Study On Key Technology And Mechanism Of Recombinant Protein Production In Pichia Pastoris

Pichia pastoris expression system is currently the most widely used eukaryoticexpression systems, nearly1,000kinds of heterologous proteins have been expressedusing this system, which has a broad market prospect. However, although with anapplication history of more than30years, no breaktrough has been achived in theoptimization study of Pichia fermentation process. Fermentation is an intermittentsteady biological process involving significant changes in the genome, proteome andmetabolome levels. Therefore, the display of internal changes during fermentationprocess at genomics levels to find the core genes, proteins or small moleculesubstrates leading the steady-state migration, may help to optimize the fermentationprocess. However, few transcriptome studies have been conducted on the Pichiafermentation process.In this study, we tried to use the self-designed gene chips to screen the key genesclosely related to the important time node during the Pichia fermentation process,digging the internal factors affecting the fermentation process and the proteinexpression levels, in order to provide the optimization of the fermentation processwith classic data base and a new perspective.To achieve the research goals, the study was carried out according to following work:1. The construction of Pichia fermentation process mode:GS115yeast strain was selected as the expressing vector because of the clearbackground and well-established fermentation technologies. Two recombinant strains were successfully constructed and were available for stably expression ofreFIP-vvo or rHSA. In comparison study during the pilot scale fermentation test，therHSA strain exhibited a stable growth curve with an more obvious trend which wasidentical to the typical characteristics of methanol inducible expression system.Therefore, the final selection of rHSA GS115was chosen for the transcriptomestudy using gene chips.2. Preparation of Pichia microarray:GeneMark method was used for predicting GS115Pichia genome exon, aftermulti-step screening, validation and optimization steps, we independently designed aGS115Pichia microarray contains5040genes, and completed the standardization andpreparation work.3. Microarray analysis of Pichia fermentation process:As it was explained above, we designed a microarray independently. The microarraywas used to investigate the transcriptomics related to the key points in the classicfermentation of Pichia pastoris in this study. The study focused on two biologicalprocesses induced by carbon source replacement and methanol-induce expression.Furthermore, concerning the importance of oxygen molecules in fermentation, therole of the oxidation-reduction-related genes were specified in the aerobic metabolicflux drift by using a new tool of building the redox function trees.This study obtained three main results as follow:1. We designed and prepared the Pichia GS115microarray, which filled with blank inthe fields at home and abroad, thus provides an important research tool on thetranscriptome study of Pichia in various areas.2. It was found that the transcriptional regulation changed most significantly in the phase of limiting carbon replacement. In this phase, the biological function oftranscription, translation, protein/nucleic acid synthesis were strongly inhibited.Moreover, the rHSA protein can be barely detected in the glycerol-feeding phase,indicating that AOX1may be induced by glycerol consistent with the recent findings.These results suggest that the traditional carbon starvation may be unnecessary andharmful to the cells. As suggested by a number of scholars recently, the glycerol/methanol mixed feeding process is more scientific. The gradual increasing proportionof additional methanol is used for the final completely replacement. This process canavoid the extreme stress during the fermentation process, and is consistent with theprinciples of the gradient fermentation design.3. Two core genes, namely PAS_chr2-1₀582and PAS_chr3₀845, which possiblyled to aerobic-anaerobic metabolic drift was screened out. PAS_chr2-1₀582may beone of most important transcription factors in Pichia aerobic metabolism regulationand control. PAS_chr3₀845possibly assists the drift between anaerobic and aerobicmetabolism through sub-function of auxiliary respiratory electron transport chain.In-depth study of the two may help to optimize the Pichia fermentation process,enhancing the expression of target proteins.In this study, the use of the microarray enabled us to investigate and demonstrate thekey genes and functions in the aerobic metabolism drift and the two biologicalprocesses, limiting carbon source replacement and methanol-induce expression,using the transcriptomic approach. The key points in the classic fermentation processof Pichia pastoris were also systematically analyzed. This study shed lights onfurther optimization of the classic fermentation process.