Overexpression of a Novel Vacuolar Serine Protease-Encoding Gene (spt1) to Enhance Cellulase Production in Trichoderma Reesei

Round 1

Reviewer 1 Report

Authors present a systematic study on the effects of vacuolar serine protease-encoding gene on cellulose production in T. Reesei. This fungus is widely used in the industry for production of cellulases, so the results presented here should have certain impact on the field. The effect of the Spt1 gene on such fugus has not been reported previously (novelty). The experiment is well-designed and the data is presented clearly including deletion strain, complementation strain and overexpression strains. However, several places have to be clarified and explained before being accepted. In conclusion, I find the article suitable for publication after a minor revision.

1.      In the last paragraph of the introduction, the authors should explicitly mention how many types of strains they are going to construct, and the what aspect of the spt1 they are willing to investigate using each strain.

2.      In the section 3.1, the authors should clarify what the purpose is for comparing T. reesei Spt 1 with its homologs in fungi in the first sentence. Even though this whole section is removed, it won’t affect the manuscript’s topic on T. reesei and novelty.

3.      Line 140-151, the authors should write out what optical instrument they use to obtain the pictures of the plate, simply a smart phone or more sophisticated camera.

4.      Line 363-366. These sentences should be put right after (B) sentence before (C).

5.      Figure 4A, B, C, the boundary of the halo and the colony is not as clear as the Figure 3. Suggest the author to add an arrow to point out the boundaries.

6.      Line 469-472, it is not coherent by suddenly mentioning Pgpd1 and Ptef1, are these two refer to the 5’ flank region mentioned in Figure A? If not, there should be a sentence talking about the 5’ flank region first and compare the cdna1 promoter with the original 5’ flank region.

7.      Line 478-479, the relationship between the SOD1 and SOD2 should be clarified. Were SOD1 and SOD2 the only strains obtained from random integration? If yes, please specify that. If not, please state why the author want to show the SOD1 data as well because SOD2 itself is enough to support “Overexpression of spt1 using the strong promoter Pcdna1 significantly enhanced the cellulase production” and better than SOD1.

8.      Line 524-536, authors listed many examples that knockout of vacuole-related functional genes could increase the extracellular protein secretion, it is not wrong, but contrary to the findings of this manuscript. Is there any examples that overexpressing such kind of genes could increase the protein secretion?

9.      Line 542-558, the authors use a whole paragraph to discuss and explain the impaired spore production of the Δspt1 strain, but missing the connection with the topic of this manuscript: cellulase production. Why would one care about spore production when improving cellulase production? The relations between spore production and cellulase production should be written out.

10.   When overexpressing the Spt1, will proteins other than extracellular cellulases also increase significantly, such as xylanases and beta-glucosidases or etc? Or the only the cellulase is increased?

Author Response

Responses to the review comments of Manuscript fermentation-2220743 entitled “Overexpression of a novel vacuolar serine protease-encoding gene (spt1) to enhance cellulase production in Trichoderma reesei”

 

Thanks for the evaluation and comments on our manuscript fermentation-2220743, which definitely improve its quality. We have made good use of these comments and suggestions to clarify the indistinct points and to enhance the manuscript. Now, we have incorporated all comments in the revised manuscript and responded to the queries point by point in the following pages. And in the revised manuscript, all the revisions are shown in blue highlight.

 

We hope that the revised manuscript is suitable for publication in Fermentation. If there are any questions, just let us know. Thanks again.

 

Sincerely yours,

 

Yaohua Zhong

Ph.D, Professor

 

State Key Laboratory of Microbial Technology, Institute of Microbial Technology

Shandong University, Qingdao 266237, P. R. China

Tel +86 531 88366118 (office); +86 13853149665 (mobile)

 

 

Responses to the review comments:

 

Reviewer #1:

Authors present a systematic study on the effects of vacuolar serine protease-encoding gene on cellulose production in T. Reesei. This fungus is widely used in the industry for production of cellulases, so the results presented here should have certain impact on the field. The effect of the Spt1 gene on such fugus has not been reported previously (novelty). The experiment is well-designed and the data is presented clearly including deletion strain, complementation strain and overexpression strains. However, several places have to be clarified and explained before being accepted. In conclusion, I find the article suitable for publication after a minor revision.

 

1. In the last paragraph of the introduction, the authors should explicitly mention how many types of strains they are going to construct, and the what aspect of the spt1 they are willing to investigate using each strain.

Response: Thanks for the comment. In the revised manuscript, we have modified the last paragraph of the introduction and introduced the strains constructed in this study as well as what aspects of the research are carried out with them (Page 6, Lines 112-122).

