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1 1224 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 陈思等 / 山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析 September 25, 2016, 32(9): Chin J Biotech, All rights reserved 工业生物技术陈思 1,2, 贾楠 1,2, 丁明珠 1,2, 元英进 ,2,,,.., 2016, 32(9): Chen S, Jia N, Ding MZ, et al. Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis. Chin J Biotech, 2016, 32(9): 摘要 : 酮古龙酸杆菌 Ketogulonigenium vulgare 是维生素 C 二步混菌发酵过程中的产酸菌山梨酮脱氢酶 (L-sorbosone dehydrogenase, 缩写为 SNDH) 作为维生素 C 直接前体 2- 酮基 -L- 古龙酸 (2-KGA) 合成的关键酶, 其作用机制并不十分清楚借助全基因组测序抽提 2 个山梨酮脱氢酶基因, 分别位于基因组 ( 缩写为 sndhg) 和质粒 ( 缩写为 sndhp) 上通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块, 比较其对 2-KGA 产量的影响研究发现 sndhg 过表达对菌株产酸影响不明显,sndhp 过表达使菌株明显产生副产物将 sndhg 和 sndhp 分别配合辅因子 PQQ 合成基因 pqqa, 分别构建 sndhg-pqqa 和 sndhp-pqqa 模块, 得到的工程菌株产酸情况与之前的结果大致相同将 4 株 K. vulgare 工程菌株分别与内生芽孢杆菌 Bacillus endophyticus 混合培养传代 50 d 后, 分离菌株进行混菌发酵, 其 2-KGA 的转化率分别提高了 15.4% 179% 0.65% 和 125% 表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性, 进而快速获得优良性状菌种的有效方法 : 酮古龙酸杆菌, 山梨酮脱氢酶, 适配性, 适应性进化, 混菌体系 Received: December 10, 2015; Accepted: January 15, 2016 Supported by: National Basic Research and Development Program of China (973 Program) (No. 2014CB745100), National Natural Science Foundation of China (No ), the Ph.D Programs Foundation of Ministry of Education of China (No ). Corresponding author: Mingzhu Ding. Tel: ; mzding@tju.edu.cn (973 ) (No. 2014CB745100) (No ) (No )

300072 1,2,,,.., 2016, 32(9): 1224 1232. Chen S, Jia N, Ding MZ, et al. Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis.

2 陈思等 / 山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析 1225 Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis Si Chen 1,2, Nan Jia 1,2, Mingzhu Ding 1,2, and Yingjin Yuan 1,2 1 Key Laboratory of Systems Bio-engineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin , China 2 Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin , China Abstract: Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation. L-sorbosone dehydrogenase (SNDH) is one of the key enzymes during the biosynthesis of 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. However, the catalytic mechanism of SNDH is unclear. According to the whole genome sequencing of K. vulgare, two genes encoding sorbosone dehydrogenases, one derived from the chromosome (named as sndhg) and one from plasmid (named as sndhp), were introduced into an industrial strain K. vulgare. The overexpression of gene sndhg had hardly effect on 2-KGA production, and the overexpression of gene sndhp produced an obvious byproduct in the fermentation broth. Combinational expression of sndhg/sndhp with pqqa (obtaining sndhg-pqqa and sndhp-pqqa modules) in K. vulgare resulted in the similar fermentation phenotype to two previous strains. After serial sub-cultivation of co-cultured Bacillus endophyticus with each engineered K. vulgare for 50 d, the conversion rate of 2-KGA increased by 15.4%, 179%, 0.65% and 125% compared with that of the parental K. vulgare with B. endophyticus. This study shows that adaptive evolution of microbial consortium is an effective strategy to increase the fitness between functional modules and chassis, thus quickly getting better strains for production of 2-KGA. Keywords: Ketogulonigenium vulgare, L-sorbosone dehydrogenase, fitness, adaptive evolution, mixed culture system C [1] VC Gluconobacter oxydans D- L- Ketogulonigenium vulgare L- VC 2- -L- (2-KGA) [2] K. vulgare L- 2-KGA [3] / L- L- C 2-KGA PQQ [4] PQQ L- / K. vulgare Cai [5] Lactococcus lactis K. vulgare K. vulgare / PQQ [6] 20% cjb@im.ac.cn

Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China Abstract: Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation.

