To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely ’UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that ’UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO₂ into excreted FAME.

为了满足未来几十年全球对生物柴油日益增长的需求，需要生产生物柴油关键成分之一（例如脂肪酸甲酯（FAME））的替代方法。藻类生物柴油已成为长期目标，因为其收割和加工成本过高。在这项工作中，我们设计了蓝细菌，将二氧化碳转化为排泄的FAME，而无需甲醇作为甲基供体。为了产生FAME，首先通过硫酯酶，即来自加利福尼亚伞形藻（Umbellularia californica）的'Uc FatB1 '，将脂肪酸生物合成途径的产物酰基ACP转化为游离脂肪酸（FFA）。接着，通过采用保幼激素酸ø -methyltransferase（DM JHAMT）从果蝇和作为甲基供体的S-腺苷甲硫氨酸（SAM）将FFA转化为相应的FAME。酯是自然分泌到细胞外的，与传统的藻类生物柴油生产相反，传统的藻类生物柴油必须首先收获并加工以用于提取的三酰基甘油（TAG）的酯交换反应，从而可以通过溶剂覆盖简单地分离产物。通过优化启动子和RBS元素，在10天之内可生产高达120 mg / L的FAME。定量分析关键蛋白和代谢产物以及过表达SAM合成酶（MetK）的构建体，表明'Uc FatB1，MetK和DmJHAMT是限制途径通量的主要因素。为了解决后者的局限性，还尝试了Dm JHAMT的两个重构祖先序列，与天然Dm JHAMT相比，菌株显示出更宽的甲酯链长度分布。总而言之，这项工作证明了将阳光直接驱动的CO ₂转化为排泄的FAME的有前途的途径。