 

2. In the section 3.1, the authors should clarify what the purpose is for comparing T. reesei Spt1 with its homologs in fungi in the first sentence. Even though this whole section is removed, it won’t affect the manuscript’s topic on T. reesei and novelty.

Response: Thanks for the suggestion. Serine protease is reported to be important for growth, sporulation and autophagy in filamentous fungi (Shi et al., 2014). However, the function of the serine protease Spt1 in T. reesei is still unclear. Thus, the sequences of the T. reesei Spt1 and its homologs in fungi were firstly compared. In the revised manuscript, these sentences have been added to clarify the purpose of comparing T. reesei Spt1 with its homologs in fungi in the "Results" section (Page 14, Lines 297-300).

 

The reference is as follows:

Shi, L.; Li, R.; Liao, S.; Bai, L.; Lu, Q.; Chen, B. Prb1, a subtilisin-like protease, is required for virulence and phenotypical traits in the chestnut blight fungus. FEMS Microbiol Lett. 2014, 359, 26–33. https://doi.org/10.1111/1574-6968.12547.

 

3. Line 140-151, the authors should write out what optical instrument they use to obtain the pictures of the plate, simply a smart phone or more sophisticated camera.

Response: Thanks for the suggestion. Photographs of the plates were obtained with a SONY DSC-HX400 camera. In the revised manuscript, the sentence was added to explain that the pictures of these plates were photographed with a SONY DSC-HX400 camera in the "Materials and Methods" section (Page 9, Line 178-179).

 

4. Line 363-366. These sentences should be put right after (B) sentence before (C).

Response: Thanks for the comment. We have placed these sentences after (B) sentence and before (C) sentence in the revised manuscript (Page 32, Lines 744-747).

 

5. Figure 4A, B, C, the boundary of the halo and the colony is not as clear as the Figure 3. Suggest the author to add an arrow to point out the boundaries.

Response: Thanks for your professional advice on picture quality. In the revised manuscript, black or white arrows have been added to indicate the boundaries of the halos and the colonies in the "Figure 4" section (Page 33, Lines 752,767-768).

 

6. Line 469-472, it is not coherent by suddenly mentioning Pgpd1 and Ptef1, are these two refer to the 5’ flank region mentioned in Figure A? If not, there should be a sentence talking about the 5’ flank region first and compare the cdna1 promoter with the original 5’ flank region.

Response: Sorry for the misunderstanding caused by the unclear words. The 5' flank region mentioned in Figure 6 actually refers to the 5' promoter region of the spt1 gene (Pspt1). Pspt1 is the promoter of the spt1 gene and can drive the expression of the gene with moderate intensity, while Pgpd1 and Ptef1 are commonly used as moderate intensity constituent promoters in T. reesei. Pcdna1, the strongest constitutive promoter in T. reesei, was found to be stronger than Pgpd1 and Ptef1 by screening the T. reesei expression cDNA library, and its activity was much higher than that of Pspt1. Therefore, Pcdna1 was used to drive higher intensity overexpression of spt1. In the revised version, we have changed “5' flank region” to “Pspt1” in the "Figure 6" section (Page 35, Lines 784). The description of the activity comparison between Pcdna1 and Pspt1 has been added into the revised manuscript (Page 21, Lines 441).

 

7. Line 478-479, the relationship between the SOD1 and SOD2 should be clarified. Were SOD1 and SOD2 the only strains obtained from random integration? If yes, please specify that. If not, please state why the author want to show the SOD1 data as well because SOD2 itself is enough to support “Overexpression of spt1 using the strong promoter Pcdna1 significantly enhanced the cellulase production” and better than SOD1.

Response: In fact, we got eight transformants at the beginning, but after three rounds of purification and three times of fermentation, only SOD-1 and SOD-2 had good strain stability and cellulase production performance, so the strains finally displayed in the manuscript were SOD-1 and SOD-2. This description has been added into the revised manuscript (Page 21, Lines 445-447).

 

8. Line 524-536, authors listed many examples that knockout of vacuole-related functional genes could increase the extracellular protein secretion, it is not wrong, but contrary to the findings of this manuscript. Is there any examples that overexpressing such kind of genes could increase the protein secretion?

Response: Thanks for the comment. Indeed, previous studies have reported that knockout of the vacuole-related genes can significantly increase protein secretion. Yoon et al. demonstrated that knockout of the vacuolar protein sorting receptor AoVps10 in A. oryzae could increase recombinant protein production (Yoon et al., 2010). It has also been reported that knockout of the vacuole-associated protein-encoding genes results in the decreased extracellular protein production (Idiris et al., 2010; Marsalek et al., 2017), similar to the spt1 deletion resulting in decreased cellulase production in this study (Figure 5). However, overexpression of the vacuole-related genes to enhance protein production has not been reported. From this perspective, the spt1 gene is a novel vacuole protease gene found in T. reesei.

 

The references are as follows:

Yoon, J.; Aishan, T.; Maruyama, J.; Kitamoto, K. Enhanced production and secretion of heterologous proteins by the filamentous fungus Aspergillus oryzae via disruption of vacuolar protein sorting receptor gene Aovps10. Appl Environ Microbiol. 2010, 76, 5718–5727. https://doi.org/10.1128/AEM.03087-09.

Idiris, A.; Tohda, H.; Sasaki, M.; Okada, K.; Kumagai, H.; Giga-Hama, Y.; Takegawa, K. Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway. Appl Microbiol Biotechnol. 2010, 85, 667–677. https://doi.org/10.1007/s00253-009-2151-0.

Marsalek, L.; Gruber, C.; Altmann, F.; Aleschko, M.; Mattanovich, D.; Gasser, B.; Puxbaum, V. Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris. Biotechnol J. 2017, 12, 10.1002. https://doi.org/10.1002/biot.201600584.

 

9. Line 542-558, the authors use a whole paragraph to discuss and explain the impaired spore production of the Δspt1 strain, but missing the connection with the topic of this manuscript: cellulase production. Why would one care about spore production when improving cellulase production? The relations between spore production and cellulase production should be written out.

Response: Thanks for the comment. It has been reported that there is a cross-talk between asexual sporulation and extracellular cellulase production in fungi, which explains the possible effect of spt1 on cellulase production in T. reesei by influencing sporulation (Yao et al., 2016; Lei et al., 2014). This description has been added into the revised manuscript (Page 24, Lines 514-518).

 

The references are as follows:

Yao, G.; Li, Z.; Wu, R.; Qin, Y.; Liu, G.; Qu, Y. Penicillium oxalicum PoFlbC regulates fungal asexual development and is important for cellulase gene expression. Fungal Genet Biol. 2016, 86, 91-102. https://doi.org/10.1016/j.fgb.2015.12.012.

Lei, Y., Liu, G., Li, Z., Gao, L., Qin, Y., Qu, Y. Functional characterization of protein kinase CK2 regulatory subunits regulating Penicillium oxalicum asexual development and hydrolytic enzyme production. Fungal Genet Biol. 2014, 66, 44-53. https://doi.org/10.1016/j.fgb.2014.02.007.

 

10. When overexpressing the Spt1, will proteins other than extracellular cellulases also increase significantly, such as xylanases and beta-glucosidases or etc? Or the only the cellulase is increased?

Response: This is a good question. As shown in Figure 6E and Figure 7E, overexpression of spt1 could significantly improve the activity of β-glucosidase. Specially, the β-glucosidase activity of the spt1 overexpression strain SOD-2 was increased by 260% compared with the parental strain QP4. In addition, the extracellular protein concentrations of the spt1 overexpression strains were also measured, and it was found that overexpression of spt1 could also significantly increase the extracellular protein concentration of T. reesei (Figure R1). In this study, we focused on the promotion of cellulose degrading enzymes, so we did not investigate the influence of spt1 overexpression on xylanase production, which can be further explored in our next studies.

  

Author Response File: Author Response.docx

Reviewer 2 Report

This work examines the influence of the vacuolar spt1 gene on cellulolytic enzyme production by Trichoderma reesei. The authors have constructed a deletion, complementation and three overexpression (SOE, SOD-1 and SOD-2) spt1 strains and analysed how this affected protease, cellobiohydrolase, endoglucanase and β-glucosidase activity. The manuscript is a well-written, clear, good structure, covering every aspect that needs to be mentioned in order to understand the matter fully. However, there are a couple of minor issues that need to be addressed before I can recommend it for publication:

1. Microorganism names should be in italics, please check the entire manuscript as well as the final section (4. Discussion) the name of T. reesei is spelt incorrectly probably due to a typing error.

2. I like things to be uniform, so please decide whether to use spt1 with a lowercase or capital letter "s".

3. Line 36: "Cellulase hydrolysis is an effective means..." check singular or plural?

4. Final paragraph of the introduction section should contain the aim of the work as it does, but it shouldn't contain the obtained results.

5. This is the main issue for me. Why was one strain (QM53) used for the deletion and complementation strain and another one (QP4) for the overexpression strains? This should be pointed out more clearly in the first paragraph of the Materials and Methods section.

6. Indexing of numbers in molecular formulas to subscripts and in cells or spores number to superscripts should be performed.

