Biotechnology for Biofuels
Transcript of Biotechnology for Biofuels
Biotechnology for Biofuels
Enhancement of docosahexaenoic acid production by overexpression of ATP-citratelyase and acetyl-CoA carboxylase in Schizochytrium sp.
--Manuscript Draft--
Manuscript Number: BBIO-D-19-00571R1
Full Title: Enhancement of docosahexaenoic acid production by overexpression of ATP-citratelyase and acetyl-CoA carboxylase in Schizochytrium sp.
Article Type: Research
Section/Category: Bacterial and fungal/yeast genetics, physiology, and metabolic engineering
Funding Information: National Natural Science Foundation ofChina(No. 31470190)
Dr. Zhi Chen
Abstract: Background: Docosahexaenoic acid (DHA) is an important omega-3 long-chainpolyunsaturated fatty acid that has a variety of physiological functions for infantdevelopment and human health. Although metabolic engineering was previouslydemonstrated to be a highly efficient way to rapidly increase lipid production, metabolicengineering has seldom been previously used to increase DHA accumulation inSchizochytrium spp.Results: Here, a sensitive β-galactosidase reporter system was established to screenfor strong promoters in Schizochytrium sp. Four constitutive promoters ( EF-1α p ,TEF-1 p , ccg1 p , and ubiquitin p ) and one methanol-induced AOX1 promoterwere characterized by the reporter system with the promoter activity ccg1 p > TEF-1p > AOX1 p (induced) > EF-1α p > ubiquitin p . With the strong constitutivepromoter ccg1 p , Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoAcarboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cellswere cultivated at 28°C and 250 rpm for 120 h with glucose as the carbon source.Shake flask fermentation results showed that the overexpression strains exhibitedgrowth curves and biomass similar to those of the wild-type strain. The lipid contents ofthe wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8,69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains wereincreased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strainand of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e.,DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%,respectively.Conclusions: Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis.ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACCcatalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate thatoverexpression of ACL and ACC enhances lipid accumulation and DHA production inSchizochytrium sp.Keywords: Schizochytrium sp., Docosahexaenoic acid, ATP-citrate lyase, Acetyl-CoAcarboxylase, β-galactosidase reporter system, Constitutive promoter
Corresponding Author: Zhi Chen, Ph.D.China Agricultural UniversityCHINA
Corresponding Author E-Mail: [email protected]
Corresponding Author SecondaryInformation:
Corresponding Author's Institution: China Agricultural University
Corresponding Author's SecondaryInstitution:
First Author: Xiao Han
First Author Secondary Information:
Order of Authors: Xiao Han
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Zhunan Zhao
Ying Wen
Zhi Chen, Ph.D.
Order of Authors Secondary Information:
Response to Reviewers: Response to ReviewersBiotechnology for BiofuelsBBIO-D-19-00571Enhancement of docosahexaenoic acid production by overexpression of ATP-citratelyase and acetyl-CoA carboxylase in Schizochytrium sp.
Reviewer #1: It's a good work with significant improvement of DHA production as wellas a tool box for further application in biofuels production in this field. There are somecomments hope authors to take into consideration.1.The work focused on the availability of carbon supply to FA synthesis, however, thedesaturation is also very important to DHA production. The whole profile dynamicinformation of FA should be provided to have a overall view on FA synthesis processother than Fig 5.Response: Thank you very much for your valuable comments and suggestions. Weagree with you that the investigation of the whole dynamic profile will provide moreinformation. Though we can determine DHA yields through gas chromatography, ourlab can not analyze the fatty acid composition due to the lack of some fatty acidstandards. Fatty acid composition analysis in the study was carried out by the detectionplatform of our university which is closed now and will not open in months due to theoutbreak of new coronavirus. So we can not do the experiment. Guo et al. (2016) havereported that the changes of fatty acid composition in Schizochytrium sp. HX-308cultured at different aeration rates. The contents of DHA in TFAs from 40-h to 120-hwere increased gradually from 38.01% to 43.59% and from 40.52% to 44.85 ataeration rates of 0.5 vvm and 1.0 vvm, respectively, and decreased gradually from39.91% to 36.61% at aeration rate of 1.5 vvm. According to the report, DHA content ofSchizochytrium sp. was slightly changed during fermentation and was affected by theoxygen supply.Reference:Guo DS, Ji XJ, Ren LJ, Li GL, Yin FW, Huang H. Development of a real-timebioprocess monitoring method for docosahexaenoic acid production by Schizochytriumsp. Bioresour Technol. 2016;216:422-427.
2.In the industrial process, the glycerol is widely used as carbon other than glucose, sothe performance with glycerol is expected.Response: Thank you very much for your suggestion. As a byproduct in the productionof biodiesel, glycerol is a cost effective carbon source for lipid production. Glycerol wasused as the carbon source for the shake flask fermentation as suggested. Although itwas reported that some Schizochytrium spp. could use glycerol efficiently, we foundhere that Schizochytrium sp. grew poorly in the glycerol medium and the production oflipid and DHA were very low (Fig. I). We were quite surprised by the results, and theshake flask fermentation experiments were repeated with new batch of glycerol, andlower amount of glycerol (60 g/L) was also used to rule out the possibility of theinhibition of high glycerol on growth. Schizochytrium sp. still grew poorly in the glycerolfermentation medium (Fig. IIa, b). Glucose fermentation medium was also used as acontrol in which Schizochytrium sp. grew well and accumulated lipid efficiently (Fig. IIc,d). Even though Schizochytrium sp. grew poorly in glycerol medium, the lipid contentsand DHA yields of ACL and ACC overexpression strains were slightly higher thanthose of the WT strain (Fig. I). Therefore, overexpression of ACL and ACC did increasethe production of lipid and DHA in Schizochytrium sp. ATCC 20888. Since cells grewpoorly in glycerol medium, we would rather not include the results of the glycerolmedium in the manuscript. We don’t know why this strain grow poorly with glycerol, butit seems Schizochytrium spp. differ very much from each other, for example, thereported genome sizes of Schizochytrium limacinum SR21 and Schizochytrium sp.CCTCC M209059 were 63 Mb and 39 Mb, respectively.
Fig. I Effect of ACL and ACC overexpression on dry cell weight (DCW), lipidaccumulation, and DHA production by Schizochytrium sp. in the glycerol medium. Cells
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were grown in fermentation medium with 100 g/L glycerol as the carbon source for 120h. a Dry cell weight (DCW, g/L). b Total lipids (% DCW). c DHA yield (g/L)
Fig. II Effect of ACL and ACC overexpression on dry cell weight (DCW) and lipidaccumulation by Schizochytrium sp. Cells were grown in fermentation medium for 120h with 60 g/L glycerol (a), 100 g/L glycerol (b), 100 g/L glucose (c, d) as the carbonsource. a, b, c Dry cell weight (DCW, g/L). d Total lipids (% DCW)
3.The expression of digital should be improved, and comma is expected to be used.Such as 1,269-bp fragment and 6,000 rpm, etc.Response: Thank you for the suggestion. We have corrected the expression of digitalin the text.
Reviewer #2I have now reviewed the manuscript, which was well-written in many aspects includingabstract, backrounds, m&m and conslusion. However, the discussion section must bere-written since it was written as a the repeatition of the results section. References,Figures and Tables are enough. Some methods are also missing. Therefore, I suggestmajor revision. After revision, it should be reconsidered. My spesific comments can befound on the text attached.Response: Thank you very much for your valuable comments and suggestions. Assuggested, we have carefully revised the manuscript, added some methods andreferences, and reorganized the discussion section. Most active tense sentences wererevised to passive tense except some (Line 120 and Line 175) in which the change willlead to inconsistent subject. Some specific concerns are as follows.
1.Line 25: Is the host or wild type? Please specify it. Line 28: What are they, pleasespecify them.Response: The specification of the strains was listed in Table 2.
2.Line 31: What is the difference between the Lipid yields and DHA yields?Response: Thank you for the question. Lipid included triacylglycerol, free fatty acids,and phospholipids, etc, which was extracted by acid-heating extraction. The fatty acidcomponents of lipids were DHA, palmitic acid (C16:0), myristic acid (C14:0), and DPA,etc (Fig. 6). For DHA yields determination, fatty acid methyl esters were firstly preparedfrom lipid samples and DHA was measured by gas chromatography.
3.Line 133, “β-galactosidase with very low activity”: please give some critical activity inthe text.Response: Only qualitative colorimetric detection was carried out on GPY plates. Theβ-galactosidase activities of the transformants in the liquid medium (seed medium)were shown in Fig. 1d.
4.Line 176, “ACL and ACC”: Italic or non-italic. Please be coherent.Response: The italic form indicates the encoding gene. And non-italic form indicatesprotein or enzyme. It was changed to be “ACL and ACC genes”.
5.Line 191: “Thus, overexpression of ACL and/or ACC did not affect cell growth ofSchizochytrium”. Any reason.Response: The results indicated that overexpression of ACL and ACC did notsignificantly affect the consumption of carbon and nitrogen sources of thetransformants, that is probably the reason why cell growth (dry cell weights) was notaffected.
6.Line 206: “Overexpression of ACL in Schizochytrium sp. WT did not significantlyaffect the percentage of TFAs represented by DHAs……”, Why? Any reason?Line 596 (Table 1): Although DHA yields, Lipid yields, and Lipid contents of therecombinant strains were higher than that of WT, DHA contents were similar to eachother. Any reason.Response: Overexpression of ACL and ACC enhanced the supply of precursors(acetyl-CoA and malonyl-CoA) for the synthesis of DHA and other fatty acids.Therefore, the increased precursors supply in the recombinant strains enhanced theproduction of DHA and other fatty acids at the same time. That is the reason of thesimilar DHA contents between WT and the recombinant strains. The related discussion
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can be found in Line 251-260.
