Accelerating Bioprocess Optimization - A series of advancements has changed the way bioprocesses are developed and optimized. - BioPharm International


Accelerating Bioprocess Optimization
A series of advancements has changed the way bioprocesses are developed and optimized.

BioPharm International
Volume 24, Issue 4, pp. 38-44

Figure 2: Mapping differentially expressed genes onto their associated metabolic pathways led to the identification of a truly essential amino acid directly involved with increased protein titers.
The real success of the microarray studies came, however, when we discovered a medium supplement that dramatically enhanced protein production for a chemically defined medium. This particular medium was characterized by high titers when monitored over time. However, at a particular stage during the fermentation process, protein yields suddenly dropped off. We carefully designed a time course microarray experiment, and mapped the resulting differentially expressed genes to their associated metabolic pathways. To our surprise, analyses of interconverting pathways led to the identification of a particular amino acid (see Figure 2). The ability to map differentially expressed genes to their associated pathways clearly made it possible to "zoom" in" and identify a key component that led to medium optimization and that targeted a truly essential amino acid. We also used microarrays to survey the dynamics of gene expression in media with varying productivities, as well as to examine process conditions that enhanced productivity. The results identified genes within the cells cultured in media yielding high product concentrations/titers that are related to growth and cell division and were expressed at significantly higher levels compared to those cells grown in media yielding lower titers. This enabled the cells to remain viable longer at the end of cultivation, when the cell concentration is highest, thus allowing more product molecules to accumulate.

Despite the utility and versatility of DNA microarray technology, it only provides for a qualitative picture of the overall transcriptome and only reveals the activity of genes for which probes are present on the array. Furthermore, based on many recent research reports for both prokaryotic and eukaryotic organisms, we know that cell physiology, as well as many functional cellular and biological processes, including cell cycle progression and induction and suppression of apoptosis, are not entirely dependant on the level of gene expression, but rather controlled by upstream regulatory regions and the increasingly important "non-coding" regions of the genome (e.g., microRNAs/small RNAs) (4,5). This is where NGG comes to the forefront.

Figure 3: A detailed breakdown of a typical mRNA-seq work flow. This work flow is for a bacterial species for which comprehensive genome information is available. Partially adapted from Wilhelm and Landry (2009).
One of the key technologies in NGG is Next Generation Sequencing (NGS). Recent technological advances in DNA sequencing have dramatically improved overall throughput and quality and have led to the development of methods to characterize whole transcriptomes of entire cell populations in a way that was never before possible (6–8). RNA–sequencing (mRNA–seq) involves the direct sequencing of complementary DNAs (cDNAs) using high throughput, massively parallel NGS technologies (Illumina's Genome Analyzer IIx; Illumina's HiSeq2000, Roche's 454 FLX system, to name a few), followed by mapping of the resulting sequencing reads to a reference genome (see Figure 3 for a detailed RNA–seq work-flow diagram).

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