Expression Of Vitreoscilla Hemoglobin Improves CALB Production In Pichia Pastori And Analysis Of Its Transcriptome

Pichia pastoris heterologous protein expression system is one of the systems for theproduction of a variety of recombinant proteins in microbiology field. It not only has thecharacteristics of simplicity of techniques needed for the molecular genetic manipulation andfast growth, but also possesses the capability of performing many eukaryoticpost-translational modifications. However, during the high density fermentation, largeamounts of oxygen will be consumed by Pichia pastoris. Although, the need of oxygen can bedecreased by improving the agitation, ventilation and regulating the methanol feed rate, itcould increase the cost of the fermentation process.To alleviate the dissolved oxygen (DO) limitation and improve CALB production duringhigh-cell density fermentation, the CALB gene and Vitreoscilla hemoglobin (VHb) gene vgbwere co-expressed in Pichia pastoris under the control of AOX1and YPT1promoter,respectively. SDS-PAGE and Western Blot analysis results indicated that VHb and CALB hadbeen successfully co-expressed in recombinant strains.Compared with the control strain, expression of CALB in GS115/VHb-CALB underoxygen limitation was improved by86.28%, while GS115/ExVHb-CALB andGS115/VHbSKL-CALB have no obvious effects. In addition, the growth ofGS115/VHb-CALB, GS115/ExVHb-CALB and GS115/VHbSKL-CALB are similar to theGS115/CALB. Further, by the RNA-Seq, we got the transcriptome of the GS115/VHb-CALBand GS115/CALB stain.899differentially expressed genes was identified from thetranscriptome. These genes involve in Electron transport chain, the pentose phosphatepathway, TCA cycle and glyoxylate pathway. NAPDH, the production of pentose phosphatepathway, make iron atoms of VHb stay in+2. Therefore, VHb maintains the ability totransport oxygen. The increase of ATP mainly results from the accelerat of electron transportchain. Some genes are upregulated in the pathway from5’-prpp to IMP. IMP is essential in thebiosynthesis of ADP. Because of the biosynthesis of ADP, the turnover of electron transportchain will speed up. Finally, the gene in Malate-aspartate shuttle route is also upregulated. Itseemly suggests that the NADH, which is generated by pentose phosphate pathway and glyoxylate pathway in Cytosolic, can be transported to Mitochondria soon.