7.The discussion section was similar to the results section or was written as arepeatition of the results. Please compare your results with the existing results in theliterature. What were the similarities and differencies of your results with the data in theliterature? Please discuss them. Also, what is the industrial significance of this study?Please also elucidate this situation. To sum up, please re-write the discussion section.Response: Thank you very much for your suggestion. In the discussion section, wesummarized the main findings in the study which is the foundation for the follow-updiscussion. These sentences have been rephrased to avoid repetition. And thediscussion section has been carefully revised as suggested.
8.Line 270 (Microorganisms and culture conditions section): There are four differentmedium in this section, please use references for each medium and explain why themedium was different?Response: The references have been added to the text. LB medium is used forcultivation of E. coli in which plasmids were constructed. The media and growthconditions for Schizochytrium sp. were according to Ling et al. [5] with modifications. Infact, we did quite a lot of work to optimize the media (especially the fermentationmedium). GPY solid medium which contained less glucose were used for rapidcultivation of Schizochytrium sp. and selection of single colony of the transformants(supplemented with 40 μg/mL zeocin). Fermentation medium which contained highconcentration of glucose was used for lipid and DHA production (high C/N helps lipidaccumulation). And seed medium which had moderate concentration of glucoseguaranteed rapid growth of Schizochytrium sp. and ample cells for fermentationmedium. Cells would grow slowly in fermentation medium if they were inoculateddirectly from GPY solid medium.
9.Line 289: Why the promoters and terminators were amplified from different sources?Please elucidate it.Response: We intended to test the promoter strengths of some commonly-usedeukaryotic promoters (ubiquitinp, EF-1αp, ccg1p, TEF-1p, and AOX1p) and screen forstrong promoters in Schizochytrium (Line 145-162). Therefore, some promoters andterminators were amplified directly from fungi and yeast expression plasmids, andsome were amplified from Schizochytrium sp. itself (endogenous promoters can berecognized by RNA polymerase more efficiently). The results indicated that ccg1p,TEF-1p, and AOX1p from the commonly-used expression plasmids displayed strongerpromoter activities, which was not unexpected because expression plasmids usuallyutilize very strong promoters to ensure the efficient expression of the target genes.Identification of promoters with different promoter strengths can provide suitablepromoters for a balanced expression of the relevant pathways in Schizochytrium.
10.The fermentations in Fig. 1d and Fig. 3a-d can be modeled kinetically using logistic,Luedeking piret and modified luedeking piret models, but not this study. It can beinteresting.Response: Thank you so much for your kind suggestion. We will try to employ thesemodels for growth and product modelling in the future study. It will be very interesting.
Reviewer #3The manuscript by Han et al. tested several constitutive promoters fromSchizochytrium sp. and used the strong ccg1p promoter to overproduce ACL and ACC,which increased the yields of DHA and total lipids.Comments:1.How many independent strains of each transformant were generated and used forthe study? It seems like there is only one strain for each construct, which is notacceptable.Response: Thank you very much for your valuable comments and the question. Aftertransformation, we have selected eight independent strains for each kind oftransformants. The transformants were cultured in fermentation broth for 5 days,stained with nile red dye for neutral lipid staining, and the fluorescence intensities weremeasured by a multifunctional plate reader. All transformants displayed higherfluorescence intensities than the wild-type strain, indicating that the transformantsaccumulated more lipids than the WT strain (Fig. IIIa, b, c). After preliminary screening,two independent strains with the highest fluorescence for each kind of transformants
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were cultured for determination of lipid and DHA yields. The same kind oftransformants displayed similar results (Fig. IIId). All fermentation experiments were atleast repeated twice. For clarity, results from one strain of each kind of transformantswere provided in the text.
Fig.III Effect of ACL and ACC overexpression on lipid accumulation by Schizochytriumsp.. a, b, c Nile red staining of WT, OACL, OACC, and OACL-ACC. d Lipid contents ofWT, OACL, OACC, and OACL-ACC. The strains were cultured in fermentation mediumfor 120 h
2.RNA or Protein levels of ACL and ACC should be examined in the overexpressingstrains.Response: Thank you very much for your suggestion. The transcriptional levels of ACLand ACC were determined by qRT-PCR in WT, OACL, OACC, and OACL-ACC whichwere cultivated in fermentation media for 2 and 4 d. The transcriptional levels of ACLand ACC were greatly increased in the corresponding transformants. The results wereadded to the text (Line 188-194) and the new Fig. 3.
3.Lipid content was increased from 54% of the WT to ~70% of the overexpressingstrains. Figure 4d, why the nile red staining shows such low level of signals in the WT?Did the expression of ACL and ACC changed the morphology of lipid bodies? i.e. sizeand numbers.Response: Thank you for the question. The nile red staining is a very efficient means toindicate the intracellular lipid accumulation. During our nile red staining preliminaryscreening, we found that the fluorescence signals had a positive correlation with thelipid contents, but the correlation was not strictly linear. The increase rates offluorescence signals were usually higher than the increase rates of lipid contents(please see Fig. III, response to comment 1). Besides, fluorescence attenuates duringmicroscopic observations. WT was the first one observed and its analysis might takelonger time than the others’. Microscopic analysis was repeated with 48-h culturedcells (it will be easier to observe the morphology of lipid bodies with “younger” cells)and all photos were taken with the same observation time. Fig. 4d was replaced withthe new figure (now as Fig. 5d), and the relative fluorescence intensities of cells wereanalyzed by ImageJ (Fig. IV), which had a good correlation with the lipid contents. Wethank you very much for your kind reminder. Since Schizochytrium cells aggregatedand produced zoospores during cultivation, it was difficult to distinguish lipid bodies.From what we saw, Schizochytrium formed a large lipid body per cell and no significantdifference of morphology wad noticed between WT and the transformants (Fig. 5d).
Fig.IV The relative fluorescence intensity of WT, OACL, OACC, and OACL-ACC
4.Explain or discuss the changes of fatty acid composition especially on the increase ofDHA in ACC and ACL-ACC.Response: Thank you for your suggestion. Please see answer to comment 6 ofReviewer 2. The discussion about the changes of fatty acid composition on theincrease of DHA in OACC and OACL-ACC strains can be found in Line 251-260.
Reviewer #4Some grammatical problems are seen in the whole manuscript which should berevised. Also, the organization of the manuscript especially in the introduction part isnot good. One more attention is needed in the discussion part. Some related paperswith for lipid extraction (with modifications) are introduced here for improvement of thelipid extraction part, determination of biomass dry weight and some other part in thematerial and method, cite these references in this part. Paper titles are in theparentheses. Add a table in discussion part and compare related studies.Best wishes1-Process Safety and Environmental Protection III (2017) 757-765http://dx.doi.org/10.1016/j.psep.2017.08.029 (Optimizing of lipid production inCryptococcus heimaeyensis through M32 array of Taguchi design)2-Process Safety and Environmental Protection III (2017) 747-756http://dx.doi.org/10.1016/j.psep.2017.08.027 (Single cell oil and its application forbiodieselproduction)
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3-Int. J. Environ. Sci. Technol. DOI 10.1007/s13762-014-0687-8 (Recycling oflignocellulosic waste materials to produce high-value products: single cell oil andxylitol)4-Int. J. Environ. Sci. Technol. (2014) 11:597-604 DOI 10.1007/s13762-013-0373-2(Improving microbial oil production with standard and native oleaginous yeasts byusing Taguchi design)5-(Medium optimization for biotechnological production of single cell oil using Yarrowialipolytica M7 and Candida sp.)6-(Selection and optimization of single cell oil production from Rodotorula 110 usingenvironmental waste as substrate)Response: Thank you very much for your kind suggestions and the recommendedrelated references. We have carefully revised the manuscript and reorganized thediscussion section. We have earnestly read the recommended references with greatinterest. Three related references have been cited in the methods (determination ofglucose and lipid extraction). We will consider the methods in the related references inthe future study.
