Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

Round 1

Reviewer 1 Report

The authors show the efficiency of the K. phaffii of the glyceraldehyde-3-phosphaste dehydrogenase promoter coupled to the S. cerevisiae MATalpha  targeting sequence in the production of truncated extracellular dextranase DEX49A from T. minioluteus. Their experimental approaches are standard and well-conducted.

I think the introduction to be shortened to make it more to the point. For example, the authors go into a deep structural description of DEX49 which has no direct bearing on the aim of the paper. They should also introduce GAP promoter in line 89 and not 88. Line 69 they use Pichia and only line 89 say that its new name is K. phaffii. Line 77 there is a beta symbol that has not converted well in pdf.

In the materials and methods, there is no description of the YPGlycerol composition, lines 130 and 142 they should use the abbreviation to make the text consistent. The authors never explain why they use a cerevisiae secretory targeting sequence instead of a phaffii which would seem more logical. They also don't explain why they use phaffi in which the endogenous dextranases have to be mutated instead of cerevisiae which is devoid of those activities in their proof of concept. Is it solely because of the more efficient secretory system?

In the results, the authors should rewrite the first 2 sentences to make more active by replacing may be by is since they are divided and it should by to. Again abbreviation line 245. Line 255 maybe add that lane 2 is undigested plasmid.

Figure 2, to what do the nulbers and letters refer to? Are they related to the zeocin concentration used in their obtention? Why are panel in roman numeral whereas all other figures panels are with letters. Homogenize the type setting style and size between figures.

Figure 4, would it be possible to improve the contrast like in figure 2 panel III.

In paragraph 3.5, the authors say that the use of their medium cheaper than complex medium and, allows to achieve higher biomass and for easier purification. I strongly doubt the 2 first points especially when using glycerol as carbon source. The third point is very valid especially for the use of this enzyme in the production of sugar. Sentence 419-423 is very hard to understand.

All in all, the paper is very interesting for the yeast community for secreted protein production and will be of interest to many industrials needing high extracellular activity.

Author Response

Reviewer (R): I think the introduction to be shortened to make it more to the point. For example, the authors go into a deep structural description of DEX49 which has no direct bearing on the aim of the paper. 

Author (A): This has been solved in the text by elimination of the paragraph.

R: They should also introduce GAP promoter in line 89 and not 88.

A: Done

R: Line 69 they use Pichia and only line 89 say that its new name is K. phaffii.

A: Solved in the text

R: Line 77 there is a beta symbol that has not converted well in pdf.

A: Solved with the elimination of the paragraph

R: In the materials and methods, there is no description of the YPGlycerol composition

A: Solved in the text

R: lines 130 and 142 they should use the abbreviation to make the text consistent.

A: Solved in the text

R: The authors never explain why they use a cerevisiae secretory targeting sequence instead of a phaffii which would seem more logical.

A: The reviewer's reasoning is logical,  but we used a commercial vector (pJexpress915 from ATUM company) which is already designed with the Saccharomyces cerevisiae prepro a-factor (MFalpha-2) signal sequence, which has proven to be very efficient for the secretion of proteins in K. phaffii. The evaluation of the secretion of this dextranase under native K. phaffii signal peptides it is something that could be done maybe in the future, but that would be matter of other manuscript.

R: They also don't explain why they use phaffi in which the endogenous dextranases have to be mutated instead of cerevisiae which is devoid of those activities in their proof of concept.

A: K. phaffii does not have to be mutated, because naturally it does not have endogenous dextranases. And we use K. phaffi because it has some other very well documented elsewhere advantages over the S. cerevisiae expression systems.

R: In the results, the authors should rewrite the first 2 sentences to make more active by replacing may be by is since they are divided and it should by to.

A: Solved in the text.

R: Again abbreviation line 245.

A: Solved in the text

R: Line 255 maybe add that lane 2 is undigested plasmid.

A: Solved in the text

R: Figure 2, to what do the numbers and letters refer to?

