Review Reports

Round 1

Reviewer 1 Report

Please to see the attached document.

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for the positive and constructive statements to the manuscript. Please find below the ponit-by-point-reply.

Referees Comments on the manuscript entitled " Regulation of the leucine metabolism in Mortierella alpina” for Journal of Fungi

 

Authors studied the regulation of the leucine metabolism in Mortierella alpina. The topic is currently important and is in good concordance with the journal. This manuscript is well-written and correctly prepared. The whole work (Introduction, Material and Methods, Results and Discussion) is appropriately organized and described. Appropriate methods are used to perform extensive biological research. The presented manuscript can be published in the Journal of Fungi, however the corrections to the manuscript are required.

 

Point 1: page 3 - Table 1 must be localized before Figure 1.

Response: We thank the reviewer for her/his keen eye on this detail. Since Figure 1 and Table 1 are topically related and are interconnected, we decided to refer to both Table 1 and Figure 1 in lane 32. We hence, kept the position of this table as is. However, if required we are fine with any positional changes by the copy editors.

 

Point 2: page 4, lines 90-97 - The purpose of the study must be reformulated in traditional manner.

Response: We rephrased this paragraph as following:

The regulation of the BCAA metabolism has been intensely studied for higher fungi. In contrast, little is known for basal fungi, which are industrially important producers of fatty acids or secondary metabolites. In this study, we investigate the transcriptional and biochemical regulation of the BCAA metabolism and its connection to the malpinin biosynthesis in M. alpina. Contrasting higher fungi, basal fungi hardly transcriptionally regulate their BCAA biosynthetic genes. Hence, we aimed at the biochemical characterization of the main enzymes to unroll their metabolic regulon. The constitutively produced BCAA aminotransferases is hardly regulated because it is required for both BCAA anabolism and catabolism. In contrast, a complex transcriptional and metabolic control was observed for the α-isopropyl malate synthase in M. alpina. This key enzyme of Leu biosynthesis is tightly regulated via inhibition by its end-product L-leucine, products of the lipid catabolism and BCAA degradation.

 

Point 3: page 4 - Starting from the fourth page, the correct pagination is not respected.

Response: The pagination has been fixed.

 

Point 4: Please to include Conclusion section. Please try to develop the major achievements presented in the manuscript and highlight the impact of those achievements on further research in the scientific field.

Response: The conclusion section has already been included int the former version of the manuscript. We improved now the conclusion section by rephrasing this paragraph with a more global view on the the topic as following:

In contrast to higher fungi, the BCAA biosynthesis pathways in basal fungi are predominantly metabolically rather than transcriptionally regulated. BCAA production is a constitutive process as it is required for protein biosynthesis, fatty acid metabolism and secondary metabolite production as well. However, it can be quickly modulated if environmental metabolite fluxes change. The main regulatory protein is the plant-derived α-isopropyl malate synthase, whose gene has been presumably introduced by horizontal gene transfer. The intimin interaction of basal fungi with bacteria or plants is a widely described phenomenon. Interspecific gene transfer provides an efficient strategy to adapt to altered environmental conditions and may have contributed to the ubiquitous spread of basal fungi in various ecological niches. From a industrial angle, the described complex inhibition loops regulating the leucine metabolism in M. alpina may help to increase the production yields of polyunsaturated fatty acids. 

Reviewer 2 Report

This manuscript describes the metabolism and regulation studies of the leucine biosynthesis in Mortierella alpine. The leucine biosynthesis is complicated compared to other biosynthesis, but as a whole, the leucine biosynthesis has been studied previously. This manuscript, however, focused on the leucine biosynthesis from fungus Mortierella alpina, and found some differences from others. Also, the regulation has been studied here using biochemistry methods, and the regulation study was intriguing. Multifaceted approaches were used to determine the regulation, and the kinetic studies were thorough. Although this discovery is not completely new, this work for the regulation is well worth to be published.  

Author Response

Comment: This manuscript describes the metabolism and regulation studies of the leucine biosynthesis in Mortierella alpine. The leucine biosynthesis is complicated compared to other biosynthesis, but as a whole, the leucine biosynthesis has been studied previously. This manuscript, however, focused on the leucine biosynthesis from fungus Mortierella alpina, and found some differences from others. Also, the regulation has been studied here using biochemistry methods, and the regulation study was intriguing. Multifaceted approaches were used to determine the regulation, and the kinetic studies were thorough. Although this discovery is not completely new, this work for the regulation is well worth to be published.

Response: We thank the reviewer for her/his opinion and this positive review.