Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica

[摘要] ENGLISH SUMMARY: Exenatide (Byetta®) is a type 2 diabetic drug which decreases the blood glucose level. Treatment using this drug is expensive due to its costly production by chemical synthesis therefore it is not an affordable drug of choice. A potential cost effective alternate for the production of exenatide is the use of recombinant production technology. Yarrowia lipolytica, a dimorphic yeast, was genetically engineered to produce the exenatide peptide (Exe, 39 amino acids) as a fusion to Lip2 (lipase) protein under the regulation of the hp4d promoter by the CSIR. The regulation of the promoter has, until recently, not been elucidated and is currently reported to be growth phase dependent. In order to optimise the conditions for the production of the fused Lip2:Exe peptide precursor, the regulation of the promoter needed to be understood.In this study, the regulation of the hp4d promoter was established and a fed-batch fermentation strategy for the production of the fused Lip2:Exe precursor was developed. A Y. lipolytica strain (YlEx-gly) producing the Lip2:Exe peptide was cell-banked (to ensure stability of the production organism and repeatability of inoculation of fermenters) and the cell-bank was validated for production of the fused peptide. A transcript profile of the recombinant strain harbouring an expression vector encoding the Lip2:Exe under control of the hp4d promoter was determined using an optimised mRNA sandwich hybridisation methodology. Batch fermentation (1.2 l) was used to monitor production profiles during growth of Y. lipolytica followed by continuous fermentation (1 l) to determine the effect of growth rate on the transcription levels of the product under regulation by the hp4d promoter. The synthetic hp4d promoter was found to be growth rate dependent which was confirmed by quantifying the amount of total protein produced during fed-batch fermentations (10 l) at different growth rates. A 60 % increase in production yields was achieved by using the optimised growth rate of 0.02 h-1. This validated that the hp4d is growth rate dependent and not growth phase dependent as reported in literature. A strategy for the recombinant production of pharmaceutical peptides and proteins, under regulation of the hp4d promoter, using Y. lipolytica as a host, was therefore established. This research has paved the way for recombinant production of proteins at a lower cost therefore impacting on the health and economy of South Africa, by providing the public with potentially cheaper, affordable pharmaceutical drugs due to an alternative production strategy.

[发布机构] Stellenbosch University