New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors are investigating the effects of gene disruption on the succinate biosynthesis pathway in Actinobacillus succinogenes, which is known for its succinate production. The authors are also disrupting genes related to sugar transport to see their effects and to infer the function of the genes.

A series of experiments have been conducted using the same methodology to monitor gene disruption and replenishment by expression of the relevant genes, as well as the production of compounds in culture.

However, the authors do not seem to have a coherent goal of what they want to clarify about the succinate synthesis pathway in this paper. The authors should explain their approach by setting objectives and providing strategies such as maximizing succinate production and maximizing CO2 intake.

Authors tried to find JEN family homolog genes in Actinobacillus succinogenes, but I can't understand why the authors believe same transport system is existing between eukaryotic yeast and prokaryotic Actinobacillus.　Besides, the yeast JEN2 gene was characterized to be a transporter of succinate uptake, but microbial fermentation produces succinate through intracellular rTCA pathway.

Minor points are as follows,

L38: PEP carboxylase (PEPC)

L142: SA was synthesized

L285-287: authors should explain why Asus_0914 is thought to be a homologue of ptsG, using identity or domain composition.

Figure 4a: graph legend should be written in English

 

 

Author Response

Reviewer 1

The authors are investigating the effects of gene disruption on the succinate biosynthesis pathway in Actinobacillus succinogenes, which is known for its succinate production. The authors are also disrupting genes related to sugar transport to see their effects and to infer the function of the genes.

A series of experiments have been conducted using the same methodology to monitor gene disruption and replenishment by expression of the relevant genes, as well as the production of compounds in culture.

However, the authors do not seem to have a coherent goal of what they want to clarify about the succinate synthesis pathway in this paper. The authors should explain their approach by setting objectives and providing strategies such as maximizing succinate production and maximizing CO₂ intake.

A1. Thank you for your comments and suggestion. Our initial goal was to genetically engineer A. succinogenes to produce higher SA. The strategies to promote SA biosynthesis reported mainly include redirecting carbon flow, balancing the redox ratio (NADH/NAD+), and optimizing CO₂ supplementation. But there is still some vague understanding of SA synthesis in A. succinogenes, we have investigated the effects of increasing CO₂ fixation pathway by expression of PEPC and PYC, reducing oxaloacetic acid outflow by inactivation of oxaloacetic acid decarboxylase, and eliminating the production of by-products acetic acid and formic acid. However, the production of SA was not as high as expected, but our works provides useful information on the mechanism of SA synthesis in A. succinogenes, which will help improve SA production performance of this microbial chassis. To facilitate understanding, we have revised the corresponding sentences as follows:

On L148-153:

The heterogeneous expression of PEPC and PYC in E. coli and C. glutamicum enhanced the C flow of SA synthesis pathway and increase SA production [31,32]. Due to the fixation of CO₂ in Corynebacterium acetoacidophilum depends that PYC catalyzes pyruvate and PEPC catalyzes phosphoenolpyruvate. Therefore, pyc and pepc genes from C. acetoacidophilum were introduced into A. succinogenes to strengthen CO₂ fixation pathway, respectively, yielding strains ZK::pyc and ZK::pepc.

 

Authors tried to find JEN family homolog genes in Actinobacillus succinogenes, but I can't understand why the authors believe same transport system is existing between eukaryotic yeast and prokaryotic Actinobacillus. Besides, the yeast JEN2 gene was characterized to be a transporter of succinate uptake, but microbial fermentation produces succinate through intracellular rTCA pathway.

A2. Thank you for your comments. To mine succinate transporters in A. succinogenes, we compared homology of all transporters reported in bacteria with the target strains. The results showed that Asuc_2056 and Asuc_0750 genes were homologous with the JEN of yeast, and we also studied these two proteins.

In addition, SA was a typical carboxylic acid, it has been reported that since the charged protons and carboxylic anions cannot exit the cell by passive diffusion, they will cause cytotoxicity if they cannot be rapidly metabolized or pumped out of the cell. The exported protons and anions again form undissociated molecules, in the low-pH environment outside the cell, and can reenter the cell to form an energy draining futile cycle. This process is believed to eventually waste a considerable portion of carbon source molecules for energy generation, thereby reducing SA synthesis. Therefore, we think that inactivation of SA importer maybe reduced influx of SA from the extracellular environment into cells during the late fermentation stage.

 

Minor points are as follows,

L38: PEP carboxylase (PEPC)

A3. Thank you for your suggestion. We have revised this sentence to “PEP carboxylase (PEPC)” on L38.

 

L142: SA was synthesized

A4. Thank you very much. We have revised this sentence to “SA was synthesized” on L142.

 

L285-287: authors should explain why Asus_0914 is thought to be a homologue of ptsG, using identity or domain composition.

A5. Thank you very much for your suggestion. Both Asuc_0914 and Asuc_1575 were homologous with ptsG. Since base editor requires specific codons and editing windows, gene Asuc_1575 do not have corresponding targets and are therefore not silenced. Therefore, the function of Asuc_1575 gene has not been studied further. We have revised the corresponding sentences as follows:

On L288-295:

Among them, Asuc_1575 gene is annotated as triosephosphate isomerase and Asuc_0914 gene is annotated as PTS system, sucrose-specific IIBC subunit. Therefore, these two genes were speculated to be involved in glucose transport. To investigate gene function, introducing a stop codon into the coding region of gene to yield gene-silenced strain by using a cytosine base editor. However, no available target site was found in coding region of gene Asuc_1575, so no Asuc_1575 gene-silenced strain was obtained. The effects of wild type and the gene-silenced strain ΔAsuc_0914 on SA synthesis were determined by shake-flask fermentation.

 

Figure 4a: graph legend should be written in English

A6. Thank you very much for your suggestion. We have revised the legend of figure 4a.

Reviewer 2 Report

Comments and Suggestions for Authors

The information written in the introduction about the production of succinic acid is sufficiently solid to address a problem regarding its  production by different microbiological strains. The routes described and figure 1 are very illustrative and I have no further comment on it.

The objective of the article is perfectly stated, in addition, the methodology used is very well understood.

The problem that I mainly detected was the sharpness and clarity of figures 2 to 8, since the graphs are not properly appreciated, they look very blurry and the results shown with them seem confusing, but they are clarified very well with the narrative in the text.

I found no signs of plagiarism and no signs of self-citation. The references used are appropriate to their extent.

Author Response

Reviewer 2

The information written in the introduction about the production of succinic acid is sufficiently solid to address a problem regarding its production by different microbiological strains. The routes described and figure 1 are very illustrative and I have no further comment on it.

The objective of the article is perfectly stated, in addition, the methodology used is very well understood.

The problem that I mainly detected was the sharpness and clarity of figures 2 to 8, since the graphs are not properly appreciated, they look very blurry and the results shown with them seem confusing, but they are clarified very well with the narrative in the text.

I found no signs of plagiarism and no signs of self-citation. The references used are appropriate to their extent.

A1. Thank you for your comments and your suggestion. We have improved the resolution of figures 2-8 to 300 dpi.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

We confirm that the authors have addressed the points raised in the revised manuscript by clearly stating the overall purpose of the experiment, including citations to previous reports.

Thank you for your response.