Confirmation of Glucose Transporters through Targeted Mutagenesis and Transcriptional Analysis in Clostridium acetobutylicum

Round 1

Reviewer 1 Report

The manuscript by Zhang et al. confirmed the glucose transporters in Clostridium acetobutylicum through targeted mutagenesis and transcriptional analyses. The study has provided some new information based on the known information from the literature concerning the major glucose transporter in Clostridium acetobutylicum. The results are sound. Some issues need to be addressed before it can be accepted for publication:

1. In this study, ClosTron has been used for gene disruption, and microarray has been used for transcriptional analyses. Both technologies are old, while both CRISPR-based gene knockout system and RNA-Seq based transcriptomic analysis have been very well established for this train. Why did the authors select to use these old technologies but not the new ones?

2. For the ClosTron gene disruption, has the intron been inserted into the gene in the sense direction or anti-sense direction?

3. Please check on Fig. S1: the Lane numbers are not correct (for example, the Marker corresponds to the Lane 3); also the PCR band sizes are not correct, either (they are not

0.3 Kb and 2.1 Kb based on the labelling in the figure).

4. Line 294-205: “mutant glcCE::int(193) even consumed slightly more glucose and produced more butanol during solventogenesis growth compared to that of wild type in the continuous fermentation”. However, based on Fig. 4, this is not correct (the shown glucose data in the figure represents the Residual glucose, correct?).

5. Lines 292, 299, 351: From the figures, you actually are not talking about the ‘yield’ in these sentences. You might want to use “titer” or “concentration”..

6. Fig. 6 Please give more details in the figure caption. For example, what 1, 2, 3 represents? What are Hpr, EI (the Figure needs to  stand alone)? What are 0386, 0570 etc. (I understand they are gene IDs, but you need to make these clearer).

7. “CAC0154 and CAC2956 maybe a tertiary glucose and secondary cellobiose transporter in C. acetobutylicum”—these are very valuable results. It would be interesting if the authors can further confirm these through gene deletion and transcriptional analyses..  

Author Response

1. In this study, ClosTron has been used for gene disruption, and microarray has been used for transcriptional analyses. Both technologies are old, while both CRISPR-based gene knockout system and RNA-Seq based transcriptomic analysis have been very well established for this train. Why did the authors select to use these old technologies but not the new ones?

> Actually, the genetic manipulation work had been done several years ago, when there were intact ClosTron vectors but not CRISPR system in our lab at that time, so all mutants were constructed by ClosTron. There were also DNA-arrayer and DNA-chip reader in our lab, so the transcriptional data were got using DNA microarray. We also use CRISPR and RNA-seq in the ongoing study.

2. For the ClosTron gene disruption, has the intron been inserted into the gene in the sense direction or anti-sense direction?

> The introns were inserted in the sense direction according to the gene sequencing results.

3. Please check on Fig. S1: the Lane numbers are not correct (for example, the Marker corresponds to the Lane 3); also the PCR band sizes are not correct, either (they are not 0.3 Kb and 2.1 Kb based on the labelling in the figure).

> I’m so sorry to ignore the problem when the supplementary file was transferred to PDF version. The correct figure had been reset.

4. Line 294-205: “mutant glcCE::int(193) even consumed slightly more glucose and produced more butanol during solventogenesis growth compared to that of wild type in the continuous fermentation”. However, based on Fig. 4, this is not correct (the shown glucose data in the figure represents the Residual glucose, correct?).

> Yes, as it mentioned in the caption of Fig. 4, the shown glucose data represents residual glucose concentration in Line 322.

5. Lines 292, 299, 351: From the figures, you actually are not talking about the ‘yield’ in these sentences. You might want to use “titer” or “concentration”.

> I have modified “yield” to “concentration” in Line 287 and 294; I have modified “yield” to “production” in Line 345.

6. Fig. 6 Please give more details in the figure caption. For example, what 1, 2, 3 represents? What are Hpr, EI (the Figure needs to  stand alone)? What are 0386, 0570 etc. (I understand they are gene IDs, but you need to make these clearer).

> I have added some details in the caption of Fig.6 in Line 392-394.

7. “CAC0154 and CAC2956 maybe a tertiary glucose and secondary cellobiose transporter in C. acetobutylicum”—these are very valuable results. It would be interesting if the authors can further confirm these through gene deletion and transcriptional analyses.

