PDF(Pubmed)
Abstract：
Biological valorization of lignin, the second most abundant biopolymer on Earth, is an indispensable sector to build a circular economy and net-zero future. However, lignin is recalcitrant to bioupcycling, demanding innovative solutions. We report here the biological valorization of lignin-derived aromatic carbon to value-added chemicals without requesting extra organic carbon and freshwater via reprogramming the marine Roseobacter clade bacterium Roseovarius nubinhibens. We discovered the unusual advantages of this strain for the oxidation of lignin monomers and implemented a CRISPR interference (CRISPRi) system with the lacI-Ptrc inducible module, nuclease-deactivated Cas9, and programmable gRNAs. This is the first CRISPR-based regulatory system in R. nubinhibens, enabling precise and efficient repression of genes of interest. By deploying the customized CRISPRi, we reprogrammed the carbon flux from a lignin monomer, 4-hydroxybenzoate, to achieve the maximum production of protocatechuate, a pharmaceutical compound with antibacterial, antioxidant, and anticancer properties, with minimal carbon to maintain cell growth and drive biocatalysis. As a result, we achieved a 4.89-fold increase in protocatechuate yield with a dual-targeting CRISPRi system, and the system was demonstrated with real seawater. Our work underscores the power of CRISPRi in exploiting novel microbial chassis and will accelerate the development of marine synthetic biology. Meanwhile, the introduction of a new-to-the-field lineage of marine bacteria unveils the potential of blue biotechnology leveraging resources from the ocean.IMPORTANCEOne often overlooked sector in carbon-conservative biotechnology is the water resource that sustains these enabling technologies. Similar to the \"food-versus-fuel\" debate, the competition of freshwater between human demands and bioproduction is another controversial issue, especially under global water scarcity. Here, we bring a new-to-the-field lineage of marine bacteria with unusual advantages to the stage of engineering biology for simultaneous carbon and water conservation. We report the valorization of lignin monomers to pharmaceutical compounds without requesting extra organic substrate (e.g., glucose) or freshwater by reprogramming the marine bacterium Roseovarius nubinhibens with a multiplex CRISPR interference system. Beyond the blue lignin valorization, we present a proof-of-principle of leveraging marine bacteria and engineering biology for a sustainable future.
摘要：
木质素的生物增值，地球上第二丰富的生物聚合物，是建设循环经济和净零未来不可或缺的部门。然而,木质素对生物循环具有顽固性，要求创新的解决方案。我们在这里报告了木质素衍生的芳香族碳对增值化学品的生物价值化，而无需通过重新编程海洋玫瑰杆菌进化枝细菌Roseovariusnubinhibens来要求额外的有机碳和淡水。我们发现了该菌株对木质素单体氧化的异常优势，并使用lacI-Ptrc诱导模块实施了CRISPR干扰（CRISPRi）系统，核酸酶失活的Cas9和可编程gRNA。这是R.nubinhibens第一个基于CRISPR的监管系统，能够精确有效地抑制感兴趣的基因。通过部署定制的CRISPRi，我们重新编程了木质素单体的碳通量，4-羟基苯甲酸酯，为了实现原儿茶酸的最大产量，一种具有抗菌作用的药物化合物，抗氧化剂，和抗癌特性，用最少的碳维持细胞生长和驱动生物催化。因此,我们通过双靶向CRISPRi系统实现了原儿茶酸产量的4.89倍提高，该系统用真实的海水进行了演示。我们的工作强调了CRISPRi在开发新型微生物底盘方面的力量，并将加速海洋合成生物学的发展。同时,海洋细菌新领域谱系的引入揭示了蓝色生物技术利用海洋资源的潜力。重要的是，碳保守生物技术中经常被忽视的部门是维持这些使能技术的水资源。类似于“食物与燃料”的辩论，人类需求和生物生产之间的淡水竞争是另一个有争议的问题，尤其是在全球水资源短缺的情况下。这里,我们为同时保护碳和水的工程生物学阶段带来了一种具有独特优势的海洋细菌新领域谱系。我们报告了木质素单体在不需要额外有机底物的情况下转化为药物化合物(例如，葡萄糖）或淡水，通过使用多重CRISPR干扰系统重新编程海洋细菌Roseovariusnubinhibens。除了蓝色木质素的增值，我们提出了利用海洋细菌和工程生物学实现可持续未来的原理证明。
