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，它们可以被有机体转化为生物活性形式的维生素。维生素A缺乏症(VAD)是一个世界性问题，尤其是在发展中国家。据估计，VAD的严重后果(失明、夜盲、死亡)影响到全世界大约三分之一的五岁以下儿童。工业规模化生产主要(90%)是通过化学合成实现的，但对天然产生的类胡萝卜素的需求正在增加。由于微生物能够通过胡萝卜素的生成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的一种有前途的替代方法。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是必不可少的。微生物生产宿主自然具有复杂的新陈代谢，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，并导致不满意的产量。通过系统地删除可有可无的基因，可以产生代谢途径减少的微生物底盘菌株，它仍然保持了亲本菌株的遗传稳定性和抗逆性。这些菌株为进一步工程实现工业生物技术的高效生产菌株奠定了良好的基础。直到最近，谷氨酸棒杆菌C1*菌株才在简化基因组文库的基础上通过组合删除而构建。C1*基因组减少了13.4%(412个缺失基因)，但在系统缩减基因组的过程中，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。该项目的重点是通过从头合成包含必需基因的基因组区域来构建谷氨酸杆菌的最小基因组。同时，β-胡萝卜素的生物合成将在已经建立的生产菌株中进行优化。随后，将这种优化的合成途径引入到谷氨酸菌底盘菌株中，首先展示了最小基因组菌株作为合成有机体的优势，同时获得了用于工业化生产维生素A的高效生产菌株。在此β-胡萝卜素平台菌株的基础上，可以进一步开发合成相关类胡萝卜素的生产菌株，如叶黄素、玉米黄质或虾青素。

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.