A biosynthesis favored high dissolved oxygen tension in the growth phase but low one in the production period. Afterwards, cultures with different impeller combinations were conducted. Growth, production and morphology differed greatly among these batches. The combination of upper three-sector-blade pitched blade turbine impeller and lower six-flat-blade disc turbine impeller led to the maximum dry biomass (13.8 g/L) and aspergiolide A production (19.3 mg/L). On another hand, feeding 0.35% (v/v) n-dodecane further improved the production by 31.0%, i.e.,25.3 mg/L in the bioreactor despite the overall cell growth was decreased.Results of further optimization of fermentation conditions showed that inoculation ratio of 14%, phosphate concentration of 1%, total antifoam addition of 2%, original pH of 6.5 and process pH between 6.0 and 6.5 are preferable to aspergiolide A production. Cultures with media of different nutrition concentration but same composition or intermittent carbon source feeding strategy showed no production improvement. Therefore, batch culture is suitable for aspergiolide A production by Aspergillus glaucus HB1-19. By combined use of the scale-up strategy, i.e., similar impeller linear velocity together with similar dissolved oxygen tension and fungus morphology, the fermentation was successfully scaled up to 30 L and 500 L finally. Aspergiolide A production in 30 L and 500 L cultures reached up to more than 37 mg/L and 30 mg/L, respectively.As for heterologous expression of Tspksl from T. stipitatus, gateway technics were adopted to construct the expression vector. D-TOPO cloning and yeast recombination were first applied to clone the intact Tspksl gene. After that, the Tspksl gene was transferred into the expression vector by LR recombination. Afterwards, the expression vectors were transformed into protoplasts of Aspergillus orazye M-2-3, and arginine defective medium was used for transformant screening. PCR experiments towards genomic DNA of the transformants indicated the vectors were effectively integrated into their genomic DNA. From the RT-PCR assays, Tspksl transcription worked well. The sequencing results showed that the intron was correctly removed in some cases. However, none of the cultivated transformants produced any positive desired compound. To investigate whether or not the translation processed correctly, the egfp gene was linked to the end of Tspksl by removing the stop coden of Tspksl. About 90% of the new transformants showed green fluorescence, which indicated the translation frame was mostly correct. Although dozens of the strongly fluorescent colonies were analyzed, none of these transformants produced desired compounds. The intron was then removed from Tspksl in the expression vector, by which means 100% of the new intron deleted transformants presented green fluorescence. LC-MS results showed positive transformants produced two new compounds 3-methylorcinaldehyde and 3,6-dimethyl-4-hydroxy-2-pyrone, which was a triketide early released from TSPKS1. These results showed that intron made great effects on heterologous expression of fungal polyketide synthase genes even in fungi host strain. Moreover, different host strains might own different intron identification mechanisms of the fungal polyketide synthase genes.