Breeding And Molecular Modification Of Acarbose High Yield Strain

α-Glucosidase inhibitors are commonly utilized to treat typeⅡdiabetes.They work by competitively and reversibly inhibiting intestinal alpha-glucosidase,which delays carbohydrate digestion.This,in turn,reduces the rate at which glucose is absorbed and helps to alleviate postprandial blood sugar levels.Acarbose is the firstα-glucosidase inhibitor to be used clinically.Its remarkable efficacy and safety have led to widespread recognition in the market.Currently,the primary method of acquiring acarbose is through fermentation production by Actinoplanes sp.SE50.However,this study aimed to enhance the yield of acarbose by conducting multiple rounds of iterative ARTP mutagenesis on Actinoplanes sp.A01.To achieve this,an efficient screening method of pore plate fermentation combined with enzymoleter detection was established.Additionally,non-targeted metabolomics analysis of the strains before and after mutagenesis was conducted.The main research of this paper is summarized as follows:（1）After conducting a comparative analysis,it was determined that24 hole plate fermentation was more effective than the traditional shaking flask fermentation.Furthermore,the conditions of the hole plate fermentation were optimized to ensure the highest level of efficiency.The findings indicate that modifying the original fermentation medium by adjusting the content of soybean cake powder,glycerin,and calcium carbonate to 3%,1.5%,and 0.4%,respectively,resulted in the highest fermentation level of acarbose.This optimization led to a 25.2%increase in acarbose yield compared to the unoptimized medium.The maximum acarbose yield of 2064.61μg/m L was achieved after 168 hours of fermentation,making this time frame the most effective for fermentation.A high-throughput screening model for the production of acarbose by Actinoplanes sp.A01 was established by combining pore plate fermentation with enzyme-label assay.（2）Breeding of acarbose-producing strain by ARTP mutagenesis of Actinoplanes sp.A01.According to the preliminary experiment,the optimal treatment time of ARTP mutagenesis was 120 s,the optimal initial screening concentration of streptomycin resistant plate is 0.1μg/m L.The acarbose yield of Actinoplanes sp.AH-02 was 3901.50μg/m L,which was higher than that of the original strain（2364.96μg/m L）acarbose yield increased by 64.97%.The p H,bacterial concentration and acarbose yield were investigated in the fermentation process of the starting strain and the high-yielding mutant Actinoplanes sp.AH-02.The optimal fermentation cycle for acarbose was 168 h,prolonging fermentation time acarbose yield began to decline.The genetic stability of the high-yield mutant Actinoplanes sp.AH-02 was investigated.The results showed that the mutant remained stable within three times of passing,but changed within 10%.After passing through the fourth generation,it began to degrade significantly.（3）For high-yielding mutant Actinoplanes sp.AH-02 with the original strain Actinoplanes sp.A01 was found by non-targeted metabolomics analysis.In the positive and negative ion mode,216 and161 different metabolites were screened respectively,indicating that ARTP mutagenesis selection is uncertain,which can not only enhance acarbose synthesis,but also change the accumulation of other metabolites.Differential metabolites are mainly divided into organic acids and their derivatives,lipids and lipid molecules,organic oxygen compounds,organic nitrogen compounds,nucleosides,nucleotides and analogues,phenylpropane and polyketones,etc.The differential metabolites included not only acarbose,but also glucose,maltose,lactose,galactose,trisaccharide,fructose,histidine,glutamic acid,lysine,proline and so on.Through the analysis of metabolites in acarbose metabolic pathway,it was found that metabolites in glycolysis pathway（EMP）,pentose phosphate pathway,ABC transporter metabolic pathway and citric acid cycle（TCA）had significant changes.The citric acid cycle,histidine metabolism,glutamate amino acid metabolism and other branches of acarbose biosynthesis were significantly up-regulated,indicating that during the process of acarbose biosynthesis,some carbon metabolic flow flowed to the citric acid cycle,which increased the level ofα-ketoglutaric acid intermediate in the citric acid cycle and promoted the synthesis of amino acids such as glutamic acid.Thus,it competes with acarbose synthesis carbon metabolic flow,which is not conducive to acarbose fermentation production.The non-targeted metabolome analysis analyzed the metabolic network of high-yielding acarbose mutant strain Actinoplanes sp.AH-02 at the metabolic level,which provided the research idea and direction for the construction of high-yielding acarbose engineered strain through subsequent metabolic engineering modification.（4）Genetic engineering strain Actinoplanes sp.AH-Δmel C2-acb S was constructed based on CRISPR/Cas9 technique.The target fragments of glycosyltransferase coding gene acb S and the upstream and downstream target fragment of the tyrosinase gene mel C2 were amplified from the whole genome of Actinoplanes sp.AH-02.The recombinant plasmid p CRISPomyces-2-Δmel C2::P_{Kas Op*}-acb S was constructed by using p CRISPomyces-2 plasmid and strong promoter P_{Kas Op*},acb S and mel C2 genes,and was introduced into Actinoplanes sp.AH-02.The knockout of tyrosinase gene mel C2 and the integration of glycosyltransferase acb S were achieved.The recombinant strain Actinoplanes sp.AH-Δmel C2-acb S was verified by shaking flask,and the acarbose yield reached 4867.21μg/m L,which was 22.99%higher than that of Actinoplanes sp.AH-02.Compared with the original strain Actinoplanes sp.A01 acarbose yield increased by 104.07%.