Abstract
Centromeres are the chromosomal sites at which the kinetochore forms and attaches to spindle microtubules, directing chromosome segregation. Plasmids based on centromeres can maintain stability and distribute accurately during cell division, supporting them as effective genetic tools for research and biotechnological applications. Here, the centromeric regions on seven chromosomes of Ogataea polymorpha, a methylotrophic non-conventional yeast with great potential in biotechnology, were located by ChIP-seq with the native Cse4. The actual centromeric sequences of chromosomes 1, 2 and 5 were characterised, which are very different from those of other eukaryotes and each unique. Although long terminal repeats (LTR) were found in these centromeres, they are 'solo LTR elements' and not important for the function of centromeres. Hence, the O. polymorpha centromeres were categorised into small regional centromeres. O. polymorpha centromeric plasmids were constructed for the first time, which exhibit high genetic stability and compatibility. Application potential for multigene or multicopy expression was validated by the production of uricase and triterpene squalene. This research elucidates the structural features of O. polymorpha centromeres and constructs new, stable centromeric plasmids, expanding the phenomenal diversity of centromeres and providing a powerful genetic toolbox for multi-node and multiple-layered engineering of cell metabolism and physiological functions.