Chen Zipeng, Wu Yanling, Qin Lingfeng, Wang Chen, Li Zhixin, Luo Xiaozhou, Wei Wei, Zhao Jing
State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China.
Shenzhen Key Laboratory for the Intelligent Microbial Manufacturing of Medicines, Key Laboratory of Quantitative Synthetic Biology, Center for Synthetic Biochemistry, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, PR China.
Synth Syst Biotechnol. 2025 Apr 10;10(3):816-826. doi: 10.1016/j.synbio.2025.04.004. eCollection 2025 Sep.
Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, plays critical roles in diverse biological processes spanning blood coagulation, immunomodulation, and post-translational protein modifications in eukaryotes. Notably, long-chain polyP (>100 phosphate units) exhibits distinct biological functionalities compared to shorter-chain counterparts. While serves as a promising microbial platform for polyP biosynthesis, the genetic regulatory mechanisms underlying polyP metabolism remain poorly elucidated. Here, we systematically investigated the genetic determinants governing intracellular polyP levels and chain length dynamics in yeast. Through screening a library of 55 single-gene knockout strains, we identified six mutants (, , , , , and ) exhibiting elevated polyP accumulation, whereas deletions of , , , , , , , , , and resulted in near-complete polyP depletion. Subsequent combinatorial deletions in the background revealed that the double mutant achieved synergistic enhancement in both polyP concentration (53.01 mg-P/g-DCW) and chain length, attributable to increased ATP availability and reduced polyphosphatase activity. Leveraging CRISPR/Cas9-mediated overexpression in , we engineered strain PP2 ( overexpression), which demonstrated a 2-fold increase in polyP yield (62.6 mg-P/g-DCW) relative to wild-type BY4741, with predominant synthesis of long-chain species. Mechanistically, qRT-PCR analysis confirmed that PP2 exhibited 46-fold up-regulation of coupled with down-regulation of polyphosphatases encoding genes, , , and . This study performed a systematic study of regulating inorganic polyphosphates production in yeast and provides a synthetic biology strategy to engineer high-yield polyP-producing strains, advancing both fundamental understanding and biotechnological applications.
无机多聚磷酸盐（polyP）是一种由正磷酸盐残基组成的线性聚合物，在真核生物的多种生物过程中发挥着关键作用，这些过程涵盖血液凝固、免疫调节和翻译后蛋白质修饰。值得注意的是，与短链多聚磷酸盐相比，长链多聚磷酸盐（>100个磷酸单位）具有独特的生物学功能。虽然[具体微生物名称]是多聚磷酸盐生物合成的一个有前景的微生物平台，但多聚磷酸盐代谢的遗传调控机制仍未得到充分阐明。在这里，我们系统地研究了控制酵母细胞内多聚磷酸盐水平和链长动态的遗传决定因素。通过筛选55个单基因敲除菌株的文库，我们鉴定出6个突变体（[具体突变体名称1]、[具体突变体名称2]、[具体突变体名称3]、[具体突变体名称4]、[具体突变体名称5]和[具体突变体名称6]）表现出多聚磷酸盐积累增加，而[具体基因名称1]、[具体基因名称2]、[具体基因名称3]、[具体基因名称4]、[具体基因名称5]、[具体基因名称6]、[具体基因名称7]、[具体基因名称8]、[具体基因名称9]和[具体基因名称10]的缺失导致多聚磷酸盐几乎完全耗尽。随后在[具体菌株名称]背景下的组合缺失表明，[具体双突变体名称]双突变体在多聚磷酸盐浓度（53.01 mg-P/g-DCW）和链长方面都实现了协同增强，这归因于ATP可用性的增加和多磷酸酶活性的降低。利用CRISPR/Cas9介导的[具体基因名称]在[具体菌株名称]中的过表达，我们构建了PP2菌株（[具体基因名称]过表达），相对于野生型BY4741，该菌株的多聚磷酸盐产量增加了2倍（62.6 mg-P/g-DCW），主要合成的是长链物种。从机制上讲，qRT-PCR分析证实PP2中[具体基因名称]的表达上调了46倍，同时编码多磷酸酶的基因[具体基因名称11]、[具体基因名称12]和[具体基因名称13]的表达下调。本研究对酵母中无机多聚磷酸盐生产的调控进行了系统研究，并提供了一种合成生物学策略来构建高产多聚磷酸盐生产菌株，推进了基础理解和生物技术应用。