3 1226 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No.9 2-KGA [7] 168 h 2-KGA 32.4 g/l [8-9] 150 d [10] 16% [11] [12] (L-sorbosone dehydrogenase SNDH) 2 ( sndhg) ( sndhp) sndhg sndhp 30.39% K. vulgare HKv604 2-KGA K. vulgare 2-KGA K. vulgare K. vulgare K. vulgare 1 材料与方法 1.1 材料 C K. vulgare HKv604 Bacillus endophyticus [13] K. vulgare pbbr1mcs2 [14] (datp dttp dgtp dctp) Taq DNA TaKaRa DNA PCR 1.2 方法 K. vulgare Hbe602 NCBI (CP CP CP012910) K. vulgare 2 L-sorbosone dehydrogenase DNA ( sndhg) 2 ( sndhp) GI Blast % ( 1) K. vulgare PQQ pqqa ( 1) K. vulgare HKv604 表 1 引物序列 Table 1 Sequence of primes Prime names Sequence (5 3 ) Restriction sites SNDHGF SNDHGR SNDHPF SNDHPR pqqaf pqqar CGGggtaccGCCACCA GCATCCAGCAA CCCaagcttATAGGCAG AGGCGTCAAAAC CGGggtaccCTGATCCA GTAAGCCACCA CCCaagcttGCCGCATT AACCGTGATAG GGactagtGCGGAGGA AACCGATCCATT ACgagctcTCAGCTATC CGCGCGTGCAT KpnⅠ Hind Ⅲ KpnⅠ Hind Ⅲ SpeⅠ SacⅠ

1 材料 C K. vulgare HKv604 Bacillus endophyticus [13] K. vulgare pbbr1mcs2 [14] (datp dttp dgtp dctp) Taq DNA TaKaRa DNA PCR 1.2 方法 1.2.1 K. vulgare Hbe602 NCBI (CP012908 CP012909 CP012910) K.

4 陈思等 / 山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析 1227 图 1 工业 K. vulgare 中 L- 山梨酮脱氢酶模块 (A) 及其与辅因子合成基因 pqqa 组合的功能模块 (B) 的构建 Fig. 1 Construction of the functional modules including (A) genes encoding L-sorbosone dehydrogenase (sndhg, sndhp) and (B) genes encoding L-sorbosone dehydrogenase with PQQ (sndhg-pqqa, sndhp-pqqa) in K. vulgare. sndhg sndhp sndhg-pqqa sndhp-pqqa 4 K. vulgare SyBE_Kv SyBE_Kv SyBE_Kv SyBE_Kv (1 L ) 3 g 3 g 3 g 1 g 10 g KH 2 PO 4 1 g MgSO g CaCO 3 1 g 0.8 L 20 g ph min 20 g/l- 0.2 L (1 L ) 15 g 12 g KH 2 PO 4 1 g MgSO g CaCO 3 2 g 0.8 L ph min 80 g 0.2 L ml ( 50 μg/ml ) r/min 48 h 24 h OD 600 = (HPLC) 1 ml r/min 5 min 0.5 mol/l HPLC Bio-Rad HPX-87H 0.5 mol/l H 2 SO ml/min K. vulgare B. endophyticus OD ml 250 ml r/min 24 h 2 ml 50 ml 50 d 1 ml r/min 5 min 2-KGA HPLC 1 ml 0.5 mol/l HCl OD K. vulgare cjb@im.ac.cn

vulgare. sndhg sndhp sndhg-pqqa sndhp-pqqa 4 K. vulgare SyBE_Kv0001116021 SyBE_Kv0001116022 SyBE_Kv00011160210 SyBE_Kv00011160220 1.2.2 (1 L ) 3 g 3 g 3 g 1 g 10 g KH 2 PO 4 1 g MgSO 4 0.

5 1228 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No.9 B. endophyticus OD 结果与讨论 2.1 山梨酮脱氢酶功能模块对 K. vulgare 的影响 K. vulgare [15-16] 2-KGA SyBE_Kv SyBE_Kv L- 20 g/l 2-KGA 2 48 h 36 h L- 2-KGA sndhp 2-KGA sndhp 2-KGA 3 24 h HPLC L min 7.87 min sndhg L- sndhp 7.40 min 87H 3 2-KGA L- [17] sndhp L- sndhp L- 2-KGA 图 2 山梨酮脱氢酶模块对 K. vulgare 耗糖 (A) 与产酸 (B) 的影响 Fig. 2 Influence of sndhg (SyBE_Kv ), sndhp (SyBE_Kv ), sndhg-pqqa (SyBE_ Kv ), sndhp-pqqa (SyBE_Kv ) modules on (A) sorbose consumption and (B) 2-KGA production of K. vulgare.