Additional Information:
Question Response
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1
Enhancement of docosahexaenoic acid production by 1
overexpression of ATP-citrate lyase and acetyl-CoA 2
carboxylase in Schizochytrium sp. 3
4
Xiao Han, Zhunan Zhao, Ying Wen, Zhi Chen* 5
6
State Key Laboratory of Agrobiotechnology and Key Laboratory of Soil Microbiology, 7
Ministry of Agriculture, College of Biological Sciences, China Agricultural University, 8
Beijing 100193, China. 9
10
*Correspondence: [email protected] 11
12
Marked Up Manuscript Click here to access/download;Manuscript;Marked UpManuscript.docx
Click here to view linked References
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Abstract 13
Background: Docosahexaenoic acid (DHA) is an important omega-3 long-chain 14
polyunsaturated fatty acid that has a variety of physiological functions for infant 15
development and human health. Although metabolic engineering was previously 16
demonstrated to be a highly efficient way to rapidly increase lipid production, 17
metabolic engineering has seldom been previously used to increase DHA 18
accumulation in Schizochytrium spp. 19
Results: Here, a sensitive β-galactosidase reporter system was established to screen 20
for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, 21
TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were 22
characterized by the reporter system with the promoter activity ccg1p > TEF-1p > 23
AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, 24
Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were 25
overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28°C 26
and 250 rpm for 120 h with glucose as the carbon source. Shake flask fermentation 27
results showed that the overexpression strains exhibited growth curves and biomass 28
similar to those of the wild-type strain. The lipid contents of the wild-type strain and 29
of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, 30
respectively, and the lipid yields of the overexpression strains were increased by 21.9, 31
30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the 32
corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields 33
of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively. 34
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Conclusions: Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. 35
ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC 36
catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate 37
that overexpression of ACL and ACC enhances lipid accumulation and DHA 38
production in Schizochytrium sp. 39
Keywords: Schizochytrium sp., Docosahexaenoic acid, ATP-citrate lyase, Acetyl-CoA 40
carboxylase, β-galactosidase reporter system, Constitutive promoter 41
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Background 43
Docosahexaenoic acid (DHA, C22:6-∆4,7,10,13,16,19) is an omega-3 long-chain 44
polyunsaturated fatty acid (LC-PUFA). As the principal omega-3 fatty acid in brain 45
gray matter, DHA has neurotrophic and neuroprotective properties that are required 46
for normal perinatal cortical maturation [1]. In addition, DHA supplementation 47
improves human health by increasing cardioprotective, anti-inflammatory, and 48
anticancer activities [2, 3]. DHA is therefore widely used as a nutritional supplement, 49
often as a nutraceutical. 50
The conventional source of DHA is fish oil obtained from cold-water marine fish. 51
Seasonal variation, overharvest, and population decline, however, prevent the steady 52
supply of DHA that is required to meet the increasing market demands. Other 53
commercial sources of DHA production are thraustochytrids, which are marine 54
microorganisms [4]. Schizochytrium spp., as well as other thraustochytrids (such as 55
species of Thraustochytrium and Ulkenia), are excellent DHA producers [5, 6]. 56
Schizochytrium spp. can produce total fatty acids (TFAs) that represent up to 70% of 57
the cell weight, with DHA representing 25–45% of TFAs [7, 8]. Owing to the 58
increasing demand for DHA, many researchers have attempted to increase DHA 59
production by Schizochytrium spp. [5, 6, 9]. To date, most studies of DHA production 60
by Schizochytrium spp. have focused on the adaptive evolution of the strains [9]; on 61
the optimization of medium composition including sources of carbon and nitrogen 62
and the addition of inorganic salts and antioxidants [5, 6, 10, 11]; and on cultivation 63
conditions and cultivation styles [12, 13]. Only a few studies have employed 64
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metabolic engineering to increase DHA accumulation in Schizochytrium. Yan et al. 65
[14], for example, introduced the Escherichia coli acetyl-CoA synthase gene into 66
Schizochytrium sp. TIO1101, which increased the biomass and TFA production of the 67
resulting transformant by 29.9% and 11.3%, respectively. Introduction of an 68
exogenous ω-3 desaturase gene into Schizochytrium sp. converted 3% 69
docosapentaenoic acid (DPA) into DHA [15]. By increasing the number of active ACP 70
domains of PUFA synthase, DHA productivity was increased by 1.8-fold in a 71
recombinant E. coli expressing Schizochytrium PUFA biosynthetic genes [16]. These 72
studies demonstrate that metabolic engineering can increase DHA production by 73
Schizochytrium spp. 74
Metabolic engineering has also been used with the oleaginous yeast Yarrowia 75
lipolytica, i.e., metabolic engineering efficiently increased the yeast’s production of 76
total lipids and ω-3 PUFAs [17-20]. By rewiring the metabolic pathways of Y. 77
lipolytica, researchers increased lipid accumulation > 60-fold, and caused lipid 78
content to approach 90% of cell mass [18]. Compared to the 10–15% lipid content in 79
wild-type (WT) Y. lipolytica [21], Schizochytrium spp. produces much higher lipid 80
levels, and ω-3 PUFA DHA represents up to 45% of TFAs. Because the genome 81
sequences of several strains of thraustochytrids (Schizochytrium, Thraustochytrium, 82
and Aurantiochytrium) are now available [22-24], metabolic engineering should be an 83
efficient way to rapidly increase their production of DHA and lipids. 84
Acetyl-CoA is precursor for fatty acid synthesis. ATP-citrate lyase (ACL) 85
catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, along 86
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with the hydrolysis of ATP [25]. ACL is present in all eukaryotes except 87
non-oleaginous yeasts. In animals and oleaginous basidiomycete yeasts, ACL is 88
encoded by a single gene [26, 27]; in plants and some filamentous fungi, ACL usually 89
consists of two subunits (ACL1 and ACL2) with homology to the N- and C-terminals 90
of the animal ATP-citrate lyase polypeptide [28]. Fatty acid content was increased in Y. 91
lipolytica by overexpression of ACL1 and ACL2 on a non-lipogenic medium in an 92
obese strain [29] or overexpression of ACL from Mus musculus [30]. Acetyl-CoA 93
carboxylase (ACC) catalyzes the synthesis of malonyl-CoA from acetyl-CoA, which 94
is the rate-limiting step in fatty acid synthesis [21]. There are two types of ACCs in 95
nature: in most bacteria and plant chloroplasts, ACC usually consists of multiple 96
subunits, including the biotin carboxylase (BC), the biotin carboxyl carrier protein 97
(BCCP), the α-carboxyltransferase (α-CT) and the β-carboxyltransferase (β-CT); but 98
in mammals, fungi, and the cytoplasm of most plants, ACC is a single multifunctional 99
polypeptide [31]. Overexpression of acetyl-CoA carboxylase in the presence of 100
thioesterase in E. coli led to a 6-fold increase in the rate of fatty acid synthesis [32]. 101
ACC overexpression increased lipid content in Y. lipolytica and free fatty acid 102
production in S. cerevisiae [21, 33]. 103
To date, very few studies have attempted to enhance DHA production in 104
Schizochytrium spp. through metabolic engineering. In this study, a sensitive 105
β-galactosidase reporter system was established in Schizochytrium sp. to screen for 106
strong promoters. Because a sufficient supply of acetyl-CoA and malonyl-CoA is a 107
prerequisite for efficient lipid accumulation, Schizochytrium ACL and ACC were 108
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overexpressed under the strong constitutive promoter ccg1p in Schizochytrium sp. 109
ATCC 20888 to enhance lipid accumulation and DHA production. 110
111
Results 112
Developing a β-galactosidase reporter system in Schizochytrium 113
Metabolic engineering involves the rewiring various metabolic pathways to redirect 114
metabolic flux towards the synthesis of target compounds. As a consequence, the 115
expression of relevant pathways must be strictly coordinated to achieve a balanced 116
expression and to avoid metabolic bottlenecks [34]. It is therefore crucial that a 117
reliable reporter system to monitor gene expression levels is established in 118
Schizochytrium. The E. coli β-galactosidase structural gene lacZ has been widely used 119
as a candidate reporter gene, providing convenient methods for qualitative 120
colorimetric detection on agars with 5-bromo-4-chloro-3-indolyl 121
β-D-galactopyranoside (X-gal) and quantitative β-galactosidase activity assays with 122
O-nitrophenyl-β-D-galactopyranoside (ONPG) [35]. To determine whether the 123
β-galactosidase reporter works in Schizochytrium, we constructed the reporter plasmid 124
pPICZαA-ubiquitinp-lacZ , in which the E. coli lacZ gene was driven by a ubiquitin 125
promoter-terminator system (Fig. 1a). pPICZαA containing lacZ without a ubiquitin 126
promoter (termed pPICZαA-AOX1p-lacZ) was also constructed as a control plasmid. 127
