莱茵衣藻持续光合放氢转基因生物反应器的构建与调控

The hydrogen production from green algae is the most promising way because the hydrogenase activity is over 100 times in green algae more than in photosynthetic bacteria or cyanobacteria. However, the hydrogenase activity can be inhibited by oxygen which is produced by photosynthesis of algal cells, so that algal cell can release hydrogen continuously for only a few seconds to a few minutes. How to make the sustained hydrogen-production in green algae? It has been an insurmountable problem which has been restricting the development of algal hydrogen-producing industry to date. Recent results showed that the duration time of producing hydrogen continuously could extend to 70 hours with sulfur-deficiency, so that algal cell was able to achieve the target of sustainable photosynthetic hydrogen-producing by intermittent culturing with or without sulfur deficiency. However, due to the separation difficulties between algae and culture medium, the finding did not promote rapidly the development of industrial hydrogen production yet. Recent results from our group found that it was also able to achieve the effect of sulfur deficiency stress by regulating the expression of microRNAs and their target genes in Chlamydomonas reinhardtii. This project intends to build a sustainable hydrogen-producing bioreacter based on microRNAs regulation by using the specific promoters in photosynthetic organism Chlamydomonas reinhardtii. Its regulatory mechanisms for sustained hydrogen release from transgenic algal cells are analyzed, the intermittent induction conditions and parameters are optimized, efficiencies of sustained hydrogen production are compared based on between microRNAs regulation and sulfur deficiency stress. It will provide a new way for the photosynthetic algal hydrogen-producing industry.

由于绿藻氢酶的活性是光合细菌和蓝藻中氢酶活性的100多倍, 所以绿藻光合放氢是最具有应用前景的生物制氢途径。然而，绿藻氢酶受光合放氧的抑制, 使得其持续放氢时间只有几秒到几分钟。如何使绿藻能够持续光合放氢？这是长期制约绿藻光合制氢技术发展的瓶颈问题。最近发现缺硫胁迫能使绿藻放氢持续70小时，使绿藻进行缺硫和不缺硫间断连续培养, 可以使绿藻达到持续光合放氢的目标。然而，由于绿藻与培养基分离困难，制约了缺硫间断培养绿藻光合制氢技术的应用。本课题组最新研究发现，通过调控microRNAs及其靶基因的表达，也能达到间断缺硫培养的效果，使绿藻持续光合产氢。本项目拟以莱茵衣藻为材料，利用衣藻的特异性启动子，构建基于microRNAs调控且能持续光合放氢的转基因绿藻生物反应器，对转基因藻持续放氢的调控机制进行研究，对间断诱导条件与参数进行优化，并对其产氢效率进行分析，为绿藻光合制氢产业发展提供新的途径。

由于绿藻氢酶的活性是光合细菌和蓝藻中氢酶活性的100多倍,所以绿藻光合放氢是最具有应用前景的生物制氢途径。然而，绿藻氢酶受光合放氧的抑制,使得其持续放氢时间只有几秒到几分钟。如何使绿藻能够持续光合放氢？这是长期制约绿藻光合制氢技术发展的瓶颈问题。最近发现缺硫胁迫能使绿藻放氢持续70小时，使绿藻进行缺硫和不缺硫间断连续培养,可以使绿藻达到持续光合放氢的目标。然而，由于绿藻与培养基分离困难，制约了缺硫间断培养绿藻光合制氢技术的应用。 本项目在研究绿藻缺硫光合放氢调控分子机制的基础上，阐述莱茵衣藻光合放氢代谢途径的调控miRNAs、靶基因与细胞产氢效率间的相关性及其调控机制，并通过基因工程手段，构建基于miRNAs 调控的莱茵衣藻持续光合放氢转基因藻株，使其转基因藻细胞能够高效、持续地产生氢气。课题组成员利用四年的时间，在对建立莱茵衣藻正常培养和缺硫培养下细胞小RNA文库的基础上，利用生物信息学与实验生物学相结合的方法，发现了大量新的调控microRNAs；通过对其功能的研究，筛选到对光合系统II活性等具有重要调控功能的microRNAs；构建了一种基于蓝光诱导的莱茵衣藻光控系统并应用于藻细胞产氢调控，取得了具有一定创新价值的研究成果。发表相关研究论文16 篇，其中6篇发表在中科院JCR大类一区、TOP期刊杂志上；完成莱茵衣藻持续光合放氢转基因生物反应器及其调控技术的探讨，申请发明专利5 项；获得能够连续高效光合放氢的莱茵衣藻基因工程新品系12 株；参加8次国内外藻类学研究大会，发表会议论文12 篇；培养硕士研究生8名。课题组完成了课题的研究计划，并达到了预期的研究目标，为进一步探索绿藻光合放氢分子机制及其调控途径提供了重要的理论依据和基础数据。