Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins

Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S. lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans, adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.


Introduction Yes
Is the length of the paper justified? Yes

Do you have any ethical concerns with this paper? No
Decision letter (RSOB-19-0013.R0)

27-Feb-2019
Dear Dr Mellado, We are writing to inform you that your manuscript RSOB-19-0013 entitled "Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins." has, in its current form, been rejected for publication in Open Biology.
The referees have recommended that major revisions are necessary but that the manuscript has potential; hence, we would like to actively encourage you to revise the manuscript accordingly, and resubmit. Nevertheless, please note that this is not a provisional acceptance.
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Please find below the comments made by the referees, not including confidential reports to the Editor, which I hope you will find useful. If you do choose to resubmit your manuscript, please upload a 'response to referees' document including details of how you have responded to the comments, and the adjustments you have made.
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Sincerely, The Open Biology Team mailto: openbiology@royalsociety.org Associate Editor, Professor J Peter Young Board Member: Comments to Author: This manuscript has been reviewed by two experts who agree that the findings are interesting and could make a good paper, but considerable revision and the addition of new material is necessary. Each of the reviewers makes a series of good points that you need to consider and respond to. Both point out that important conclusions are drawn from Western blots that are not presented in the manuscript. They must be included. Furthermore, both reviewers are concerned that you do not present any evidence for direct interactions between Fkbp and the substrates, and they point out a number of ways in which the results could be generated by indirect effects. You must provide additional data to address this issue, but I leave it to you to decide on the best experiments that will convince the reviewers that your proposed mechanism is correct.
Reviewer ( Comments to the Author(s) Vicente et al describe the identification of a FKBP-like protein in Streptomyces lividans that plays a role in the production of increased amounts of active overproduced secreted proteins. This is well written paper with a straightforward observation. From a biotechnological perspective the findings will be useful in protein secretion applications. What the paper is missing is sufficient biological insight that the authors could provide. Currently the paper is phenomenological without any direct molecular explanation as to what causes the changes they see and even if they are direct. Major 1. The authors mention that western blots (not shown) showed no enhancement in levels of the secreted enzymes but only in activities. I presume this is why they attribute their findings to a direct effect of the FKBP-like protein on folding of the already secreted proteins. However, their data do not provide any direct evidence for a direct effect on the secreted reporters and are rather phenomenological. What is the evidence that the FKBP-like protein does not operate indirectly by modifying a secretion component for example or another chaperone?? The same is true for the deletion experiment. What is the evidence that FKBP-like protein is not needed for asome generic cell structure/export job that is compromised? 2. What is the effect of deleting or over-expressing the FKBP-like protein on the secretome overall?? Can it be claimed for its role to be rather generic or is it specialized?? 3. I am afraid there is no justification for the structures in Fig. 4 as the indicated Pro residues are completely speculative. In their current state these figures belong to a supplement. Otherwise, the authors need to mutate these residues and demonstrate folding effects. 4. The discussion essentially repeats the results. Its should stick to real discussion issues and be significantly trimmed. 5. How are the units normalized in the experiments? Are the activities expressed per cell?? Per fixed DCW?? Do the cells grow to the same extent when the FKBP-like protein gene is either deleted or over-expressed?
Minor grammar editing 249 were subtracted FROM those obtained in the presence of pIJ486 protoplasts and pIJFKBP 261 the secretion stress response S. lividans TK21, which consists OF the induction of the 268 genes was determined by qRT-PCR analyses and compared TO that of the wild type cell. A 278 extracellular alpha-amylase severely decreased WITH respect to that of the wild type ( Fig  2A).
282 mutant when compared to that of the wild type (Fig 2B)

