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. 2023 Jan 31;11(2):e04361-22. doi: 10.1128/spectrum.04361-22

Engineering the β-Carotene Metabolic Pathway of Microalgae Dunaliella To Confirm Its Carotenoid Synthesis Pattern in Comparison To Bacteria and Plants

Hao-Hong Chen a,b,#, Ming-Hua Liang a,#, Zhi-Wei Ye c,#, Yue-Hui Zhu d, Jian-Guo Jiang a,✉
Editor: Cesar de la Fuente-Nuneze
PMCID: PMC10100976  PMID: 36719233

ABSTRACT

Dunaliella salina is the most salt-tolerant eukaryote and has the highest β-carotene content, but its carotenoid synthesis pathway is still unclear, especially the synthesis of lycopene, the upstream product of β-carotene. In this study, DsGGPS, DsPSY, DsPDS, DsZISO, DsZDS, DsCRTISO, and DsLYCB genes were cloned from D. salina and expressed in Escherichia coli. A series of carotenoid engineering E. coli strains from phytoene to β-carotene were obtained. ZISO was first identified from Chlorophyta, while CRTISO was first isolated from algae. It was found that DsZISO and DsCRTISO were essential for isomerization of carotenoids in photosynthetic organisms and could not be replaced by photoisomerization, unlike some plants. DsZDS was found to have weak beta cyclization abilities, and DsLYCB was able to catalyze 7,7′,9,9′-tetra-cis-lycopene to generate 7,7′,9,9′-tetra-cis-β-carotene, which had not been reported before. A new carotenoid 7,7′,9,9′-tetra-cis-β-carotene, the beta cyclization product of prolycopene, was discovered. Compared with the bacterial-derived carotenoid synthesis pathway, there is higher specificity and greater efficiency of the carotenoid synthesis pathway in algae. This research experimentally confirmed that the conversion of phytoene to lycopene in D. salina was similar to that of plants and different from bacteria and provided a new possibility for the metabolic engineering of β-carotene.

IMPORTANCE The synthesis mode of all trans-lycopene in bacteria and plants is clear, but there are still doubts in microalgae. Dunaliella is the organism with the highest β-carotene content, and plant-type and bacterial-type enzyme genes have been found in its carotenoid metabolism pathway. In this study, the entire plant-type enzyme gene was completely cloned into Escherichia coli, and high-efficiency expression was obtained, which proved that carotenoid synthesis of algae is similar to that of plants. In bacteria, CRT can directly catalyze 4-step continuous dehydrogenation to produce all trans-lycopene. In plants, four enzymes (PDS, ZISO, ZDS, and CRTISO) are involved in this process. Although a carotenoid synthetase similar to that of bacteria has been found in algae, it does not play a major role. This research reveals the evolutionary relationship of carotenoid metabolism in bacteria, algae, and plants and is of methodologically innovative significance for molecular evolution research.

KEYWORDS: Dunaliella salina, carotenoid metabolic pathway, lycopene, β-carotene, gene expression, plasmid construction

INTRODUCTION

Dunaliella salina, a marine unicellular green alga, is the most salt-tolerant eukaryote with the highest natural β-carotene content known at present (up to 10% of the dry weight) and is a model organism both for osmoregulation and carotenoid metabolism research (1–3). However, the identification and functional verification of its β-carotene pathway synthases are still incomplete.

The biosynthesis of carotenoids is an important branch of the isoprenoid metabolic pathway. Phytoene is the first carotenoid in this pathway, and lycopene is the center and branch point of this system (Fig. 1). Lycopene gradually forms more than 700 known carotenoids after a series of branching metabolism by alpha- and beta-cyclization (4–6).

FIG 1.

FIG 1

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Biosynthesis of carotenoids in bacteria, fungi, cyanobacteria, algae, and plants. Bacteria can synthesize C40 carotenoids via different biosynthetic pathways. Bacterial CrtI can catalyze the three-step or four-step desaturations, while fungal CrtI can catalyze the four-step or five-step desaturations.

In bacteria and fungi, the conversion from phytoene to lycopene is catalyzed by a CrtI-type phytoene dehydrogenase to complete a four-step continuous dehydrogenation (7, 8). In plants, four key enzymes are involved in this process: phytoene desaturase (PDS) catalyzes the dehydrogenation of 15-cis-phytoene twice to successively produce 9,15-cis-phytoene and 9,15,9′-tri-cis-ζ-carotene. Subsequently, 9,15,9′-tri-cis-ζ-carotene is converted to 9,9′-di-cis-ζ-carotene by 15-cis-ζ-carotene isomerase (ZISO) or photoisomerization. ζ-Carotene desaturase (ZDS) then dehydrogenates 9,9′-di-cis-ζ-carotene to 7,9,9′-tri-cis-neurosporene and 7,7’,9,9’-tetra-cis-lycopene successively. Finally, carotenoid isomerase (CRTISO) catalyzes the production of all-trans-lycopene from 7,7′,9,9′-tetra-cis-lycopene (prolycopene) (4).

