CN114854648A - 能够耐受高光强的集胞藻驯化菌株的驯化方法 - Google Patents
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Abstract
本发明公开了一种获得耐受高光强集胞藻菌株的定向驯化方法，步骤：确定集胞藻野生型菌株的初始敏感光照强度；在培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04，在初始敏感光照强度下，培养至平台期；转接入培养基，继续驯化，以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm≥0.8；逐步提高光照条件，继续驯化，以此连续传代培养至光照强度到2000μmol光子m‑2s‑1时集胞藻在平台期的酶标仪测定OD750nm≥0.8，本发明获得的驯化菌株能在2000μmol光子m‑2s‑1高光条件下生长7天：OD750nm、叶绿素a、类胡萝卜素、干重和糖原含量高于野生型菌株。
Description
技术领域
本发明属于工业微生物领域，具体涉及能够耐受高光强的集胞藻驯化菌株的驯化方法。
背景技术
化石燃料是不可再生的资源，过多使用会释放大量的温室气体到大气中，导致全球变暖。因此，探索可再生和绿色的生物能源以替代传统的化石能源并减轻环境污染是我们迫切需要解决的问题。经由光合作用，每年约2580亿吨CO2被固定为有机物，而这其中陆地植物贡献约为30％，其余70％由海洋光合微生物贡献。其中，蓝细菌是唯一可以进行光合作用并释放氧气的原核生物，其可贡献全球固碳总量的25％左右。除了作为初始生产者的传统角色之外，近几十年来，借助先进的合成生物学技术，蓝细菌已被成功地改造为“光驱动细胞工厂”，可直接利用二氧化碳生产数十种可再生燃料和化学物质，包括乙醇、丁醇、丁二醇、丙二醇、丙酮、蔗糖、柠檬烯和3-羟基丙酸等。以上研究充分证明了基于光合蓝细菌开发可持续生产系统的可行性。其中模式蓝细菌集胞藻PCC 6803是蓝藻门、集胞藻属，繁殖方式为二分裂形式的一种单细胞非固氮球形蓝藻，在1968年首次从淡水湖中分离得到。作为模式菌株，集胞藻6803的全基因组测序在1996年已经完成，其基因组全长为3573kb，GC含量为47.7％，约含3264个蛋白编码基因。
光作为蓝细菌进行光合作用的能量来源，在蓝细菌的生长过程中是必不可少的。但是，早期研究表明，过多的光能会破坏蓝细菌的能量供应和消耗之间的平衡，从而导致细胞内的毒性活性氧(ROS)积累，包括过氧化氢(H2O2)、超氧阴离子(O2 -)、羟基自由基(OH·)和单线态氧(1O2)。过多的ROS进一步破坏其他关键细胞成分，如核酸、脂质、色素和蛋白质，最终导致细胞死亡。具体来说，光合作用可以被认为是一系列有序的氧化还原反应。当光能供应等于消耗时，光合作用的氧化还原平衡得以维持，但当收集过多的光时，平衡将被破坏，从而导致电子传递链的过度还原。此外，光系统II比光系统I更容易受到高光强度的影响，高光对光系统II反应中心极易产生光损伤。而高光造成的光损伤无疑将会限制蓝细菌的光合作用效率及碳固定能力。另外，为了具有成本效益，蓝细菌的工业或大规模培养必须基于户外培养利用天然太阳能。对于户外培养，保持持续光照成本很高，而且蓝细菌在自然界中不可避免地遭受高光胁迫。在自然环境中，太阳光最强可达2000μmol光子m-2s-1，对细胞造成严重的损伤并大大降低其光合效率。因此，为了促进高效光合细胞工厂的建设及其工业应用，构建高光耐受底盘以及解析蓝细菌的高光耐受机制是我们亟待解决的问题。
发明内容
本发明的目的是克服现有技术的不足，提供能够耐受高光强的集胞藻驯化菌株的驯化方法。
本发明的技术方案概述如下：
能够耐受高光强的集胞藻驯化菌株的驯化方法，包括如下步骤：
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度；
(2)在液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750 nm为0.04，在初始敏感光照强度下，培养至平台期；
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基，在初始敏感光照强度下继续驯化，以此连续传代培养至集胞藻在平台期的酶标仪测定OD750 nm大于或等于0.8；
(4)逐步提高光照条件，继续驯化，以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750 nm大于或等于0.8，得到能够耐受高光强的集胞藻驯化菌株。
菌种优选：集胞藻(Synechocystis sp.PCC 6803)ATCC 27184。
逐步提高光照条件为光照强度从初始敏感光照强度至2000μmol光子m-2s-1。
初始敏感光照强度为100μmol光子m-2s-1。
本发明的优点：
本发明的方法获得的集胞藻驯化菌株能够耐受2000μmol光子m-2s-1的光强。本发明的方法获得的能够耐受高光强的集胞藻驯化菌株的能够在2000μmol光子m-2s-1高光条件下生长7天：OD750 nm可达到野生型的2.55倍，叶绿素a含量可达到野生型的1.61倍，类胡萝卜素含量可达到野生型的3.87倍，干重可达到野生型的2.13倍，糖原含量可达到野生型的1.82倍。
附图说明
图1为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的生长曲线。
图2为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的叶绿素a含量。
图3为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的类胡萝卜素含量。
图4为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的干重。
图5为野生型菌株和驯化菌株在2000μmol光子m-2s-1条件下的糖原含量。
具体实施方式