Do you have any ethical concerns with this paper? No
Comments to the Author Major Comments 1. Line 321: Inhibition of the Fkbp protein is discussed. However, the data isn't represented anywhere. Why has this not been included in Fig1 (b) for example? Its highly relevant data. 2. Line 337: qRT-PCR analysis is discussed. However, the data isn't represented anywhere in the tables/figures. Why has this not been included in the main body or as supplementary data? 3. Line 355 and Line 369: the sentence is slightly misleading, as it implies that Fig 2 in the main text shows growth rate. This is only shown clearly in Supplementary Fig 2. 4. Line 371 and 383: The respective words 'require' and 'need' are strong here. Considering Tatproteins are pre-folded and clearly do not require PKBP activity for normal functioning (shown in Fig 2). Consider a milder term such as overexpressed amylase 'benefits' from PKBP. 5. Section starting Line 416-458: This section needs to be re-written, in its current form its very confusing. Please explain what is meant by the Sli-PKBP control and why is FKBP detected in the mutant fkbp strain (5509 relative abundance?). Line 433: Sentence starting 'in some cases' doesn't make sense. Line 445: It is not clear to me why S. lividans TK24 and TK21 are discussed here and what the annotation means, what is meant by strain of origin? Table 1: (1) Could you show the actual ratio of relative abundance between the strains instead of only the p-value?
Minor comments 1. Line 287: Heading says Characterisation -data discussed is more identification of fkbp gene. 2. Line 345: This paragraph suddenly starts taking about amylase and agarase, whereas previous paragraph discussed secretion stress response. It feels like a heading is missing here. 3. Line 399: This is the first mention of molecular dynamics in the main text, introduce what MD stands for ease of the reader. 4. Line 468: Figure 1 doesn't show inhibition, this is misleading sentence construction. (See major comment 1) 5. Line 480, 492: The use of 'required and 'need' . These sentences are misleading in that it implies Agarase requires the action of FKBP for maturation. This is a Tat-dependent substrate and under normal conditions is secreted in its mature form without FKBP action. Consider a milder term Decision letter (RSOB-19-0201.R0)

22-Sep-2019
Dear Dr Gullón, We are pleased to inform you that your manuscript RSOB-19-0201 entitled "Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins." has been accepted by the Editor for publication in Open Biology. The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the reviewer(s)' comments and revise your manuscript.
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Minor comments 1. Line 287: Heading says Characterisation -data discussed is more identification of fkbp gene. 2. Line 345: This paragraph suddenly starts taking about amylase and agarase, whereas previous paragraph discussed secretion stress response. It feels like a heading is missing here.
3. Line 399: This is the first mention of molecular dynamics in the main text, introduce what MD stands for ease of the reader. 4. Line 468: Figure 1 doesn't show inhibition, this is misleading sentence construction. (See major comment 1) 5. Line 480, 492: The use of 'required and 'need' . These sentences are misleading in that it implies Agarase requires the action of FKBP for maturation. This is a Tat-dependent substrate and under normal conditions is secreted in its mature form without FKBP action. Consider a milder term Author's Response to Decision Letter for (RSOB-19-0201.R0) Decision letter (RSOB-19-0201.R1)

