Application of a Pyruvate-Producing Escherichia coli Strain LAFCPCPt-accBC-aceE: A Case Study for d-Lactate Production

Round 1

Reviewer 1 Report

The manuscript "Application of a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE: a case study for D-lactate production" was written well. The study is well-done and the approach is interesting.
The authors should make the following minor changes:
From my point of view, authors should emphasize the possible practical applications of the new LAC strain, in the discussion and conclusions.
In the caption of figure 3 it is necessary to add the description of the symbols.

Author Response

Dear, Reviewer #1

>> Thank you for your review. Explanations to your comments are as follows.

1. The authors should make the following minor changes: From my point of view, authors should emphasize the possible practical applications of the new LAC strain, in the discussion and conclusions.

>> Thank you for your suggestion. As your comment, we added the description about the possible practical applications of the new LAC strain in the discussion (L216-218, L245-246) and the conclusions (L269-271).

2. In the caption of figure 3 it is necessary to add the description of the symbols.

>> As your comment, we added the description of the symbols in the caption of Figure 4 in the revised manuscript (L166-167).

Reviewer 2 Report

The manuscript by Wada and coworkers presents the application of the pyruvate-producing E. coli (PYR) to lactate production by expressing the gene encoding lactate dehydrogenase (DLDH). The authors constructed LAC strain by introducing DLDH gene into PYR strain, and the new LAC strain achieved product conversion from pyruvate to lactate. The progress has been made by three investigations: 1) employing T7 promoter for DLDH gene, 2) optimizing the aeration condition, and 3) optimizing induction period of DLDH gene. This manuscript is clear and focused. Experimental details are provided suitable to be repeated. Comparisons, limitations, and future directions of the LAC strain are fairly discussed. This manuscript can expand the knowledge of the lactate production by E. coli. Overall, I feel the manuscript become suitable for publication. However, I have one major concern.

Major concern:

1. Expression of DLDH gene and specific activity should be shown.

The present manuscript changed the promoter from tac to T7 in expectation of the enhancement of expression level and specific activity of DLDH. Indeed, changing promoter contributed to the lactate production. However, the manuscript Section 3.1 did not provide the direct evidence that the synthesis and specific activity of DLDH in LAC were enhanced compared to those in old-LAC. Section 3.3 also related to the DLDH gene expression by optimizing gene induction period.

The relationship between productivity and expression level with the specific activity of DLDH must be discussed.

Minor comments:

2. Please consider providing the schematic metabolic pathway for lactate production. Which pathways were disrupted in wildtype? How the respiration influences the lactate production? This can help readers understanding.
3. Please state the differences between the authors’ approach and other researches to lactic acid fermentation by E. coli. What is the advantage over the ref. 22?
4. (Figure 2 and Table 2) Why did pyruvate production by NULL decrease in comparison to PYR? PYR and NULL seem to have the same genomic background for pyruvate production.

Author Response

Dear Reviewer #2

>> Thank you for your review. Explanations to your comments are as follows.

1. Expression of DLDH gene and specific activity should be shown. The present manuscript changed the promoter from tac to T7 in expectation of the enhancement of expression level and specific activity of DLDH. Indeed, changing promoter contributed to the lactate production. However, the manuscript Section 3.1 did not provide the direct evidence that the synthesis and specific activity of DLDH in LAC were enhanced compared to those in old-LAC. Section 3.3 also related to the DLDH gene expression by optimizing gene induction period. The relationship between productivity and expression level with the specific activity of DLDH must be discussed.

>> Thank you for your meaningful suggestion. In the previous report, the effects of T7 and trc promoters on E. coli production of various proteins were investigated [Tegel et al., 2011, FEBS J 278, 729–739] (The expression activities of trc promoter and tac promoter are similar [Brosius et al., 1985, J Biol Chem 260(6), 3539–3541]). According to the report by Tegel et al., protein production under the control of the T7 promoter gives the larger amount of target protein. These data imply that the activity of DLDH in the LAC strain is higher than that in the old-LAC strain. We added the description about promoter selection in Section 3.1 (L130-131).

>> In addition, as you mentioned, the maximum titer observed at 36 h by the LAC strain (4.1 ± 0.4 g L⁻¹, Figure 3b) was higher than the maximum and final titer by the old-LAC strain (1.1 ± 0.0 g L⁻¹, Ref. 17). These results were obtained by cultivation of the both strains under the same conditions, suggesting that the stronger activity of DLDH has a positive effect on D-lactate production.

>> Regarding Section 3.3, we showed the specific D-lactate production rates after 36 h by the LAC strain under the air-regulated and the combined conditions instead of the specific activity of DLDH (newly added Figure S1). The specific D-lactate production rate of the both conditions were same level, indicating that the increase of final D-lactate concentration was caused by cell density. Hence, we consider that no significant change of the specific activity of DLDH was occurred under the air-regulated and the combined conditions. We added the explanation and Figure S1 in Section 3.3 (L187-189) and discussion (L235-237).

2. Please consider providing the schematic metabolic pathway for lactate production. Which pathways were disrupted in wildtype? How the respiration influences the lactate production? This can help readers understanding.

>> As your comment, we added a schematic figure (new Figure 2).

3. Please state the differences between the authors’ approach and other researches to lactic acid fermentation by E. coli. What is the advantage over the ref. 22?

>> Thank you for your meaningful suggestion. In Ref. 22 (Ref. 23 in the revised manuscript), the engineered E. coli strain produced some kinds of organic acids as by-products. On the other hand, since the genes related to the production of major organic acids were already disrupted (Figure 2), almost no by-products were produced by the LAC strain (Figure 4). Hence, in the future industrial D-lactate production by the LAC strain, the cost required for the process of removing by-products will be able to be reduced, so that the superiority of the LAC strain can be stated (L216-218 and L245-246).

