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Metabolic engineering of Bacillus amyloliquefaciens for efficient production ofα-glucosidase inhibitor1-deoxynojirimycin

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Synthetic and Systems Biotechnology 2023年第3期8卷 378-385页

作者： Xujie Li Meng Zhang Yu Lu Ningyang Wu Jian’gang Chen Zhixia Ji Yangyang Zhan Xin Ma Junyong Chen Dongbo Cai Shouwen Chen State Key Laboratory of Biocatalysis and Enzyme Engineering Environmental Microbial Technology Center of Hubei ProvinceCollege of Life SciencesHubei UniversityWuhan430062China State Key Laboratory of Agricultural Microbiology Huazhong Agricultural UniversityWuhan430070China Department of Urology Zhuhai People’s Hospital(Zhuhai Hospital Affiliated to Jinan University)Zhuhai519000China Wuhan Jun’an Biotechnology Co. Ltd.Wuhan430070China

Owing to the feature of strongα-glucosidase inhibitory activity,1-deoxynojirimycin(1-DNJ)has broad application prospects in areas of functional food,biomedicine,etc.,and this research wants to construct an efficient strain for 1-DNJ production,basing on Bacillus amyloliquefaciens HZ-12.Firstly,using the temperature-sensitive shuttle plasmid T2(2)-Ori,gene ptsG in phosphotransferase system(PTS)was weakened by homologous recombination,and non-PTS pathway was strengthened by deleting its repressor gene iolR,and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold,reached 110.72 mg/L.Then,to increase precursor fructose-6-phosphate(F-6-P)supply,phosphofructokinase was weaken,fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement,moreover,regulator gene nanR was deleted,1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold.Subsequently,promoter of 1-DNJ synthetase cluster was optimized,as well as 5′-UTRs of downstream genes in synthetase cluster,and 1-DNJ produced by the final strain reached 478.62 mg/L.Last but not the least,1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter,which was the highest yield of 1-DNJ reported to date.Taken together,our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis,this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

关键词： 1-deoxynojirimycin Bacillus amyloliquefaciens Metabolic engineering Glucose transport pathway Fructose 6-phosphate 1-DNJ synthetase Gene cluster

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1-deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-deoxynojirimycin in Silkworm Powder

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Food and Nutrition Sciences 2011年第2期2卷 87-89页

作者： Kazuhisa Yatsunami Kohji Murata Tsutomu Kamei 不详

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

关键词： Silkworm Bombyx Mori 1-deoxynojirimycin Alfa-Glucosidase Inhibitor Heat Stability

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Transcriptomics analysis hemolymph response to 1-deoxynojirimycin & Molecular characterization and functional analysis of a Ferritin heavy chain subunit from the Eri-Silkworm,Samia cynthia ricini

Transcriptomics analysis hemolymph response to 1-Deoxynojiri...

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中国蚕学会第九届青年学术研讨会

作者：张尚志 Hai-Zhong Yu Jia-Ping Xu

The Samia Cynthia ricini(Lepidoptera:Saturniidae) is an important commercial silk-producing insect,not the same as silkworm,mulberry leaves show toxicity to it because of the component 1-deoxynojirimycin(DNJ).To insight into the molecular mechanism of eri-silkworm hemolymph responses to DNJ,transcriptomics of eri-silkworm hemolymph was conducted to analyze the genes related to different metabolic pathways.A total of 865 differentially expressed genes(DEGs) were identified,among which 577 DEGs had up-regulation and 288 DEGs showed down-regulation in 2% DNJ group compared to control(ddHO) at the 12 h.Functional analysis indicated that these DEGs were associated with ribosomes,glycolysis,N-Glycan biosynthesis,and oxidative phosphorylation.Especially,according to KEGG analysis,138 DEGs were involved in energy metabolism,glycometabolism and lipid metabolism,and nine of them were confirmed by reverse transcription quantitative PCR(RT-qPCR).In addition,we identified a ferritin heavy chain subunit named ScFerHCH in transcriptome database.The full-length S.c.ricini ferritin heavy chain subunit(ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 k Da.Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits.Higher ScFerHCH expression levels were found in the silk gland,fat body and midgut of S.c.ricini by RT-q PCR and Western blotting.Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut,fat body and hemolymph,indicating that ScFerHCH may contribute to the host‘s defense against invading pathogens.Moreover,the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined.Taken together,our study indicated that DNJ could cause a significant metabolic difference in eri-silkworm hemolymph.These results will lay a foundation for researching toxicological effect of DNJ using eri-silkworm as a

关键词： 1-deoxynojirimycin eri-silkworm transcriptomics ferritin immune response iron binding capacity anti-oxidation activity

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Optimization of microwave-assisted technology for extracting 1-deoxynojirimycin from mulberry tea by response surface methodology

Optimization of microwave-assisted technology for extracting...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Chao Liu Chuan-hong Wang Jing Liu Li Xu Wei Xiang yuan-cheng Wang College of Biotechnology Southwest University

In order to improve the utilization ratio of mulberry tea 1-deoxynojirimycin（DNJ）, mulberry tea was prepared based on the common production process of green tea, and corresponding experimental design and statistical analysis were set using Design-Expert software. The results suggested microwave power 602.28 W and processing time 11.41 min were found to be optimal for microwave-assisted extraction DNJ. The confirmatory experiment showed the coincidence rate of detection was 99.58%. It provided a reference for efficient use of mulberry tea DNJ for people who would drink mulberry tea to control blood glucose levels or to prevent diet-induced obesity.

