Efficient And Novel Technologies For Lovastatin Production With Aspergillus Terreus In Solid State Fermentation

Lovastatin,an important blood-lipid-lowering medicine,is commercially produced by Apergillus terreus in submerged fermentation.Based on the mycelia growth features of A.terreus and its intracellular product nature of lovastatin,the solid-state fermentation(SSF)of A.terreus for the production of lovastatin was proposed and studied systematically.It is an important and unsolved problem to measure biomass precisely during solid-state fermentation.In this work,three kinds of cell components(nucleic acid,ergosterol and glucosamine)were examined to evaluate their capability as indirect index in estimating the biomass of A.terreus ZJU-510 in SSF.The results indicated that the linear relationships between biomass and three kinds of cell components did exist respectively.Because of the obvious affection of medium composition and/or culture time,neither ergosterol nor glucosamine was suitable for biomass estimation.Nucleic acid content in biomass was almost maintained constant regardless of the culture time or medium composition,therefore,was the best indicator to estimate the amount of biomass during SSF process.The method was successfully applied in the measurement of biomass of A.terreus ZJU-510 and Monascus rubber M7 during the SSF process for lovastatin production.The biological properties of A.terreus ZJU-510 were examined.With the ability to produce some amylase and cellulase,A.terreus could grow on the solid substrates such as starch and cellulose.It was found that the morphology was different when it was cultured on the plates with different solid medium.The mycelia growth was relative weak and spores formed easily when grew on the PDA medium,whereas grew healthily and produced less spores on Cazpek's medium or malt-wort medium.When cultured in steamed rice,the relatively easy formation of spores affected the mycelia growth and the utilization ratio of the substrate.The culture conditions and medium components of the SSF for producing lovastatin by A. terreus ZJU-510 were studied by single-factor test method in flask.At the optimized conditions and with a two-step fermentation strategy(cultivated at 32℃in first 3 days and then lowered to 28℃for another 8 days),the lovastatin yield reached 3.36 mg/g.The fermentation parameters, such as lovastatin yield,moisture content and pH value during SSF process were measured and the fermentation kinetics was studied.The results indicated that both biomass and lovastatin yield increased quickly in first 5 days and then slowly in the following days,and reached the highest in the 11^thday.Although lovastatin was a kind of secondary metabolite,it was obvious that its intracellular accumulation was partially related to the growth rate of A.terreus ZJU-510.In order to improve lovastatin production further,the response surface methodology(RSM)was employed to study the effect of culture medium on the production of lovastatin.The optimized culture conditions were:cooked rice with additional CH₃COONa 0.54%,glucose 3.36%,peptone 1.5%, initial water content of 45%and inoculums ratio of 25%.The lovastatin yield reached 3.99 mg/g, which was 18.8%higher than that at initial conditions.A small-scale fermentor for SSF was designed and used for lovastatin production by A.terreus ZJU-510.The effects of solid substrate load,substrate turnover and aeration on lovastatin production were studied.The results were compared with those of tray bioreactor culture and flask culture.A mathematical model considering mycelium growth,lovastatin production and substrate consuming was proposed based on the experimental results.The model parameters were correlated from the experiment data by MATLAB software.The good agreement between the calculated results and the experiment data indicated that the model was capable to simulate the batch SSF process.The comparison between A.terreus ZJU-510 and Monascus rubber M7 showed that the lovastatin productivity of A.terreus ZJU-510 was higher than that of Monascus strains,whereas the amylase activity of A.terreus ZJU-510 was lower which could result in poorer mycelia growth, less substrate utilization and easier spore formation.M.rubber M7 does have the ability to produce lovastatin and with higher amylase activity and better mycelia growth.Based on this,the mix-culture of A.terreus ZJU-510 and M.rubber M7 was explored.With the mixed cultivation of A.terreus ZJU-510 and M.rubber M7 in the SSF process,the higher amylase activity was a supplement to A.terreus ZJU-510 and beneficial for its growth,therefore,higher lovastatin yield was observed.The genome shuffling was applied for the breeding of A.terreus ZJU-510 for high yield lovastatin strains.With the lovastatin grads plates and starch agar plates,the strains with lovastatin resistant ability and high amylase activity were picked out and then the lovastatin yield was evaluated in shake bottle fermentation.After UV+LiCl and DES mutation,6 high yield strains were screened out which were used as parent strains for genome shuffling.Through three rounds of shuffling and at the optimized culture conditions of the SSF process,a high-yield stable strain, named GS-185,was obtained and its lovastatin yield reached as high as 8.17 mg/g,which was more than 2-fold increase in comparing with the original strain(3.99mg/g).