7. How did you know when to add uracil, i.e. when needed?

8. Line 218: "108 spores..."?

9. Section heading of 3.5., 3.6. and 3.7. are formulated more as a sentence than a heading. Please rephrase.

Author Response

Responses to the review comments of Manuscript fermentation-2220743 entitled “Overexpression of a novel vacuolar serine protease-encoding gene (spt1) to enhance cellulase production in Trichoderma reesei”

 

Thanks for the evaluation and comments on our manuscript fermentation-2220743, which definitely improve its quality. We have made good use of these comments and suggestions to clarify the indistinct points and to enhance the manuscript. Now, we have incorporated all comments in the revised manuscript and responded to the queries point by point in the following pages. And in the revised manuscript, all the revisions are shown in blue highlight.

 

We hope that the revised manuscript is suitable for publication in Fermentation. If there are any questions, just let us know. Thanks again.

 

Sincerely yours,

 

Yaohua Zhong

Ph.D, Professor

 

State Key Laboratory of Microbial Technology, Institute of Microbial Technology

Shandong University, Qingdao 266237, P. R. China

Tel +86 531 88366118 (office); +86 13853149665 (mobile)

 

 

Responses to the review comments:

 

Reviewer #2:

This work examines the influence of the vacuolar spt1 gene on cellulolytic enzyme production by Trichoderma reesei. The authors have constructed a deletion, complementation and three overexpression (SOE, SOD-1 and SOD-2) spt1 strains and analysed how this affected protease, cellobiohydrolase, endoglucanase and β-glucosidase activity. The manuscript is a well-written, clear, good structure, covering every aspect that needs to be mentioned in order to understand the matter fully. However, there are a couple of minor issues that need to be addressed before I can recommend it for publication:

 

1. Microorganism names should be in italics, please check the entire manuscript as well as the final section (4. Discussion) the name of T. reesei is spelt incorrectly probably due to a typing error.

Response: Thanks for the comment. The PDF generated by the website contains formatting and spelling errors of T. reesei, which do not match the manuscript we uploaded. The spelling and format of T. reesei in the manuscript we uploaded are correct. In the revised manuscript, we have checked the format and spelling of Trichoderma reesei throughout the whole manuscript and have determined that it is shown in italics and abbreviated to “T. reesei”.

 

2. I like things to be uniform, so please decide whether to use spt1 with a lowercase or capital letter "s".

Response: Thanks for the comment. The generated PDF has formatting errors, and the italics of spt1 cannot be displayed correctly. In our submission, “Spt1” stands for the Spt1 protein or protease, and “spt1” stands for the spt1 gene. In the revised manuscript, we have tried to unify the usage and changed some “Spt1” to “spt1” (Page 19, Lines 407; Page 23, Lines 497).

 

3. Line 36: "Cellulase hydrolysis is an effective means..." check singular or plural?

Response: The word "means" has the same singular and plural forms, and is understood as "method" when used as a noun. Here, "Cellulase hydrolysis is an effective means..." can be used in this way. In the revised manuscript, we have changed “means” to “method” in order to avoid misunderstanding (Page 3, Lines 48).

 

4. Final paragraph of the introduction section should contain the aim of the work as it does, but it shouldn't contain the obtained results.

Response: Thanks for the suggestion. In the revised manuscript, we have modified the final paragraph of the introduction and fully reflected the purpose of our work (Page 6, Lines 112-122).

 

5. This is the main issue for me. Why was one strain (QM53) used for the deletion and complementation strain and another one (QP4) for the overexpression strains? This should be pointed out more clearly in the first paragraph of the Materials and Methods section.

Response: Thanks for the comment. The strain QM53 lacking the mus53 gene has high targeted integration frequency, so the spt1 gene can be efficiently knocked out and the spt1 deletion strain can be obtained by using QM53 as the parental strain. However, QM53 cannot undergo non-homologous recombination, which cannot be used as the parental strain to overexpress the spt1 gene by introducing multiple copies. The strain QP4, as an uracil defective strain, can introduce multiple copies through non-homologous recombination and its transformants can be quickly screened on the MM plate without uracil. Therefore, QP4 was used as the parental strain to construct the spt1 overexpression strain. This description has been added into the revised manuscript (Page 6-7, Lines 130-133).

 

6. Indexing of numbers in molecular formulas to subscripts and in cells or spores number to superscripts should be performed.

Response: Thanks for the comment. There are errors in the superscripts and subscripts of the numbers in the PDF generated by the website, which are inconsistent with the manuscript we uploaded. The superscripts and subscripts of the numbers in the manuscript we uploaded are correct. In the revised manuscript, the superscripts and subscripts of the numbers have been rechecked to make sure they are correct.

