Isobutanol production freed from biological limits using synthetic biochemistry

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L−1 h−1, a titer of 275 g L−1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

The manuscript "Isobutanol production freed from biological limits using synthetic biochemistry" described about production of isobutanol by multiple enzymatic reactions without cells.
The authors responded to all the issues pointed out in the peer review process [redacted] and it is considered that the current version of the manuscript is suitable for publication in the journal Nature Communications.

Comments:
As the author commented, so far, the cell-free production of isobutanol is not cost competitive compared to cell-based fermentation and further studies are required for real application (listed in Response to reviewers). However, I agree that this work can be a milestone towards cell-free biomanufacturing and thus I feels that the manuscript should attract much attention to the broad reader of Nature Communications.
Minor point. In vitro production of isobutanol was performed at 30 °C in both small scale reaction and bioreactor. According to the supplementary Fig.2, the activity of SmilvD gradually decreased at 25 °C and SmilvD showed almost no activity over 37 °C within 12 h. Based on this thermal tolerance results, I expect that SmilvD has low thermal stability and it will lose its activity at 30 °C more rapidly than that at 25 °C. However, according to the Fig.5c, the residual activity of SmilvD is almost 45 %. In case of KivD-S, although it had higher thermal tolerance than SmilvD, it showed almost no activity after 70 hours of incubation (Fig.5c). In addition, AlsS-p also almost lost its activity after 70 hours of incubation (Fig.5c) despite of its high thermal tolerance.

Response to Reviewer Comments
Reviewer #1 (Remarks to the Author): I was reviewer #1 in the previous iteration. The authors have done an excellent job addressing the concerns and comments made. I believe this manuscript is ready for publication and will be widely cited as a pivotal study as the cell-free field continues to develop.
Reviewer #2 (Remarks to the Author): The manuscript "Isobutanol production freed from biological limits using synthetic biochemistry" described about production of isobutanol by multiple enzymatic reactions without cells. The authors responded to all the issues pointed out in the peer review process [redacted] and it is considered that the current version of the manuscript is suitable for publication in the journal Nature Communications.
Reviewer #3 (Remarks to the Author): As the author commented, so far, the cell-free production of isobutanol is not cost competitive compared to cell-based fermentation and further studies are required for real application (listed in Response to reviewers). However, I agree that this work can be a milestone towards cell-free biomanufacturing and thus I feels that the manuscript should attract much attention to the broad reader of Nature Communications.
Minor point. In vitro production of isobutanol was performed at 30 °C in both small scale reaction and bioreactor. According to the supplementary Fig.2, the activity of SmilvD gradually decreased at 25 °C and SmilvD showed almost no activity over 37 °C within 12 h. Based on this thermal tolerance results, I expect that SmilvD has low thermal stability and it will lose its activity at 30 °C more rapidly than that at 25 °C. However, according to the Fig.5c, the residual activity of SmilvD is almost 45 %. In case of KivD-S, although it had higher thermal tolerance than SmilvD, it showed almost no activity after 70 hours of incubation (Fig.5c). In addition, AlsS-p also almost lost its activity after 70 hours of incubation (Fig.5c) despite of its high thermal tolerance.
The reviewer raises an important issue as it seems clear that more needs to be done to understand why enzymes inactivate more or less rapidly under the conditions of the bioreactor compared to our test conditions. We discuss this issue at the end of the results section: