Heterologous protein expression enhancement of Komagataella phaffii by ammonium formate induction based on transcriptomic analysis

Abstract

Heterologous protein expression by Komagataella phaffii (K. phaffii) attracts attention of researchers and engineers for high level of heterologous protein, strict pattern of alcohol oxidase 1 promoter (PAOX1), and high cell density fermentation. It is urgent to increase level of heterologous protein by K. phaffii using formate, the only substitute of methanol as inducer, because methanol is toxic and explosive, especially, in industrial scale. A recombinant K. phaffii expressing green fluorescent protein (GFP) was constructed and sequenced by RNA-seq technology based on methanol and ammonium formate (AF) optimization. The transcriptomes were analyzed using COG, GO, KEGG databases with the result of 1172 identical up-regulated genes at both conditions of methanol and AF induction. The transcription levels of 173 genes and 144 genes at AF induction were higher and lower significantly than that of methanol induction respectively within 1172 identical genes. Low energy generation pathway was considered as the reason of low GFP expression level at AF induction based on analysis of 173 and 144 genes. This result was confirmed by nutrients addition obtaining 30.6 % increment of GFP intensity at 2 g/L ATP addition. This result provided a bright future of high-level heterologous protein production by K. phaffii of AF induction.