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1 微生物学通报 Microbiology China Jan. 20, 2017, 44(1): DOI: /j.microbiol.china 研究报告枯草芽孢杆菌基因修饰生产核黄素张续 1 班睿 1* 刘露 1 张然 ( ) ( ) 2 摘要 : 目的研究枯草芽孢杆菌核黄素合成途径木糖代谢相关基因修饰对核黄素合成的影响方法单独过表达或共同过表达核黄素操纵子中的基因过表达木糖代谢相关基因构建相应的重组菌株通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量, 表征各个基因修饰的效应采用摇瓶和 5 L 罐发酵, 考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响结果 riba 基因单独过表达, 使核黄素产量提高 99%, 但生物量降低 30%, 出现细胞自溶现象 riba-ribh 基因共表达, 使核黄素产量提高 280%, 并且无细胞自溶和生物量下降现象 1.5% 蔗糖与 6.5% 木糖作为碳源,5 L 发酵罐发酵 70 h, 核黄素产量达到 3.6 g/l, 与 8% 蔗糖为碳源的发酵相比, 核黄素产量提高 80% 木糖代谢相关基因过表达, 均明显降低核黄素产量结论与 riba 基因单独过表达相比,ribA-ribH 基因共表达可有效避免细胞自溶现象, 并能进一步提高核黄素产量蔗糖与木糖共代谢, 能够改善前体物供给, 有利于提高核黄素产量关键词 : 枯草芽孢杆菌, 核黄素,ribH 基因, 基因表达, 木糖, 共代谢 Riboflavin production by a genetically modified Bacillus subtilis ZHANG Xu 1 BAN Rui 1* LIU Lu 1 ZHANG Ran 2 (1. Key Laboratory of Systems Biotechnology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin , China) (2. Tianjin Medical College, Tianjin , China) Abstract: [Objective] We modified genes responsible for xylose metabolism and riboflavin biosynthesis in Bacillus subtilis. [Methods] Genes responsible for riboflavin biosynthesis were overexpressed or co-overexpressed with genes of xylose metabolism in Bacillus subtilis. Recombinant strains were evaluated by measuring the riboflavin yield and biomass. Fermentation was done in shake flask and fermenter to produce riboflavin with sucrose as sole carbon source or a mixture of sucrose/xylose. Meanwhile, riboflavin yield, xylose consumption and biomass were analyzed. [Results] Overexpression of riba gene increased riboflavin yield by 99% and reduced maximum biomass by 30% because of autolysis. With riba and ribh genes co-overexpression, Foundation item: National High-Tech R&D Program of China (863 Program) (No. 2012AA02A701) *Corresponding author: banrui@tju.edu.cn Received: March 31, 2016; Accepted: May 18, 2016; Published online ( June 21, 2016 基金项目 : (863 ) (No. 2012AA02A701) * 通讯作者 : banrui@tju.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 60 微生物学通报 Microbiol. China 2017, Vol.44, No.1 riboflavin yield increased by 280% without biomass decrease and autolysis. Through a 70 h batch fermentation with 6.5% xylose and 1.5% sucrose as carbon source in a 5 L fermenter, riboflavin yield of 3.6 g/l was obtained, 80% higher than that with 8% sucrose as carbon source. Riboflavin yield decreased significantly by overexpressing the genes involved in xylose metabolism. [Conclusion] Co-overexpression of riba and ribh genes avoided the cytotoxicity effectively and further increased riboflavin production. Keywords: Bacillus subtilis, Riboflavin, ribh gene, Gene expression, Xylose, Co-metabolism (Riboflavin) B (Bacillus subtilis) [1-2] (ribdeaht) [3] (ribc) [4] [5] 7 ribdeaht 4 [6] riba GTP Ⅱ/3, [3,7] riba 25% [8] ribh 6,7- -8-(D- ) 5- [9] [10] 5- ( 1) [11-13] LXZ1 rib 图 1 枯草芽孢杆菌中 5- 磷酸核酮糖合成与核黄素合成途径 Figure 1 Ribulose-5-phosphate synthesis and riboflavin synthesis pathway in B. subtilis 1 材料与方法 1.1 菌株质粒和引物 1 PCR ( 2) 1.2 主要试剂和仪器 Taq Hifi DNA dntps cdna Thermo scientific RNA TIANGEN RT-PCR Roche Sigma YP101