The corresponding transformants of Schizochytrium sp. ATCC 20888 were selected on 128
glucose-peptone-yeast extract (GPY) plates with zeocin. DNA fragments of 5.0 and 129
3.3 kb were amplified from the genomic DNAs of the ubip-lacZ and AOX1p-lacZ 130
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transformants, respectively; the sizes of the fragments corresponded with the sizes of 131
the lacZ expression cassettes (Fig. 1b), indicating that both plasmids were integrated 132
into chromosomes. 133
The transformants of ubip-lacZ and AOX1p-lacZ grew normally on GPY agar. 134
When the substrate X-gal was added to GPY plates, Schizochytrium sp. WT produced 135
orange colonies with a very slight blue color, indicating that the WT possessed 136
endogenous β-galactosidase with very low activity. AOX1p-lacZ produced colonies 137
that were similar to those of the WT, while ubip-lacZ produced blue colonies (Fig. 1c). 138
β-galactosidase enzymatic activity was much higher in the lysate of the ubip-lacZ 139
transformant than in the AOX1p-lacZ transformant (Fig. 1d). These results show that, 140
although Schizochytrium WT possesses endogenous β-galactosidase activity, 141
β-galactosidase is able to serve as a sensitive reporter system in Schizochytrium. 142
143
Selection of strong promoters in Schizochytrium 144
The use of four commonly-used eukaryotic promoters (EF-1αp, ccg1p, TEF-1p, and 145
AOX1p) in addition to ubiquitinp was characterized in the β-galactosidase reporter 146
system. In pPICZαA-AOX1p-lacZ, a methanol-induced AOX1p [36] is present 147
upstream of the lacZ gene. On X-gal plates, AOX1p-lacZ produced orange colonies 148
like those of the WT, but the transformants with other promoters produced blue 149
colonies, and the transformants with ccg1p or TEF-1p produced the bluest colonies. 150
β-galactosidase activity in AOX1p-lacZ without methanol induction treatment was 151
similar to that in the WT (Fig.1c), indicating that AOX1p is inactive without induction 152
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9
by methanol. The enzymatic activities driven by ccg1p, TEF-1p, EF-1αp, or ubiquitinp 153
were much higher than that of WT, with promoter activity of ccg1p > TEF-1p > 154
EF-1αp > ubiquitinp (Fig. 1d). These findings indicate that the four promoters are 155
strong, constitutively-expressed promoters. When methanol was added to a 12-h 156
culture of AOX1p-lacZ to a final concentration of 1% (vol/vol), the β-galactosidase 157
activity increased substantially and remained at a high level, with the induction 158
strength between TEF-1p and EF-1αp, indicating that AOX1p can be recognized by 159
Schizochytrium RNA polymerase and induced by methanol. Therefore, AOX1 160
promoter can serve as a methanol-induced promoter in Schizochytrium, although the 161
induction time and strength require optimization. 162
163
Construction of ACL and ACC-overexpression strains in Schizochytrium sp. 164
In cytoplasm, ATP-citrate lyase converts intracellular citrate to acetyl-CoA in an 165
ATP-dependent manner, and acetyl-CoA carboxylase catalyzes the synthesis of 166
malonyl-CoA from acetyl-CoA [25]. Acetyl-CoA and malonyl-CoA are the precursors 167
for fatty acids synthesis [21, 29]. A BLAST search of the Schizochytrium sp. CCTCC 168
M209059 genome [22] revealed one putative ACL-encoding gene and one putative 169
ACC-encoding gene (Additional file 2: Table S1). The putative ACL (422 aa) contains 170
an ATP citrate (pro-S)-lyase domain at the N-terminus, which is homologous to 171
ATP-citrate lyase subunit 1, and a citrate-binding domain at the C-terminus, which is 172
homologous to ATP-citrate lyase subunit 2 (Fig. 2a). Therefore, Schizochytrium 173
ATP-citrate lyase functions as a single-subunit ACL. Like ACC in most fungi, ACC 174
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10
(2,352-aa) in Schizochytrium is a single multifunctional polypeptide, containing a 175
biotin carboxylase (BC) domain, a biotin carboxyl carrier protein (BCCP) domain, 176
and a carboxyl transferase (CT) domain (Fig. 2a). 177
To promote lipid biosynthesis and DHA accumulation in Schizochytrium sp. 178
ATCC 20888, we developed transformants that overexpressed Schizochytrium 179
ATP-citrate lyase and acetyl-CoA carboxylase. ACL and ACC genes were amplified 180
from the cDNA of Schizochytrium sp. ATCC 20888 and were cloned separately or 181
together into pPICZαA, in which the cloned gene was driven by the ccg1 promoter 182
and terminator (Additional file 1: Figure S1). After transformation, 2.8-, 8.7-, and 183
12.0-kb PCR fragments containing the corresponding expression cassettes 184
(ccg1p-ACL-ccg1t, ccg1p-ACC-ccg1t, and ccg1p-ACL-ccg1t-ccg1p-ACC-ccg1t) were 185
amplified from the genomic DNAs of pPICZαA-ACL, pPICZαA-ACC, and 186
pPICZαA-ACL-ACC transformants (termed OACL, OACC, and OACL-ACC) (Fig. 187
2b), indicating that the plasmids were integrated into the chromosomes. The 188
transcription levels of ACL and ACC were examined by qRT-PCR in WT, OACL, 189
OACC, and OACL-ACC cultivated in fermentation broth for 2 and 4 d. Compared to 190
WT, the expression of ACL were increased in OACL and OACL-ACC, and the 191
expression of ACC were increased in OACC and OACL-ACC at both time points, 192
indicating that transcription levels of ACL and ACC were increased in the 193
corresponding overexpression strains (Fig. 3). 194
195
ACL and ACC overexpression enhanced lipid accumulation 196
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Shake-flask fermentation results showed that the WT strain and the overexpression 197
transformants did not significantly differ in their consumption of carbon and nitrogen 198
sources, pH values, or dry cell weights (DCW) (Fig. 4). DCW reached their maximum 199
values on day 5, which were 23.7, 22.7, 23.9, and 24.3 g/L for WT, OACL, OACC, 200
and OACL-ACC, and then decreased slightly with further cultivation (Fig. 4d, 5a). 201
Thus, overexpression of ACL and/or ACC did not affect cell growth of 202
Schizochytrium. 203
After 5 days of cultivation, lipid yields were lowest for the WT (12.8 g/L), 204
highest for OACL-ACC (17.7 g/L), and intermediate for OACL (15.6 g/L), OACC 205
(16.7 g/L) (Table 1). A similar pattern was evident for lipid content, i.e., lipid content 206
was substantially higher in the overexpression strains than in the WT (Fig. 5b). 207
Compared to the WT strain, the lipid yields were increased by 21.9% in OACL, by 208
30.5% in OACC, and by 38.3% in OACL-ACC. Microscopic observation revealed an 209
increased intensity of lipid droplet staining in the overexpression strains (Fig. 5d). The 210
findings indicated that overexpression of ACL and ACC increased lipid production in 211
Schizochytrium sp., probably by increasing the supply of acetyl-CoA and 212
malonyl-CoA. 213
214
ACL and ACC overexpression promoted DHA production 215
Gas chromatography analysis showed that the main fatty acid components of 216
Schizochytrium sp. ATCC 20888 were DHA, palmitic acid (C16:0), myristic acid 217
(C14:0), and docosapentaenoic acid (DPA, C22:5) (Fig. 6). Overexpression of ACL in 218
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12
Schizochytrium sp. WT did not significantly affect the percentage of TFAs represented 219
by DHAs (36.4% for the WT, and 36.2% for OACL) (Fig. 6), but slightly decreased 220
the percentage represented by palmitic acid and slightly increased the percentage 221
represented by C16:1. In ACC-overexpression strains, the percentage of TFAs 222
represented by DHAs increased slightly for OACC (37.6%) and OACL-ACC (37.9%), 223
and the percentage represented by oleic acid and DPA decreased slightly (Fig. 6). 224
Compared to the DHA yield of the WT (4.3 g/L), the DHA yields of OACL, OACC, 225
and OACL-ACC strains were 5.3, 6.1, and 6.4 g/L, increased by 23.3, 41.9, and 226
48.8%, respectively (Fig. 5c). These results indicated that overexpression of ACL and 227
ACC greatly increased DHA production in Schizochytrium sp. ATCC 20888. 228
229
Discussion 230
In this study, a sensitive β-galactosidase reporter system in Schizochytrium was 231
developed and used to compare the strengths of some commonly-used eukaryotic 232
promoters. Although endogenous β-galactosidase is present in Schizochytrium, the 233
reporter system was able to detect different levels of β-galactosidase activity with the 234
LacZ-reporter driven by different promoters. ccg1p, TEF-1p, EF-1αp, and ubiquitinp 235
promoters are constitutive promoters and the AOX1p promoter is inducible by 236
methanol in Schizochytrium. More work can be carried out for subsequent research, 237
such as screening for more endogenous constitutive promoters with different 238
expression intensities and optimization of the induction conditions for the 239
methanol-induced AOX1p promoter. The β-galactosidase reporter system will facilitate 240
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13
characterization of novel genetic elements and will help identify promoters for 241
fine-tuning gene expression in Schizochytrium. 242
Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis [37]. 243
Previous studies have shown that increasing the substrates supply significantly 244
enhanced the synthesis of fatty acids and lipids in bacterium, yeast and fungi [30, 32, 245
38]. In the study, lipid and DHA production were greatly increased by overexpression 246
of ACL and ACC. ACL converts intracellular citrate to acetyl-CoA [29], and ACC 247
catalyzes the synthesis of malonyl-CoA from acetyl-CoA [21]. It follows that 248
overexpression of ACL and ACC evidently promoted production of intracellular 249
