A Combined Bio-Chemical Synthesis Route for 1-Octene Sheds Light on Rhamnolipid Structure

Round 1

Reviewer 1 Report

The present contribution by Blank and co-workers is a new instalment of their previous studies on rhamnolipid-type derivatives (see for example:  Applied and Environmental Microbiology (2020), 86(6), e02317; Journal of Chromatography A (2016), 1465, 90; Journal of Chromatography A (2016), 1455, 125). In this case, the authors studied a chemoenzymatic process for the conversion of carbohydrates into higher olefins by the combination of the biological conversion of glucose to unsaturated fatty acids using a whole-cell biocatalyst with a crossed metathesis catalyzed by Grubbs-type ruthenium complexes. In general the manuscript is well-written in an easy to follow manner. However, there are very important points that the authors should clarify before publication in Catalysis:

1.- The main concern of this reviewer is related with the fact that the authors never commented in the main text of the manuscript the plethora of previous combinations of Grubbs-type catalyzed metathesis processes with different biotransformations (see for example: Angew. Chem. Int. Ed. 2016, 55, 14823; ChemCatChem. 2018, 10, 1192; Front. Chem., 2020, 8, 139). Thus, it is mandatory that the authors clearly comment which is the state of the art of this combination.

2.- The authors should comment that other chemoenzymatic process have been recently reported. Please also add some revisions in this field (for example: Chem. Eur. J. 2018, 24, 1755; Catalysts 2018, 8, 75)

3.- One of the main problems of the reported chemoenzymatic process is related with the fact that two purification steps are needed after the biocatalyzed procedure. It is not possible to use surfactants to prevent this problem?? See previous work in the field by Lipshutz (Nat. Commun., 2019, 10, 2169)

4.- Line 150 “several similar species were present”. Authors should clearly enumerate and identify all the species that they observe experimentally.

5.- line 203 “as can be assume from the spectrum of the produced HAA”. For clarity, the authors should present in the main text of the manuscript this spectrum.

Thus, I can only recommend publication of this work in Catalysts after major revisions

Author Response

We thank Referee 1 for the general comment, that the manuscript is well-written and easy to follow. The referee recommends publication of this work, after the following points have been addressed.

Comment: The main concern of this reviewer is related with the fact that the authors never commented in the main text of the manuscript the plethora of previous combinations of Grubbs-type catalyzed metathesis processes with different biotransformations (see for example: Angew. Chem. Int. Ed. 2016, 55, 14823; ChemCatChem. 2018, 10, 1192; Front. Chem., 2020, 8, 139). Thus, it is mandatory that the authors clearly comment which is the state of the art of this combination.

Addressed. We thank the referee for raising the attention to the combination of olefin metathesis with different biotransformations. We added the following paragraph to the introduction:

“Besides other metal-based catalysts [41-44], the relatively high stability of the Grubbs-Hoveyda type catalysts allows the combination of metal catalysis and proteins or enzymes in one pot in a concurrent or sequential fashion. Thereby, novel synthetic routes are investigated that enable the efficient synthesis of compounds starting from different resources such as renewables. The biorthogonality of the olefin metathesis reaction does not interfere with in the reaction or with co-substrates of the enzymes. Nonetheless, compatibility challenges need to be overcome. Commonly, cell- or media components inhibit metal catalysis [42,43,45]. Overcoming these challenges involves the utilization of compartmentalization strategies [42,46], utilization of biphasic systems [47], generation of artificial metalloproteins capable of catalyzing the olefin metathesis reaction [48-52] or, if the enzyme allows, the utilization of co-solvents [53-55].”

This paragraph contains the suggested references of the referee now as references 46 (Angew. Chem. Int. Ed. 2016, 55, 14823), 47 (ChemCatChem. 2018, 10, 1192) and 55 (Front. Chem., 2020, 8, 139). Additionally, we added 11 more references (References 38 to 52) to give a balanced overview of the combinations of Grubbs-Hoveyda type catalysts and proteins/enzymes.

Comment: The authors should comment that other chemoenzymatic process have been recently reported. Please also add some revisions in this field (for example: Chem. Eur. J. 2018, 24, 1755; Catalysts 2018, 8, 75)

Addressed. We added the following sentence in line 69 of the introduction:

“Besides other metal-based catalysts [41-44], the relatively high stability of the Grubbs-Hoveyda type catalysts allows the combination of metal catalysis and proteins or enzymes in one pot in a concurrent or sequential fashion.”

The suggested references from the referee can be found under reference 43 (: Chem. Eur. J. 2018, 24, 1755) and 41 (Catalysts 2018, 8, 75). Additionally, we added two more reviews under reference 42 and 44, respectively, to highlight recent efforts in the area of combining metal and enzymatic catalysis.

Comment: One of the main problems of the reported chemoenzymatic process is related with the fact that two purification steps are needed after the biocatalyzed procedure. It is not possible to use surfactants to prevent this problem?? See previous work in the field by Lipshutz (Nat. Commun., 2019, 10, 2169)

We thank the referee for bringing attention to the work of Lipschutz and coworkers. Indeed, performing steps in a one-pot fashion would likely decrease the effort and costs of purification of intermediates. However, in our present manuscript, we need to purify the intermediate, because the amount of the C10:12-1 congener containing the double bond is unfortunately relatively low; we are currently working on strategies, to improve the yield of this particular congener. For the reaction itself, we are testing biphasic conditions or other compartmentalization techniques.

