<p>Methanol is a one-carbon compound that has emerged as a promising carbon source for microbial bioproduction due to its abundance and sustainability. Despite the development of synthetic methylotrophic cell factories, challenges, such as the accumulation of toxic intermediates and suboptimal growth rates, have hindered their industrial application. This review summarizes the engineering strategies for methylotrophic cell factory synthesis, including pathway engineering for methanol assimilation optimization, detoxification methods targeting formaldehyde accumulation, and optimization of cell resource utilization. Future challenges and prospects of advancing microbial methanol assimilation in biotechnological applications are also highlighted.</p>