Reconstruction And Application Of Acarbose Producing Strain Actinoplanes Sp.SE50/110 Genome-scale Metabolic Model

Because of its advantages of mild and persistent therapeutic effect and nontoxicity, acarbose is the widely used drug for Diabetes mellitus type 2. Actinoplanes sp. was the common strain for industrial fermentation, but the research was focus on conventional random mutation, screening, and optimization of media composition and culture conditions, scarcely anything is known about the overexpression or knockout targets to improve acarbose production from the systems-level. And the major bottleneck is not researched by now. Therefore, a systematic understanding of physiological features and a global understanding of metabolism is in great need. In this research, a genome-scale metabolic model of Actinoplanes sp. SE 50/110 was reconstructed. The metabolism of Actinoplanes sp. SE 50/110 was analyzed. The strategies of improving acarbose production were simulated and the batch fermentation was conducted based on the in silico simulation. The main results were:1 Based on the method of Model SEED automatic reconstruction and a local sequence similarity search and combined with literature mining, a genome-scale metabolic model i YLW1028 of Actinoplanes sp. SE 50/110 was reconstructed. Model i YLW1028 consists of 1028 genes, 1219 reactions, 1128 metabolites, and the ORF coverage is 12.5%. The 1219 reactions were located into two cellular compartments（intracellular and extracellular） and 90% were gene–associated. Actinoplanes sp. SE50/110 could utilize 30 different carbon sources and 16 different nitrogen sources as sole carbon/nitrogen source for cell growth, and the match of 92% between in silico and in vivo was obtained. Batch fermentation of different substrate uptake rates was conducted to evaluate model i YLW1028 quantitatively. The growth rate of in silico agrees with the fermentation data.2 The physiological metabolism was analyzed based on model i YLW1028.（1） The essential genes to cell growth under acarbose-synthesis medium and CPC medium were 122 genes（11.9%） and 81 genes（7.9%）, respectively.（2） The number of essential genes for acarbose synthesis on acarbose-synthesis medium was 133（12.9%）.（3） Based on gene annotation and literature mining, the features of acarbose metabolic pathway, which contains of 38 reactions, were illustrated in model i YLW1028. 31 reacions were biochemical reactions and the other 7 were transport reactions.（4） Three key metabolic nodes for acarbose production were identified by the comparison of carbon source flux distribution between cell growth phase and acarbose production phase. They were pentose phosphate pathway, glycolysis pathway and glutamic acid synthetic pathway catalyzed by aminotransferase.3 The strategies of improving acarbose production based on model i YLW1028 were predicted.（1） Through addition of nicotinic acid, the acarbose producing rate increased from 0.00511 mmol×g DCW-1×h-1 to 0.00553 mmol×g DCW-1×h-1, with an increment of 8.2%.（2） The effects of amino acids on cell growth and acarbose production were simulated. Results indicated that addition of amino acids had a good effect on cell growth and acarbose production, arginine increased cell growth rate by 13.2% and histidine increased acarbose producing rate by 58.7%.（3） Robustness analysis of proton demonstrated that acarbose producing rate was more sensitive to p H than cell growth rate, a neutral p H promotes the accumulation of acarbose.（4） Robustness analysis of oxygen demonstrated a relatively low dissolved oxygen level（0.1917 mmol×g DCW-1×h-1） was good for acarbose production.（5） Genes ACPL₁861, ACPL₆461, ACPL₁328 and ACPL₆750 were predicted to be candidate to over-express via FSEOF.（6） The deletion of gene tre Y contrubited to elimination of component C.4 Based on strategies to improve acarbose production of model i YLW1028, batch fermentation experiments were conducted.（1） Nicotinic acid（5 mg×L-1） increased acarbose production by 53.5%.（2） Among the addition experiments of arginine, histidine and glutamine, only histidine increased DCW by 9.4%. Glutamic acid, cysteine, lysine, glutamine and asparagine lead to acarbose production increased 29.6%, 26.5%, 26.3%, 11.8% and 9.2%.（3） Ca2+ and Fe3+ promoted the accumulation of acarbosse respectively by 38.0% and 21.1% than without addition.（4） The neutral p H increased acarbose production by 7.2% and 15.3% than uncontrolled control and acid environment.（5） Excessive oxygen exposure（> 0.5 vvm） during fermentation goes against acarbose accumulation and a relatively low dissolved oxygen level was good for acarbose production, a aeration rate of 0.5 vvm lead to a highest acarbose production, which was 1.11 g×L-1.