acetyl-CoA and malonyl-CoA, resulting in enhanced biosynthesis of fatty acids, 250
which in turn promoted lipid accumulation. The percentage of TFAs represented by 251
DHAs in ACL-overexpression strain was similar to that in the WT, indicating that 252
overexpression of the ACL gene led to increased intracellular acetyl-CoA pools, which 253
promoted the production of both saturated fatty acids and polyunsaturated fatty acids. 254
In ACC-overexpression strains, the percentage of TFAs represented by DHAs 255
improved slightly, while the percentage of TFAs represented by oleic acid and DPA 256
decreased slightly. Thus, the increased malonyl-CoA pool in ACC-overexpression 257
strains increased DHA production more than saturated fatty acid production. The 258
findings suggested that the supply of malonyl-CoA is the limiting factor of DHA 259
overproduction in Schizochytrium. In oleaginous microorganisms, efficient fatty acid 260
synthesis requires not only an abundant supply of acetyl-CoA and malonyl-CoA, but 261
also an ample NADPH supply [38, 39]. Therefore, combining this strategy with other 262
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14
strategies rewiring the metabolic flux of Schizochytrium towards precursors and 263
NADPH accumulation might significantly improve its lipogenesis capability and 264
DHA productivity. 265
Schizochytrium spp. are excellent producers of ω-3 PUFA: they can synthesize 266
DHA de novo and DHA represents up to 45% of TFAs [7, 8]. To date, most studies of 267
Schizochytrium spp. have focused on the optimization of fermentation media and 268
cultivation conditions and the adaptive evolution of the strains [5, 6, 9-12]. Very few 269
studies have employed metabolic engineering to improve DHA production of 270
Schizochytrium spp. [14-16], mainly because the genetic background of 271
Schizochytrium remains poorly understood. Metabolic engineering has been proven to 272
be a highly efficient way to increase total lipids and ω-3 PUFA accumulation in the 273
yeast Y. lipolytica [17-20]. In the current investigation, we demonstrated that 274
metabolic engineering is an efficient way for increasing lipid accumulation and DHA 275
production in Schizochytrium. The study provided a sensitive reporter system to 276
monitor gene expression levels in Schizochytrium and a genetically engineered 277
Schizochytrium sp. for industrial production of DHA. 278
279
Conclusions 280
A strain of Schizochytrium sp. ATCC 20888 that overexpressed ACL and ACC under 281
the strong constitutive promoter ccg1p was constructed and thereby produced high 282
quantities of DHA. Under shake flask culture conditions, OACL, OACC, and 283
OACL-ACC strains attained a dry cell weight of 22.7, 23.9, and 24.3 g/L, respectively. 284
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15
Compared to the WT, total lipid content of OACL, OACC, and OACL-ACC strains 285
reached 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression 286
strains were increased by 21.9, 30.5, and 38.3%, respectively. A final DHA yield of 287
6.4 g/L in OACL-ACC was achieved, which was 48.8% higher than that of the WT. 288
Next, fermentation control of OACL-ACC in fermentors will be optimized to make it 289
more suitable for industrial application. 290
291
Methods 292
Microorganisms and culture conditions 293
Strains and plasmids used in the study are listed in Table 2. Media and growth 294
conditions of Schizochytrium sp. were according to Ling et al. [5] with modifications. 295
Schizochytrium sp. was cultured at 28°C on solid GPY medium containing per liter 20 296
g of glucose, 10 g of peptone, 5 g of yeast extract, 20 g of sea crystal, and 20 g of agar. 297
Transformants were selected and cultured on GPY supplemented with 40 μg/mL 298
zeocin. For lipid and DHA production, 250-mL flasks containing 50 mL of seed 299
medium (containing per liter 30 g of glucose, 10 g of peptone, 5 g of yeast extract, 300
and 20 g of sea crystal) were inoculated with Schizochytrium sp. cells and incubated 301
for 24 h at 28°C on a rotary shaker (230 rpm). The seed culture was inoculated at 5% 302
(vol/vol) into 50 mL of fermentation medium (containing per liter 100 g of glucose, 5 303
g of yeast extract, 3.94 g of NaCl, 0.264 g of KCl, 0.5 g of (NH4)2SO4, 1 g of KH2PO4, 304
1.43 g of MgSO4, 0.04 g of CaCl2, 10 g of sodium glutamate, 0.001 g of vitamin B1, 305
and 0.001 g of vitamin B12) and was then incubated at 28°C on a rotary shaker (250 306
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16
rpm) for 168 h. For qualitative colorimetric and quantitative detection of 307
β-galactosidase, cells were cultured on GPY plates with 40 μg/mL X-gal and in seed 308
medium, respectively. E. coli was grown in LB medium at 37°C [35]. 309
310
Construction of β-galactosidase reporter plasmids and of ACL-, and 311
ACC-overexpression plasmids 312
To construct β-galactosidase reporter plasmids, a 3,075-bp fragment containing the 313
coding sequence of the lacZ gene was amplified from pMC1403 [35] by PCR using 314
primer pair lac-Fw and lac-Rev (Additional file 2: Table S2). The promoters and 315
terminators of EF-1α [40] and ubiquitin [15] were amplified from Schizochytrium sp. 316
ATCC 20888; ccg1 promoter and terminator were amplified from the Neurospora 317
expression vector pCCG.N-3xMyc [41]; and the TEF-1 promoter and CYC-1 318
terminator were amplified from the yeast expression vector pPICZαA [42] using the 319
primer pairs listed in Additional file 2: Table S2. After purification, the lacZ gene was 320
digested with KpnI/NotI, the promoters were digested with EcoRI/KpnI, and the 321
terminators were digested with NotI/XbaI; the digested gene, promoters, and 322
terminators were then simultaneously ligated into EcoRI/XbaI-digested pPICZαA to 323
generate reporter plasmids. Ligation reactions were performed overnight at 16°C 324
using T4 DNA Ligase (TaKaRa, Japan). 325
To construct the ACL overexpression plasmid, a 1,269-bp fragment of the ACL 326
gene was amplified from Schizochytrium sp. ATCC 20888 cDNA. After purification, 327
the KpnI/NotI-digested ACL gene, EcoRI/KpnI-digested ccg1p, and 328
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17
NotI/XbaI-digested ccg1t were simultaneously ligated into EcoRI/XbaI-digested 329
pPICZαA to generate the overexpression plasmid pPICZαA-ACL. For construction of 330
the ACC overexpression plasmid, a 7,059-bp ACC fragment was amplified from the 331
same cDNA and purified. The ACC gene and the ccg1 promoter and terminator were 332
inserted into EcoRI-digested pPICZαA to generate pPICZαA-ACC using the 333
Seamless assembly cloning kit (Clone Smarter, USA) following the manufacture’s 334
protocol. To construct the ACL and ACC co-overexpression plasmid, the 2,611-bp 335
ccg1p-ACL-ccg1t expression cassette was amplified from plasmid pPICZαA-ACL and 336
was inserted into EcoRI-digested pPICZαA-ACC to generate pPICZαA-ACL-ACC by 337
Seamless assembly cloning. 338
339
Transformation of Schizochytrium sp. 340
Transformation of Schizochytrium was performed as described previously with 341
modification [15]. Schizochytrium sp. cells were cultured in seed medium for 24 h to 342
the logarithmic growth phase, and were harvested by centrifugation (5,900 g, 4°C, 10 343
min) (HITACHI CF16RXⅡ, Japan), washed with ice-cold sterile water, washed with 344
1 M sorbitol, and then suspended in 1 M sorbitol. The plasmids were linearized with 345
restriction enzyme BamHI before transformation. The competent cells and 5 μg of 346
linearized plasmid DNA were placed in a 0.1-cm-gap cuvette. The parameters of 347
electroporation were 0.75 kV, 200 Ω, 50 μF, twice. After electroporation, 1 mL of seed 348
medium was added to the mixture, which was incubated at 28°C for 4 h. The 349
transformants were spread on GPY plates with 40 μg/mL zeocin and grown at 28°C. 350
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18
351
Genomic PCR analysis of transformants 352
Genomic DNAs of putative transformants were extracted according to Lippmeier et al. 353
[43]. To confirm Schizochytrium sp. transformants, the incorporation of the expression 354
cassette into the genome was verified by PCR using primers AOX-Fw and AOX-Rev 355
(Additional file 2: Table S2). PCR reactions were set up using Taq DNA polymerase 356
(TaKaRa, Japan) following the manufacture’s protocol. PCR amplification parameters 357
were as follows: 5 min at 95°C; followed by 30 cycles of 50 s at 95°C, 50 s at 55°C, 358
and 5 min at 72°C; and a final extension for 10 min at 72°C. 359
360
β-galactosidase activity assay 361
Schizochytrium sp. cells cultured in seed medium were collected by centrifugation at 362
the indicated time, washed with phosphate buffer saline solution (PBS, 38.7 mM 363
Na2HPO4•12H2O, 11.3 mM NaH2PO4•2H2O, and 150 mM NaCl), resuspended in 1 364
mL PBS, and disrupted with Mini Bead Beater (Biospec Mini-Bead-Beater-16 Model 365
607EUR, USA) for 50 s each for 5 times. After centrifugation, 500 μL of supernatant 366
was transferred to a new tube. A 500-μL volume of buffer Z (60.0 mM 367
Na2HPO4•12H2O, 39.7 mM NaH2PO4•2H2O, 10.0 mM KCl, 1.0 mM MgSO4•7H2O, 368
and 2.7 mL/L β-mercaptoethanol) and 200 μL of 13.3 mM ONPG were added to the 369
supernatant, and the mixture was incubated at 37°C for 15 min; the reaction was 370
stopped by adding 500 μL of 1 M Na2CO3. The OD420 was recorded with an 371
ultraviolet spectrophotometer for the determination of β-galactosidase activity. The 372
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19
