High Throughput Screening Of High-yield Gentamicin C_1a Strain And The Feeding Strategy Optimization

Gentamicin C1a(GM C1a),which is produced by Micromonospora purpurea through fermentation,belongs to an aminoglycoside antibiotic.It has broad-spectrum antibacterial activity to gram-negative bacteria.GM C1a is an important precursor for the synthesis of etimicin(ETM).In this paper,a method of rapidly detecting GM-C1a was established at the first,and subsequently,producing strain mutagenesis and high throughput screening were conducted to obtain high-yield strains.Then the relationship of specific growth rate and specific productivity was investigated through different fermentation models,and the feeding strategy was optimized as well as scaled up preliminarily to the industrial production.Firstly,a rapid and simple detection method for GM C1a in the broth was established and integrated into the high throughput screening model.The sodium phosphotungstate was adopted to react with GM C1a to produce an white precipitate,and then the absorbance of the supernatant at 248 nm was linearly related to the GM C1a titer with a correlated coefficient of 0.9978±0.0005.Through comparing to the results of high performance liquid chromatography(HPLC)detection,a maximum deviation of 3.98%was found in this rapid detection method.For high throughput cultivating system,the fermentation results of 24 wells microtiter plate(MTP)with 3 mL working volume exhibited good relationship with that in shake flask.Secondly,in order to obtain high yield GM C1a strains,different mutation methods of atmospheric room temperature plasma(ARTP),microwave,LiCl and ARTP+LiCl with above mentioned high throughput cultivating and detecting systems were used.A total of 2336 mutants were tested in the preliminary screening,and 221 mutants performed a titer improvement of more than 10%.Among them,a mutant of AL324 could enhance the titer by 52%in the shake flask,compared to the parent strain.By the following rescreening and genetic stability verification processes,only 4 mutants still had better fermentation performances.Moreover,the titer improvement further reached to 81.3%in the 5 L fermentation.Thirdly,the relationship between the cell growth and the product formation was studied with different fermentation models(batch,fed-batch and semi-continuous cultivations)in 50 L bioreactors.In the batch fermentation,starch was found to be beneficial for GM C1a synthesis,however in the fed-batch fermentation,continuous glucose feeding strategy could reach the GM C1a titer to 1322 U/mL,101.2%higher than that with pulse starch feeding strategy.Furthermore,the results of the semi-continuous fermentation indicated that the best specific growth rate for GM C1a synthesis was 0.020±0.005 h-1,at which the specific productivity could achieve 1.50±0.03 U/(g dw)/h.Finally,The preliminary scale-up experiments was carried out in a 60 m3 industrial bioreactor with the process regulation of increasing agitation speed and feeding diluted medium,which contained a low soybean powder content of 3%and a relatively high corn steep liquor content of 1.2%,to increase the oxygen supply in the late phase.A GM C1a titer of 1198 U/mL was attained,and the total unit increased by 66.2%with a 35.4%decrease of carbon source consumption.