Xueqian Sun

Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously

Aspergiolide A is a novel anti-tumor anthraquinone derivant produced by marine-derived fungus Aspergillus glaucus. To identify its biosynthetic pathway and further improve the production, the effects of biosynthetic pathway specific inhibitors and precursors were investigated. Cerulenin and iodoacetamide, the specific inhibitors of polyketide pathway, could completely inhibit the aspergiolide A accumulation. Putative precursors of polyketide pathway could increase aspergiolide A production greatly, such as 6 mM acetate increased production by 135%. Simvastatin and citrate, the inhibitors of mevalonate pathway, stimulated the production by 63% and 179%, respectively. Considering that acetyl-CoA is the common starter unit in both polyketide and mevalonate pathway, a novel strategy was designed to stimulate the aspergiolide A accumulation. Combinations of 12 mM acetate with 0.3 mM simvastatin could increase the production by 151%, while the supplementation with 12 mM acetate and 12 mM citrate brought a 262% increase of aspergiolide A production. The strategy might be very useful to enhance the production of other secondary metabolites derived from polyketide pathway.

Enhancing aspergiolide A production from a shear-sensitive and easy-foaming marine-derived filamentous fungus Aspergillus glaucus by oxygen carrier addition and impeller combination in a bioreactor.

Production enhancement of a novel antitumor compound aspergiolide A from shear-sensitive and easy-foaming marine-derived fungus Aspergillus glaucus HB1-19 in a 5-l stirred bioreactor was investigated. Two types of impellers, i.e., six-flat-blade disc turbine impeller (DT) and three-sector-blade pitched blade turbine impeller (PB) were used in this work. In cultures with fermentation medium, the combination of upper PB and lower DT led to the maximum dry biomass (13.8 g/l) and aspergiolide A production (19.3 mg/l). However, two PBs brought the highest aspergiolide A yield coefficient (1.9 mg/g dry biomass) despite it produced the lowest dry biomass (5.3 g/l). By contrast, two DTs and the upper DT and lower PB showed insignificant results. Feeding 0.35% (v/v) n-dodecane in cultures with upper PB and lower DT further improved aspergiolide A production by 31.0%, i.e., 25.3 mg/l, which is also 322% higher than that in the ordinary cultures with two DTs.

Efficient strategy for enhancing aspergiolide A production by citrate feedings and its effects on sexual development and growth of marine-derived fungus Aspergillus glaucus.

Aspergiolide A production enhancement by citrate and its effects on growth and sexual development of marine-derived fungus Aspergillus glaucus HB1-19 were investigated. In agar plate culture, 15 mM 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 mM 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. Differently, sodium citrate made no obvious or even opposite effects. Citrate and low pH could significantly improve pyruvate accumulation but inhibit succinate and fumarate production. Moreover, low pH was favorable to citrate utilization. Organic acids changes were closely related to aspergiolide A biosynthesis. Comparing to pH controls, effects of citric acid comprised pH decrease solicitation and citrate utilization enhancement.

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris

Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop‐in/pop‐out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirΔkex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirΔkex1 while it was only 1.1 g/l in GS115Hir. Copyright

Significant stimulation of o-phthalic acid in biosynthesis of aspergiolide A by a marine fungus Aspergillus glaucus.

The effect of o-phthalic acid (o-PA) on the production of the anti-tumor polyketide compound aspergiolide A by the marine fungus Aspergillus glaucus was investigated. o-PA at 12mM increased the aspergiolide A production by 77%. A combination of 12mM acetate with 12mM o-PA increased the production by 126%, i.e., from 27.1mg/l to 60.9mg/l, which was 27% and 75% higher than adding either 12mM o-PA or 12mM acetate, respectively. It was also found that A. glaucus could use o-PA as the sole carbon source on Minimal Medium. Ninety percent of o-PA was degraded by the fungus strain, especially when 12mM acetate was supplemented simultaneously. In culture medium supplemented with o-PA addition, sugar consumption decreased with increasing o-PA concentration, at the same time, the oxalacetate and pyruvate levels increased and the concentration of fumarate decreased. The results indicated that o-PA could stimulate biosynthesis of aspergiolide A effectively by regulating the metabolic pathway.