State Key Laboratory of Microbial Technology, Shandong Universitygrid.27255.37, Qingdao, People's Republic of China.
School of Food Science and Engineering, South China University of Technologygrid.79703.3a, Guangzhou, People's Republic of China.
mSystems. 2022 Apr 26;7(2):e0136621. doi: 10.1128/msystems.01366-21. Epub 2022 Mar 1.
Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid mutagenesis. The combination of growth-based screening with mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis.
丙二酰辅酶 A（malonyl-CoA）是生产各种化学品的重要前体，但由于其可用性较低，限制了酿酒酵母中下游产物的合成。由于代谢的复杂性，需要进行进化工程来开发具有改进的丙二酰辅酶 A 合成能力的菌株。在这里，我们使用之前构建的生物传感器，开发了一种基于生长的筛选系统，该系统将丙二酰辅酶 A 的可用性与细胞生长联系起来。将该系统与连续诱变相结合，能够快速生成全基因组规模的突变文库并筛选出具有更高丙二酰辅酶 A 可用性的菌株。通过全基因组测序和转录组分析对突变株进行分析。组学分析表明，碳通量重新分配到储存碳水化合物和氨基酸合成会影响丙二酰辅酶 A 代谢。通过反向工程，发现新的过程，特别是减少赖氨酸和精氨酸的合成，有助于提高丙二酰辅酶 A 的合成。我们的研究为具有改进代谢物合成能力的突变株的其他高通量筛选方法提供了有价值的补充工具，并提高了我们对丙二酰辅酶 A 合成代谢调控的理解。丙二酰辅酶 A 是生产各种有价值化学品的关键前体。尽管已经进行了合理的工程设计来提高酿酒酵母中丙二酰辅酶 A 的合成，但由于代谢的复杂性，需要进化菌株并分析新的机制来提高丙二酰辅酶 A 通量。在这里，我们开发了一种基于生长的筛选系统，该系统将丙二酰辅酶 A 的可用性与细胞生长联系起来，并操纵 DNA 复制以进行快速诱变。基于生长的筛选与诱变的结合使具有更高丙二酰辅酶 A 可用性的菌株能够快速进化。突变株的全基因组测序、转录组分析以及反向工程表明，削弱赖氨酸和精氨酸合成以及储存碳水化合物的碳通量有助于丙二酰辅酶 A 合成。我们的工作为同时进行菌株筛选和连续进化以提高代谢中间产物提供了指导，并确定了提高丙二酰辅酶 A 下游产物合成的新目标。
