| Improvement of microbial strains for the high-production of industrial products has been the hallmark of all commercial fermentation processes. The construction of high-throughput screening strategy was pivotal for industrial fermentation technology. In order to realize the scale down cultivation by using microtiter plates (MTP), computational fluid dynamics (CFD) was used to simulate and compare the water superficial velocity and shear force between microtiter plates and shake flask. We solved the key problems encountered in microculture for aerobic microorganism, such as oxygen transfer rate and mixing time. In this thesis, filamentous fungi Acremonium chrysogenum was used to establish the flexible and universal high-throughput screening platform, two other aerobic microorganisms Saccharopolyspora erythraea and Monascus purpureus were used to verify the accuracy, feasibility and extensibility of the established screening platform.First, A.chrysogenum was employed to construct the high efficiency high-throughput screening strategy. The low probability was the big drawback existed in the traditional screening method. Therefore, the mixture cultivation was effectively proposed and used for the first time to realise the whole mutant library being screened after mutagenesis, which avoided those unselected mutants being unscreened. The high-yield mixture in one well of the microtiter plate was screened out first. The subsequent isolation of the desirable high-yield colonies was further selected and screened from the high-yield mixture. This new screening approach greatly increased probability of obtaining the high-yield strain compared with the conventional method, by which only the parts of treated suspension had chance to be screened by tranditional dilution-plate method. As a result, the high-yield strain W-6was successfully screened out and the cephalosporin C (CPC) titer was nearly two-fold higher than that of the wild strain in50L bioreactor. In order to decipher the mechanism of high yield strain, the performance of the two strains were compared and analysed from the perspective of metabolic properties, mycelium morphology, enzymology, transcription and metabolic flux analysis based on multi-scale study approach. Glucose-6-phosphate dehydrogenase, citrate synthase, isocitrate lyase and malate synthetase are the four key enzymes involved in the primary metabolism. Their enzyme activities were analysed and compared between two strains.The results showed that the higher activities of isocitrate lyase and malate synthetase in primary metabolism might be the reason leading to the high yield of CPC production. Therefore, it can be speculated that the glyoxylate cycle played a pivotal role in the increasement of the metabolic flux leading to CPC biosynthesis, when soybean oil was added. ACV synthetase, expandase/hydroxylase and acetyltransferase were three important enzymes involved in CPC synthesis The genes encoding these three enzymes were pcbAB, cefEF and cefG perspectively. The transcription level of these three genes was compared between strain W-6and1-D1. The mRNA transcriptional levels of pcbAB, cefEF and cefG of W-6was5,8,1.2times of1-D1at72h,5,14,1.5times of1-D1at96h and4,50,7times of1-D1at120h. The results revealed that cefEF encoding expandase/hydroxylase might be the most important gene affecting CPC biosynthesis. The metabolic flux analysis indicated that glyoxylate cycle of high-yield strain was43%higher than the parent strain, so more oxaloacetic acid could be generated and entered into the replenishment pathway, which provided more NADPH and precursor amino acid for CPC synthesis.Second, a rapid high-throughput microplate bioassay based on turbidimetry was developed for erythromycin quantification in the fermentation broth. The optimum detection conditions were obtained as follows:25μl certain concentration of samples were added into the225μl bioactivity assay medium mixed with5%inoculation volume of indicator bacteria suspension(OD1.5), incubated for5h at37℃. Thousands of samples could be determined simultaneously. Meanwhile, another rapid high-throughput assay based on colorimetric was established for the measurement of erythromycin chemical titer. The material of reaction medium, reaction volume and dehydrating agent were the three differences existed between microassay and colorimetric, but did not affect the outcome.Last, a simple high-throughput screening system was developed for medium optimization. Strain D39-4was screened out and the pigment production was265.8U/ml fermented in shake flask. This high-throughput screening approach was designed to optimize medium composition combined with statistical methods. The analysis revealed that the optimum concentrations of glucose, peptone, NaNO3, KH2P04were51.42,4.91,1.00,1.00g/L. The fermentation medium optimized was verified in shake flasks, the pigment production could be enhanced from206.5U/ml in un-optimized medium to265.8U/ml, giving nearly1.30-fold increase in production.In the paper, we also designed the screening device matched to high-throughput technology, included various kinds MTPs and corresponding’sandwich’lids to guarantee the minimization of fluid evaporation rate while maintain suitable aeration and gas exchange rate without cross-contamination. Different productions could select different device to construct their own high-throughput screening platform. We believe that the HTS field continues to be promising and dynamic in the future. |
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