Page 45 - Advances in bioenergy (2016)

Numerous nonrational techniques are also available that extend the reach of traditional strain
improvement: high throughput screening, directed evolution, gene shuffling, and combinatorial
engineering. These techniques increase the efficiency of strain improvement by sampling a
much larger phenotypic search space, opening up the possibility to select for changes in less-
intuitive or distal targets. An interesting example is the use of transcriptional engineering to
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achieve phenotypic diversity. This technique involves mutagenesis of transcription factors to
produce global changes in the transcription and expression of genes in the cell, which would
not be feasible through isolated point mutations. 17

Finally, optimizing to maximize flux through the production pathway is often the most difficult
engineering task. Tuning the expression of genes many times is necessary as intermediates and
cofactors need to be balanced within the pathway. Furthermore, troubleshooting the pathway
often involves alleviating any number of potential bottlenecks that may impede flux, such as
competing pathways, lack of enzymatic driving force, cofactor imbalances, insufficient enzyme
activity, unbalanced enzyme expression, transport issues, enzyme regulation, and toxicity.

Because of its interdisciplinary nature, metabolic engineering will be limited to the available
tools and technologies of the fields from which it draws. However, as the state of the art
progresses and development improves at the interface of these related disciplines, metabolic
engineers will have increased power to evaluate, design, and manipulate cells to produce the
desired products that will be in demand in the future. These abilities will most certainly be
necessary for the development of the next generation of biofuels.