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1 1286 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 孙溢华等 / 光质对工程聚球藻 7002 生长及 psba 启动子的调控 September 25, 2016, 32(9): Chin J Biotech, All rights reserved 生物育种与工艺优化 7002 psba 孙溢华 1, 张春莉 2, 施定基 3, 贾晓会 1, 贾睿 1, 何培民 Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str.3, D Frankfurt am Main, Germany ,,, psba., 2016, 32(9): Sun YH, Zhang CL, Shi DJ, et al. Effects of light quality on cell growth and psba promoter of engineered Synechococcus sp. PCC7002. Chin J Biotech, 2016, 32(9): 摘要 : 工程聚球藻的 psba 基因及其所用载体的启动子 PpsbA 均受光质调控, 利用该机制可通过光质调控提高聚球藻的光合效率及其外源基因表达率以转 vp28 基因聚球藻 7002 为实验材料, 通过优化光强温度及 ph, 解除光限制因素并提高光能利用率通过改变白光红光及蓝光的比例, 调控光质组成及单色光的光强, 检测细胞生长外源基因的表达及 psba 基因的转录研究结果表明 : 高比例蓝光下,vp28 基因的表达率达到 2.4%, 是纯白光下的 3 倍, 重组蛋白 VP28 的积累量提高至 2 倍高比例红光抑制了 psbaii psbaiii 基因及外源基因的表达, 但促使生物量在 3 d 内突破 1.5 g/l 本研究为蓝藻的生物制药和工程蓝藻代谢产物的产业化提供了理论基础 : 光生物反应器, 聚球藻 7002,psbA 启动子,VP28, 光质调控,psbA Effects of light quality on cell growth and psba promoter of engineered Synechococcus sp. PCC7002 Yihua Sun 1, Chunli Zhang 2, Dingji Shi 3, Xiaohui Jia 1, Rui Jia 1, and Peimin He 1 1 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai , China 2 Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str.3, D Frankfurt am Main, Germany 3 Institute of Botany, Chinese Academy of Sciences, Beijing , China Abstract: Light quality can regulate both psba genes and vector promoter psba of the engineered Synechococcus. Through light regulation, we tried to improve yield of the recombinant protein for vp28 gene-expressed Synechococcus sp. PCC7002. To drive photon-capturing efficiently, three limiting factors (irradiance, temperature and ph) were optimized by measuring net photosynthesis. High cell density cultures were performed with variant ratios of white, red and blue light in a 5-L photo-bioreactor. Yields of biomass, expressions of vp28 and transcription levels of psba were compared. High ratio blue light-induced vp28 transcription had tripled and the relative accumulation of VP28 protein was doubled. The relative expressions of psbaii and psbaiii had positive correlations with higher ratio of blue light, not the red light. With high ratio red light inducing, dry biomass reached 1.5 g/l in three days. Therefore, we speculated that red light accelerated biomass accumulation of the transgenic strain and blue light promoted transcription for PpsbA and psba. These results provided useful information for mass production of cyanobacteria and its secondary metabolites. Keywords: biophotoreactor, Synechococcus sp. PCC7002, psba promoter, VP28, light quality regulation, psba Received: January 3, 2016; Accepted: January 29, 2016 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2014AA093506). Corresponding author: Rui Jia. Tel: ; rjia@shou.edu.cn (863 ) (No. 2014AA093506)