3 : 61 表 1 菌株 Table 1 Strains Strains Genetic markers and instructions Source B. subtilis LXZ-1 trpc2, ΔaraR::P ara -neo, rib+(gsib), ΔguaC, ΔpurA, ΔpurR, pur+(cryiiia), ribc Laboratory B. subtilis TD-234m trpc2, ΔaraR::P ara -neo, Δhom::P cdd -pyrh-dn-cat-arar, ΔxylR::P AE -prs-dn-cat-arar Laboratory B. subtilis LXZ-2 B. subtilis LXZ1, ΔsacB::P cdd -riba This work B. subtilis LXZ-3 B. subtilis LXZ1, ΔsacB::P cdd -riba-ribh This work B. subtilis LXZ-31 B. subtilis LXZ1, ΔsacB::P cdd -ribe-riba This work B. subtilis LXZ-32 B. subtilis LXZ1, ΔsacB::P cdd -ribh This work B. subtilis LXZ-4 B. subtilis LXZ3, ΔyolA::P cdd -arae This work B. subtilis LXZ-5 B. subtilis LXZ3, xyl + (P AE ) This work B. subtilis LXZ-6 B. subtilis LXZ3, Δhom::P AE -rpe This work B. subtilis LXZ-3/pMX45 B. subtilis LXZ1, ΔsacB::P cdd -riba-ribh This work pmx45 rib Laboratory 表 2 Table 2 PCR 引物 PCR primers Primers Primer sequences (5 3 ) Location Au1 CTCCTCCAGCAAGATGATT pbpe Au2 CAAGAGCGTGATGTTTGTCGTGGTAGCCGTGATAGTT sacb Ap1/Ap2 GACAAACATCACGCTCTTG/GTGTAAATTCCTCCCTTACCT pyrh riba1 AGGTAAGGGAGGAATTTACACATGTTTCATCCGATAGAAGAAG riba riba2 CAAGCCACATCAATGTCATT ribh Ad1 AATGACATTGATGTGGCTTGCTTGATCCTAACGATGTAACC sacb Ad2 GAAGAATCCGCTTGTGTAA yveb Ac1 TTACACAAGCGGATTCTTCTCTTCAACTAAAGCACCCAT cat Ac2 TTATTCATTCAGTTTTCGTG arar Ag1 CACGAAAACTGAATGAATAACAGCCACATTTACATCTGAC sacb Ag2 CGTAATGCCGTCAATCG sacb AH2 AGGTTACATCGTTAGGATCAAGAATGTCCAGTCTGCTATTAAC ribt EA1 AGGTAAGGGAGGAATTTACACATGTTTACAGGAATTATCGAAGA ribe UPA2 ATTCCTCCTTTTGTCCTTATTGATTAGAAATGAAGTAAATGACCTAG riba ribd1 CAATAAGGACAAAAGGAGGAAT ribd ribd2 AGGTTACATCGTTAGGATCAAGTCGCCAAGATGAACATCC riba Aupa1 TGTTCGTCTGTCTCTAATCTT yral arae1 AGGTAAGGGAGGAATTTACACATGAAGAATACTCCAACTCAAT arae arae2 CCGTAGAAAGGGCTGTTT arae Adcrg1 AAACAGCCCTTTCTACGGGGACCTTGGCGATTTCAT csn Adcrg2 TTGTTCTCCTTGGCGATAG csn Rupa1 TACGGCATTCACATCAGG yuth Rupa2 GAATAGATGGAGCAACCTTTATCATTCTTTACCCTCTCCTTT pyrh rpe1/rpe2 ATAAAGGTTGCTCCATCTATTC/TGTGTTCATCCGTATAGCC rpe/ylos Rdcrg1 CGGCTATACGGATGAACACAACTGTACGCAGCACATATC hom Rdcrg2 ACTTCTTGAACGACTTCCA hom Xu1/Xu2 GCGGTAACGATGTTGAATC/GCTCAATCTCATTCCAGTTC xyla Xp1 GAACTGGAATGAGATTGAGCCATTCTTTACCCTCTCCTTT prs Xp2 ATGGAGTGGATGAAGTGGA prs Xdcrg1 TCCACTTCATCCACTCCATTGTCCTCCATTGTGATTGAT xylr Xdcrg2 CTATTCCTGTGCTGATACTTAC xylr RT-ribA1/RT-ribA2 TTGGCTCTCATCGCTGTG/TCATTGGCTTCTACGGTGTC riba RT-araE1/RT-araE2 TCCTGTAACAAGAAGCCATT/CCACAACTCCTCCAATCATA arae RT-rpe1/RT-rpe2 AAGGTTGCTCCATCTATTCT/GGACCGCTTCTACAATCA rpe RT-xylA1/RT-xylA2 GCTCAATCTCATTCCAGTTC/TCCGCCGATTACTTCTTG xyla RT-ccpA1/RT-ccpA2 ACGAGCAGTGGCGGAAT/GATAGCGACTGACGGTGT ccpa

