Substitution of the 5'-untranslated region in the alcohol oxidase 1 promoter of Komagataella phaffii alleviated the repression in the presence of glycerol.

Komagataella phaffii (K. phaffii) alcohol oxidase 1 promoter (PAOX1) is repressed in the presence of glycerol, and induced by methanol when methanol is the only carbon source, which is considered as the core feature of this cell factory for recombinant protein production. It was generally accepted that the 5´ untranslated region (5´ UTR) was involved in post-transcription processing and RNA transport. In this study, the 5' UTR of PAOX1 was replaced by 5´ UTR of constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) which was not inhibited by glycerol. Modification of PAOX1 alleviated its repression at the condition of mixed carbon sources of glycerol and methanol, especially at 0.67 - 1 % glycerol addition and 1 % methanol. Presence of protein encoded by PAS_chr4_0626 was considered as the key part of PAOX1 repression in glycerol according to sequence analysis by JASPAR. Therefore, the substitution of the 5´ UTR or knock out of PAS_chr4_0626 attenuated PAOX1 inhibition by glycerol, demonstrating the potential application of heterologous protein production under the condition of mixed carbon sources of methanol and glycerol.