Enhancement of Triterpenoid Synthesis in Antrodia cinnamomea through Homologous Expression of the Key Synthetic Pathway Genes AcLSS and AcERG4
Round 1
Reviewer 1 Report
In this manuscript, the key genes involved in the MVA pathway and subsequent group modification pathway were determined in Antrodia cinnamomea by correlation analysis under different fermentation cultural conditions. Then the AcLSS and AcERG4 gene were homologous expressed by PEG-CaCl2-mediated transformation, and the recombinant strains achieved higher triterpenoid content than wild type. In view of previous studies on Antrodia cinnamomea triterpenoid content enhancement, this manuscript explored the method of molecular breeding. However, several confusions and/or errors were found in the current version. I think this manuscript could be published by this journal after revision.
1) Please make sure that the gene name AcERG4 matches with the enzyme name sterol C-24 reductase.
2) In Section 2.4, the draft of the plasmid should be list to introduce the elements of the vector.
3) How did you calculate the number of protoplasts in section 2.6?
4) Line 179: The references of “previous reports were suggested to list.
5) Line 158: the primer name of hygR should be in italic.
Author Response
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Author Response.pdf
Reviewer 2 Report
The manuscript is interesting because it aims to improve the production of triterpenes in the filamentous fungi Antrodia cinnamomea (AC) by overexpressing two different genes of the pathway. That is important since genetic modifications for metabolite production improvement is not usual yet and the authors could confirm the efficiency of the applied transformation method. Besides, it is very well written and figures are ok. It is manuscript suitable for publication in Fermentation.
However, the authors worked with two general enzyme coding sequences (LSS and ERG4) of the primary metabolism of fungi, which are present in the pathway to the essential molecule of ergosterol. Also, they did not mention which triterpenes are so important in AC and produced after genes overexpression. The genes overexpression could be improving only ergosterol production, but it was not investigated. Is lanosterol and/or ergosterol the precursor or intermediated in their biosynthesis? Or the biosynthesis of the claimed important triterpene is a competitive path to ergosterol, which is essential for fungi cell wall?
Also, there are other important observation to adjust in the manuscript, as follows:
- In line 63, the authors wrote "during recent decades" and in line 64 they give a reference of 1974 (number 27) as example. A reference of the 70s is not recent.
- Line 82: please give the meaning of YM medium.
- Line 107: a reference for the vanillin-glacial acetic acid method should be given.
- Line 125: it is missing the denaturation step in the 40 cycles of qPCR.
- item 2.4., line 131: when working with a plasmid genetic description should be given: is it for episomal expression or integrative? which is the selection marker? Which is the promoter for cloned genes? Is this plasmid coding eGFP? under which promoter?
- Line 133: change "od" for "of"
- Item 3.3: a triterpene pathway squeme could contribute enormously to the manuscript understanding.
- Lines 204 to 207: the expression level of genes in ergosterol biosynthesis can be affected by adaptative responses in the cell and have regulatory proteins involved (as showed in doi: 10.3390/genes11070795 and https://doi.org/10.1007/s11274-019-2673-2). A simple conclusion as given in those lines, based only in a single qPCR experiment, can be superficial.
-Item 3.4: to confirm what is described in that part of the manuscript, it is important to state the plamid size, LSS and ERG4 gene sizes. Also, besides the band corresponding to plasmid, there are three other small bands at lane 1 and two small bands at lane 2. Why?
- Item 3.5. Lines 225 to 227: as understood, LSS and ERG4 coding sequences were cloned from AC species and an extra copy was integrated in the genome. Therefore, AC without LSS and ERG4 also have the natural LSS and ERG4 genes. So, endogenous and natural LSS and ERG4 could also amplify from cDNA synthetized from AC mRNA. Why are the authors sure those amplicons in Figure 4B are from the transformed sequences and not from the natural genes?
- Line 238: I would like to have it clear in the manuscript if oleanolic acid in culture was not a interferent in triterpene quantification method.
- Line 246: when the authors stated "ACT synthesis was strongly promoted" which triterpenes? Is not only ergosterol and their intermediates?
- Line 259: please give examples of the mentioned studies in "however, most studies have focused on effects of culture conditions on gene expression levels"
I recommend the authors to consider the comments and adjust the manuscript.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Manuscript has been revised and improved very much. Added figures and schemes will contribute to the manuscript success.
As said before, it is very interesting a manuscript about metabolic engineering in a non-model fungi which is also a filamentous fungi.
However, it remains unknown which triterpenes were produced in higher amount when LSS and ERG4 were overexpressed. Therefore, it is reasonable that authors clearly state in the manuscript that lanosterol and ergosterol are the known triterpenes produced in fungi by those enzymes overexpressed and they could be the ones with increased production.
Also, in Figure 3B it should be explained the secondary bands in lanes 1 and 2, as the answer given in the point-to-point response.
It is also intriguing why primers to confirm LSS and ERG4 genome integration did not amplify the same genes naturally ocurring in the fungi. The answer previously given to this comment is not suitable. If genomic or cDNA were used as template, there should be amplification of endogenous LSS and ERG4 although they have introns and primers were designed considering that. Besides, authors stated in Figure 4 legend that "(B) Amplification of AcLSS and AcERG4 genes from AC transformant genomes." It is ok if the primers only amplified integrated LSS and ERG4 because the sequences were diferent from the endogenous ones; or it is ok if primers annealed to a sequence in the vector. But the reason should be presented in the text.
After those minor explanation, I will consider the manuscript ok for publication.
Author Response
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Author Response File: Author Response.pdf