87 min sndhg L- sndhp 7.40 min 87H 3 2-KGA L- [17] sndhp L- sndhp L- 2-KGA 图 2 山梨酮脱氢酶模块对 K. vulgare 耗糖 (A) 与产酸 (B) 的影响 Fig.

6 陈思等 / 山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析 1229 图 3 Fig. 3 改造 K. vulgare 发酵产物的 HPLC 检测 HPLC analysis of the engineered strains. 2.2 山梨酮脱氢酶与辅因子 PQQ 组合对 K. vulgare 及混菌发酵的影响 PQQ PQQ PQQ [18] Deinococcus radiodurans PQQ [19] K. vulgare PQQ PQQ 70.5 g/l SyBE_Kv g/l sndhg 66.3 g/l sndhg-pqqa 67.6 g/l ( 4) sndhp pqqa SyBE_Kv sndhp SyBE_Kv h NADH NADPH [20-21] sndhg SyBE_Kv h g 2-KGA 9.83 g/l sndhg-pqqa SyBE_ Kv g 2-KGA 9.68 g/l 120 h SyBE_Kv 图 4 山梨酮脱氢酶模块及其与辅酶合成模块组合对混菌发酵生产 2-KGA 的影响 Fig. 4 Effects of sndh modules on the 2-KGA production and L-sorbose consumption in the consortium cjb@im.ac.cn

6 g/l ( 4) sndhp pqqa SyBE_Kv00011160220 sndhp SyBE_Kv0001116022 48 h NADH NADPH [20-21] sndhg SyBE_Kv0001116021 48 h 0.402 g 2-KGA 9.83 g/l sndhg-pqqa SyBE_ Kv00011160210 0.365 g 2-KGA 9.

7 1230 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No g 2-KGA 5.75 g/l sndhp-pqqa SyBE_Kv g 2-KGA 6.55 g/l 13.9% sndhp 70.5 g/l 69.1 g/l sndhp 7.41 g/l sndhp-pqqa 7.42 g/l sndhp 2.3 混菌配合适应性进化对混菌发酵的影响 [22] [23-24] K. vulgare 5 50 d 4 2-KGA C K. vulgare [9] [25] K. vulgare [26] SyBE_Kv KGA 15.4% SyBE_Kv KGA 179% SyBE_Kv KGA SyBE_Kv KGA 125% ( 6) 图 5 混菌传代 50 d 过程中 2-KGA 产量 Fig. 5 The 2-KGA production of mixed culture in the process of sub-cultivation for 50 d.

vulgare 5 50 d 4 2-KGA C K. vulgare [9] [25] K. vulgare [26] SyBE_Kv0001116021 2-KGA 15.

8 陈思等 / 山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析 1231 REFERENCES 图 6 传代菌株混菌发酵产酸图 Fig. 6 2-KGA production of the sub-cultivated K. vulgare and B. endophyticus consortium. 3 结论 K. vulgare L- sndhg sndhp sndhg-pqqa sndhp-pqqa K. vulgare sndhg-pqqa sndhg sndhg-pqqa sndhp-pqqa sndhp 13.9% K. vulgare B. endophyticus 50 d 2-KGA 2-KGA 15.4% 179% 0.65% 125% [1] Chauhan AS, Ramteke RS, Eipeson WE. Properties of ascorbic acid and its applications in food processing: a critical appraisal. J Food Sci Technol, 1998, 35(5): [2] Yin GL, He JM, Ren SX, et al. Production of vitamin C precursor-2-keto-l-gulonic acid from L-sorbose by a novel bacterial component system of SCB329-SCB933. Ind Microbiol, 1997, 27: 1 7. [3] Ma Q, Zhang WW, Zhang L, et al. Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection. PLoS ONE, 2012, 7(2): e [4] Wang X, Zhang WC. Advances in biosynthesis of pyrroloquinoline quinone. Lett Biotechnol, 2007, 18(3): ,.., 2007, 18(3): [5] Cai L, Yuan MQ, Li ZJ, et al. Genetic engineering of Ketogulonigenium vulgare for enhanced production of 2-keto-L-gulonic acid. J Biotechnol, 2012, 157(2): [6] Du J, Bai W, Song H, et al. Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-bacillus cereus consortium. Metab Eng, 2013, 19: [7] Gao LL, Hu YD, Liu J, et al. Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol. Metab Eng, 2014, 24: [8] Ma Q, Zhou J, Zhang W, et al. Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C cjb@im.ac.cn

65% 125% [1] Chauhan AS, Ramteke RS, Eipeson WE. Properties of ascorbic acid and its applications in food processing: a critical appraisal. J Food Sci Technol, 1998, 35(5): 381 392.