4 62 微生物学通报 Microbiol. China 2017, Vol.44, No.1 PCR DNAEngine Light Cycler 480 PCR Roche Thermo Scientific Q Exactive Focus Orbitrap Thermo Scientific Bio Quest BIOTECH 培养基 LB (g/l) NaCl 15.0 ph % A 15 mg/l 6 mg/l (g/l) MgSO 4 7H 2 O 0.5 ph 7.2 (g/l) ( ) MgSO 4 7H 2 O ph 基因的整合过表达方法 [14] riba sacb TD-234m PCR riba (A bp) cdd (P cdd 232 bp) [15] sacb (U bp) (G 581 bp) (CR bp) (D 958 bp) PCR (50 μl) 37.5 μl 10 Buffer 5 μl dntps (2.5 mmol/l) 4 μl 1 μl (10 μmol/l) 1 μl TransTaq HiFi DNA polymerase (5 U/μL) 0.5 μl PCR 94 C 5 min 94 C 30 s 30 s 72 C 1 min/kb C 10 min PCR [16] UP cdd ADCRG (6 302 bp) UP cdd ADCRG LXZ-1 Spizizen [17] PCR 5 ml LB 37 C 220 r/min 4.5 h PCR DNA P cdd -riba sacb LXZ-2 sacb P cdd -ribh LXZ-32 P cdd -riba-ribh P cdd -ribe-riba P cdd -riba-ribd LXZ-3 LXZ-31 csn P cdd -arae LXZ-4 hom rpe LXZ-6 LXZ-3 xyl TD-234 m P AE [18] xyl LXZ qrt-pcr 分析 Light Cycler 480 PCR PCR (qrt-pcr) mrna CcpA ccpa qrt-pcr qrt-pcr bp LB h RNA cdna qrt-pcr C t 2 Ct mrna [19] 胞内代谢物的质谱检测方法 LB 10 ml ( kpa 0.45 μm ) 2 ml 2.6% NaCl 30 s 20 C 1.5 ml ( : : =40:40:20 ) 3

: 63 4 C 13 000 r/min 5 min 0.5 ml 4 C 13 000 r/min 5 min 80 C [20] Thermo Scientific Q Exactive Focus Orbitrap 1.