amount of enzyme that releases 1 µmol of ONP per minute is defined as one unit of 373
enzyme activity. 374
375
Determination of dry cell weight, pH and glucose and nitrogen concentrations 376
Determination of dry cell weight was performed as described previously with 377
modification [10]. A 40-mL volume of fermentation broth was centrifuged at 5,900 g 378
for 10 min, and dry cell weight was determined after freeze-drying for 24 to 48 h to a 379
constant weight. For measurement of pH, glucose and nitrogen concentrations, 1 mL 380
of broth was centrifuged (Heraeus BIOFUGE pico, Germany) at 13,523 g for 10 min, 381
and the supernatant was used for determination. The pH was measured by a laboratory 382
pH meter (METTLER TOLEDO FiveEasy, Switzerland). The concentration of 383
glucose was determined by the 3, 5-dinitrosalicylic acid (DNS) method [44, 45]. NH4+ 384
concentration was measured by the indophenol blue spectrophotometric method [46]. 385
386
Microscopic analysis 387
Nile red staining of cells was conducted as described previously with modification 388
[18]. A 1-mL volume of a culture grown in fermentation medium for 48 h was 389
collected by centrifugation, washed twice with PBS solution, and resuspended in 1 390
mL of PBS. Cells were stained with nile red dye (0.5 mg/L) and were incubated for 5 391
min in the dark. Fluorescence images were captured with a LEICA TCS SP8 392
microscope equipped with an oil immersion objective (×1,000 magnification). 393
394
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20
Lipid extraction and fatty acid composition analysis 395
Lipids were extracted as described previously with some modification [47-49]. About 396
0.3 g of a freeze-dried Schizochytrium sp. pellet was mixed with 6 mL of 4 M HCl for 397
30 min and then incubated in boiling water for 8 min before 16 mL of 398
methanol/chloroform (1:1, vol/vol) was added. The preparation was mixed vigorously, 399
and then centrifuged at 129 g for 10 min. The lower phase was transferred to a 400
pre-weighed glass tube and evaporated under a stream of nitrogen. 401
Fatty acid methyl esters (FAMEs) were prepared according to Ren et al. [50] with 402
some modifications. About 30 mg of lipid sample was transferred to a glass tube 403
before 1 mL of internal standard (methyl nonadecanoate, C19:0, 1 mg/mL) and 1 mL 404
of 0.5 M KOH in methanol were added; the mixture was incubated at 65°C in a water 405
bath for 15 min. After the mixture had cooled to room temperature, 2.1 mL of 406
methanol and 0.9 mL of 45% BF3-ether were added to the tube, which was incubated 407
at 65°C for 5 min. Then 1 mL of hexane and 2 mL of saturated sodium chloride 408
solution were added; the preparation was mixed vigorously and allowed to stand for 409
10 min. The upper layer of the solution was transferred to a new tube and used for 410
analysis of fatty acid composition. FAMEs were separated by gas chromatography 411
(WUFENG GC522) with an Agilent J & W DB23 capillary column (30 m×0.25 mm 412
i.d.). Nitrogen was used as the carrier gas at a flow rate of 2 mL/min. The injector was 413
at 250°C. The column temperature was increased from 150°C to 200°C at the rate of 414
5°C per min, was kept at 200°C for 1 min, was then raised to 230°C at the rate of 4°C 415
per min, and was maintained at 230°C an additional 9 min. 416
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21
417
RNA preparation and quantitative real-time PCR analysis (qRT-PCR) 418
Schizochytrium sp. cells cultured in fermentation medium were collected at 2 and 4 d, 419
frozen in liquid nitrogen, and ground to fine powder. Total RNA was extracted with 420
TRIzol reagent (Tiangen, China) according to the manufacturer’s protocol. cDNA was 421
synthesized by M-MLV (RNase H-; TaKaRa) with oligo-dT18 from 4 µg of total RNA. 422
qRT-PCR analysis was performed using FastStart Universal SYBR Green Master 423
(ROX) with primers listed in Table S2. PCR included a 10 min preincubation at 95°C, 424
followed by 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 425
60°C for 30 s. The relative expression levels were determined according to the 426
comparative Ct method, using actin as the internal control. 427
428
Statistical analysis 429
All experiments were performed with three biological replicates. Statistical analyses 430
were performed using one-way ANOVAs and Duncan’s multiple range tests or 431
two-tailed Student’s t-tests. And it was considered indicative of statistical significance 432
at p < 0.05. 433
434
Abbreviations 435
ACC: acetyl-CoA carboxylase; ACL: ATP-citrate lyase; DCW: dry cell weight; DHA: 436
docosahexaenoic acid; DNS: 3,5-dinitrosalicylic acid; DPA: docosapentaenoic acid; 437
FAMEs: Fatty acid methyl esters; LC-PUFA: long chain polyunsaturated fatty acid; 438
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
22
ONPG: O-nitrophenyl-β-D-galactopyranoside; PBS: phosphate buffer saline; TFAs: 439
total fatty acids; WT: wild type; X-gal: 5-bromo-4-chloro-3-indolyl 440
β-D-galactopyranoside. 441
442
Declarations 443
Authors’ contributions 444
ZC and XH designed the study. XH and ZZ performed the experiments. YW helped 445
with analysis and discussion of results. XH and ZC wrote the manuscript. All authors 446
read and approved the finalized manuscript. 447
Acknowledgements 448
The authors are grateful to Prof. B. Jaffee for English editing of the manuscript. 449
Competing interests 450
The authors declare that they have no competing interests. 451
Availability of supporting data 452
All data supporting the conclusions of this article are included in the manuscript and 453
in the additional information. 454
Consent for publication 455
All authors have read and approved the final manuscript. 456
Ethics approval and consent to participate 457
Not applicable. 458
Funding 459
This work was financially supported by the National Natural Science Foundation of 460
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23
China (No. 31470190). 461
462
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621
622
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
32
Figure Captions 623
624
Fig. 1 Development of a β-galactosidase reporter system and selection of strong 625
promoters in Schizochytrium sp. a Construction of reporter plasmid pPICZαA-lacZ. b 626
PCR verification of transformants. +: positive control, reporter plasmid was used as 627
template; -: negative control, WT genomic DNA was used as template; AOX1p-lacZ, 628
ubip-lacZ, EF-1αp-lacZ, ccg1p-lacZ, and TEF-1p-lacZ: the transformants of 629
pPICZαA-AOX1p-lacZ, pPICZαA-ubiquitinp-lacZ, pPICZαA-EF-1αp-lacZ, 630
pPICZαA-ccg1p-lacZ, and pPICZαA-TEF-1p-lacZ. c Phenotypes of the 631
corresponding transformants. Cells were grown for 96 h on GPY plates with 40 632
μg/mL X-gal. d β-galactosidase enzymatic activities of the transformants. Cells were 633
grown in seed medium for 64 h 634
635
Fig. 2 Overexpression of ATP-citrate lyase and acetyl-CoA carboxylase in 636
Schizochytrium sp. a Schematic diagram of Schizochytrium sp. ACL and ACC. The 637
proposed enzymatic domains are marked in different colors. b PCR verification of the 638
transformants. +: positive control, the plasmid was used as template; -: negative 639
control, WT genomic DNA was used as template; OACL, OACC, and OACL-ACC: 640
genomic DNA from the transformants of pPICZαA-ACL, pPICZαA-ACC, and 641
pPICZαA-ACL-ACC was used as template 642
643
Fig. 3 qRT-PCR analysis of the transcription levels of ACL and ACC in WT, OACL, 644
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
33
OACC, and OACL-ACC. RNAs were isolated from WT, OACL, OACC, and 645
OACL-ACC grown in fermentation media for 2 and 4 days. P values were determined 646
by Student’s t-test. ***, P < 0.001; NS, not significant 647
648
Fig. 4 Time course of fermentation profiles of Schizochytrium sp. WT, OACL, OACC, 649
and OACL-ACC. a Glucose (g/L); b NH4+ (g/L); c pH; d Dry cell weight (DCW, g/L) 650
651
Fig. 5 Effect of ACL and ACC overexpression on lipid accumulation and DHA 652
production by Schizochytrium sp. a Dry cell weight (DCW, g/L). b Total lipids (% 653
DCW). c DHA yield (g/L). Cells were cultured in fermentation medium for 120 h. d 654
Imaging analyses of WT, OACL, OACC, and OACL-ACC. The 48-h cultured cells 655
were stained with nile red dye for neutral lipid staining. Scale bar, 10 µm. Error bars: 656
SD from three independent experiments. Data were analyzed by one-way ANOVAs 657
and Duncan’s multiple range tests in SPSS version 23.0. In a, b, and c, columns with 658
different lowercase letters are significantly different at P < 0.05 659
660
Fig. 6 Effect of ACL and ACC overexpression on fatty acid composition (TFA, %) of 661
Schizochytrium sp. Cells were cultured in fermentation medium for 120 h 662
663
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
34
Tables 664
Table 1 Fermentation characteristics of strains of Schizochytrium sp. 665
Strains DCW (g/L) Lipid yield
(g/L)
Lipid content
(%)
DHA yield (g/L) DHA content
(%)
WT 23.7 ± 0.6a 12.8 ± 0.5d 53.8c 4.3 ± 0.1c 36.4b
OACL 22.7 ± 1.0a 15.6 ± 0.4c 68.8b 5.3 ± 0.2b 36.2b
OACC
OACL-ACC
23.9 ± 1.1a
24.3 ± 1.1a
16.7 ± 0.7b
17.7 ± 0.3a
69.8b
73.0a
6.1 ± 0.1a
6.4 ± 0.2a
37.6a
37.9a
Cells were cultured in fermentation medium for 120 h. Data were analyzed by one-way ANOVAs and Duncan’s 666
multiple range tests in SPSS version 23.0. Values with different lowercase letters are significantly different at P < 667
0.05 668
669
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
35
Table 2 Strains and plasmids used in this study 670
671
672
Strain or plasmid Description Source or reference
Schizochytrium sp.