 

7. How did you know when to add uracil, i.e. when needed?

Response: Thanks for the suggestion. When the strain used in the experiment is the uracil defective strain, such as QP4, it is necessary to add 0.1% uracil in the medium during culture or fermentation to ensure the normal growth of the strain. This description has been added into the revised manuscript (Page 8, Lines 158-160).

 

8. Line 218: "108 spores..."?

Response: In the revised manuscript, we have changed “108 spores” to “10⁸ spores” in the "Materials and Methods " section (Page 12, Lines 247).

 

9. Section heading of 3.5., 3.6. and 3.7. are formulated more as a sentence than a heading. Please rephrase.

Response: Thanks for the suggestion. In the revised manuscript, the headings of 3.5, 3.6 and 3.7 have been changed in the "Results" section. The heading of section 3.5 has been changed from “Deletion of spt1 resulted in the decreased cellulase production in T. reesei” to “Deletion of spt1 resulting in the decreased cellulase production in T. reesei” (Page 18, Lines 393). The heading of section 3.6 has been changed from “Overexpression of spt1 by introducing multiple copies could improve cellulase production” to “Overexpression of spt1 with multiple copies improving cellulase production” (Page 19, Lines 411). The heading of section 3.7 has been changed from “Overexpression of spt1 using the strong promoter Pcdna1 significantly enhanced the cellulase production” to “Overexpression of spt1 with the strong promoter Pcdna1 enhancing the cellulase production” (Page 20, Lines 433-434).

Author Response File: Author Response.docx

Reviewer 3 Report

1- Original raw data for assays must be provided.
2- licences for software used must be clarified.
3- Certificate for extensive English editing must be provided.

 

4- Detailed statistical analyses must be performed and given.

Author Response

Responses to the review comments of Manuscript fermentation-2220743 entitled “Overexpression of a novel vacuolar serine protease-encoding gene (spt1) to enhance cellulase production in Trichoderma reesei”

 

Thanks for the evaluation and comments on our manuscript fermentation-2220743, which definitely improve its quality. We have made good use of these comments and suggestions to clarify the indistinct points and to enhance the manuscript. Now, we have incorporated all comments in the revised manuscript and responded to the queries point by point in the following pages. And in the revised manuscript, all the revisions are shown in blue highlight.

 

We hope that the revised manuscript is suitable for publication in Fermentation. If there are any questions, just let us know. Thanks again.

 

Sincerely yours,

 

Yaohua Zhong

Ph.D, Professor

 

State Key Laboratory of Microbial Technology, Institute of Microbial Technology

Shandong University, Qingdao 266237, P. R. China

Tel +86 531 88366118 (office); +86 13853149665 (mobile)

 

Responses to the review comments:

 

Reviewer #3:

1- Original raw data for assays must be provided.

Response: Thanks for the suggestion. In the manuscript, the original data have been provided, such as the photographs of the plates and microscopic observation in Figure 3, the photographs of the plates in Figure 4, etc (Page 32, Lines 741; Page 33, Lines 752).

 

2- licences for software used must be clarified.

Response: Thanks for the suggestion. In the manuscript, the software we use is downloaded from formal official channels, and the version of the software used has been clarified (Page 9, Lines 186-189).

 

3- Certificate for extensive English editing must be provided.

Response: Thanks for the suggestion. In the revised manuscript, we have revised the sentences. For example, we have changed “Deletion of spt1 resulted in the decreased cellulase production in T. reesei” to “Deletion of spt1 resulting in the decreased cellulase production in T. reesei”, “Overexpression of spt1 by introducing multiple copies could improve cellulase production” to “Overexpression of spt1 with multiple copies improving cellulase production” and “Overexpression of spt1 by introducing multiple copies could improve cellulase production” to “Overexpression of spt1 with the strong promoter Pcdna1 enhancing the cellulase production” (Page 18, Lines 393; Page 19, Lines 411; Page 20, Lines 433-434).

 

4- Detailed statistical analyses must be performed and given.

Response: Thanks for the suggestion. The values in the manuscript are averages of three repeated measurements from at least three parallel experiments. The error bar refers to standard deviation (SD). The differences between the parental strain and the recombinant strain were analyzed ANOVA followed by Turkey test. In the manuscript, the methods and results of detailed statistical analysis have been described (Page 31, Lines 736-739; Page 32, Lines 744-747; Page 33, Lines 768-771; Page 34, Lines 779-782; Page 35, Lines 791-794; Page 36, Lines 805-808).

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Raw data of this manuscript must be provided as drive file to check them.