关键词： mulberry tea microwave-assisted extraction 1-deoxynojirimycin response surface methodology

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Comparative analysis of 1-deoxynojirimycin contribution degree to α-glucosidase inhibitory activity and physiological distribution in Morus alba L

Comparative analysis of 1-deoxynojirimycin contribution degr...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Chao Liu Wei Xiang Yan Yu Zhengqin Shi Xianzhi Huang Li Xu College of Biotechnology Southwest University

Natural-glucosidase inhibitors offer an attractive strategy to manage postprandial hyperglycemia in patients with type 2 diabetes. 1-deoxynojirimycin（DNJ）, the main hypoglycemic constituents in Morus alba L., has been extensively researched. Here, for different parts of the mulberry tree, the DNJ content and the IC50 values of DNJ and ethanol extracts have been determined and compared. Possible metabolic pathways and storage sites for DNJ in mulberry trees have been investigated, and the kinetics of inhibition of-glucosidase by DNJ and ethanol extracts of different parts of the mulberry tree have been measured. DNJ content was found to be highest in trunk bark, whereas-glucosidase inhibitory activity was highest in both twig and trunk bark. Buds and roots are likely to be the topmost and bottommost sites of DNJ biosynthesis. DNJ is an insect antifeedant（not including silkworms） and may itself have antiviral activity, or be the precursor to a compound with antiviral activity. Ethanol extracts of twig, trunk and root bark are mixed-type inhibitors of-glucosidase;extracts from other parts of the tree are competitive inhibitors. The study provides useful information about the physiological distribution of important active components, especially DNJ, in the mulberry tree and suggests how best to develop and use the crude drug resources provided by the tree.

关键词： 1-deoxynojirimycin gical distribution α-Glucosidase inhibitory activity Mulberry Ethanol extracts

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Biotransformation Effect of Bombyx Mori L. May Play An Important Role in Treating Diabetic Nephropathy

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Chinese Journal of Integrative Medicine 2016年第11期22卷 872-879页

作者：张蕾张腊李茵郭新峰刘旭生 Department of Nephrology Guangdong Provincial Hospital of Chinese Medicine Guangzhou University of Chinese Medicine Evidence-Based Medicine and Clinical Research Service Team Guangdong Provincial Hospital of Chinese Medicine

Compared with herbal drugs, medicine processed from animals(animal medicine) was thought to have more bioactive substances and higher activities. Biotransformation effect often plays an important role in their effect. However, researches about effect of animal medicine on diabetic nephropathy and applying animal medicine as natural bio-transformer were seldom reported. The purpose of this paper was to reveal the use of Bombyx Mori L. on diabetic nephropathy from ancient to modern times. The classical literature indicated that Saosi Decoction(缫丝汤), which contains Bombyx Mori L. or silkworm cocoon, was applied to treat disorders congruent with modern disease diabetic nephropathy from the Ming to Qing Dynasty in ancient China. Modern studies showed that Bombyx Mori L. contains four main active constituents. Among these, 1-deoxynojirimycin(1-DNJ) and quercetin showed promising potential to be new agents in diabetic nephropathy treatment. The concentrations of 1-DNJ and the activities of quercetin in Bombyx Mori L. are higher than in mulberry leaves, because of the biotransformation in the Bombyx Mori L. body. However, these specific components need further human and mechanistic studies to determine their therapeutic potential for this challenging condition.

关键词： diabetic nephropathy Bombyx Mori L 1-deoxynojirimycin quercetin biotransformation Chinese medicine

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A New Alkaloid from Bombycis Feculae and Its α-Glucosidase Inhibitory Activity

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Chinese Herbal Medicines 2011年第1期3卷 64-65,74页

作者： ZHU Yuan-yuan1,WANG Zhi-hong2,QI Hui1,BAI Gang1 1.Tianjin Key Laboratory of Molecular Drug Research,College of Pharmacy,Nankai University,Tianjin 300071,China 2.College of Chemistry,Nankai University,Tianjin 300071,China

Objective To study the chemical constituents of Bombycis Feculae.Methods Chemical constituents were isolated by HPLC-ELSD.The structures of the isolated compounds were determined by spectral means.Results Two compounds were isolated and identified as 1-deoxynojirimycin(1)and(2R,3R,5R)-2-(hydroxymethyl) piperidine-3,5-diol,named as 1,3-dideoxygalatonojirimycin(2).Conclusion Compound 2 is a new alkaloid.The extract of Bombycis Feculae,compound 1 and compound 2 show inhibitory activities againstα-glucosidase.

关键词： alkaloid Bombycis Feculae 1-deoxynojirimycin 1,3-dideoxygalatonojirimycin α-glucosidase inhibitor

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