2 孙溢华等 / 光质对工程聚球藻 7002 生长及 psba 启动子的调控 1287 [1-3] [4] Ⅱ psba [5] psba [6] PpsbA [7] psba [8] psba PpsbA [9] [10] psba PpsbA ( ph) (SDJ ) ph LED ( nm nm) [11] LED Nannochloropsis sp. [12] Chlorella sp. [13] LED (WSSV) WSSV 70 [14] Witteveldt [15] VP28 (WSSV ) WSSV Jia [10] vp28 WSSV Synechococcus sp. [16] vp [17] 1 材料与方法 1.1 转 vp28 基因聚球藻的摇床培养 vp28 S. PCC ml BG-11 [18] 250 ml r/min 50 μmol/(m 2 s) 1.2 净光合作用的单因素试验 7002 (1) [19] P = (A 678 A 750 ) (A 720 A 750 ) (1) P (μg/ml) A 750 A 720 A nm 720 nm 678 nm 10 μg (Hansatech UK) (Julubo Germany) ( ph) [20] (2) NPR = S Q /P (2) NPR (μmol O 2 /mg Chlah) (Net photosynthetic rate) S (/min) Q (mol O 2 /ml) P (mg/ml) min 转 vp28 基因聚球藻的光生物反应器培养 SDJ-5L ( ) LED ph ( ph) ((300±10) μmol/(m 2 s) 37 ph 7.5) 3.5 L A ± L/min CO 2 0.5% 72 h LED (3) [21] π 2 av ( ( ) 0 ) 2I I = 1 cosθexp 2RKcosθ (3) πrk I av (μmol/(m 2 s)) I (μmol/(m 2 s)) K R (m) 7002 (4) [22] K = ( A 750 ) (4) K A 750 White Red Blue Balance 4 ( 1) (3) ((300±10) μmol/(m 2 s)) 1.4 vp28 表达率测定及 VP28 蛋白检测 Trizol vp 表 1 光质配比及平均光强 Table 1 The proportions of light quality and average irradiance Item White light: Red light: Blue light I av (μmol/(m 2 s)) White 6:0: Red 3:2: Blue 3:1: Balance 2:2: cjb@im.ac.cn

1288 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech September 25, 2016 Vol.32 No.9 RNA RNA cdna(tiangen China) vp28 [17] vp28 qpcr ( 2) 16S rrna [23] 20 μl PCR 2 μl cdna 0.

gov/) psbai (ID 501262777) psbaii (ID 501264014) psbaiii (ID 501264746) tblastn 7002 (CP000951) psbai (region 1489906-1490985) psbaii (region 161804-162883) psbaiii (region 2245055-2246134) qpcr ( 2)

3 1288 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No.9 RNA RNA cdna(tiangen China) vp28 [17] vp28 qpcr ( 2) 16S rrna [23] 20 μl PCR 2 μl cdna 0.6 μl 10 μl SYBRFast qpcr Master Mix (2X) (KAPA USA) Jia [10] vp28 Western blotting 1.5 psba 的表达分析 NCBI ( psbai (ID ) psbaii (ID ) psbaiii (ID ) tblastn 7002 (CP000951) psbai (region ) psbaii (region ) psbaiii (region ) qpcr ( 2) ( ) 24 h White Red Blue qpcr 结果与讨论 2.1 ph 光强温度对藻细胞光合作用的影响 ph [24] ph Ⅱ ( ) 35 ph 7.3 ( μmol/(m 2 s)) ( 1A) 400 μmol/(m 2 s) 320 μmol O 2 /mg Chlah 281 μmol O 2 /mg Chlah 300 μmol/(m 2 s) ph 7.3 (15 50 ) ( 1B) μmol O 2 /mg Chlah μmol/(m 2 s) 35 ph 表 2 本实验所用引物 Table 2 Primers used in this study Primer name Primer sequence (5 3 ) vp28-fw AACCTCCGCATTCCTGTGACTG vp28-rv TCCGCATCTTCTTCCTTCATCTGTG psbai-fw CGTATTCCAGGCAGAGCACAACAT psbai-rv GCAGTGAACCAGATACCGACTACAG psbaii-fw ATGTTGTGCTCTGCCTGGAATACG psbaii-rv TCACTGCCACCACCTGCTTCA psbaiii-fw GCGACTGCTGTGCTCTTGGTT psbaiii-rv TCCGTTTCTGTGGTCTCCCGAAT 图 1 光强温度 ph 对净光合速率的影响 Fig. 1 Effect of irradiance, temperature and ph on net photosynthetic rates. (A) Irradiance. (B) Temperature. (C) ph. ( 1C) ph ph μmol O 2 /mg Chlah 210 μmol O 2 /mg Chlah 2.2 藻细胞干生物量的积累 ( ) 3 d 1 g/l [22] [26] ( 1) [11] White Red Blue Balance 72 h 1.48 g/l 1.68 g/l 1.22 g/l 1.38 g/l 3 36 h 4 36 h Red Blue Balance Red Red Blue Blue White Balance [22,25] 3 d 1.2 g/l 2.3 vp28 基因的表达量与 VP28 蛋白的积累 [27] PpsbA PpsbA IPTG Ptac 1.17 PpsbA vp

孙溢华等/光质对工程聚球藻 7002 生长及 psba 启动子的调控效率只有 0.

