Optimisation of aurodox production by Streptomyces goldiniensis.
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Aurodox is a specialised metabolite of Streptomyces goldiniensis originally identified for its anti-streptococcal and anti-staphylococcal activity. More recently, there has been a renewed focus on the study of aurodox as the compound has been shown to inhibit virulence in both Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC/EHEC). Aurodox is able to inhibit host colonization in EPEC and EHEC infections via the downregulation of the Type III Secretion System – a major virulence factor in these pathogens. To further investigate activity, increased quantities of the compound are required for in-depth mechanism of action studies. To identify suitable aurodox production conditions, a number of relevant fermentation media were tested for enhanced aurodox yield, followed by bioassay and Liquid Chromatography Mass Spectrometry analysis. Glucose Yeast Extract medium (GYM) was identified as the leading production medium tested. Further analysis of S. goldiniensis growth in GYM medium established the optimum temporal range for aurodox production. A minimum growth period of 154 hours is required for detectable aurodox production in S. goldiniensis fermentations in liquid GYM medium. To improve overall aurodox titres, a heterologous expression (HE) approach was taken and four potential HE hosts were investigated. These data indicated that the aurodox biosynthetic gene cluster can be heterologously expressed in both S. collinus Tü365 and S. coelicolor M1152, with increased aurodox titres compared to those of S. goldiniensis. This work demonstrates how traditional physiological analysis and modern genetic techniques can be combined to improve natural product production for detailed mode-of-action studies.
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Comments to Author
The manuscript reports the optimization of media and heterologous strains of Streptomyces to improve aurodox production. In general, the work presented has some deficiencies that should be corrected to consider this work suitable for publication. 1. It is necessary to improve the introduction to put the work in context with respect to the information already available, in this way it will facilitate understanding by non-specialized readers. For example, does aurodox have an antibacterial effect against grambacteria? If so, it should be reported against which gram- bacteria it is effective and references provided. If it is only effective against gram+, it should have no effect against Klebsiella. Is there anything previously described in the literature about the effect of aurodox on klebsiella? 2. …
Comments to Author
The manuscript reports the optimization of media and heterologous strains of Streptomyces to improve aurodox production. In general, the work presented has some deficiencies that should be corrected to consider this work suitable for publication. 1. It is necessary to improve the introduction to put the work in context with respect to the information already available, in this way it will facilitate understanding by non-specialized readers. For example, does aurodox have an antibacterial effect against grambacteria? If so, it should be reported against which gram- bacteria it is effective and references provided. If it is only effective against gram+, it should have no effect against Klebsiella. Is there anything previously described in the literature about the effect of aurodox on klebsiella? 2. Section corresponding to Figure 1 should be discussed more depth: - There are discrepancies between the detection of the peaks and their correspondence with the antibacterial activity. For example, in the NB and AP medium there is activity, and no peak is indicated in Figure 1C. How do the authors explain the discrepancy? - There is a lack of information on the culture conditions in the different media, incubation time, etc. This is relevant since in the following section the production in different phases of growth is compared. - Lack of information on the source of the reagents including the aurodox standard procedence. 3. Section corresponding to Figure 2: - What is the terminal time? in the growth curve and in the text, it is indicated 70 hours but for the extracts is 154h - in the previous section it is indicated that the aurodox production has a level of purity comparable to that of aurodox standard, but the HPLC result of this section is different, what is the reason for this difference? - Have these extracts been tested for antimicrobial activity? - The discussion of this section is missing. 4. Regarding the heterologous expression (Figure 3): - Figure 3C is not cited in the text nor in the figure legend. - In the main text it is stated that sensitivity tests for heterologous strains were performed and that the results are shown in Figure S1, but this figure does not correspond to these results, and they are not shown in any figure. - This section should be better explained and discussed because it is not well understood. It should be discussed what could be the reason for the antimicrobial activity in the control strains, as well as the inhibition of endogenous activity in the case of S venezuelae. - Figure 3, if possible, should include the LCMS analysis of culture extracts from the controls. 5. In the Conclusions section: Authors indicated that the titre has been improved but, if the conclusion is that the best condition is to use S. colelicolor in GYM, then these conditions are not new, but are the same as in their previous article (ref 11). Therefore, the conclusions should be better stated. Other comments: - Pag 4, L84: reference (11) must be wrong because it does not correspond to old fermentation protocols. - In pag 6 where it says table S1 should be table S2 - Pag 6 L 138: where it says Figure 1B should be 1C
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: Not applicable
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In summary, the study presents a detailed description of the optimisation for the production of aurodox used by the authors for a previous work. After the peer-review process, several concerns have emerged that need to be addressed prior to publication. Please, consider all the reviewers’ suggestions and comments thoroughly, especially those concerning: • Including more context and information in the introduction to give a complete background to the non-specialised reader. • Major changes in the figures: missing data, scales, discrepancies with antibacterial activity of the fractions, legends, citations within the text. • Information about the source of reagents and clarity of the culture conditions. • Clarity in the description of the results and depth in their interpretation and discussion. Please provide a revised …
In summary, the study presents a detailed description of the optimisation for the production of aurodox used by the authors for a previous work. After the peer-review process, several concerns have emerged that need to be addressed prior to publication. Please, consider all the reviewers’ suggestions and comments thoroughly, especially those concerning: • Including more context and information in the introduction to give a complete background to the non-specialised reader. • Major changes in the figures: missing data, scales, discrepancies with antibacterial activity of the fractions, legends, citations within the text. • Information about the source of reagents and clarity of the culture conditions. • Clarity in the description of the results and depth in their interpretation and discussion. Please provide a revised manuscript containing all changes and a point-by-point response to all the reviewers’ comments within 2 months.