> We would further confirm them in the following study.

Reviewer 2 Report

Dear editor and authors:

        This article focused on identifying the glucose uptake transporter of C. acetobutylicum. To some extent, the discovery in study and results obtained are useful for the further metabolic engineering and synthetic biology in C. acetobutylicum. However, several issues should be addressed before the acceptance of this manuscript.

1.       Glucose transporters are not limited in PTS system. Whether the non-PTS systems of C. acetobutylicum are responsible for glucose uptake and the relative research advances are not mentioned at all. If the non-PTS systems are also responsible for glucose uptake, it is necessary to be knocked out for confirming the function of glcCE. If not, at least it should be mentioned in the introduction and discussion.

2.       Why C. acetobutylicum glcG::int(1224) and C. acetobutylicum glcG::int(1224)-glcCE::int(193) showed higher cell growth than wildtype in batch fermentations? An explanation should be given for this reverse result. Maybe it caused by the neglection of non-PTS systems.

3.       In line 345, “Therefore, the cells were washed out of the fermenter in continuous culture.” If dilution rate of 0.075 h^-1 is too high, transcription data will also be unreliable. The dilution rate should be reduced to obtain stable experimental conditions. Please give the results of other lower dilution rate.

4.       In Figure 6, the exchange of phosphate group between different components is clearly not equal. The upper and lower positions of HBr/HBr~P and pyruvate/phosphoenolpyruvate should be swapped and the order of other reactions involved also should be modified.

5.       Please provide the new Figure S1 with more clear background. The bands in lane 1 and 2 cannot be seen in the current version. In addition, the lane 4 is missing.

Author Response

1. Glucose transporters are not limited in PTS system. Whether the non-PTS systems of C. acetobutylicum are responsible for glucose uptake and the relative research advances are not mentioned at all. If the non-PTS systems are also responsible for glucose uptake, it is necessary to be knocked out for confirming the function of glcCE. If not, at least it should be mentioned in the introduction and discussion.

>According to the previous study, hexose sugars are mainly carried by PTS while non-PTS are used to transport pentose sugars. I have added some introduction in Line 50-52.

2. Why C. acetobutylicum glcG::int(1224) and C. acetobutylicum glcG::int(1224)-glcCE::int(193) showed higher cell growth than wildtype in batch fermentations? An explanation should be given for this reverse result. Maybe it caused by the neglection of non-PTS systems.

>During the initial 12h, the mutants glcG::int(1224) and glcCE::int(193) consumed more glucose than WT and the mutant glcG::int(1224)-glcCE::int(193) (Fig. 1B) because of their high transcriptional levels of the genes glcCE and glcG, respectively (Table 1&2), so they got higher growth rate. In addition, the mutant glcG::int(1224)-glcCE::int(193) consumed almost identical glucose to the WT during the initial 12h, whereas WT produced more solvent (Fig. S3), so the mutant glcG::int(1224)-glcCE::int(193) showed higher cell growth to survive. Then the growth of mutant glcG::int(1224)-glcCE::int(193) declined a lot maybe because the non-PTS could only transport a small amount of glucose.

3. In line 345, “Therefore, the cells were washed out of the fermenter in continuous culture.” If dilution rate of 0.075 h-1 is too high, transcription data will also be unreliable. The dilution rate should be reduced to obtain stable experimental conditions. Please give the results of other lower dilution rate.

>The dilution rate of 0.075 h^-1 means a volume of medium is replaced per 13.3 h, which had been verified to be a suitable rate by previous study (Holger et. al, J Bacteriol, 2012; Christina et. al, J Mol Microbiol Biotechnol, 2010; Daniel et. al, Appl Microbiol Biotechnol, 2014) and I have added the references in Line 135.

4. In Figure 6, the exchange of phosphate group between different components is clearly not equal. The upper and lower positions of HBr/HBr~P and pyruvate/phosphoenolpyruvate should be swapped and the order of other reactions involved also should be modified.

>I have modified all the phosphorylation reactions in Fig.6.

5. Please provide the new Figure S1 with more clear background. The bands in lane 1 and 2 cannot be seen in the current version. In addition, the lane 4 is missing.

> I’m so sorry to ignore the problem when the supplementary file was transferred to PDF version. The correct figure had been reset.

Round 2

Reviewer 2 Report

The manuscript was revised well and can be accepted.