均受到蓝光的诱导作用然而外源蛋白 (VP28) 的积累量 (图 4) 差异不如外源基因表达差异明显 (图 3) 这可能是 DNA 转录与 mrna 翻译的时空差异造成的图 3 中 White Red Blue Balance 四组 vp28 基因表达率总体均呈现上升趋势前 36 h 内 4 组 vp28 表达率都较低其中 Blue 与 Balance 组表达率较高 White 与

11% 出现于 12 h 图 4 中总蛋白条带在 PAGE 上无明显差别而 VP28 条带在 PVDF 上差异较为明显灰度值从少到多分别为 Red White Balance Blue 说明 Blue 组蛋白积累量最高 Red 组蛋白积累量最少并且 Blue 组与 Red 组差异明显 2.

3 Variations of vp28 transcription with various proportions of light quality. 010-64807509 图 4 不同光质组成下 VP28 的相对含量 Fig. 4 Relative accumulation of VP28 in engineered Synechococcus sp.

4 孙溢华等/光质对工程聚球藻 7002 生长及 psba 启动子的调控效率只有 0.1% 1%[10] 转基因聚球藻 7002 所用表达载体为 prl-489 启动子为 PpsbA[17] 本文中高比例蓝光促进了 vp28 基因的表达并促进了 psbaii psbaiii 的转录这与 Tsinoremas 等[9]研究转基因聚球藻 7942 结果一致图 3 与图 5 的结果证明载体启动子 PpsbA 与光合基因 psba 的转录在时空效应上具有一致性均受到蓝光的诱导作用然而外源蛋白 (VP28) 的积累量 (图 4) 差异不如外源基因表达差异明显 (图 3) 这可能是 DNA 转录与 mrna 翻译的时空差异造成的图 3 中 White Red Blue Balance 四组 vp28 基因表达率总体均呈现上升趋势前 36 h 内 4 组 vp28 表达率都较低其中 Blue 与 Balance 组表达率较高 White 与 Red 组表达率较低 36 h 后前两组与后两组相比差异明显 Blue 与 Balance 组在前 36 h 及 60 h 后差异不显著而在 36 h 至 60 h 之间差异明显 White 与 Red 组在 72 h 内差异都不明显 Blue 组中 vp28 基因表达率最高达到 2.41% 出现于第 72 h Red 组中 vp28 基因表达率最低达到 0.11% 出现于 12 h 图 4 中总蛋白条带在 PAGE 上无明显差别而 VP28 条带在 PVDF 上差异较为明显灰度值从少到多分别为 Red White Balance Blue 说明 Blue 组蛋白积累量最高 Red 组蛋白积累量最少并且 Blue 组与 Red 组差异明显 2.4 psba 的表达差异图 5 显示接种后光能利用率较高 psba 转录效率比接种前提高了 50% 75% White 与 Red 组图 2 不同光质组成下干生物量积累曲线 Fig. 2 Variations of dry biomass with various proportions of light quality. 图 3 不同光质组成下 vp28 表达率变化曲线 Fig. 3 Variations of vp28 transcription with various proportions of light quality 图 4 不同光质组成下 VP28 的相对含量 Fig. 4 Relative accumulation of VP28 in engineered Synechococcus sp. PCC7002 with various proportions of light quality. (A) SDS-PAGE analysis of total protein. (B) Western blotting of VP28. M: marker; 1: wild type; 2: red; 3: white; 4: balance; 5: blue. 图 5 不同光质组成下 psba 的相对表达量 Fig. 5 Relative transcription of psba genes with various proportions of light quality. psba 的总 mrna 表达量高于 Blue 组随时间变化总 psba mrna 含量呈下降趋势 psbai psbaii psbaiii 在 3 组中都呈下降趋势其中 psbaii psbaiii 在 White 与 Red 组中下降速度较快 psbaii 分别从第 1 天的下降至第 3 天的 psbaiii 分别从下降至而在 Blue 组中 psbaii 与 psbaiii 的下降速度明显较慢从第 1 天的分别下降至第 3 天的 Blue 组的 psbaii 与 psbaiii 始终高于 White 与 Red 组而 Red 组的 psbaii 与 psbaiii 始终处于较低水平与纯白光相比高比例红光并不影响 psba 基因总 mrna 含量但最终提高了转基因聚球藻 7002 的干生物量积累高比例蓝光抑制了 psba 总 mrna 的含量从而抑制了干生物量的积累但促进了 psbaii 与 psbaiii 的转录这可能是由于不同比例的红光蓝光影响了 Rubisco 与碳酸酐酶的活性[28] 从而影响了碳循环途径本文中高比例红光促进了生物量的积累这可能是因为红光诱导了一种以钙调蛋白为二级信使的信号通路[29] 并促进了 psbai 的表达 cjb@im.ac.cn 1289