01-Oct-2019
Dear Dr Gullón We are pleased to inform you that your manuscript entitled "Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins." has been accepted by the Editor for publication in Open Biology.
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Point 1. What is the evidence that the FKBP-like protein does not operate indirectly by modifying a secretion component for or another chaperone? What is the evidence that FKBP-like protein is not needed for asome generic cell structure/export job that is compromised? Response: Recently, it has been reported the S. lividans TK24 secretome showing that SLIV_29545 is the only extracytoplasmic PPIase. We performed targeted mass spectrometry analyses (SRM/MRM) to study the relative protein abundance of a SLIV_18490 that has been annotated in the secretome of S. lividans TK24 as chaperone protein and the extracytoplasmic secretion components involved in the folding previously identified in our group ([24, 58]; using supernatants of the Δsli-fkbp, the single-copy sli-fkbp and the sli-fkbp overexpressing strains. Results are summarised in a new Table1 and Table S1 and in the Results section of the new version of the manuscript page 21 lines 451-458 and in the Discussion section of the new version of the manuscript page 24-25 lines 539 to 555. Briefly, secretion is not compromised in a mutant lacking FKBP, although the amount of correctly folded proteins seems to be. Point 2. What is the effect of deleting or over-expressing the FKBP-like protein on the secretome over-all? Can it be claimed for its role to be rather generic or is it specialized? Response: We have performed targeted mass spectrometry (SRM/MRM) analyses to study the effect of the deletion or overexpression of SLI-FKBP over a group of selected proteins potentially isomerised by Sli-FKBP showing that Sli-FKBP affects a relevant group of secreted proteins. The results are summarised in a new Table 1, Table S1  According to this, the equivalent amount of protein loaded onto the SDS-PAGE acrylamide gel was corrected by the bacterial dry weight in each case. A new sentence has been added in the Methods section page 8 lines 167-169. Alpha-amylase activity was normalized per mg of protein and agarase activity was normalized per mg of dry weight.
Point 7. Minor grammar editing Line 249 Thank you for the correction of has been replaced by from Line 261 This sentence has been removed according to reviewer 1 suggestion. Line 268 "to" has been added. Line 278 "with" has been added. Line 282 "in" has been removed and by "to a lesser extent than" has been added. Line 288 "with respect to" has been added. Line 302 "it" has been included. 2. Novelty-PrsA does similar things. Its not explained why this protein is particular is novel. Response: Analysis of the S. lividans secretome [59 of the new version of the manuscript] shows that the only extracytoplasmic PPIase in S. lividans is this one. Sli-FKBP is an homolog to FKBP and thus should be classified in the family of FK506 binding proteins, while PrsA is a parvulin, hence belonging to a different family in a different organism. The findings in B. subtilis therefore, cannot be applied to S. lividans where no parvulin-like proteins have been identified in the secretome. This is the first report of such an extracytoplasmic PPIase involved in the folding of secreted proteins in Streptomyces and the results obtained provide useful information for use in engineering Streptomyces strains for homologous or heterologous secretory protein overproduction.
3. Line 253: An experiment describing the inhibition of FKBP using FK506 is described but the data for this is not shown. This is an important conclusion and needs to be included in the results section. It is also mentioned in the discussion Line 311, however, since this information is not actually presented it shouldn't be used to make such conclusions. Response: Data from inhibition assays has now been included in the results section of the new version of the manuscript page 14 lines 321-328.
4. Line 276-283. The activity of amylase and agarase are measured and shown to decrease when Fkbp is absent, activity assays are excellent methods of showing that the deleted Fkbp is linked to the activity of Amylase and Agarase. However, it is implied that the reason the Agarase/amylase activity is decreased is due to an increase in misfolded protein in the absence of the Fkbp. However, its not clear that in the absence of Fkbp the same amount of Sec/Tat substrate is actually secreted. Western blot assays for Amylase and Agarase could show whether the amount of protein present is altered. Response: Thank you for the suggestion. A new figure 2 including western blot of the ∆sli-fkbp mutant overproducing alpha-amylase and agarase has been included showing that the alpha-amylase secretion pattern has been severely affected.
5. Line 294 and Line 304. The authors say that Western blot assays were performed and showed that increased Amylase/Agarase production remained the same with or without Fkbp. The reference another paper and do not shown the data. Western blot showing this information is essential to making this statement, further this is an exceptionally important observation that needs to be incorporated into the results. Response: A new figure 3 that compares the Sli-FKBP and agarase or alphaamylase overproducing strains with their isogenic strains, TK21pAMI11pFDT (alpha-amylase) and TK21pAGAs5pFDT (agarase) has been added to the manuscript. It reveals that overproduction of Sli-FKBP improves alpha-amylase secretion although there is no significant increase in extracellular production of agarase despite the increase of activity.
6. The absence of Fkbp has an effect on Sec and Tat secreted proteins. However, direct interactions between Fkbp and these substrates have not been shown. This is particularly important with regard to the Tat substrate, as these are secreted in a folded form. The effect that Fkbp has on Agarase activity might not be directly associated with Fkbp but rather an indirect effect of Fkbp on membrane protein stability/decrease stress response (a more stable environment results in more active protein). This is not addressed in the results section or the discussion. In Line 301 it's also said that Fkbp isomerase action improves Tat-dependent agarase folding. Direct protein-protein interaction studies such as co-precipitation studies would show this interaction. Response: We do agree that more experiments showing protein-protein interactions are needed but at present this is beyond our possibilities. The new version of the manuscript presents the study based on agarase modelling (new figure S4) as a potential explanation without asserting it. New text has been included in Results section page 17-18 lines 396-415 and Discussion section page 24 lines 521 and 527.