5 : 63 4 C r/min 5 min 0.5 ml 4 C r/min 5 min 80 C [20] Thermo Scientific Q Exactive Focus Orbitrap 1.7 发酵方法 30 ml/250 ml 200 r/min 37 C 14 h 3% 30 ml/250 ml 37 C 220 r/min 60 h h 0.5 ml 5% 37 C 500 r/min 1.5 VVM ph MPa 1.8 定量分析方法 3,5- (DNS ) [21] 0.05 mol/l NaOH r/min 2 min - (ph 5.0) nm (g/l)= (OD )/ 结果与分析 2.1 riba 和 ribh 基因单独表达对核黄素合成的影响 LXZ-1 riba ribh LXZ-2 LXZ-32 qrt-pcr LXZ-2 riba mrna LXZ-1 34 LXZ-32 ribh 25 ( 2A) LXZ-2 60 h 0.47 g/l LXZ-1 99% LXZ-32 riba ribh ( 2B) LXZ CFU/mL LXZ-1 LXZ CFU/mL LXZ-2 LXZ-2 Figure 2 图 2 riba 和 ribh 基因分别过表达对核黄素产量 (A) 及生物量 (B) 的影响 The effect of riba and ribh genes overexpression on riboflavin production (A) and biomass (B)

6 64 微生物学通报 Microbiol. China 2017, Vol.44, No.1 Figure 3 图 3 两个基因共同过表达对核黄素产量 (A) 及活菌数 (B) 的影响 The effect of genes co-overexpression on riboflavin production (A) and biomass (B) 2.2 riba 与 ribh 基因共表达的效应 LXZ-1 riba-ribd riba-ribh riba-ribe riba-ribh LXZ-3 riba-ribe LXZ-31 riba-ribd ribd riba-ribd (D- ) LXZ g/l LXZ-1 280% riba LXZ-2 91% ( 3A) CFU/mL ( 3B) riba-ribe LXZ-31 riba LXZ-2 LXZ-3 LXZ (D- ) (m/z) ( 4) LXZ-3 m/z riba (D- ) riba-ribh (D- ) 2.3 木糖作为碳源对核黄素发酵的影响 rib pmx45 LXZ-3 LXZ-3/pMX45 8% ( ) 60 h ( 5A B) 1.5% 6.5% 1.6 g/l 图 4 5- 氨基 -6-(D- 核糖氨基 ) 尿嘧啶的质谱图 Figure 4 The mass spectrum of 5-amino-6-(1-D-ribitylamino) uracil

4 木糖代谢相关基因修饰及其效应 LXZ-3 AraR [22] LXZ-3 5- LXZ-3 arae rpe LXZ-4 LXZ-6 P AE xyl LXZ-5 qrt-pcr arae rpe xyl 99 51 42 1.

7 : 65 Figure 5 图 5 不同碳源发酵 60 h 核黄素产量 (A) 及菌体生长曲线 (B) The yield (A) of riboflavin in 60 h and the biomass (B) with different carbon source 1.4 g/l CFU/mL 1.5% CFU/mL 2.4 木糖代谢相关基因修饰及其效应 LXZ-3 AraR [22] LXZ-3 5- LXZ-3 arae rpe LXZ-4 LXZ-6 P AE xyl LXZ-5 qrt-pcr arae rpe xyl % 6.5% arae ( 6B) arae rpe xyl Figure 6 图 6 木糖代谢相关基因过表达对核黄素产量 (A) 及木糖消耗 (B) 的影响 The effect of genes overexpression on riboflavin production (A) and xylose consumption (B)

8 66 微生物学通报 Microbiol. China 2017, Vol.44, No.1 ( 6A) AraR arae rpe xyl 2.5 木糖和蔗糖混合糖作为碳源的发酵罐发酵 1.5% 6.5% 5 L LXZ-3/pMX45 8% ( 7A) 60 h 2 g/l / 45 h 3.6 g/l 70 h 80% / 15 h ( 7B) / 3 讨论 riba GTP Ⅱ/3, [8] riba (D- ) riba (D- ) riba-ribd (D- ) riba-ribh (D- ) riba ribh / 5-5- PP 5- PP 5- / LXZ-3 AraR arae rpe xyl 图 7 5 L 发酵罐中的核黄素发酵过程曲线 Figure 7 The riboflavin fermentation profile in fermenter