ATCC 20888
AOX1p-lacZ
ubip-lacZ
TEF-1p-lacZ
EF-1αp-lacZ
ccg1p-lacZ
wild-type strain (WT)
WT strain carrying pPICZαA-AOX1p-lacZ
WT strain carrying pPICZαA-ubiquitinp-lacZ
WT strain carrying pPICZαA-TEF-1p –lacZ
WT strain carrying pPICZαA-EF-1αp-lacZ
WT strain carrying pPICZαA-ccg1p-lacZ
ACL overexpression strain
American Type Culture
Collection
This study
This study
This study
This study
This study
This study OACL
OACC
OACL-ACC
ACC overexpression strain
ACL and ACC co-overexpression strain
This study
This study
E. coli
JM109 General cloning host for plasmid manipulation Laboratory stock
Plasmids
pPICZαA Yeast expression vector [42]
pPICZαA-AOX1p-lacZ
pPICZαA-ubiquitinp-lacZ
pPICZαA-TEF-1p-lacZ
pPICZαA-EF-1αp-lacZ
pPICZαA-ccg1p-lacZ
lacZ reporter vector using AOX1 promoter and terminator
lacZ reporter vector using ubiquitin promoter and terminator
lacZ reporter vector using TEF-1 promoter and terminator
lacZ reporter vector using EF-1α promoter and terminator
lacZ reporter vector using ccg1 promoter and terminator
This study
This study
This study
This study
This study
pPICZαA-ACL ACL overexpression vector based on pPICZαA This study
pPICZαA-ACC
pPICZαA-ACL-ACC
ACC overexpression vector based on pPICZαA
ACL and ACC co-overexpression vector based on pPICZαA
This study
This study
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
36
Additional files 673
674
Additional file 1: Figure S1. Physical map of overexpression plasmid 675
pPICZαA-ACL. 676
677
Additional file 2: Table S1. The open reading frames of ACL and ACC genes in 678
Schizochytrium sp. Table S2. Primers used in this study. 679
680
681
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Fig. 1
BackFront
a
bp
AOX1p ubiquitinp EF-1αp ccg1p TEF-1p
M + - + - + - + - + -
5000 3000 2000
1000
WT
ccg1p-lacZ
b
c
d
0 8 1 6 2 4 3 2 4 0 4 8 5 6 6 4
0
3 0
6 0
9 0
1 2 0
1 5 0 W T
A O X 1 p -la c Z
E F -1 p - la c Z
c c g 1 p - la c Z
T E F -1 p - la c Z
u b ip - la c Z
A O X 1 p -la c Z -M e th a n o l
T im e (h )
-g
ala
cto
sid
as
e a
ctiv
ity
(U
/mL
)
revised Figures Click here to access/download;Figure;revisedFigures.pptx
Fig. 2
ATP-citrate lyase
ATP citrate-lyase domain Citrate-binding domain
1 422
ACL ACC ACL-ACC
bp
10000
6000
3000
2000
79 356 411
Acetyl-CoA carboxylase
1 2226 2352
332
Biotin carboxylase Biotin carboxyl carrier protein Carboxyl transferase
35 531 670 735 1669
8000
M + - + - + -
a
b
Fig. 3
2 4
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
A C L
D a y s
Re
lativ
e m
RN
A e
xp
re
ss
ion
W T
O A C L
O A C C
O A C L -A C C
***
N S
****** ***
N S
a b
2 4
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
A C C
D a y s
Re
lativ
e m
RN
A e
xp
re
ss
ion
W T
O A C L
O A C C
O A C L -A C C
***
N S
*** ***
***
N S
Fig. 4
b a
c d
0 1 2 3 4 5 6 7
0
5
1 0
1 5
2 0
2 5
3 0
D a y s
Dr
y c
ell
we
igh
t (
g/L
)
W T
O A C L
O A C C
O A C L -A C C
0 1 2 3 4 5 6 7
0 .0 0
0 .0 3
0 .0 6
0 .0 9
0 .1 2
0 .1 5
D a y s
NH
4
+ (
g/L
)
W T
O A C L
O A C C
O A C L -A C C
0 1 2 3 4 5 6 7
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
D a y s
Glu
co
se
(g
/L)
W T
O A C L
O A C C
O A C L -A C C
Fig. 5
d
c ba
WT
OA
CL
OA
CC
OA
CL
-AC
C
0
2
4
6
8
DH
A y
ield
(g
/L)
aa
b
c
WT
OA
CL
OA
CC
OA
CL
-AC
C
0
1 0
2 0
3 0
Dr
y c
ell
we
igh
t (
g/L
) aaaa
WT OACL OACC OACL-ACC
488 nm
Light
Merge
Fig. 6
Additional file 1
Click here to access/downloadSupplementary Material
Additional file 1.pptx
revised Additional file 2
Click here to access/downloadSupplementary Material
revised Additional file 2.docx
Response to Reviewers
Biotechnology for Biofuels
BBIO-D-19-00571
Enhancement of docosahexaenoic acid production by overexpression of ATP-citrate
lyase and acetyl-CoA carboxylase in Schizochytrium sp.
Reviewer #1: It's a good work with significant improvement of DHA production as
well as a tool box for further application in biofuels production in this field. There are
some comments hope authors to take into consideration.
1. The work focused on the availability of carbon supply to FA synthesis, however,
the desaturation is also very important to DHA production. The whole profile
dynamic information of FA should be provided to have a overall view on FA
synthesis process other than Fig 5.
Response: Thank you very much for your valuable comments and suggestions. We
agree with you that the investigation of the whole dynamic profile will provide more
information. Though we can determine DHA yields through gas chromatography, our
lab can not analyze the fatty acid composition due to the lack of some fatty acid
standards. Fatty acid composition analysis in the study was carried out by the
detection platform of our university which is closed now and will not open in months
due to the outbreak of new coronavirus. So we can not do the experiment. Guo et al.
(2016) have reported that the changes of fatty acid composition in Schizochytrium sp.
HX-308 cultured at different aeration rates. The contents of DHA in TFAs from 40-h
to 120-h were increased gradually from 38.01% to 43.59% and from 40.52% to 44.85
at aeration rates of 0.5 vvm and 1.0 vvm, respectively, and decreased gradually from
39.91% to 36.61% at aeration rate of 1.5 vvm. According to the report, DHA content
of Schizochytrium sp. was slightly changed during fermentation and was affected by
the oxygen supply.
Reference:
Guo DS, Ji XJ, Ren LJ, Li GL, Yin FW, Huang H. Development of a real-time
For reviewers Click here to access/download;Personal Cover;Response toReviewers.docx
bioprocess monitoring method for docosahexaenoic acid production by
Schizochytrium sp. Bioresour Technol. 2016;216:422-427.
2. In the industrial process, the glycerol is widely used as carbon other than glucose,
so the performance with glycerol is expected.
Response: Thank you very much for your suggestion. As a byproduct in the
production of biodiesel, glycerol is a cost effective carbon source for lipid production.
Glycerol was used as the carbon source for the shake flask fermentation as suggested.
Although it was reported that some Schizochytrium spp. could use glycerol efficiently,
we found here that Schizochytrium sp. grew poorly in the glycerol medium and the
production of lipid and DHA were very low (Fig. I). We were quite surprised by the
results, and the shake flask fermentation experiments were repeated with new batch of
glycerol, and lower amount of glycerol (60 g/L) was also used to rule out the
possibility of the inhibition of high glycerol on growth. Schizochytrium sp. still grew
poorly in the glycerol fermentation medium (Fig. IIa, b). Glucose fermentation
medium was also used as a control in which Schizochytrium sp. grew well and
accumulated lipid efficiently (Fig. IIc, d). Even though Schizochytrium sp. grew
poorly in glycerol medium, the lipid contents and DHA yields of ACL and ACC
overexpression strains were slightly higher than those of the WT strain (Fig. I).
Therefore, overexpression of ACL and ACC did increase the production of lipid and
DHA in Schizochytrium sp. ATCC 20888. Since cells grew poorly in glycerol
medium, we would rather not include the results of the glycerol medium in the
manuscript. We don’t know why this strain grow poorly with glycerol, but it seems
Schizochytrium spp. differ very much from each other, for example, the reported
genome sizes of Schizochytrium limacinum SR21 and Schizochytrium sp. CCTCC
M209059 were 63 Mb and 39 Mb, respectively.
Fig. I Effect of ACL and ACC overexpression on dry cell weight (DCW), lipid accumulation, and
DHA production by Schizochytrium sp. in the glycerol medium. Cells were grown in fermentation
medium with 100 g/L glycerol as the carbon source for 120 h. a Dry cell weight (DCW, g/L). b
Total lipids (% DCW). c DHA yield (g/L)
Fig. II Effect of ACL and ACC overexpression on dry cell weight (DCW) and lipid
accumulation by Schizochytrium sp. Cells were grown in fermentation medium for 120 h with
60 g/L glycerol (a), 100 g/L glycerol (b), 100 g/L glucose (c, d) as the carbon source. a, b, c
a b c
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3. The expression of digital should be improved, and comma is expected to be used.
Such as 1,269-bp fragment and 6,000 rpm, etc.
Response: Thank you for the suggestion. We have corrected the expression of digital
in the text.
Reviewer #2
I have now reviewed the manuscript, which was well-written in many aspects
including abstract, backrounds, m&m and conslusion. However, the discussion
section must be re-written since it was written as a the repeatition of the results
section. References, Figures and Tables are enough. Some methods are also missing.
Therefore, I suggest major revision. After revision, it should be reconsidered. My
spesific comments can be found on the text attached.
Response: Thank you very much for your valuable comments and suggestions. As
suggested, we have carefully revised the manuscript, added some methods and
references, and reorganized the discussion section. Most active tense sentences were
revised to passive tense except some (Line 120 and Line 175) in which the change
will lead to inconsistent subject. Some specific concerns are as follows.
1. Line 25: Is the host or wild type? Please specify it. Line 28: What are they, please
specify them.
Response: The specification of the strains was listed in Table 2.
2. Line 31: What is the difference between the Lipid yields and DHA yields?
Response: Thank you for the question. Lipid included triacylglycerol, free fatty acids,
and phospholipids, etc, which was extracted by acid-heating extraction. The fatty acid
components of lipids were DHA, palmitic acid (C16:0), myristic acid (C14:0), and
DPA, etc (Fig. 6). For DHA yields determination, fatty acid methyl esters were firstly
prepared from lipid samples and DHA was measured by gas chromatography.
3. Line 133, “β-galactosidase with very low activity”: please give some critical
activity in the text.
Response: Only qualitative colorimetric detection was carried out on GPY plates. The
β-galactosidase activities of the transformants in the liquid medium (seed medium)
were shown in Fig. 1d.
4. Line 176, “ACL and ACC”: Italic or non-italic. Please be coherent.
Response: The italic form indicates the encoding gene. And non-italic form indicates
protein or enzyme. It was changed to be “ACL and ACC genes”.
5. Line 191: “Thus, overexpression of ACL and/or ACC did not affect cell growth of
Schizochytrium”. Any reason.
Response: The results indicated that overexpression of ACL and ACC did not
significantly affect the consumption of carbon and nitrogen sources of the
transformants, that is probably the reason why cell growth (dry cell weights) was not
affected.
6. Line 206: “Overexpression of ACL in Schizochytrium sp. WT did not significantly
affect the percentage of TFAs represented by DHAs……”, Why? Any reason?
