Biotechnological Assessment of Culture Conditions on the Stress-Induced Carotenoid Production of Dunaliella salina and Growth Kinetics of Chlorophyceae Microalgae Strains

Abstract Microalgae are potential sources of a wide range of bioproducts. It is essential to choose the proper microalgae strain and culture condition to achieve an efficient production. The production yield of carotenoids by Dunaliella salina under the stress-induced culture conditions of nitrogen deprivation and excessive light intensity was evaluated. Also, a survey at laboratorial scale of the growth kinetics under different culture conditions of photoperiod, aeration, and agitation was performed for the seven species of green microalgae Ankistrodesmus fusiformis, Chlamydocapsa bacillus, Desmodesmus brasiliensis, Kirchneriella lunaris, Pseudokirchneriella subcapitata, and Scenedesmus obliquus. As a result, aeration of atmospheric air is enough to improve the growth kinetics of the seven species studied. Production of carotenoids was enhanced under stress by excessive light intensity. Although D. salina does not grow effectively under nitrogen deprivation, this stress condition may be used to quickly stimulate carotenoid production once the culture reaches a high cellular population.

Effect of exogenous stress factors on the biosynthesis of carotenoids and lipids by Rhodotorula yeast strains in media containing agro-industrial waste.

In this study, we aimed to determine the effect of exogenous stress factors (sodium chloride as osmotic stressor, hydrogen peroxide as an inducer of oxidative stress, white light irradiation, and low temperature) on the biosynthesis of carotenoids and lipids by red yeast (Rhodotorula glutinis, R. mucilaginosa, and R. gracilis) during cultivation in media containing potato wastewater and glycerol. According to our results, the yeast were able to grow and biosynthesize lipids and carotenoids in the presence of the applied stress factors. Low temperature caused an increase in the biosynthesis of intracellular lipids and carotenoids. R. gracilis synthesized lipids (21.1 g/100 gd.w.) and carotenoids (360.4 µg/gd.w.) in greater quantities than that of other strains. Under these conditions, there was also an increase in the content of unsaturated fatty acids, especially linoleic and linolenic acids. The highest percentage of polyunsaturated fatty acid (PUFA) (30.4%) was synthesized by the R. gracilis yeast after cultivation at 20°C. Their quantity was 2.5-fold greater than that of the biomass grown in control conditions. The contribution of individual carotenoid fractions depended both on the yeast strain and the culture conditions. Induction of osmotic stress and low temperature intensified the biosynthesis of ß-carotene (up to 73.9% of the total carotenoid content). In oxidative stress conditions, yeast synthesized torulene (up to 82.2%) more efficiently than under other conditions, whereas white light irradiation increased the production of torularhodin (up to 20.0%).

Effect of growth temperature on biosynthesis and accumulation of carotenoids in cyanobacterium Anabaena sp. PCC 7120 under diazotrophic conditions.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) ß-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of ß-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of ß-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between ß-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Construction of Escherichia coli cell factories for crocin biosynthesis.

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Acyl-CoA synthetases from Physcomitrella, rice and Arabidopsis: different substrate preferences but common regulation by MS188 in sporopollenin synthesis.

MAIN CONCLUSION: ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.

Simultaneous Production of Lipids and Carotenoids by the Red Yeast Rhodotorula from Waste Glycerol Fraction and Potato Wastewater.

AbstractThe objective of this study was to determine the possibility of simultaneous biosynthesis of lipids and carotenoids by the Rhodotorula yeast strains in media with waste glycerol and deproteinized potato wastewater and to determine the level of pollution reduction by media. On the basis of results obtained during the yeast microcultures in the Bioscreen C system, it was found that potato wastewater and glycerol can be used as components of media for Rhodotorula glutinis, Rhodotorula mucilaginosa, and Rhodotorula gracilis yeast strains. The amount of glycerol added to media higher than 10% significantly decreased the growth rate of yeast. The results of yeast culture in the laboratory shaker flasks showed a possibility of simultaneous production of lipids and carotenoids by R. glutinis, R. mucilaginosa, and R. gracilis yeast strains during cultivation in media containing only waste glycerol and deproteinized potato wastewater. A higher intracellular lipid content (approximately 15 g/100 gd.w.) was obtained for R. mucilaginosa and R. gracilis yeast biomass after cultivation in experimental media with waste glycerol and potato wastewater. In conclusion, the yeast grown in media with potato wastewater supplemented with 3% or 5% glycerol synthesized carotenoids, and their content in biomass did not exceed 230 µg/gd.w.