A: Numbers and letters refer to the designation of the different clones for which the dextranase enzymatic activity was quantified and compare each other after biomass normalization. 

R: Are they related to the zeocin concentration used in their obtention?

A: Numbers and letters as I mentioned above are not related with the zeocin concentration

R: Why are panel in roman numeral whereas all other figures panels are with letters.

A: I used roman letters in figure panels every time in the figures were used numerals and letters as part of the figure itself.

R: Homogenize the type setting style and size between figures.

A: Solved in the text

R: Figure 4, would it be possible to improve the contrast like in figure 2 panel III.

A: Maybe, but I certainly don´t know hoe to do it

R: In paragraph 3.5, the authors say that the use of their medium cheaper than complex medium and, allows to achieve higher biomass and for easier purification. I strongly doubt the 2 first points especially when using glycerol as carbon source.

A: Solved in the text

R: Sentence 419-423 is very hard to understand

A: Solved in the text

Reviewer 2 Report

In this study, the authors generated a new variant of the Talaromyces minioluteus endodextranase, expressed in K. phaffii (P. pastoris), an important yeast in biotechnology ideally suited to the large-scale expression of recombinant enzymes. By replacing the N-terminal region with the signal sequence from MF-alpha-2, the authors demonstrate expression of large quantities of active dextranase in the supernatant, in a manner scalable from plates to large 5L fermenter cultures. Overall, the paper is clear, with a well-written introduction that highlights well the biotechnological importance of dextranases in the sugar industry. The results show that this recombinant enzyme has promising potential.

The paper should mention briefly that this type of strategy (fusion to MF-alpha) is inspired from earlier work in budding yeast, see notably (Emr et al, 1983, PNAS), which has been adapted to K. phaffii (Ahmad et al, 2014, Appl Microbiol & Biotechnol). These two papers should likely be cited, to provide some background on available secretion systems in the field.

Line 94 is a good statement of the problem: the enzyme yields are still low. But the authors should mention what they are already in this section, so that a quantified estimate is given and allows the readers to form an informed opinion. (given in line 320, and corresponding to a minimum of 2.7x improvement, and in certain conditions up to 25x as line 414).

Fig 2-III should ideally include a negative control (supernatant of non-transformed K. phaffii PSS 9010), although the t=0 result of Fig 4A is convincing and makes this not essential.

line 357-358: “will be discussed later” – this formulation is vague and does not make it clear that the authors explain the most probable cause in Fig 7. The sentence should include the mention “(see below, section 3.7 and Fig 7)”. Furthermore, Fig 7 should include labels indicating apparent MW sizes, especially at the level of drawn lines. Dextran-binding is an attractive explanation for the 70 to 130kDa shift in MW on gels in Fig 4.

Minor comments:

Fig 1 uses ABC for panels, while Fig 2 uses I,II,III: the authors should modify this to be consistent throughout the manuscript, using one of the systems (most likely ABC as it is more standard).

line 40: “In the health arena” was probably meant to be “In the health area” or “In health sciences,”.

line 77: a symbol is missing and replaced by spirals.

line 239-241: the authors have not removed the default “Results” text from the template. Same comment for lines 514-516 in the “Bibliography” section.

line 425: “somehow” – the authors can speculate on this. Auto-inhibitory domains are frequent in enzymes, this could be a potential cause (among several others).

Fig 7: “p/v” (peso/volumen) should be translated to “w/v” like in the legend.

line 249-250: “the synthetic DNA fragment was verified by sequencing”: clarify if this was Sanger sequencing, or other technique. We encourage the authors to submit the sequence to a repository database, and provide the accession number in either this section, or at the end of the vector construction segment of Methods.

Author Response

Reviewer (R): The paper should mention briefly that this type of strategy (fusion to MF-alpha) is inspired from earlier work in budding yeast, see notably (Emr et al, 1983, PNAS), which has been adapted to K. phaffii (Ahmad et al, 2014, Appl Microbiol & Biotechnol). These two papers should likely be cited, to provide some background on available secretion systems in the field.

