Formighieri Cinzia, Melis Anastasios
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA, 94720-3102, USA,
Planta. 2014 Aug;240(2):309-24. doi: 10.1007/s00425-014-2080-8. Epub 2014 May 20.
Successful application of the photosynthesis-to-fuels approach requires a high product-to-biomass carbon-partitioning ratio. The work points to the limiting amounts of heterologous terpene synthase in cyanobacteria as a potential barrier in the yield of terpene hydrocarbons via photosynthesis. Cyanobacteria like Synechocystis sp. can be exploited as platforms in a photosynthesis-to-fuels process for the generation of terpene hydrocarbons. Successful application of this concept requires maximizing photosynthesis and attaining a high endogenous carbon partitioning toward the desirable product. The work addressed the question of the regulation of β-phellandrene synthase transgene expression in relation to product yield from the terpenoid biosynthetic pathway of cyanobacteria. The choice of strong alternative transcriptional and translational cis-regulatory elements and the choice of the Synechocystis genomic DNA loci for transgene insertion were investigated. Specifically, the β-phellandrene synthase transgene was expressed under the control of the endogenous psbA2 promoter, or under the control of the Ptrc promoter from Escherichia coli with the translation initiation region of highly expressed gene 10 from bacteriophage T7. These heterologous elements allowed for constitutive transgene expression. In addition, the β-phellandrene synthase construct was directed to replace the Synechocystis cpc operon, encoding the peripheral phycocyanin rods of the phycobilisome antenna. Results showed that a 4-fold increase in the cellular content of the β-phellandrene synthase was accompanied by a 22-fold increase in β-phellandrene yield, suggesting limitations in rate and yield by the amount of the transgenic enzyme. The work points to the limiting amount of transgenic terpene synthases as a potential barrier in the heterologous generation of terpene products via the process of photosynthesis.
光合作用制燃料方法的成功应用需要高的产物与生物质碳分配比。这项工作指出，蓝藻中异源萜烯合酶的限量是通过光合作用生产萜烯碳氢化合物产量的潜在障碍。像集胞藻属这样的蓝藻可被用作光合作用制燃料过程中生产萜烯碳氢化合物的平台。这一概念的成功应用需要使光合作用最大化，并使内源性碳更多地分配到所需产物中。这项工作解决了与蓝藻萜类生物合成途径产物产量相关的β-水芹烯合酶转基因表达调控问题。研究了强替代性转录和翻译顺式调控元件的选择以及转基因插入集胞藻基因组DNA位点的选择。具体而言，β-水芹烯合酶转基因在集胞藻内源性psbA2启动子的控制下表达，或在来自大肠杆菌的Ptrc启动子与噬菌体T7高表达基因10的翻译起始区域的控制下表达。这些异源元件允许转基因组成型表达。此外，β-水芹烯合酶构建体旨在取代集胞藻cpc操纵子，该操纵子编码藻胆体天线的外周藻蓝蛋白棒。结果表明，β-水芹烯合酶细胞含量增加4倍，β-水芹烯产量增加22倍，这表明转基因酶的量限制了反应速率和产量。这项工作指出，转基因萜烯合酶的限量是通过光合作用异源生产萜烯产物的潜在障碍。