Critical applications of Mucor circinelloides within a biorefinery context.

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.

Cycles of external dependency drive evolution of avian carotenoid networks.

All organisms depend on input of exogenous compounds that cannot be internally produced. Gain and loss of such dependencies structure ecological communities and drive species' evolution, yet the evolution of mechanisms that accommodate these variable dependencies remain elusive. Here, we show that historical cycles of gains and losses of external dependencies in avian carotenoid-producing networks are linked to their evolutionary diversification. This occurs because internalization of metabolic controls-produced when gains in redundancy of dietary inputs coincide with increased branching of their derived products-enables rapid and sustainable exploration of an existing network by shielding it from environmental fluctuations in inputs. Correspondingly, loss of internal controls constrains evolution to the rate of the gains and losses of dietary precursors. Because internalization of a network's controls necessarily bridges diet-specific enzymatic modules within a network, it structurally links local adaptation and continuous evolution even for traits fully dependent on contingent external inputs.

Multi-species transcriptome analyses for the regulation of crocins biosynthesis in Crocus.

BACKGROUND: Crocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation. RESULTS: A large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation. CONCLUSIONS: Our study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level.

Biosynthetic Pathway of Carotenoids in Rhodotorula and Strategies for Enhanced Their Production.

Rhodotorula is a group of pigment-producing yeasts well known for its intracellular biosynthesis of carotenoids such as ß-carotene, γ-carotene, torulene and torularhodin. The great potential of carotenoids in applications in food and feed as well as in health products and cosmetics has generated a market value expected to reach over $2.0 billion by 2022. Due to growing public concern over food safety, the demand for natural carotenoids is rising, and this trend significantly encourages the use of microbial fermentation for natural carotenoid production. This review covers the biological properties of carotenoids and the most recent findings on the carotenoid biosynthetic pathway, as well as strategies for the metabolic engineering methods for the enhancement of carotenoid production by Rhodotorula. The practical approaches to improving carotenoid yields, which have been facilitated by advancements in strain work as well as the optimization of media and fermentation conditions, were summarized respectively.

Challenges and tackles in metabolic engineering for microbial production of carotenoids.

Naturally occurring carotenoids have been isolated and used as colorants, antioxidants, nutrients, etc. in many fields. There is an ever-growing demand for carotenoids production. To comfort this, microbial production of carotenoids is an attractive alternative to current extraction from natural sources. This review summarizes the biosynthetic pathway of carotenoids and progresses in metabolic engineering of various microorganisms for carotenoid production. The advances in synthetic pathway and systems biology lead to many versatile engineering tools available to manipulate microorganisms. In this context, challenges and possible directions are also discussed to provide an insight of microbial engineering for improved production of carotenoids in the future.

Phytoene Accumulation in the Novel Microalga Chlorococcum sp. Using the Pigment Synthesis Inhibitor Fluridone.

Carotenoids are lipophilic pigments found in plants and algae, as well as some bacteria, archaea, and fungi that serve two functions-(1) as light harvesting molecules-primary carotenoids, and (2) as antioxidants, acting against reactive oxygen species⁻secondary carotenoids. Because of their strong antioxidant properties, they are also valuable for the development of anti-aging and photo-protective cosmetic applications. Of particular interest is the carotenoid phytoene, for its colorless and UV absorption characteristics. In this study, we targeted a reduction of phytoene desaturase (PDS) activity with the pigment-inhibiting herbicide 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridin-4-one (fluridone), which leads to the over-accumulation of phytoene in the recently characterized microalgal strain Chlorococcum sp. (UTEX B 3056). After post-incubation with fluridone, phytoene levels were measured at ~33 ug/mg cell tissue, as opposed to non-detectable levels in control cultures. Hence, the novel microalga Chlorococcum sp. is a viable candidate for the production of the high-value carotenoid phytoene and subsequent applications in cosmeceuticals, as well as more obvious nutraceutical and pharmaceutical applications.