5 1290 ISSN CN /Q Chin J Biotech September 25, 2016 Vol.32 No.9 3 结论 7002 vp28 3 d 1.2 g/l VP28 PpsbA vp28 psba REFERENCES [1] Aguilera J, Francisco J, Gordillo L. Light quality effect on photosynthesis and efficiency of carbon assimilation in the red alga Porphyra leucosticta. J Plant Physiol, 2000, 157(1): [2] Niizawa I, Heinrich JM, Irazoqui HA. Modeling of the influence of light quality on the growth of microalgae in a laboratory scale photo-bio-reactor irradiated by arrangements of blue and red LEDs. Biochem Eng J, 2014, 90(5): [3] Rashid N, Rehman MSU, Han JI. Enhanced growth rate and lipid production of freshwater microalgae by adopting two-stage cultivation system under diverse light and nutrients conditions. Water Environ J, 2015, 29(4): [4] Nesbit AD, Whippo C, Hangarter RP. Translation initiation factor 3 families: what are their roles in regulating cyanobacterial and chloroplast gene expression? Photosynth Res, 2015, 126(1): [5] Zouni A, Witt HT, Kern J, et al. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature, 2001, 409(6821): [6] Tsinoremas NF, Schaefer M, Golden SS. Blue and red light reversibly control psba expression in the cyanobacterium Synechococcus sp. strain PCC J Biol Chem, 1994, 269(23): [7] Ma WM, Shi DJ, Wang QX, et al. Exogenous expression of the wheat chloroplastic fructose-1,6-bisphosphatase gene enhances photosynthesis in the transgenic cyanobacterium, Anabaena PCC7120. J Appl Phycol, 2005, 17(3): [8] Bergsland KJ, Haselkorn R. Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoc1 gene of Anabaena sp. strain PCC7120. J Bacterial, 1991, 173(11): [9] Tsinoremas NF, Kawakami A, Christopher DA. High-fluence blue light stimulates transcription from a higher plant chloroplast psba promoter expressed in a cyanobacterium, Synechococcus (sp. strain PCC7942). Plant Cell Physiol, 1999, 40(4): [10] Jia XH, Zhang CL, Shi DJ, et al. 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Evaluation of photosynthetic efficiency in microalgal cultures using averaged irradiance. Enzyme Microb Tech, 1997, 21(5): [22] Kang RJ, Zhou WQ, Cai ZL, et al. Photoautotrophic cultivation of Synechococcus sp. PCC7002 in photobioreactor. Chin J Biotech, 2000, 16(5): (in Chinese).,,, , 2000, 16(5): [23] Pinto F, Pacheco CC, Ferreira D, et al. Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria. PLoS ONE, 2012, 7(4): e [24] Moore LR, Goericke R, Chisholm SW. Comparative physiology of synechococcus and prochlorococcus influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Mar Ecol Prog Ser, 1995, 116: [25] Zeng WL, Zhao FF, Cao ZG, et al. Medium optimization by response surface method for transgenic Synechococcus sp. PCC 7002 with mouse metallothionein-i gene. Chin J Biotech, 2008, 24(1): (in Chinese).,,,. -I 7002., 2008, 24(1): [26] Camacho-Rubio F, Padial-Vico A, Martinez-Sancho ME. The effect of the mean intensity of light on the cultivation of Chlorella pyrenoidosa. Int Chem Eng, 1985, 25(2): [27] Ning WY, Wu XM, Wang CM, et al. Efficiency comparison of promoters Ptac & PsbA driving hg-csf expression in Anabaena sp. PCC7120. J Mirobiol, 2014, 34(3): (in Chinese).,,,. Ptac PsbA 7120 hg-csf., 2014, 34(3): [28] Roscher E, Zetsche K. The effects of light quality and intensity on the synthesis of ribulose-1,5-bisphosphate carboxylase and its mrnas in the green alga Chlorogonium elongatum. Planta, 1986, 167(4): [29] Alizadeh D, Cohen A. Red light and calmodulin regulate the expression of the psba binding protein genes in Chlamydomonas reinhardtii. Plant Cell Physiol, 2010, 51(3): ( 本文责编郝丽芳 )

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%