Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System

Round 1

Reviewer 1 Report

Dear Authors of the ,
Your manuscript is well written. The introduction is appropriate and fun. The methods are well described, although SDS PAGE description is missing and needs to be completed. In my opinion, the results are well described. The discussion, in my opinion, may be more detailed.

Author Response

Dear reviewer 1,

Thanks for your kind and helpful comments. We have added some description on SDS-PAGE analysis and also some detailed discussion in the revised manuscript. We hope this revised version will meet with your opinion.

The detailed revision was as follows:

“As shown in the SDS-PAGE analysis of the fermentation supernatant of strain KM71/pGAP-integrated-pGCW14-episomal in 3.6 L fermentor (Fig. 5), a protein of the predicted size (43.0 kDa) for XynA was the main band, which also showed obvious xy-lanase activity. It could be confirmed that XynA was the primary expression product during the whole period.”

Reviewer 2 Report

This paper describes higher expression of xylanase activity in Pichia pastoris, using a combination of integrated and autonomously replicating vectors for gene expression. P. pastoris is a useful system for production of recombinant proteins, and improvement of activity without using a methanol-inducible system is of interest for constitutive enzyme production.

Comments for the authors’ consideration:

1. The manuscript would benefit from light polishing for English language. In particular: in the abstract, text, figure legends, and Figure 6D caption, correct the spelling of integrative and integrating and integrated. Abstract Line 20: change protain to protein
2. For secreted enzymes such as xylanase, the enzyme activity on a volume basis in the culture supernatant is useful as stated in the manuscript. In addition, the enzyme activities should be expressed as specific activities, i.e. activities on a protein basis.
3. A related question: What was the final cell density of the cell cultures? Could any of the difference in enzyme activity in the supernatants be due to differences in cell growth? I don’t see where that is reported for Tables 1 and 3.
4. Line 373: was the band in Figure 5 actually shown to have XynA activity? If not, it would be correct to say a protein of the predicted size (how many kDa?) for XynA was the primary expression product.
5. If I understand the text correctly, some of the strains contain the xynA gene both integrated into the genome and on a replicating plasmid. It seems possible that the two copies of xynA would lead to recombination and possibly disruption of expression. Was this examined?
6. Sections 2. 7 and 2.10: was 1 ml the enzyme culture supernatant or was the enzyme partially purified?
7. Line 239: I don’t understand the first two sentences in this section as they are written. Is precipitate the correct word here? Maybe something like “prepare” or “inoculate” would be better. Or is there a centrifugation step missing that was used to collect a cell pellet? Liquid nitrogen was added to what precipitate? Was that a cell pellet?
8. The text in Figures 1 and 2 (plasmid names etc.) is not readable when the hard copy is printed at 100% scale. Please increase the font size.
9. Line 249: provide reference or accession # for GAPDH
10. Line 253: was the enzyme really pre-denatured? Or were the reaction tubes pre-incubated or was the DNA (not enzyme) denatured?
11. Line 178: please clarify the meaning of “blown gently”
12. Lines 284 and 289: change lossing to losing; Line 440 change indicateing to indicating; Line 478 change obivious to obvious

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

As the authors stated that methanol is a potential safety hazard in methanol induced yeast expression. This work constructed a novel dual plasmids system in yeast using a combination of strategy of genomic integrating and episomal expression, which not only significantly reduced methanol production but significantly enhanced the target protein yield. Thus, this work is novel and of great importance. It can be accepted with minor revision. The revisions have been marked in the PDF file.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 3,

Thanks for your kind and helpful comments. We have revised the manuscript according to your suggestions point by point. We hope this revised version will meet with your opinion.