Engineering Corynebacterium glutamicum towards high yield vitamin A production

在人体内，维生素A对生长发育、维持免疫系统和良好的视力起着至关重要的作用。它指的是一组维生素，最著名的是β-胡萝卜素，它可以被生物体转化为维生素的生物活性形式。维生素A缺乏症（VAD）是一个世界性的问题，特别是在发展中国家。据估计，VAD（失明、夜盲症、死亡）的严重后果影响到全世界大约三分之一的五岁以下儿童。工业化大规模生产主要（90%）通过化学合成实现，但对天然生产的类胡萝卜素的需求正在增加。由于微生物能够通过生成胡萝卜素形成不同的类胡萝卜素，因此微生物生产过程可能是合成维生素a的一个有前途的替代方法。为了提供大量的β-胡萝卜素，降低生产成本和实现更高的生产率至关重要。微生物生产宿主天生具有复杂的代谢，这在生物反应器的非自然环境中是不可取的，因为它会对发酵过程产生负面影响，导致产量不理想。通过系统地删除不必要的基因，可以产生代谢途径减少的微生物底盘菌株，这些菌株仍然保持遗传稳定性和与亲本菌株一样的耐受性。这些菌株为工业生物技术的高效生产菌株的进一步工程设计提供了有希望的基础。仅在最近，菌株谷氨酰胺棒状杆菌C1*是通过基于还原基因组文库的组合缺失构建的。C1*基因组减少了13.4%（412个缺失基因），但在系统地减少基因组的过程中发现一些基因簇缺失是不相容的。因此，进一步的底盘优化需要替代策略。本项目旨在通过包含必需基因的基因组区域的重新合成，构建谷氨酰胺最小基因组。与此同时，β-胡萝卜素的生物合成将在已经建立的生产菌株中得到优化。随后，将优化后的合成途径引入C. glutamicum底盘菌株，首先展示最小基因组菌株作为合成生物的优势，同时获得维生素a工业生产的高效生产菌株。基于该β-胡萝卜素平台菌株，可以进一步开发合成叶黄素、玉米黄质或虾青素等相关类胡萝卜素的生产菌株。

In the human body, Vitamin A plays an essential role for growth and development, the maintenance of the immune system and good vision. It refers to a group of vitamers, most notably β-carotene, which can be converted into the bioactive form of the vitamin by organisms. Vitamin A deficiency (VAD) is a worldwide issue, especially in developing countries. The severe consequences of VAD (blindness, night blindness, death) are estimated to affect approximately one third of all children younger than five around the world. Industrial large-scale production is mainly (90%) realized by chemical synthesis, but the demand for naturally produced carotenoids is increasing. As microorganisms are capable of forming different carotenoids via the carotenogenesis, a microbial production process could represent a promising alternative for synthesizing vitamin A. In order to provide high amounts of β-carotene, it is essential to reduce production costs and achieve higher productivity. Microbial production hosts are naturally equipped with a complex metabolism, which is undesirable in the non-natural environment of a bioreactor, as it can negatively impact fermentation processes and lead to unsatisfactory yields. By systematic deletion of dispensable genes, microbial chassis-strains with reduced metabolic pathways can be generated, which still maintain the genetic stability and stress tolerance as the parental strain. These strains represent a promising basis for further engineering towards highly efficient production strains for industrial biotechnology. Only recently, the strain Corynebacterium glutamicum C1* was constructed by combinatorial deletions based on a library of reduced genomes. The C1* genome is reduced by 13.4% (412 deleted genes), but in the course of systematically reducing the genome some gene cluster deletions were found to be incompatible. For this reason, further chassis optimization requires alternative strategies. The proposed project focuses on the construction of a minimal genome of C. glutamicum by de novo synthesis of genomic regions containing essential genes. In parallel, β-carotene biosynthesis will be optimized in an already established production strain. Subsequently, this optimized synthesis pathway should be introduced into the C. glutamicum chassis strain, to first demonstrate the advantages of a minimal genome strain as synthesis organism and at the same time obtain an efficient production strain for the industrial production of vitamin A. Based on this β-carotene platform strain, further production strains for the synthesis of relevant carotenoids like lutein, zeaxanthin or astaxanthin can be developed.