Line 596 (Table 1): Although DHA yields, Lipid yields, and Lipid contents of the
recombinant strains were higher than that of WT, DHA contents were similar to
each other. Any reason.
Response: Overexpression of ACL and ACC enhanced the supply of precursors
(acetyl-CoA and malonyl-CoA) for the synthesis of DHA and other fatty acids.
Therefore, the increased precursors supply in the recombinant strains enhanced the
production of DHA and other fatty acids at the same time. That is the reason of the
similar DHA contents between WT and the recombinant strains. The related
discussion can be found in Line 251-260.
7. The discussion section was similar to the results section or was written as a
repeatition of the results. Please compare your results with the existing results in
the literature. What were the similarities and differencies of your results with the
data in the literature? Please discuss them. Also, what is the industrial significance
of this study? Please also elucidate this situation. To sum up, please re-write the
discussion section.
Response: Thank you very much for your suggestion. In the discussion section, we
summarized the main findings in the study which is the foundation for the follow-up
discussion. These sentences have been rephrased to avoid repetition. And the
discussion section has been carefully revised as suggested.
8. Line 270 (Microorganisms and culture conditions section): There are four
different medium in this section, please use references for each medium and
explain why the medium was different?
Response: The references have been added to the text. LB medium is used for
cultivation of E. coli in which plasmids were constructed. The media and growth
conditions for Schizochytrium sp. were according to Ling et al. [5] with modifications.
In fact, we did quite a lot of work to optimize the media (especially the fermentation
medium). GPY solid medium which contained less glucose were used for rapid
cultivation of Schizochytrium sp. and selection of single colony of the transformants
(supplemented with 40 μg/mL zeocin). Fermentation medium which contained high
concentration of glucose was used for lipid and DHA production (high C/N helps lipid
accumulation). And seed medium which had moderate concentration of glucose
guaranteed rapid growth of Schizochytrium sp. and ample cells for fermentation
medium. Cells would grow slowly in fermentation medium if they were inoculated
directly from GPY solid medium.
9. Line 289: Why the promoters and terminators were amplified from different
sources? Please elucidate it.
Response: We intended to test the promoter strengths of some commonly-used
eukaryotic promoters (ubiquitinp, EF-1αp, ccg1p, TEF-1p, and AOX1p) and screen for
strong promoters in Schizochytrium (Line 145-162). Therefore, some promoters and
terminators were amplified directly from fungi and yeast expression plasmids, and
some were amplified from Schizochytrium sp. itself (endogenous promoters can be
recognized by RNA polymerase more efficiently). The results indicated that ccg1p,
TEF-1p, and AOX1p from the commonly-used expression plasmids displayed stronger
promoter activities, which was not unexpected because expression plasmids usually
utilize very strong promoters to ensure the efficient expression of the target genes.
Identification of promoters with different promoter strengths can provide suitable
promoters for a balanced expression of the relevant pathways in Schizochytrium.
10. The fermentations in Fig. 1d and Fig. 3a-d can be modeled kinetically using
logistic, Luedeking piret and modified luedeking piret models, but not this study.
It can be interesting.
Response: Thank you so much for your kind suggestion. We will try to employ these
models for growth and product modelling in the future study. It will be very
interesting.
Reviewer #3
The manuscript by Han et al. tested several constitutive promoters from
Schizochytrium sp. and used the strong ccg1p promoter to overproduce ACL and
ACC, which increased the yields of DHA and total lipids.
Comments:
1. How many independent strains of each transformant were generated and used for
the study? It seems like there is only one strain for each construct, which is not
acceptable.
Response: Thank you very much for your valuable comments and the question. After
transformation, we have selected eight independent strains for each kind of
transformants. The transformants were cultured in fermentation broth for 5 days,
stained with nile red dye for neutral lipid staining, and the fluorescence intensities
were measured by a multifunctional plate reader. All transformants displayed higher
fluorescence intensities than the wild-type strain, indicating that the transformants
accumulated more lipids than the WT strain (Fig. IIIa, b, c). After preliminary
screening, two independent strains with the highest fluorescence for each kind of
transformants were cultured for determination of lipid and DHA yields. The same
kind of transformants displayed similar results (Fig. IIId). All fermentation
experiments were at least repeated twice. For clarity, results from one strain of each
kind of transformants were provided in the text.
b a
d c
Fig.III Effect of ACL and ACC overexpression on lipid accumulation by Schizochytrium sp.. a, b,
c Nile red staining of WT, OACL, OACC, and OACL-ACC. d Lipid contents of WT, OACL,
OACC, and OACL-ACC. The strains were cultured in fermentation medium for 120 h
2. RNA or Protein levels of ACL and ACC should be examined in the
overexpressing strains.
Response: Thank you very much for your suggestion. The transcriptional levels of
ACL and ACC were determined by qRT-PCR in WT, OACL, OACC, and OACL-ACC
which were cultivated in fermentation media for 2 and 4 d. The transcriptional levels
of ACL and ACC were greatly increased in the corresponding transformants. The
results were added to the text (Line 188-194) and the new Fig. 3.
3. Lipid content was increased from 54% of the WT to ~70% of the overexpressing
strains. Figure 4d, why the nile red staining shows such low level of signals in the
WT? Did the expression of ACL and ACC changed the morphology of lipid
bodies? i.e. size and numbers.
Response: Thank you for the question. The nile red staining is a very efficient means
to indicate the intracellular lipid accumulation. During our nile red staining
preliminary screening, we found that the fluorescence signals had a positive
correlation with the lipid contents, but the correlation was not strictly linear. The
increase rates of fluorescence signals were usually higher than the increase rates of
lipid contents (please see Fig. III, response to comment 1). Besides, fluorescence
attenuates during microscopic observations. WT was the first one observed and its
analysis might take longer time than the others’. Microscopic analysis was repeated
with 48-h cultured cells (it will be easier to observe the morphology of lipid bodies
with “younger” cells) and all photos were taken with the same observation time. Fig.
4d was replaced with the new figure (now as Fig. 5d), and the relative fluorescence
intensities of cells were analyzed by ImageJ (Fig. IV), which had a good correlation
with the lipid contents. We thank you very much for your kind reminder. Since
Schizochytrium cells aggregated and produced zoospores during cultivation, it was
difficult to distinguish lipid bodies. From what we saw, Schizochytrium formed a large
lipid body per cell and no significant difference of morphology wad noticed between
WT and the transformants (Fig. 5d).
Fig.IV The relative fluorescence intensity of WT, OACL, OACC, and OACL-ACC
4. Explain or discuss the changes of fatty acid composition especially on the
increase of DHA in ACC and ACL-ACC.
Response: Thank you for your suggestion. Please see answer to comment 6 of
Reviewer 2. The discussion about the changes of fatty acid composition on the
increase of DHA in OACC and OACL-ACC strains can be found in Line 251-260.
Reviewer #4
Some grammatical problems are seen in the whole manuscript which should be
revised. Also, the organization of the manuscript especially in the introduction part is
not good. One more attention is needed in the discussion part. Some related papers
with for lipid extraction (with modifications) are introduced here for improvement of
the lipid extraction part, determination of biomass dry weight and some other part in
the material and method, cite these references in this part. Paper titles are in the
parentheses. Add a table in discussion part and compare related studies.
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Best wishes
1- Process Safety and Environmental Protection III (2017) 757-765
http://dx.doi.org/10.1016/j.psep.2017.08.029 (Optimizing of lipid production in
Cryptococcus heimaeyensis through M32 array of Taguchi design)
2- Process Safety and Environmental Protection III (2017) 747-756
http://dx.doi.org/10.1016/j.psep.2017.08.027 (Single cell oil and its application for
biodieselproduction)
3- Int. J. Environ. Sci. Technol. DOI 10.1007/s13762-014-0687-8 (Recycling
of lignocellulosic waste materials to produce high-value products: single cell oil and
xylitol)
4- Int. J. Environ. Sci. Technol. (2014) 11:597-604 DOI
10.1007/s13762-013-0373-2 (Improving microbial oil production with standard and
native oleaginous yeasts by using Taguchi design)
5- (Medium optimization for biotechnological production of single cell oil
using Yarrowia lipolytica M7 and Candida sp.)
6- (Selection and optimization of single cell oil production from Rodotorula
110 using environmental waste as substrate)
Response: Thank you very much for your kind suggestions and the recommended
related references. We have carefully revised the manuscript and reorganized the
discussion section. We have earnestly read the recommended references with great
interest. Three related references have been cited in the methods (determination of
glucose and lipid extraction). We will consider the methods in the related references
in the future study.
Dear editor Hallenbeck,
Thank you very much for your decision letter of BBIO-D-19-00571 (Enhancement of
docosahexaenoic acid production by overexpression of ATP-citrate lyase and
acetyl-CoA carboxylase in Schizochytrium sp.). We have carefully revised the
manuscript to address all the comments of the editor and reviewers. New data
including qRT-PCR analysis of ACL and ACC expression (Fig. 3) were added to the
revised manuscript. Some methods and references were added to the text, and the
discussion section was reorganized. The modifications are marked in yellow in the
marked-up manuscript. Details of our revision are included in our point-by-point
“Response to Reviewers”. Some illustrated figures are added to the “Response to
Reviewers”, and the “Response to Reviewers” file has been submitted in the
Biotechnology for Biofuels system.
We would like to thank you and the reviewers for careful reviewing and valuable
comments which helped to improve the manuscript.
Sincerely yours,
Zhi Chen
State Key Laboratory of Agrobiotechnology and Key Laboratory of Soil Microbiology,
Ministry of Agriculture, College of Biological Sciences, China Agricultural University,
Beijing 100193, China
Cover letter for revised Click here to access/download;Personal Cover;Cover letter forrevised.docx