Construction Of High Throughput Screening Platform And Its Applications

The aim of this study is to establish an efficient platform for high throughput screening and apply it to the increasing the yield of cephalosporin C (CPC), which was one of β-lactam antibiotics, through nitrogen source optimization as well as preliminary exploration on high throughput screening of high-yield isoleucine producer strains..In this study, the following problems are to be resolved.(1) Selecting the type of microtiter plates to study. The types of mirotiter plates often used were24-well,48-well,96-well microtiter plates. Microtiter plates can be chosen based on the oxygen transfer coefficient (KLa) and liquid evaporation (%).(2) Optimizing the filling volume of microtiter plates and testing the feasibility of microtiter plates for cultivation and for turbidimetric assay.(3) Investigating of parallelity among wells in24-well and48-well microtiter plates.(4) Optimizing cultural conditions such as inoculum age, inoculum volume and fermentation time. In addition, the effect of different concentration starch on production of CPC in24-well microtiter plates was studied.(5) Establishing the high throughput methods for optimization medium, especially nitrogen source.(6) Use of Genipin color reaction with the amino acid L-isoleucine for high throughput screening of high-yield isoleucine producer strains.Through the study, the platform for high throughput screening was successfully established. It was found that24-well microtiter plates and48-well microtiter plates were more appropriate for cultivate aerobic microorganism than widely used96-well microtiter plates. There were higher oxygen transfer coefficient and larger filling volume enough for analyzing of the broth after fermentation. In addition, the water evaporation ratio in24-well is smaller than in48-well and the water evaporation ratio in48-well is smaller than in96-well microtiter plates. Hence,24-well and48-well microtiter plates were suggested for fermentation of aerobic microorganism-Cephalosporium acremonium.The filling volumes of24-well and48-well microtiter plates were1000μL and1900μL, respectively, in which the biomass weight was same as in shake flasks. the smallest average deviation in24-well and48-well microtiter plates was2.52%,-0.66%, respectively. The oxygen transfer coefficient of24-well and48-well was larger and close to the oxygen transfer coefficient of shake flasks. The production of CPC in48-well microtiter plates had good correlation with that in500mL shake flasks (r=0.9781, p<0.01,n=8). So, we can use the microtiter plates instead of shake flasks to culture Cephalosporium acremonium for producing CPC. Others, the production of CPC were analyzed by turbidimetric assay was good correlation with HPLC assay (r=0.9885, p<0.01, n=8). Therefore, the turbidimetric assay was used to instead of HPLC assay. The parallelity among wells of24-well and48-well microtiter plates was very good. After fermentation, the sample of each well was analyzed CPC (U/mL) concentration by HPLC and biomass (g/mL) by weighing. The strain of this experiment was1-D1. The RSD (%) of CPC on each well of24-well microtiter plates was4.38%, the RSD (%) of biomass on each well of24-well microtiter plates was3.22%.The RSD (%) of CPC and biomass on each well of48-well microtiter plates were6.93%,5.06%, respectively.The established high throughput screening platform was employed to the application of media recipe optimization on CPC production. The optimized cultural conditions in this study were68h of inoculum age,10%of inoculum volume, six-days of fermentation time. In addition, the optimized concentration starch in microtiter plates was5%. Based on the established method for high throughput medium optimization, the production of CPC was increased from121.04%to138.28%of range. The cottonseed meal was selected as the best nitrogen sources for cultivation Cephalosporium acremonium and producing CPC.In this study, a preliminary exploration on highthrouput screening for high-yield isoleucine producer strain was also carried out. It was found that genipin with amino acids in principle under certain conditions, be able to generate gardenia blue color reaction, thus, the establishment of a new, rapid detection method for amino acid concentrations are feasible. Reaction system was determined as follows: Genipin working concentration of0.05g/L, the volume of500microliters of the reaction solution, and the reaction temperature was80℃, reaction time2h, the detection of the amino acid concentration of0.1-1.0g/L. Since the method is the color reaction, the reaction can be conveniently detected, the absorbance can be determined by detecting the content of amino acids in different samples, therefore it is possible to apply this method to a subsequent high-throughput screening for high-yield isoleucine producer strains.