Date Published: October 22, 2011
Author(s): Ashish K Sharma, Shubhashree Mahalik, Chaitali Ghosh, Anuradha B Singh, Krishna J Mukherjee.
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.
The wide variability in the expression levels of recombinant proteins in Escherichia coli remains a major challenge for biotechnologists. While some proteins are routinely expressed at 30-40% of total cellular protein (TCP) (Joly and Swartz 1997; Kim et al. 2003; Suzuki et al. 2006), others may reach a maximum of only 5% of TCP (Kiefer et al. 2000). The uses of strong promoters, removal of codon bias and media design are favored strategies for improving recombinant protein yield (Acosta-Rivero et al. 2002; Hale and Thompson 1998). It is important to note that most scale up strategies involving high cell density cultures tend to increase biomass concentrations and hence volumetric product concentrations rather than the specific product yield in terms of product formed per unit biomass (Yp/x). This yield remains an intrinsic property of the host-vector-gene combination used for expression. Improvements in host vector systems has tended to focus on developing high copy number plasmids with strong tightly regulatable promoters (Bowers et al. 2004; Jones et al. 2000; Wild and Szybalski 2004) along with protease free and recombination deficient strains (Meerman and Georgiou 1994; Ratelade et al. 2009). The focus has thus primarily been on enhancing the metabolic flux of the recombinant protein expression pathway, with few studies on analyzing how the gene products interact with the host cell machinery to depress its own expression.
In this work rhIFN-β was expressed as an inclusion body whereas xylanase and GFP were expressed as soluble proteins. While rhIFN-β and xylanase were expressed under a strong promoter (T7) in E.coli BL21 (DE3) cells, GFP was expressed under the ara promoter in an E.coli DH5α strain. Cells were grown exponentially in the bioreactor at a specific growth rate of 0.3 h-1 by using an exponential feed of complex media and induction was done at an OD between 20-25. At this point the feed rate was ~40 ml/h and the OUR was 0.27 moles/l/h, with a Respiratory Quotient (RQ) of 1.1. Since the biomass yield (Yx/s) on glycerol, while using complex nitrogen sources had been previously determined to be between 1-1.1 g/g. The above results matched stoichiometrically and demonstrated complete consumption of substrate feed. A continuous fall in the specific growth rate was observed which dropped to zero within 4 hours of induction. In the post induction phase continuous increase in the OUR was observed which necessitated oxygen supplement of the inlet air after 1 h of induction. From the on-line metabolic activity measurement we could identify 3 phases in the metabolic activity of the culture. In the first phase from the point of induction till 2 hours the activity as measured by OUR, CER and RPM2 kept increasing, even though there was continuous decline in specific growth rate. Clearly a large part of this metabolic activity was diverted towards maintenance (Russell and Cook 1995). The specific product formation rate was high during this period. Since the metabolic activity doubled in this period, the post-induction feed was also increased concomitantly (Ramalingam et al. 2007). In the second phase between 2 to 4 hours the feed was kept constant since the on-line measurement indicated a constant metabolic activity. Finally after 4 hours there was a decline in metabolic activity and the specific product formation rate declined to reach zero in 6 hours. Samples were collected to represent these three phases 2, 4 and 6 hour (post-induction). Figure 2 shows the SDS-PAGE gel picture of rhIFN-β, xylanase and GFP expression profile post induction.
It was observed that the cellular response to the diversion of metabolites for product formation, is at multiple levels directed both at growth rate and protein production. Since growth rate and protein synthesis share common pathways, this stress response hits both processes simultaneously, affirming previous reports on the growth associated nature of recombinant protein production (Bentley et al. 1990; Shin et al. 1998). The stress response first affects the carbon uptake by down-regulating various transporters and this phenomenon was observed for all the conditions irrespective of the nature and level of recombinant protein expression. Simultaneously the carbon utilization and energy generation pathways starting from Glycolysis, TCA to electron transport chain were severely repressed resulting in decreased growth yield, product formation and viability of the cell population as has been shown by Hardiman et al. (2007).
The authors declare that they have no competing interests.