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Review
. 2019 Dec:60:221-229.
doi: 10.1016/j.copbio.2019.05.003. Epub 2019 Jun 14.

A critical comparison of cellular and cell-free bioproduction systems

Nico J Claassens  1 , Simon Burgener  2 , Bastian Vögeli  2 , Tobias J Erb  3 , Arren Bar-Even  4
Affiliations
Review

A critical comparison of cellular and cell-free bioproduction systems

Nico J Claassens et al. Curr Opin Biotechnol. 2019 Dec.

Abstract

Conversion of biological feedstocks into value-added chemicals is mostly performed via microbial fermentation. An emerging alternative approach is the use of cell-free systems, consisting of purified enzymes and cofactors. Unfortunately, the in vivo and in vitro research communities rarely interact, which leads to oversimplifications and exaggerations that do not permit fair comparison of the two strategies and impede synergistic interactions. Here, we provide a comprehensive account for the advantages and drawbacks associated with each strategy, and further discuss recent research efforts that aim to breach the limits of cellular and cell-free production. We also explore emerging hybrid solutions that integrate the benefits of both worlds and could expand the boundaries of biosynthesis.

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Figures

None
Graphical abstract
Figure 1
Figure 1. Key advantages of cellular and cell-free bioproduction.
Advantages connected to both strategies exemplify features that can be at least partially obtained in both cellular and cell-free systems.
Figure 2
Figure 2. Isobutanol production exemplifies advantages of cellular and cell-free approaches.
Key advantages and pathways are color-marked green for cellular production and purple for cell-free production. Isobutanol production in engineered in vivo in E. coli reaches a titer of 23 g/L and 86% of the theoretical yield [78]. The first in vitro demonstration of isobutanol production was based on an ATP-independent glycolysis pathway, including the promiscuous gluconate and glycerate dehydrogenase activity of di-hydroxy-acid dehydratase (DHAD) [12]. A more recent in vitro system achieved dynamic cofactor balancing by a glycolytic ‘rheostat’ that combines a non-phosphorylating, NADPH-regenerating GapN with a mutant GapDH that can regenerate NADPH while generating ATP [31]. This design enabled dynamic balancing of cofactor consumption and regeneration during isobutanol production and supported a high isobutanol titer of 24 g/L at 91.5% theoretical yield. While isobutanol toxicity was a major limitation of cellular production, in vitro production could sustain higher titer with little adversary effects. However, the cell-free system is stable for 48 days due to enzyme inactivation, emphasizing the advantage of cellular production, in which the catalysts are produced at low cost.
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