Author (A): Solved in the text

R: Line 94 is a good statement of the problem: the enzyme yields are still low. But the authors should mention what they are already in this section, so that a quantified estimate is given and allows the readers to form an informed opinion. (given in line 320, and corresponding to a minimum of 2.7x improvement, and in certain conditions up to 25x as line 414).

A: I think it is solved in the text, although, I don´t understand very well what the reviewer suggest.

R: Fig 2-III should ideally include a negative control (supernatant of non-transformed K. phaffii PSS 9010), although the t=0 result of Fig 4A is convincing and makes this not essential.

A: It is solved as is said by the reviewer

R: line 357-358: “will be discussed later” – this formulation is vague and does not make it clear that the authors explain the most probable cause in Fig 7. The sentence should include the mention “(see below, section 3.7 and Fig 7)”

A: Solved in the text

R: Furthermore, Fig 7 should include labels indicating apparent MW sizes, especially at the level of drawn lines. Dextran-binding is an attractive explanation for the 70 to 130kDa shift in MW on gels in Fig 4.

A: I think we can not include the apparent MW sizes because the Marker used is for SDS-PAGE under denaturing conditions, and we worked under native conditions without SDS. So, we can not be sure the protein bands will move according to the sizes reported by the manufacturer. The most important in that Fig. is dextran does not affect the mobility of the Marker´s proteins, but it shift the mobility of dextranase.

R: 

Minor comments:

Fig 1 uses ABC for panels, while Fig 2 uses I,II,III: the authors should modify this to be consistent throughout the manuscript, using one of the systems (most likely ABC as it is more standard).

A: Solved in the text but using roman letters.

R: line 40: “In the health arena” was probably meant to be “In the health area” or “In health sciences,”.

A: Solved in the text.

R: line 77: a symbol is missing and replaced by spirals.

A: Solved in the text

R: line 239-241: the authors have not removed the default “Results” text from the template. Same comment for lines 514-516 in the “Bibliography” section.

A: Solved in the text

R: Auto-inhibitory domains are frequent in enzymes, this could be a potential cause (among several others).

A: Solved in the text

R: line 249-250: “the synthetic DNA fragment was verified by sequencing”: clarify if this was Sanger sequencing, or other technique.

A: Solved in the text

R: We encourage the authors to submit the sequence to a repository database, and provide the accession number in either this section, or at the end of the vector construction segment of Methods.

A: Yes, it is a typical procedure. But in our case even when we worked with a synthetic gene, we did not do mayor changes in the sequence, only a deletion. We did not do codon optimization on the original gene. So, we kept the original sequence except the deletion.

Round 2

Reviewer 1 Report

Line 77 the authors should remove Pichia pastoris
Lines 90-95 they mention 30 amino acids deleted and lines 130-132 34 but partially explain the reason. Which is it? in the same paragraph they mention a 200 amino acid deletion to what purpose or than to confuse the reader.
Line 125 it should be 1827 not 1826 since it can't be divided by 3
Line 132 the authors delete 16 amino acids of the mature form of their dextranase but fail to give the rational.
Lines 271-273, the sentence gives the impression that K phaffi does have dextranases and that the strain they are using has been deleted of them which is in contradiction with lines 77-79.
Lines 307-319 should be rewritten so has to explain why they can convert 273U/ml to 273 mg/l and add in the latter case that they are talking about proteins. To this effect line 317 should be moved to line 310.
Lines 338, 350, 351 and 457 keep panel references consistent.
Finally lines 444-447, simplify the sentence by saying that dashed lines correspond to the same molecular weight in the different gels presented.