Developing a Novel Two-Stage Process for Carotenoid Production by Cordyceps militaris (Ascomycetes).

Natural carotenoids are attracting increasing interest, but their widespread use is limited because of poor production. Cordyceps militaris, a traditional Chinese mushroom, contains a large amount of carotenoids, and this study aimed to increase carotenoid production by C. militaris by optimizing a liquid-state cultivation system. We developed and optimized a novel 2-stage process, including cultivation under shaking in darkness and under static irradiation on a flat panel, using response surface methodology, and we compared this process to common shake-flask cultivation. In addition, we examined the effects of different inducers (chitosan, peanut oil, tomato juice, yeast, and metal ions) on carotenoid production. Results showed that under optimal conditions (4 days of shaking in darkness, 10 days of static irradiation with a 100-mL flat panel volume), a maximum of 1217.5 ± 115.9 µg/g carotenoids were produced; only 662.9 µg/g were produced by common shake-flask cultivation. Only a large amount of chitosan (8 mg) could significantly increase carotenoid content; some of the other inducers showed inhibitory effects. This study demonstrated that this 2-stage process could effectively increase the natural carotenoid content in C. militaris, making it a potential source for commercial exploitation.

Light quality-induced changes of carotenoid composition in pak choi Brassica rapa ssp. chinensis.

Carotenoids as part of the photosystems are crucial for their assembly, light-harvesting, and photoprotection. Light of different wavelengths impacts the composition and structure of photosystems, thus offering the possibility to influence the carotenoid concentrations and composition in photosystems by illumination with specific narrow-banded light spectra. Key components involved in the regulation of gene transcription are still poorly characterized, particularly in leafy vegetables as compared to model plants. In particular, the effect of different light qualities and its connection to redox control mechanisms, which also determine the photosystem composition and structure, is not yet well understood. Furthermore, light quality effects are species-dependent, and thus, increase the need to perform research on individual vegetable species such as pak choi Brassica rapa ssp. chinensis. Here, we investigated the carotenoid concentrations and composition of pak choi sprouts grown for 6 days under blue, red, or white light emitting diodes (LEDs) as light source. After 6 days, the total carotenoid content was the highest under white and slightly reduced under blue or red LEDs. Blue, red, and white light differently affected the carotenoid composition mainly due to variations of the ß-carotene content which could be correlated to changes in the transcript levels of ß-carotene hydroxylase 1 (ß-OHASE1). Further investigations implied a redox controlled gene expression of ß-OHASE1. In addition, transcription factors related to light signaling and the circadian clock differed in their transcriptional abundance after exposure to blue and red light. RNA-Seq analysis also revealed increased transcript levels of genes encoding the outer antenna complex of photosystem II under red compared to blue light, indicating an adjustment of the photosystems to the different light qualities which possibly contributed to the alternations in the carotenoid content and composition.

Light emitting diodes applied in Synechococcus nidulans cultures: Effect on growth, pigments production and lipid profiles.

Researches about light emitting diodes (LEDs) as energy source in microalgae cultivations has been growing in recent years due to its spectral quality, durability and reduced energy consumption. In this study, green, red and yellow LEDs were evaluated as energy source in Synechococcus nidulans LEB 115 cultures. Productivities and specific growth rates were up to 2.5 times greater than in cultures using fluorescent light. The different LED colors evaluated did not influence the chlorophyll, carotenoid or lipid productions. Biomass cultivated with LEDs showed high amounts of saturated fatty acids (above 48%), which is desirable for biodiesel production. In addition to the Synechococcus nidulans LEB 115 growth stimulation, the application of green, red and yellow LEDs in the cultivations produces potential biomass for biodiesel synthesis and other industrial interest biomolecules utilization.

Exploring natural biodiversity to expand access to microbial terpene synthesis.

BACKGROUND: Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established-mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes. RESULTS: The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule. CONCLUSION: IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.

Optimization of fermentation conditions for carotenoid production in the radiation-resistant strain Deinococcus xibeiensis R13.