9 : (tkt) (ywjh) PP L / 45 h PP tkt [23] / 5- 参考文献 [1] Stahmann KP, Revuelta JL, Seulberger H. Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production[j]. Applied Microbiology and Biotechnology, 2000, 53(5): [2] Abbas CA, Sibirny AA. Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers[j]. Microbiology and Molecular Biology Reviews, 2011, 75(2): [3] Perkins JB, Sloma A, Hermann T, et al. Genetic engineering of Bacillus subtilis for the commercial production of riboflavin[j]. Journal of Industrial Microbiology & Biotechnology, 1999, 22(1): 8-18 [4] Karelov DV, Kreneva RA, Lopes L É, et al. Mutational analysis of the ribc gene of Bacillus subtilis[j]. Russian Journal of Genetics, 2011, 47(6): [5] Shi SB, Shen Z, Chen X, et al. Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis[j]. Biochemical Engineering Journal, 2009, 46(1): [6] [7] Hümbelin M, Griesser V, Keller T, et al. GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production[j]. Journal of Industrial Microbiology & Biotechnology, 1999, 22(1): 1-7 [8] Birkenmeier M, Neumann S, Röder T. Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions[j]. Biotechnology Letters, 2014, 36(5): [9] Duan YX, Chen T, Chen X, et al. Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis[j]. Applied Microbiology and Biotechnology, 2010, 85(6): [10] Sauer U, Cameron DC, Bailey JE. Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid[j]. Biotechnology & Bioengineering, 1998, 59(2): [11] Krispin O, Allmansberger R. The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars[j]. Journal of Bacteriology, 1998, 180(12): [12] Franco IS, Mota LJ, Soares CM, et al. Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon[j]. Nucleic Acids Research, 2007, 35(14): [13] Dahl M K, Schmiedel D, Hillen W. Glucose and Glucose-6-Phosphate Interaction with Xyl Repressor Proteins from Bacillus spp. May Contribute to Regulation of Xylose Utilization[J]. Journal of Bacteriology, 1995, 177(19): [14] Liu SH, Endo K, Ara K, et al. Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter[j]. Microbiology, 2008, 154(9): [15] Bai LL, Ma RJ, Yang SM, et al. Effect of TatAdCd translocases overexpression on the secretion of lipase in Bacillus subtilis[j]. Microbiology China, 2016, 43(1): 2-8 (in Chinese),,,. TatAdCd [J]., 2016, 43(1): 2-8 [16] Shevchuk NA, Bryksin AV, Nusinovich YA, et al. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously[j]. Nucleic Acids Research, 2004, 32(2): e19 [17] Anagnostopoulos C, Spizizen J. Requirements for transformation in Bacillus subtilis[j]. Journal of Bacteriology, 1961, 81(5): [18] Zhu H, Yang SM, Yuan ZM, et al. Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis[j]. Microbial Cell Factories, 2015, 14: 54 [19] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Research, 2001, 29(9): e45 [20] Bennett BD, Yuan J, Kimball EH, et al. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach[j]. Nature Protocols, 2008, 3(8): [21] Wang JL, Nie GX, Cao XL, et al. Effects of different DNS reagents in determination of xylose content[j]. Food Research and Development, 2010, 31(7): 1-4 (in Chinese),,,. DNS [J]., 2010, 31(7): 1-4 [22] Mota LJ, Tavares P, Sá-Nogueira I. Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis[j]. Molecular Microbiology, 1999, 33(3): [23] Gershanovich VN, Aia K, Galushkina ZM, et al. Transketolase mutation in riboflavin-synthesizing strains of Bacillus subtilis[j]. Molekuliarnaia Genetika Mikrobiologiia I Virusologiia, 2000(3): 3-7

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