9 1232 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No.9 PLoS ONE, 2011, 6(10): e [9] Du J, Zhou J, Xue J, et al. Metabolomic profiling elucidates community dynamics of the Ketogulonicigenium vulgare-bacillus megaterium consortium. Metabolomics, 2012, 8(5): [10] Zou Y, Hu ML, Lv YJ, et al. Enhancement of 2-keto-gulonic acid yield by serial subcultivation of co-cultures of Bacillus cereus and Ketogulonigenium vulgare. Bioresource Technol, 2013, 132: [11] Ding MZ, Zou Y, Song H, et al. Metabolomic analysis of cooperative adaptation between co-cultured Bacillus cereus and Ketogulonicigenium vulgare. PLoS ONE, 2014, 9(4): e [12] Ma Q, Zou Y, Lv YJ et al. Comparative proteomic analysis of experimental evolution of the Bacillus cereus-ketogulonicigenium vulgare co-culture. PLoS ONE, 2014, 9(3): e [13] Jia N, Du J, Ding MZ, et al. Genome sequence of Bacillus endophyticus and analysis of its companion mechanism in the Ketogulonigenium vulgare-bacillus strain consortium. PLoS ONE, 2015, 10(8): e [14] Kovach ME, Phillips RW, Elzer PH, et al. pbbr1mcs: a broad-host-range cloning vector. Biotechniques, 1994, 16(5): 800. [15] Asakura A, Hoshino T. Isolation and characterization of a new quinoprotein dehydrogenase, L-sorbose/L-sorbosone dehydrogenase. Biosci Biotech Biochem, 1999, 63(1): [16] Miyazaki T, Sugisawa T, Hoshino T. Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid. Appl Environ Microb, 2006, 72(2): [17] Zou W, Liu LM, Zhang J, et al. Reconstruction and analysis of a genome-scale metabolic model of the vitamin C producing industrial strain Ketogulonicigenium vulgare WSH-001. J Biotechnol, 2012, 161(1): [18] Blattner FR, Plunkett G, Bloch CA, et al. The complete genome sequence of Escherichia coli K-12. Science, 1997, 277(5331): [19] Shrivastava M, Rajpurohit YS, Misra HS, et al. Survival of phosphate-solubilizing bacteria against DNA damaging agents. Can J Microbiol, 2010, 56(10): [20] San KY, Bennett GN, Berrios-Rivera SJ, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metab Eng, 2002, 4(2): [21] Heux S, Cachon R, Dequin S. Cofactor engineering in Saccharomyces cerevisiae: expression of a H 2 O-forming NADH oxidase and impact on redox metabolism. Metab Eng, 2006, 8(4): [22] Portnoy VA, Bezdan D, Zengler K. Adaptive laboratory evolution--harnessing the power of biology for metabolic engineering. Curr Opin Biotech, 2011, 22(4): [23] Hong KK, Nielsen J. Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose. Metab Eng, 2013, 16: [24] Peng BY, Shen Y, Li XW, et al. Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae. Metab Eng, 2012, 14(1): [25] Zhang J, Liu J, Shi ZP, et al. Manipulation of B. megaterium growth for efficient 2-KLG production by K. vulgare. Process Biochem, 2010, 45(4): [26] Zhou J, Ma Q, Yi H, et al. Metabolome profiling reveals metabolic cooperation between Bacillus megaterium and Ketogulonicigenium vulgare during induced swarm motility. Appl Environ Microb, 2011, 77(19): ( 本文责编陈宏宇 )

Enhancement of 2-keto-gulonic acid yield by serial subcultivation of co-cultures of Bacillus cereus and Ketogulonigenium vulgare. Bioresource Technol, 2013, 132: 370 373.

Microsoft Word - 1263-1268gc100083张静.doc

Microsoft Word - 1263-1268gc100083张静.doc 生物工程学报 Chin J Biotech 2010, September 25; 26(9): 1263 1268 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn 2010 CJB, All rights reserved. 发酵工程分阶段 ph 调控提高 2-