Dynamic metabolic engineering: New strategies for developing responsive cell factories

Abstract

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up- and downregulation of genes to redistribute steady-state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high-throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade-offs between growth and production to be better managed and can help avoid build-up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.

Keywords: Gene expression; Metabolic engineering; Metabolite sensors; Synthetic biology.

Figure 1

Typical implementations of dynamic control in metabolic engineering. For “pathway balancing”, buildup of an undesired intermediate is used to trigger repression of upstream enzymes and activation of downstream enzymes. The sensing of intermediate buildup may be direct (binding of the target small molecule) or indirect (sensing of cofactor imbalance, growth inhibition). “Pathway redirection” is typically associated with splitting intermediate flux between cellular growth and energy production and a pathway for a desired product. In this case, some information about cellular state, such as biomass concentration, is used to trigger knockdown of enzyme(s) in the cell’s native metabolism and/or upregulation of the pathway toward the desired product.

Figure 2

Strategies for dynamically modulating enzyme activity. After sensing of a relevant condition or small molecule, control of enzyme activity can be exerted at the transcriptional, post-transcriptional, and post-translational level. At the transcriptional level, interactions between transcription factors and relevant small molecules can be exploited to activate or repress gene expression. At the post-transcriptional level, use of RNA aptamers can provide a method for controlling translation of the relevant mRNA. Control at the post-translational level is possible in some cases using strategies such as engineered allostery.