Photoperiodic and circadian clock mediated control of hydrocarbon production in botryococcus braunii.

2008年12月，欧盟同意，到2020年，成员国必须用可再生资源满足10%的运输燃料需求。藻类是一种很有希望的第三代生物燃料来源，尤其是因为藻类可以在边际土地上生长，不会与农业粮食作物争夺空间。尤其令人感兴趣的浮游藻类是布氏芽孢杆菌，因为它合成和分泌高达80%的干重作为长链(C30-C40)碳氢化合物(Banerjee等人。(2002)Crit.生技诺尔牧师。22,245-79)。然而，布氏巴氏杆菌产生碳氢化合物(HC)的分子控制和时间尚不清楚。这一知识对于有效评估布氏杆菌作为生物燃料的可持续来源至关重要，因为在微藻中，就像在高等植物中一样，关键代谢过程的时间，如糖分配或脂肪生产，既受光周期控制，也受内源生物钟产生的细胞节律控制(Johnson，C.H.(2001)Annu)。菲西奥尔牧师。63,695-728；Mittag(2001)Int.赛托尔牧师。206、213-47)。该项目将通过埃克塞特大学和普利茅斯海洋实验室(PML)的专家之间的动态合作，研究布氏双胞菌中HC的产生受光周期和生物钟调节的假说。工作方案紧张，结合了各种复杂的技能，包括分子生理学、生物信息学、建模和分析化学。我们预计，该学生将至少花16个月的时间借调到工业合作伙伴那里。因此，申请的学龄为4年。在第一年，学生将描述布氏芽孢杆菌Guadeloupe菌株在不同光照周期、8h光照、16h黑暗(8L/16D)、12L/12D、16L/8D、20L/4D和恒定光照下的生长和HC产生情况。样本将在30天内每天采集。生长将通过培养物的干生物量和叶绿素含量以及用尼罗红荧光分析监测的HC产生来量化。每隔5天，总HC将被提纯，并用气相色谱(GC)进行分析。光周期对布氏杆菌生长、HC产量和组成的影响将被关联和建模，以确定潜在的生物燃料生产流的最佳培养条件。在二年级和三年级，学生将在整个一天的周期中详细描述布劳尼岛的碳氢化合物生产情况。布氏杆菌在8L/16D、12L/12D、16L/8D和20L/4D条件下生长到线状或静止相。将使用尼罗红试验监测HC产量，每30分钟监测一次，持续至少28小时，并与生物量相关。此外，每隔2小时从布氏杆菌细胞中提取HC，并用气相色谱分析其成分。为了研究HC的产生是否受生物钟的控制，布氏假单胞菌将被置于最合适的光周期中，转移到昼夜自由运行(CFR；恒定的光或恒定的黑暗)，并在选定的时间点监测HC的产生和组成至少5天。时钟的自由运行周期将被确定。主观的夜间和白天休息时间将被用来确定昼夜节律的相位响应曲线。在3年级和4年级，学生将构建和测试布氏杆菌生物钟的生物信息学模型。我们已经对布氏杆菌转录组进行了测序和注释。学生将通过与已识别的昼夜节律系统进行电子比较来识别时钟基因同源物，并使用这些数据构建布氏杆菌昼夜节律时钟的分子模型。模型中基因之间的功能关系将使用从特定光周期和CFR中生长的布氏芽孢杆菌提取的RNA的qRT-PCR进行经验性测试。这个模型将能够比较布氏杆菌和其他微藻的昼夜节律系统，并能够预测藻类生物燃料生产的最佳产量。

In December 2008, the European Union agreed that, by 2020, member states must satisfy 10% of their transport fuel needs from renewable resources. Algae are a promising source of 'third generation' biofuel, not least because algae can be grown on marginal lands and do not compete for space with agricultural food crops. The planktonic alga Botryococcus braunii is particularly interesting because it synthesises and secretes up to 80% of its dry mass as long-chain (C30-C40) hydrocarbons (Banerjee et al. (2002) Crit. Rev. Biotechnol. 22, 245-79). However, the molecular control and timing of hydrocarbon (HC) production in B. braunii is not known. This knowledge is essential for a valid assessment of B. braunii as sustainable source of biofuel because in microalgae, as in higher plants, the timing of key metabolic processes such as sugar allocation or lipid production are controlled both by photoperiod and through cellular rhythms generated by the endogenous circadian clock (Johnson, C.H. (2001) Annu. Rev. Physiol. 63, 695-728; Mittag (2001) Int. Rev. Cytol. 206, 213-47). This project will investigate the hypothesis that HC production in B. braunii is modulated by photoperiod and the circadian clock via a dynamic collaboration between experts at the University of Exeter and the Plymouth Marine Laboratory (PML). The programme of work is intense and combines diverse and complex skills, including molecular physiology, bioinformatics, modelling and analytical chemistry. We anticipate that the student will spend at least 16 months seconded to the industrial partner. Consequently, the requested duration of the studentship is for 4 years. In year 1, the student will characterise growth and HC production of B. braunii, strain Guadeloupe, grown in batch culture, in different photoperiods; 8 h light, 16 h darkness (8L/16D), 12L/12D, 16L/8D, 20L/4D and in constant light. Samples will be harvested daily over a 30 day period. Growth will be quantified by dry biomass and chlorophyll content of the cultures and HC production monitored using a Nile red fluorescence assay. Every 5 days, total HC will be purified and analysed using gas chromatography (GC). The effect of photoperiod on B. braunii growth, HC yield and composition will be correlated and modelled to determine optimal culture conditions for a potential biofuel production stream. In years 2 & 3, the student will perform a detailed characterisation of HC production in B. braunii over an entire diurnal cycle. B. braunii will be grown to linear or stationary phase in 8L/16D, 12L/12D, 16L/8D and 20L/4D. HC yields will be monitored using the Nile red assay, every 30 min for at least 28 h and related to biomass. In addition, every 2 h, HC's will be extracted from B. braunii cells and the composition analysed by GC. To investigate whether HC production is controlled by the circadian clock, B. braunii will be entrained in the most appropriate photoperiod, transferred to circadian free-run (CFR; constant light or constant darkness) and HC production and composition monitored at selected time-points for at least 5 days. The free running period of the clock will be determined. Subjective night- and day-breaks will be used to determine the phase response curve of the circadian rhythm. In years 3 & 4, the student will construct and test a bioinformatic model of the B. braunii circadian clock. We have sequenced and are annotating the B. braunii transcriptome. The student will identify clock gene homologues by in silico comparison with identified circadian systems, and use that data to construct a molecular model of the B. braunii circadian clock. The functional relationships between the genes in the model will be empirically tested using qRT-PCR of RNA extracted from B. braunii grown in specified photoperiods and in CFR. This model will enable comparisons between the circadian system in B. braunii and other microalgae, and allow predictions of optimal yields in algal biofuel production.