The biggest challenge we are currently facing is the utilization of ethylene within the cascade. Since we do not perform standard cross-metathesis with, e.g., disubstituted butene derivatives like cis-2-butene-1,4-diyl diacetate, the utilization of ethylene is mandatory. The problem is, that the reaction needs to be performed under a certain pressure, because the solubility of ethylene in aqueous media is relatively low. To this end, applying gas pressure (e.g., 5 bar or more) within a cultivation setup that requires a certain amount of oxygen for the proper growth of cells is simply impossible. To increase the solubility of ethylene, co-solvent (e.g., THF) might help, but again, this is highly contra productive for cell growth. Nonetheless, we will try to include this valuable suggestion of the referee in our current study, where we try to increase the C10:12-1 congener content.

Comment: Line 150 “several similar species were present”. Authors should clearly enumerate and identify all the species that they observe experimentally.

Done. We added the following paragraph in lines 161-165:

“This mixture is comprised of the two ethenolysis products owning now terminal double bonds as well as the cleaved styrene derivative after initiation of the metathesis reaction. Furthermore, cross-metathesis reaction of the reaction partners after release of ethylene cannot be excluded, even though the catalyst was quenched with ethoxyethene.”

Comment: line 203 “as can be assume from the spectrum of the produced HAA”. For clarity, the authors should present in the main text of the manuscript this spectrum.

Addressed. We shifted the figure S1 from the supporting information into the main-text and can be found now as Figure 4 in line 154.

Reviewer 2 Report

The manuscript reports 1-octene synthesis via ethenolysis of rhamnolipids. The subject is topic because it describes the way of alkene generation from renewable resources. I recommend its publication after minor revision.

• When speaking about the application of olefin metathesis for fine chemicals and fuel components preparation from renewable resources following references are worth to be added: Green Chem. 16 (2014) 4728 (jojoba oil metathesis), Catal. Today 304 (2018) 127 (cardanol metathesis), and Energy 116 (2016) 177 (lignocellulosic biomass utilization).
• Some experimental details are missing:

1. the source of G.-H. catalysts;
2. the purity of ethylene;
3. the temperature of ethenolysis in NMR tube.

• 5 – within 80 min the HAA is completely consumed, however, 1-octene production continues till about 140 min. How is it possible?

Author Response

Referee 2 recommends publication after the minor revisions indicated:

Comment: When speaking about the application of olefin metathesis for fine chemicals and fuel components preparation from renewable resources following references are worth to be added: Green Chem. 16 (2014) 4728 (jojoba oil metathesis), Catal. Today 304 (2018) 127 (cardanol metathesis), and Energy 116 (2016) 177 (lignocellulosic biomass utilization).

Added in the manuscript. The references mentioned by the referee have been added in to the manuscript as reference 38 (Energy 116 (2016) 177 (lignocellulosic biomass utilization)), 39 (Catal. Today 304 (2018) 127 (cardanol metathesis)) and 40 (Green Chem. 16 (2014) 4728 (jojoba oil metathesis)).

Comment: Some experimental details are missing:

1. the source of G.-H. catalysts;
2. the purity of ethylene;
3. the temperature of ethenolysis in NMR tube.

Added in the manuscript. The missing details have been added:

1. source of the G.-H. catalysts (line 325);
2. the purity of ethylene (line 328);
3. the temperature of ethenolysis in NMR tube (line 188).

Comment: Within 80 min the HAA is completely consumed, however, 1-octene production continues till about 140 min. How is it possible?

Addressed. The integration of NMR signals is very challenging and may have faults, especially during the conversion process when several similar species occur. Additionally, the sensitivity of NMR is relatively low compared to GCMS. For clarity, we added the different species in line 163:

“This mixture is comprised of the two ethenolysis products owning now terminal double bonds as well as the cleaved styrene derivative after initiation of the metathesis reaction. Furthermore, cross-metathesis reaction of the reaction partners after release of ethylene cannot be excluded, even though the catalyst was quenched with ethoxyethene.”

And additionally, we added a comment after the figure describing the time-conversion analysis to comment on the concern raised by the referee (line 193):

“Under these conditions, conversion of the rhamnolipid was complete within 80 min corresponding to quantitative conversion of the substrate. Noteworthy, due to overlapping signals of the mixtures of products during the conversion of the rhamnolipid, the end of octene formation was observed after 130 minutes”.

Round 2

Reviewer 1 Report

This manuscript reported by Blank and et al. presents the design of a new chemoenzymatic process for the conversion of carbohydrates into higher olefins by the combination of the biological conversion of glucose to unsaturated fatty acids using a whole-cell biocatalyst with a crossed metathesis catalysed by Grubbs-type ruthenium complexes. As previously commented, the manuscript is well-written and the results are clearly presented. The authors have answered/commented all the previous question pointed out by the referee. Thus, this new version of the manuscript is now suitable for publication in Catalysts.