Optimization of Fermentation Conditions for Elevating Limonene Production with Engineered Rhodosporidium toruloides
Round 1
Reviewer 1 Report
The paper by Zhao et al. presents fermentation data for elevating limonene production using Rhodosporidium toruloides strain. The effects of carbon source, inoculation, working volume, temperature, pH, dodecane coverage, and media on limonene production were investigated. The manuscript is well written, organized and adds new understanding to the fermentation processes to obtain valuable products. I have only one minor note:
The manuscript contains no information about the possible industrial implementation of the fermentation processes under study. What carbon source is recommended in this case?
I do not have any major concerns, and recommend consideration for inclusion in FERMENTATION.
Author Response
Comment 1: The manuscript contains no information about the possible industrial implementation of the fermentation processes under study. What carbon source is recommended in this case?
Response: Thanks for your comments! At the current stage, the performance of the engineered strains should be further improved via in-depth metabolic engineering. Actually, Rhodosporidium toruloides can utilize a wide range of low-cost feedstock such as lignocellulosic hydrolysates, waste glycerol from oleochemical industry, food waste and so on. Of course, sugarcane juice could be utilized as a clean carbon source for limonene production.
Reviewer 2 Report
This research aimed to enhance limonene production by engineered strains of R. toruloides. The effects of carbon source, inoculum, working volume, temperature, pH, dodecane coverage, and media were explored. Single-factor and orthogonal experiments were conducted to improve the limonene titer. The study contents are not novel at all, in addition, the text is not well organized. The draft should be totally reworked. Its present status is not worthy of publication. Several comments are listed below.
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1. Introduction, “To date, microbes include Escherichia coli [11], Saccharomyces cerevisiae [9,12–14], Yarrowia lipolytica and Rhodotorula toruloides [15,16] have been employed for limonene production. However, the maximum titer was 3.63 g/L with engineered E. coli BL21 (DE3) in a bioreactor”, To the reviewer, there are already lots of papers concerning limonene production, and the production levels have reached 3g/L. Why the author select to use their low production level strain for study?
2. The engineered strains of R. toruloides (carrying PPGK-Ble-Tnos-Pxyl-CltLS1-NPPS- tHMGR-Thsp and pZPK-PPGKNtc-Tnos-Pxyl-MmMK-EfMvaS-EfMvaE-Thsp). To the reviewer, the construction and characteristics of this strain should be introduced in detail. It is hard to understand what the values are for trying to use this low production engineering strain for limonene production.
3. In section 3.2.6. The Effect of the Medium on Limonene Production: The media YPD, SD, MM, NL studies are quite common practices in the first place. The study should begin with media study for selection of a better media, then to test the carbon and nitrogen sources and their concentrations. The draft seems to reverse the study order.
4. Figure 2a, how to determine the error bars significance. Figure 2b, loss of y-axis titles.
5. Opitimization of Limonene Production through Orthogonal Experiments, this test is too simple and not able to obtain the real optimal condition. The response surface methodology should be applied to obtain the real optimization conditions.
Should be revised carefully by a native English speaker editor.
Author Response
Reviewer #2
Comment 1: 1. Introduction, “To date, microbes include Escherichia coli [11], Saccharomyces cerevisiae[9,12–14], Yarrowia lipolytica and Rhodosporidium toruloides[15,16] have been employed for limonene production. However, the maximum titer was 3.63 g/L with engineered E. coli BL21 (DE3) in a bioreactor”, To the reviewer, there are already lots of papers concerning limonene production, and the production levels have reached 3 g/L. Why did the author select to use their low production level strain for study?
Response: Thank you for your interesting comments. Rhodosporidium toruloides is a non-traditional yeast that can utilize a wide range of low-cost carbon source, and has been explored for various terpenoids production. Limonene in a typical monoterpene that featured with divers applications in food, medicine, agriculture, and so on. Here, we want to provide information for further tapping the potential of R. toruloides as the workhorse for terpenoids production. The results in the manuscript demonstrated the possibility of R. toruloides for elevated limonene production under optimized fermentation. However, we have to admitted that the red yeast should be systematically rewired with metabolic engineering to give a more desirable performance.
Comment 2: The engineered strains of R. toruloides (carrying PPGK-Ble-Tnos-Pxyl-CltLS1- NPPS-tHMGR-Thsp and pZPK-PPGKNtc-Tnos-Pxyl-MmMK-EfMvaS-EfMvaE-Thsp). To the reviewer, the construction and characteristics of this strain should be introduced in detail. It is hard to understand what the values are for trying to use this low production engineering strain for limonene production.
Response: Thank you for your suggestion. The details for the construction of characteristics of the engineered strain has been added in the revised manuscript. Rhodosporidium toruloides is a non-traditional yeast that can utilize a wide range of low-cost carbon source, and has been explored for various terpenoids production. The results in the present study demonstrated the possibility of R. toruloides for elevated limonene production under optimized fermentation. However, it should be further rewired with metabolic engineering to give a more desirable performance.
New text: The carotenogenesis-defcient R. toruloides NP11 was kindly provided by Prof. Zongbao kent Zhao from Dalian Institute of Chemical Physics, CAS, and used as the parental strain. The R. toruloides engineered strain was constructed through the Agrobacterium-mediated transformation (ATMT) [16]. The ATMT experiment was conducted as follows [16]. First, the vector carrying Agrobacterium tumefaciens cells were cultivated in LB medium supplemented with 50μg/mL kanamycin at 28 ℃ for 15 h, and R. toruloides NP11 cells in YPD medium at 30 ℃for 15 h. Second, cells were collected by centrifugation at 8,000 g for 30 s, then washed twice and diluted with sterilized water to an optical density 0.6 (OD600). For transformation, 100 μL of each cell suspension was mixed, and spread onto the IM agar plates (MM medium with 200 μM acetosyringone and 20 g/L agar) , and incubated at 25 ℃ for 2 days. Then, the transformant was transferred onto the selection plate to incubate until colony generated. The obtained transformants were streaked on selection YPD for five successive generations to verify their phenotypic stability. Finally, the resultant tranformants were subjected for testing limonene production efficiency and stability.
