Application of  Biotechnology Tools to Biofuels Processes

Technologies for biofuel production vary in the degree of process maturity from highly refined corn ethanol and FAME biodiesel production to emerging technologies for production of ethanol and thermochemical biodiesel, not to mention nascent renewable gasoline production schemes. Therefore, these technologies provide varying degrees of potential improvement by advanced biotechnology tools collectively called system biology tools. A system biology approach essentially examines a biological process holistically at the system or total biological process level, including the system’s genetic content (genome), gene expression, enzyme and protein production, flow of metabolic intermediates and resulting interaction with the genome and enzymatic processes. An example of application of system biology tools is the examination of differential expression of the genome by transcriptomic analysis using whole genome microarray methods where theoretically the degree of differential expression of every gene can be examined. Such an approach provides potential insights into the interaction of multiple gene systems and pathways. A companion biological tool is examination of the proteins present within a biological system, called a proteome, using advanced protein processing and analysis tools.

Combining the transcriptome and the proteome data and analysis of results can provides unparalleled information needed to begin to understand the functioning of a biological system. Critical to most of these system biology tools is access to the genomic sequence data as they form the foundation of gene expression and proteome analysis. Fortunately, genome sequence technology is advancing at an immense pace where bacterial genome sequencing is measured in hours and genome maps can be constructed and genes identified in weeks. This genomic information is used to construct microarrays using selected oligonucleotides for coding regions of the genome. The oligonucleotides are attached to a solid support and used for hybridization studies with labeled cDNA produced from RNA from selected biological samples. Due in part to the vagaries of nucleic hybridization, differential hybridization is often used with RNA from two samples from different biological conditions labeled with different molecular tags. The microarray is able to detect difference in expression of specific genes by the ratio of hybridization by competing labeled RNAs produced from two different experimental conditions. Use of this technology is growing rapidly, spawning numerous companies and supportive products with improved data sets.
Analysis of proteins expressed by a biological system, called proteomics, can provide valuable information regarding the biological process functioning in a specific environment especially regarding the presence of specific enzymes for known biological processes. Like transcriptomics, proteomic analysis of protein expression requires knowledge of the system’s genome sequence, whether it is a complex biological system with multiple (micro)organisms or a biological process functioning with a single (micro)organism. Early proteomic analysis examined proteins using two-dimensional gel analyses, which is a particularly challenging technique to obtain acceptable separation of the thousands of proteins present in microbial, let alone eukaryotic, cells. Gel-based proteomic analysis has largely been replaced with mass spectrometric analysis aided by various initial sample separation technologies such as liquid chromatography. With advanced separation and detection instrumentation, detection of nearly all the soluble proteins present in a microorganism, called the ‘whole proteome’, can be accomplished.
However, the proteome analysis can miss poorly expressed proteins and hard to capture hydrophobic and membrane proteins. Of particular interest are the alterations in the enzymes and other proteins present in a biological system as conditions change and the biological system responds to a changing environment. However, both proteomics and transcriptomics are limited to the degree of accuracy of gene calling where the gene function has been assigned to open reading frames in the genome. Additionally, the function of numerous theoretical genes or open reading frames remains unknown even within the genome of E. coli, the most studied organism, limiting full understanding of a biological system. An additional challenge is that the
reason for the change in thousands of genes seen by differential transcriptomic or proteomic analysis is often unknown due the lack of holistic knowledge of the gene expression cascades at the organism level.

During biofuel production, the cell’s metabolic content, called metabolome, changes in response to the fermentation conditions. Through knowledge of the internal and external biochemicals present, which are the result of ongoing metabolic activity, much can be learned about the overall operation and functioning of the biological system under investigation. With temporal evaluation of metabolism, particularly after a perturbation or substrate addition, the flow of biological intermediates and precursor–product relationships can be determined. This analysis of the flow of metabolic intermediates, or fluxomics, is valuable both to understand the functions of a biological system and to determine metabolic responses to environmental or process changes or stimuli. A true systems biology analysis of a biological process will involve all the aforementioned ‘omics’ analysis tools to understand an ongoing biological process. A prime example of systems biology tools application is the process of bacterial sporulation, where known environmental stimuli (such a nitrogen deficiency) lead to a cascade of gene and protein expression culminating in the construction of biological survival system, the bacterial endospore. All these ‘omics’ tools have the potential also to delineate the biological processes under way during biofuels production, thus potentially providing knowledge whose use may provide keys to making significant improvements in the microorganism and engineered processes, yielding more economical biofuels production.