Author Response

Reviewer 1 (R1): Line 77 the authors should remove Pichia pastoris

Author (A): Solved in the text

R1: Lines 90-95 they mention 30 amino acids deleted and lines 130-132 34 but partially explain the reason. Which is it? in the same paragraph they mention a 200 amino acid deletion to what purpose or than to confuse the reader.

A: We removed the first 30 aa considering ORF-4, starting at M35. This ORF-4 seems to be the coding region that it is really working according to the experimental facts reported by [15]. In this case, the real signal peptide is MGTTXNTXXGADFXTW (16 aa), plus the next 14 aa, is the 30 aa region we removed in the variant we are reporting.

Why this 14 aa? As we say in the Introduction (line 93-95), the Beta-sandwich N-terminal domain (200 aa) in GH49 have not been identified any specific function, for the protein. So, we decided to delete it partially to engineer DEX49A and to see its effect on the enzyme production and activity. As it is shown in the manuscript this deletion does not affect the enzyme and even could enhance its production for a more efficient use in the sugar industry or elsewhere. 

In the same idea, we have also removed the whole N-terminal domain but we got a non active enzyme (unpublished result). But in this sense, we plan to design some other deletion variants to see up to which point it is necessary that domain for the function of the enzyme.

I would like to remark that we don´t mention a 200 aa deletion. What we say is "the 200-residues b-sandwich N-terminal domain in GH49, can be partially deleted to..." that is what we did, a partial deletion.

R1: Line 125 it should be 1827 not 1826 since it can't be divided by 3

A: Solved in the text

R1: Line 132 the authors delete 16 amino acids of the mature form of their dextranase but fail to give the rational.

A: I think we have solved the problem in the text, but sometimes it is hard to find a rational reason when you are in an unknown area as is the case of the function of this domain. For example, in the design of the variant that we deleted the whole N-terminal domain (200 aa) (unpublished) we modeled the truncated variant and performed Molecular Dynamic Simulations and everything looked good, but when we went for it in vitro, it did not work. So, most of the time what you can do is trail and error.

Anyway, we are open to find the best way to say if we did not make it this time with the modification we did.

R1: Lines 271-273, the sentence gives the impression that K phaffi does have dextranases and that the strain they are using has been deleted of them which is in contradiction with lines 77-79.

A: In this case, I´m not agree with the reviewer since dextranase in not a sucrose-transforming enzyme. We don´t say K. phaffii does have dextranases.

What we say in 77-79 is that K. phaffii is a good host for crude dextranase preparations for sugar industry due to it does not produce sucrose-transforming enzymes.

And in 271-273 we say that K. phaffii PSS 9010 lacks dextranolytic activity or endogenous dextranase enzyme. I insist dextranase is not a sucrose transforming enzyme. In brief, in 77-79 we say K. phaffii is good for one reason and in 271-273 we use K. phaffii PSS 9010 for other reason.

Anyway we did some changes in the text, which potentially answer the comment of the reviewer.

R1: Lines 307-319 should be rewritten so has to explain why they can convert 273U/ml to 273 mg/l and add in the latter case that they are talking about proteins. To this effect line 317 should be moved to line 310.

A: Solved in the text

R1: Lines 338, 350, 351 and 457 keep panel references consistent.

A: Solved in the text

R1: Finally lines 444-447, simplify the sentence by saying that dashed lines correspond to the same molecular weight in the different gels presented.

A: Solved in the text.

Reviewer 2 Report

The authors have addressed the points from the previous review. However, there is a mistake in the bibliography, with both Emr et al and Lin-Cereghino et al as ref 25, instead of being refs 25 and 26; this offsets every ref number. This should be fixed and the authors should carefully double-check every reference number in the text and in the bibliography.

Author Response

Reviewer 2: The authors have addressed the points from the previous review. However, there is a mistake in the bibliography, with both Emr et al and Lin-Cereghino et al as ref 25, instead of being refs 25 and 26; this offsets every ref number. This should be fixed and the authors should carefully double-check every reference number in the text and in the bibliography.

Author: Solved in the text