Comment 3: In section 3.2.6. The Effect of the Medium on Limonene Production: The media YPD, SD, MM, NL studies are quite common practices in the first place. The study should begin with media study for selection of a better media, then to test the carbon and nitrogen sources and their concentrations. The draft seems to reverse the study order.
Response: Thank you for your suggestion. We cannot agree with you more that the study should begin with media study for selection of a better media, then to test the carbon and nitrogen sources and their concentrations. As an unconventional, R. toruloides usually grows faster in YPD medium, accumulates more cell mass and carotenoids. Thus, at first, we considered the accumulation of cell mass as the priority. To our surprise, however, although the cell mass from the YPD medium was much higher than those from other media, the limonene production was best with MM medium. The result might be attributed to different the characteristics of the target products. Therefore, once more desirable genetically engineered strains produced, we will first begin with MM medium for fermentation optimization in the future investigations.
Comment 4: 4. Figure 2a, how to determine the error bars significance. Figure 2b, loss of y-axis titles.
Response: Thank you for your comments. The y-axis title for Figure 2b has been added. The significance analysis was conducted through the t-test in SPSS. For Fig. 2a the significance analysis was conducted, respectively, for each strain based on their final titers of three-round fermentation.
Comment 5: Opitimization of Limonene Production through Orthogonal Experiments, this test is too simple and not able to obtain the real optimal condition. The response surface methodology should be applied to obtain the real optimization conditions.
Response: Thanks for your constructive suggestions! Indeed, the response surface methodology is a much better strategy than the orthogonal design to obtain the real optimization conditions. We would definitely optimize the fermentation system with the response surface methodology in the future as long as we obtained a much more efficient producer. Based on the current results, we could conclude that optimization of fermentation conditions is very helpful for enhancing the limonene production, however, the red yeast should be further redressed at genetic and metabolic level to achieve desired titers.
Reviewer 3 Report
In general, the manuscript was well prepared and fit well the scope of the journal. However, there are still some fields to work on. The comments are as follows:1. Please specify reagent and chemicals manufacturers.
2. The Latin name of the microbes should be written in italic, please check throughout the manuscript.
3. Too many prepositions in the sentences. For example, In Materials and Methods the description of Orthogonal test should be revised, "an orthogonal design of an". Please revise the whole manuscript.
4. The analytical method is too brief, please give more details.
5. In part 3.2.6, the words “microbial host” should not be in italic.
6. Was the original titer of limonene before optimization 52.5 mg/L or 52.2 mg/L? Please check it.
Author Response
Comment 1: Please specify reagent and chemicals manufacturers.
Response: Thank you for your suggestion. We have provided the related information in the revised manuscript.
New text: Limonene standard was purchased from Sigma-Aldrich (Shanghai, China), and 3, 5-dinitrosalicylic acid, hexane, and dodecane were purchased from Shanghai Kefeng Industry Co., Ltd. (Shanghai, China). All the other reagents were obtained locally.
Comment 2: The Latin name of the microbes should be written in italic, please check throughout the manuscript.
Response: Thank you for your advice. The problem has been fixed throughout the manuscript.
New text: Optimization of Fermentation Conditions for Elevating Limonene Production with Engineered Rhodosporidium toruloides.
Comment 3: Too many prepositions in the sentences. For example, In Materials and Methods the description of Orthogonal test should be revised, "an orthogonal design of an". Please revise the whole manuscript.
Response: Thank you for your advice. In Materials and Methods, the “an orthogonal design of an L9 (34) array was····” has been revised to “array of L9 (34) was conducted to ····”. In 3.3. Opitimization of Limonene Production through Orthogonal Experiments, “a L9 (34) orthogonal experiments were conducted······” has been revised to “the L9 (34) orthogonal experiment was conducted······”.
New text:
Based on the single-factor experiments above, an orthogonal array of L9 (34) was conducted to optimize the limonene fermentation. To optimize the fermentation system, the L9 (34) orthogonal experiment was conducted with limonene titer as the investigation index.
Comment 4: The analytical method is too brief, please give more details.
Response: Thank you for your comment. The detailed information for “analytical method” has been added in the revised manuscript.
New text: All experiments were performed in triplicates, and the data were presented as mean and standard deviation. Statistical significance was determined using one-way ANOVA followed by Tukey multiple ranged tests (SPSS22.0 software, Chicago, IL, USA), and statistical differences between the means were evaluated using least significant difference (LSD) analysis at P=0.05. Histograms were drawn using Datagraph 4.3.0 (USA).
Comment 5: In part 3.2.6, the words “microbial host” should not be in italic.
Response: Thank you. The problem has been fixed.
New text: Monoterpenes usually pose certain cytotoxicity on microbial host. The bi-phasic fermentation system is a common practice to facilitate the extraction of monoterpenes and the elimination of its toxicity.
Comment 6: Was the original titer of limonene before optimization 52.5 mg/L or 52.2 mg/L? Please check it.
Response: Sorry for the typing mistake. The original titer was 52.5 mg/L.
Round 2
Reviewer 2 Report
This paper is now acceptable for publication.