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Comments to Author
Overview The aim of this study was to optimise small scale production of the aurodox antibiotic for laboratory studies. The authors did this by growing the native producing strain in different growth media and identified GYM as the best for production in liquid (in stationary phase). They also tested four heterologous host strains by introducing the auredox gene cluster in trans. One of these - S. coelicolor M1152 - they have shown in their previous study to be the best host. The conclusions of this work are that their existing heterologous host, S,. coelicolor M1152, is the best of the four hosts they tested and that GYM is the best production medium for batch cultures. Points to be addressed by the authors: Introduction: "Historic fermentation protocols described in the early papers largely use …
Comments to Author
Overview The aim of this study was to optimise small scale production of the aurodox antibiotic for laboratory studies. The authors did this by growing the native producing strain in different growth media and identified GYM as the best for production in liquid (in stationary phase). They also tested four heterologous host strains by introducing the auredox gene cluster in trans. One of these - S. coelicolor M1152 - they have shown in their previous study to be the best host. The conclusions of this work are that their existing heterologous host, S,. coelicolor M1152, is the best of the four hosts they tested and that GYM is the best production medium for batch cultures. Points to be addressed by the authors: Introduction: "Historic fermentation protocols described in the early papers largely use poorly defined media components and processes failed to take advantage of developments in extraction methods, analysis.." It is difficult to see from this intro what advance this study has on existing methods, the previous work on aurodox and the "poorly defined media components…" should be introduced with clear reference to how they have improved upon this (is GYM a defined medium?). Results: If there is no detection of any aurodox ion (as stated on line 133 by the authors) to support the corresponding (weak) retention time then aurodox cannot be confirmed to be present. They should state this clearly. Did they detect aurodox ions in any of the other growth media? The authors state on line 154 that aurodox was detected in positive and negative mode from the MS analysis - these data should be shown. This is not an isolated example - all LCMS data should be presented in full. Showing only the LC data from an LC-MS study and photographs of chromatograms as poor quality jpegs is insufficient. The authors must publish their data (.raw) using FAIR principles, The following edits should be made so that fair and informative conclusions can be drawn based on a sufficient quantity and quality of data: Line 123/124 'yields of aurodox from shake flask fermentations of S. goldiniensis were poor' - data and evidence to support this is missing. Figure 1A does not add to the manuscript and should be removed. Without blank media controls these images are meaningless Figure 1C needs to be revised - it is illegible in size and the absorbance units are misleading - ranging from 4 to 25. The TICs should be aligned all together or at the very least be on the same scale. Only the chromatogram is shown - this only shows peaks eluting at certain retention times. The corresponding MS data showing the peak being attributed to the aurodox ion should also be shown (standard in comparison to various media). Retention times are insufficient evidence of aurodox production. Figure 2B: as in Fig 1C this needs to be revised - it is illegible in size, the absorbance units are misleading - ranging from 4 to 25. The TICs should be aligned all together or at the very least be on the same scale and the corresponding MS data showing the peak being attributed to the aurodox ion should be shown. If this is a PDA detector, as you have the known concentration of standard you should be able to quantify peak area. The objective of the paper was to 'increase titer', you should therefore be quantitative in your results. Figure 3B - all the points already raised for the other chromatograms (1C and 2B), re: show the MS data, standardise the scales etc. Minor corrections: Line 68. Should be "exert" not "exhibit" Line 121. "…and this is known as…" Line 127. "…to determine the optimum…" Line 130. Give full names of growth media at first mention. Line 142. The word in should not be in italics Line 164. "to further investigate heterologous expression" Line 177. …and kirromycin, Given (should be a full stop not comma). Line 187. Should be "two host strains" not "multiple hosts"
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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