Climate change and population growth has created a higher demand for new sustainable ways to produce energy, food, and materials (i.e. polymers). Single carbon compounds, such as CO2 and CH4, are main greenhouse gases, which contribute to climate change. Yet, because both CO2 and CH4 are virtually unlimited carbon sources, they can represent novel feedstock for industrial bioconversions, and can be exploited as such to establish novel technologies to address 21st century sustainability constraints. This work is centered on Methylomicrobium alcaliphilum 20ZR, a model methanotrophic bacterium, which can take a variety of single carbon compounds as a sole source of carbon. The codon usage bias, ribosomal binding sites (RBSs) and promoters in M. alcaliphilum 20ZR were investigated to have a better understanding of the transcription and translation process in M. alcaliphilum 20ZR. Optimal codon usage bias, a set of strong RBSs and promoter sequences were identified, and validated via heterologous expression of sfGFP (superfolder-green fluorescent protein). The results show that sfGFP-codon usage bias 2 (sfGFP-CUB2), which represents a set of highly expressed proteins in M. alcaliphilum 20ZR, showed the highest levels of expression and thus, it can be recommended for heterologous protein synthesis. Furthermore, the study revealed that using the RBS2 (PtacI) sequence yields a high protein expression, and that using Pslp or Pbhr as a promoter of choice could give the highest protein expression. The newly established mechanisms of protein production in M. alcaliphilum 20ZR will help engineer the system for optimal production of protein-based materials.