>> Another advantageous point of our strain is using the PYR strain as a basal strain. The PYR strain was engineered to produce high amount of pyruvate. Hence, it is possible to construct various chemical-producing strain by changing the introducing genes (L56-68).

4. (Figure 2 and Table 2) Why did pyruvate production by NULL decrease in comparison to PYR? PYR and NULL seem to have the same genomic background for pyruvate production.

>> There are two genetic differences between the PYR and the NULL strains: (i) the NULL strain has a gene encoding T7 RNA polymerase in its chromosome; and (ii) the NULL strain harbors a plasmid pETIK. Since IPTG was added into the medium of the NULL strain, the expression of the gene under T7 promoter on pETIK was induced. In the multi-cloning site of pETIK, a 144-bp sequence (encoding 47 amino acids which include 6×His-tag and enterokinase recognition sequence) is contained between the start and stop codons. Hence, we consider that the expression of the unexpected product might by affected to growth of the NULL strain, resulting different culture profile to the PYR strain. We added the description (L140-141 and L145-146).

Reviewer 3 Report

Report is attached.

Referees Comments

on the manuscript entitled "Application of pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE: a case study for D-lactate production” for Fermentation

Authors demonstrated the possibility of the effective conversion biosynthetic pyruvate into D-lactate by a new D-lactate-producing strain, based on the pyruvate-producing strain, by introducing the DLDH-encoding gene under the control of a T7 expression system. It is well written and easy to read. The presented manuscript can be published in the Fermentation, however corrections to the manuscript are required.

Point 1: Page 2, lines 75-79 - The purpose of the study must be reformulated in traditional manner. It is recommended that the results be removed from the Introduction section.

Point 2: Page 4, lines 126-136 - In my opinion, in Figure 2, the original old-LAC strain is best designated simply as a CONTROL (not NULL).

Point 3: Page 4, line 146 – It is necessary to replace "in waste medium" with "in culture medium".

Author Response

Dear, Reviewer #3

>> Thank you for your review. Explanations to your comments are as follows.

1. Page 2, lines 75-79 - The purpose of the study must be reformulated in traditional manner. It is recommended that the results be removed from the Introduction section.

>> As your comment, the last paragraph of introduction was modified (L78-81).

2. Page 4, lines 126-136 - In my opinion, in Figure 2, the original old-LAC strain is best designated simply as a CONTROL (not NULL).

>> Thank you for your meaningful suggestion. To obtain the LAC strain, we introduced a gene encoding T7 RNA polymerase in PYR’s chromosome and changed the expression vector from pMAL-c5X/D-LDH to pETIK-DLDH. Thus, the genetic background of the LAC strain is different from that of the old-LAC strain. Therefore, we selected the NULL strain, which has a same genetic background with the LAC strain, as a control. We added the description (L140-141).

>> For your information, the selection marker gene is different between the expression vectors. The marker gene of pMAL-c5X/D-LDH is β-lactamase which confers ampicillin resistance; however, ampicillin is not suitable for long-term cultivation because ampicillin is hydrated over time by β-lactamase. This might be cause of the unstable culture profile of the old-LAC strain (please see SD values in Table 2). Thus, we constructed a new vector pETIK-DLDH that can be maintained by kanamycin which is not easily degraded even in latter phase of cultivation.

3. Page 4, line 146 – It is necessary to replace "in waste medium" with "in culture medium".

>> As your comment, it was changed.

>> in the spent medium => in the culture medium (L151-152)

Round 2

Reviewer 2 Report

Thank you for addressing my comments. I feel the revised manuscript has been significantly improved. Considering other reviewers' comments to this manuscript, this is maybe suitable for publication in the journal.

However, I still have a major concern about DLDH expression and activity in LAC strain. The authors provided the report by Tegel et al., instead of providing experimental results.

I believe that clarifying the relationship between DLDH activity per unit cell weight and overall titer for lactate production is key to explain the mechanism that LAC strain exhibits higher productivity than old-LAC strain. Further, this will contribute to expanding the knowledge of this research field and also to further improvement of the LAC strain.

Author Response

Dear Reviewer #2

>> Thank you for your review. Explanations to your comments are as follows.

Thank you for addressing my comments. I feel the revised manuscript has been significantly improved. Considering other reviewers' comments to this manuscript, this is maybe suitable for publication in the journal.

However, I still have a major concern about DLDH expression and activity in LAC strain. The authors provided the report by Tegel et al., instead of providing experimental results.

I believe that clarifying the relationship between DLDH activity per unit cell weight and overall titer for lactate production is key to explain the mechanism that LAC strain exhibits higher productivity than old-LAC strain. Further, this will contribute to expanding the knowledge of this research field and also to further improvement of the LAC strain.

>> Thank you for your valuable suggestion. We compared the DLDH activities and d-lactate productivities in the old-LAC and the LAC strains at 36 h under the basal conditions. The specific activity (μmol mg-protein⁻¹ min⁻¹) of DLDH in the LAC strain was 33-fold that in the old-LAC strain. The d-lactate productivity of the LAC strain was 9.0-fold higher than that of the old-LAC strain. From these observations, it seems that the increased DLDH activity is correlated with an increase of the d-lactate productivity.

>> Based on the above results, we changed the manuscript as follow:

1. The method of measurement of DLDH activity was added in materials and methods section (L127-137).
2. The results about the DLDH activities in the LAC strain and the old-LAC strain were added (L161-163).
3. The d-lactate concentration of the old-LAC strain at 72 h (1.07 g L⁻¹) was changed to that at 36 h (0.45 g L⁻¹) (L160).

Round 3

Reviewer 2 Report

Thank you for addressing my comment.

I recommend acceptance of this manuscript.