A synthetic biology platform for sustainable, climate-friendly conversion of CO2 to products using cyanobacteria

蓝细菌是最简单和最容易遗传的生物体，能够进行光合作用，使用二氧化碳和阳光作为唯一的碳和能源。它们的光合产量和生长速度与生长最快的微藻相似，并且大于陆地植物。因此，蓝细菌在光驱动的、负碳的生物过程中作为全细胞生物催化剂具有巨大的潜力，将大气或废物CO2转化为感兴趣的产品，同时避免与食物链竞争。蓝细菌经过基因改造，可以合成从日用化学品和生物燃料到高价值产品的各种非天然化合物。挑战：由于目前使用化石碳和排放CO2的经济成本较低，因此使用蓝细菌的生产与传统方法的生产在商业上还没有竞争力。合理地设计编码最佳代谢途径的DNA也是困难的。如果表达太高、太低或“平衡”差，则会导致生产率低和/或遗传不稳定性。解决方案：现代DNA组装允许构建许多途径编码构建体变体的大型文库，组合地改变每种酶的表达。高性能变体可以通过筛选来鉴定。这种方法已经得到了很好的证明，但只有少数模式生物。我们最近开发了一个用于组合构建和优化蓝藻代谢途径编码构建体的平台，并将其应用于集胞藻PCC 6803中从CO2生产番茄红素，成功获得了具有高产和遗传稳定途径的菌株，克服了这一关键问题。目的：该新的组合代谢途径构建平台将被应用和发展，方法：使用DNA合成，外源途径酶和天然“瓶颈”酶的编码序列，以及宿主适当的表达控制部分（启动子，核糖体结合位点，终止子等）将被格式化用于启动-停止组装，这是我们最近发表的针对代谢工程优化的DNA组装系统。具有受控部分混合物的分级多部分组装将产生用于插入宿主细胞中并使用分析方法（例如GC/LC/MS/NMR）进行筛选的组合途径库。A）宿主：该平台已经针对标准菌株集胞藻属PCC 6803进行了验证，允许工作快速开始并完全集中在途径上，而不需要平台开发。新描述的，快速生长，高产，但很少研究的菌株聚球藻PCC 11901具有很大的潜力，首先需要开发表达控制部分。B）产品：广泛的天然和非天然产品可以生物合成，包括醇类、有机酸、脂肪酸、脂肪醇、烷烃、萜类、生物碱、肽、糖、香料、芳香剂、专门的代谢物、药物前体、营养品、药物和疫苗。C）与其他方法和技术的整合：组合途径优化将与经典的代谢工程相结合，敲除竞争途径，以生理学理解和/或代谢建模为指导，根据学生的兴趣。最后，我们新的专利高通量酶进化系统可以应用于重要途径中的问题酶，如烷烃生物合成。

Cyanobacteria are the simplest and most genetically-tractable organisms capable of oxygenic photosynthesis, using CO2 and sunlight as sole carbon and energy sources. Their photosynthetic yield and growth rate are similar to the fastest-growing microalgae, and greater than terrestrial plants. Consequently, cyanobacteria have great potential as whole-cell biocatalysts in light-driven, carbon-negative bioprocesses converting atmospheric or waste CO2 to products of interest, while avoiding competition with the food chain. Cyanobacteria have been genetically modified to synthesise a wide range of non-native compounds from commodity chemicals and biofuels to high-value products.CHALLENGE:Production using cyanobacteria is not yet commercially competitive with production by conventional approaches due to the current low economic cost of using fossil carbon and emitting CO2. It is also difficult to rationally design DNA encoding an optimal metabolic pathway. If expression is too high, too low, or poorly 'balanced', then low productivity and/or genetic instability results.SOLUTION:Modern DNA assembly allows construction of large libraries of many pathway-encoding construct variants, varying the expression of each enzyme combinatorially. High-performance variants can be identified by screening. This approach has been well proven, but in only a few model organisms. We recently developed a platform for combinatorial construction and optimisation of metabolic pathway-encoding constructs in cyanobacteria, applied it to production of lycopene from CO2 in Synechocystis sp. PCC 6803, and successfully obtained strains with pathways that were both productive and genetically stable, overcoming this key problem.AIM:The new combinatorial metabolic pathway construction platform will be applied and developed to generate photoautotrophic cell factories with highly-productive, genetically stable engineered metabolic pathways, using key strains.APPROACH:Using DNA synthesis, coding sequences for foreign pathway enzymes and native 'bottleneck' enzymes, as well as host-appropriate expression control parts (promoters, ribosome-binding sites, terminators, etc) will be formatted for Start-Stop Assembly, our recently-published DNA assembly system optimised for metabolic engineering. Hierarchical multi-part assembly with controlled part mixtures will generate combinatorial pathway libraries for insertion into host cells and screening using analytical methods e.g. GC/LC/MS/NMR.A) HOSTS:The platform is already validated for the standard strain Synechocystis sp. PCC 6803, allowing work to start quickly and focus entirely on pathways, without need for platform development. The newly-described, fast-growing, high-producing, but little-studied strain Synechococcus sp. PCC 11901 has great potential, and will first require the development of expression control parts. Similarly, the platform will be transferred to Spirulina, a safe, food-grade strain suitable for nutraceutical and medical products.B) PRODUCTS:A wide range of natural and non-natural products could be biosynthesised including alcohols, organic acids, fatty acids, fatty alcohols, alkanes, terpenoids, alkaloids, peptides, sugars, flavours, fragrances, specialised metabolites, pharmaceutical precursors, nutraceuticals, drugs and vaccines. The high-throughput assembly allows multiple different products to be targeted in parallel.C) INTEGRATION WITH OTHER APPROACHES & TECHNIQUES:Combinatorial pathway optimisation will be combined with classical metabolic engineering, knocking out competing pathways, guided by physiological understanding and/or metabolic modelling, subject to the student's interests. Finally, our new, patented, high-throughput enzyme evolution system can be applied to problematic enzymes in important pathways, such as alkane biosynthesis.