Deinococcus xibeiensis R13 was isolated from an extreme environment in Xinjiang, China, and can resist gamma-radiation and UV-irradiation. In this study, D. xibeiensis R13 was shown to be capable of efficiently producing carotenoids in culture, and factors influencing its productivity were identified. The maximum carotenoid yield was observed at an initial temperature of 30 °C and pH 7.0 in the presence of fructose, tryptone at a C/N ratio of 1:5, and 10 µM Fe2+. The carotenoid yield under modified culture conditions was 6.64 mg/L after fermentation for 48 h, representing an increase of 84% compared to the original conditions. The biomass reached 7.22 g/L, which was 2.19-fold higher than under non-optimized conditions. The produced carotenoids were extracted from R13 and analyzed by UPLC-MS. This is the first study of carotenoid production by the new strain D. xibeiensis R13, which provides a new source for the microbial fermentation of natural carotenoids, and also provides a good reference for industrial production of other carotenoids and other terpenoid products.

Enhancement of Torularhodin Production in Rhodosporidium toruloides by Agrobacterium tumefaciens-Mediated Transformation and Culture Condition Optimization.

Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box-Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.

Light induces carotenoids accumulation in a heterotrophic docosahexaenoic acid producing microalga, Crypthecodinium sp. SUN.

In the present study, the effect of various light conditions on carotenoid accumulation in a novel heterotrophic microalga, Crypthecodinium sp. SUN was investigated. The results showed that C. sp. SUN mainly produced γ-carotene and ß-carotene. The total carotenoid content could reach to 12.8 mg g-1 dry weight under high light intensity (100 µmol m-2 s-1), which was >100-fold higher than that under dark condition. Besides, along with the light intensity increased, the ROS level in vivo was decreased at 48 h and 72 h. Further study showed that, light could efficiently promote the gene expression of PSY and LCYb, which explain the molecular mechanisms of carotenoids accumulation under light conditions. Meanwhile, slightly inhibited fatty acids accumulation could promote the carotenoids yield. The present work proposed that C. sp. SUN could be a potential carotenoid producer, and provided valuable insight for carotenoids biosynthesis.

Exploring the differential mechanisms of carotenoid biosynthesis in the yellow peel and red flesh of papaya.

BACKGROUND: Red-fleshed papaya is a good material to study the different carotenoids accumulation mechanism in the peel and flesh. Although the peel and flesh of papaya closely integrated into one body, the flesh coloration changing from white to red, while the exocarp coloration changing from green to yellow. In this study, the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes in the process of papaya fruit ripening were studied, and the carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were investigated. RESULTS: The carotenoid composition in papaya flesh and peel were different. The major carotenoids were lutein and ß-carotene in the peel, while lycopene in the flesh. The accumulation of carotenoids, including lycopene, ß-carotene, and ß-cryptoxanthin were considered to cause the orange-red color of papaya cv. 'Daqing No.10' flesh. The color of peel changed from green to yellow because of the fast degradation of chlorophyll and the appearance of carotenoids such as lutein and ß-carotene. Thirteen genes that encode enzymes in the carotenoid biosynthetic pathway were detected in papaya fruit transcriptome: two phytoene synthase (PSY1, PSY2), two phytoene desaturase (PDS1, PDS2), one ζ-carotene desaturase (ZDS), four lycopene cyclase (CYCB, LCYB1, LCYB2, LCYE), one ß-carotene hydroxylase (CHYB), one carotene ε-monooxygenase (LUT1), one violaxanthin de-epoxidase (VDE), and one zeaxanthin epoxidase (ZEP). The results of RNA-Seq and RT-qPCR showed the expression of carotenoid biosynthetic pathway genes was consistent with the change of carotenoid content. Carotenoid biosynthetic pathways in the yellow peel and red flesh of papaya were analysed based on the major carotenoids accumulation and the expression patterns of key carotenoid biosynthesis pathway genes. There was only a ß-branch of carotenoid biosynthesis in the flesh of papaya, while there were both α- and ß-branch of carotenoid biosynthesis in papaya peel. In the process of papaya fruit ripening, the α-branch was inhibited and the ß-branch was enhanced in the peel. CONCLUSIONS: The differential carotenoid accumulation and biosynthesis pathway genes expression in peel and flesh, lay a foundation for further study and provide further insights to control fruit color and improve fruit quality and appearance.