本发明各实施例使用集胞藻(Synechocystis sp.PCC 6803)，该菌购自美国菌株保藏中心ATCC，菌株代码ATCC 27184。菌株购买于2012.5.30。
下面的实施例是为了使本领域的技术人员更好地理解本发明但并不用于限制本发明。下面结合具体实施例对本发明作进一步说明：
配制液体BG11培养基配方:NaNO3 1.5g，K2HPO4·3H2O 0.04g，MgSO4·7H2O0.075g，EDTA 0.001g，Na2CO3 0.02g，H3BO3 2.86g，MnCl2·4H2O 1.81g，ZnSO4·7H2O0.222g，NaMoO4·5H2O 0.390g，CuSO4·5H2O 0.079g，Co(NO3)2·6H2O 0.0494g，CaCl2·2H2O0.036g，柠檬酸铁铵0.006g，加水至1L而成，pH为7.5，121℃，20min高压锅灭菌；
实施例1
能够耐受高光强的集胞藻驯化菌株的驯化方法，包括如下步骤：
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度；具体步骤是：在25mL液体BG11培养基中接种集胞藻野生型菌液(集胞藻PCC 6803菌液)使酶标仪测定OD750nm为0.04，在50-2000μmol光子m-2s-1光照条件下(分别为：50、100，再以100为一个梯度，至2000μmol光子m-2s-1)培养集胞藻野生型菌株，确定集胞藻野生型菌的初始敏感光照强度为100μmol光子m-2s-1；
(2)在25mL液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04，在100μmol光子m-2s-1光照条件下，培养至平台期；
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基，在100μmol光子m-2s-1光照条件下继续驯化，以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm大于0.8(也可以等于0.8)；
(4)在100μmol光子m-2s-1至2000μmol光子m-2s-1范围逐步提高光照条件，继续驯化，以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750nm大于0.8(也可以等于0.8)，得到能够耐受高光强的集胞藻驯化菌株。
检测：
1.实施例1获得的能够耐受高光强的集胞藻驯化菌株(简称驯化菌株)和集胞藻野生型(集胞藻PCC 6803，简称野生型菌株)生长曲线：
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04，每组设置三个平行。在2000μmol光子m-2s-1光照条件下培养，摇床参数为160rpm，30℃，每隔24h取200μL菌液使用酶标仪测定OD750nm，并绘制生长曲线，见图1。
2.驯化菌株和野生型菌株干重特性的分析：
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04，每组设置三个平行，摇床参数为160rpm，30℃。在2000μmol光子m-2s-1条件下培养培养至7天，取20mL细胞液，7500rpm，5min离心收集菌体，使用真空冷冻干燥机冻干2-3天，称取对应菌体的重量，得到的菌体重量除以体积即对应菌株的干重数据；见图4。
3.驯化菌株和野生型菌株的叶绿素a含量和类胡萝卜素含量的测定：
在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04，每组设置三个平行，摇床参数为160rpm，30℃，在2000μmol光子m-2s-1条件下培养7天。取1mL OD750nm为0.4的细胞培养液，7500rpm，5min离心收集菌体，加入1mL的纯甲醇(4℃，预冷)重悬菌体，4℃萃取20min。12000，5min离心后取1-2mL上清液，使用紫外分光光度计测定OD665nm、OD470nm和OD720nm，通过公式叶绿素a含量[μg/ml]＝稀释倍数*12.9447*(OD665nm-OD720nm)；类胡萝卜素含量[μg/ml]＝稀释倍数*[1,000*(OD470nm-OD720nm)-2.86*(叶绿素a含量)]/221计算叶绿素a和类胡萝卜素含量；见图2和图3。
4.驯化菌株和野生型菌株糖原含量的测定：在25mL液体BG11培养基中接种驯化菌株和野生型菌株使酶标仪测定OD750nm为0.04，每组设置三个平行，摇床参数为160rpm，30℃，在2000μmol光子m-2s-1条件下培养7天。取1mL酶标仪测定OD750nm为0.4的细胞培养液，按照biobox的糖原测定试剂盒说明书进行糖原含量的测定，见图5。
Claims (4)
1.能够耐受高光强的集胞藻驯化菌株的驯化方法，其特征是包括如下步骤：
(1)通过体外敏感实验确定集胞藻野生型菌株的初始敏感光照强度；
(2)在液体BG11培养基中接种集胞藻野生型菌液使酶标仪测定OD750nm为0.04，在初始敏感光照强度下，培养至平台期；
(3)将步骤(2)培养至平台期的细胞转接入液体BG11培养基，在初始敏感光照强度下继续驯化，以此连续传代培养至集胞藻在平台期的酶标仪测定OD750nm大于或等于0.8；
(4)逐步提高光照条件，继续驯化，以此连续传代培养至光照强度到2000μmol光子m-2s-1时集胞藻在平台期的酶标仪测定OD750nm大于或等于0.8，得到能够耐受高光强的集胞藻驯化菌株。
2.根据权利要求1所述的驯化方法，其特征在于所述菌种为集胞藻PCC 6803。
3.根据权利要求1所述的驯化方法，其特征在于所述逐步提高光照条件为光照强度从初始敏感光照强度至2000μmol光子m-2s-1。
4.根据权利要求1或3所述的驯化方法，其特征在于所述初始敏感光照强度为100μmol光子m-2s-1。
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