It is generally believed that the carotenoid pathway of green algae is consistent with that of higher plants, but there are still many problems to be solved (9–11). In plant genetic engineering, a bacterial CrtI gene can be used to replace the four genes of PDS, ZISO, ZDS, and CRTISO of plants to achieve carotenoid accumulation. The bacterial CrtI gene is introduced in carotenoid-rich “golden rice” to achieve the conversion of phytoene to lycopene (12, 13). It has not been confirmed whether green algae have the four genes that can be replaced by the bacterial CrtI gene or whether these four enzymes have the same function as plants.

The aim of this study was to clone all the key enzyme genes of the β-carotene metabolic pathway in D. salina, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), PDS, ZISO, ZDS, CRTISO, and lycopene β-cyclase (LYCB), and construct these genes into Escherichia coli to express them completely. By observing the expression efficiency of β-carotene and various intermediate carotenoids upstream, the application potential of β-carotene engineering E. coli strains constructed by D. salina’s carotenoid synthesis gene was judged. At the same time, according to the intermediate carotenoid products, consistencies or differences between lycopene synthesis in green algae and plants were confirmed. The functions of ZISO and CRTISO in green algae were further elucidated by comparing light and dark conditions.

RESULTS AND DISCUSSION

Isolation of the carotenoid genes from D. salina.

D. salina can grow in saturated saline. Its optimum salinity is 1.5 to 2.0 M NaCl. When the salinity exceeds 3.5 M NaCl, a large amount of β-carotene will accumulate, making the cells yellowish red (Fig. 2).

FIG 2.

FIG 2

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(A) Images of cells at 1.5 M NaCl (top) and 3.5 M NaCl (bottom). (B) Carotenoid analysis of D. salina CCAP 19/18 at 1.5 M and 3.5 M NaCl. The following are the peak assignments: a, lutein; b, zeaxanthin; c, α-cryptoxanthin; d, α-carotene; e, all-trans-β-carotene; e1, 9-cis-β-carotene; f, γ-carotene. mAu, arbitrary unit.

To identify the DsGGPS, DsPSY, DsPDS, DsZISO, DsZDS, DsCRTISO, and DsLYCB genes in D. salina, homology searches of the genomic sequences of D. salina were performed on the Phytozome platform using the corresponding amino acid sequences in Arabidopsis thaliana (14, 15). Candidate crts (carotenogenic enzymes) genes were found and isolated from the cDNAs of D. salina, and the results are shown in Table S1 available at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. The function of ZISO in Euglena has been identified, but not in Chlorophyta, while CRTISO was first isolated and identified from algae. The initial ATG of each gene was verified by 5′ rapid amplification of cDNA ends (RACE) PCR, and the termination codon of each gene was verified by 3′ RACE PCR.

The length of the coding DNA sequence (CDS) of the crts gene, the length of the coding amino acids, and the similarity with Arabidopsis-related genes are listed in Table S1 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. Among them, DsGGPS, DsPSY, DsZISO, DsZDS, DsCRTISO-homo1, DsCRTISO-homo2, and DsLYCB were consistent with the sequence information provided by the Phytozome platform, while DsPDS and DsCRTISO were incomplete in Phytozome. Compared with the functional Crts enzymes of A. thaliana, the deduced amino acid sequences of D. salina were highly conserved (50 to 70%), except for the N-terminal region, which implied potential functional similarities among these proteins.

Two transmembrane domains of each enzyme were predicted using the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/), which were present in typical plastid-targeting proteins (http://www.cbs.dtu.dk/services/ChloroP/), indicating the plastid localization of D. salina Crts protein.

Construction and expression of the D. salina β-carotene synthesis pathway in E. coli.

The function of D. salina Crts was confirmed by using a heterologous E. coli expression system. Fig. 3A shows a series of strains that were cultured under dark conditions at 28°C and centrifuged after isopropyl-β-d-thiogalactopyranoside (IPTG) induction for 48 h. It can be seen that the absence of DsPSY, DsPDS, and DsZISO all formed pale yellow bacteria, The ΔZDS strain produced beige bacteria, the ΔCRTISO strain formed golden yellow bacteria, the CRTS strain formed red bacteria, and the CRT+LYCB strain formed yellowish bacteria, which indicated that these enzymes could be normally expressed in E. coli and had catalytic activity. It could also be seen from Fig. 3B that a lack of any one of the crt genes could not produce β-carotene.

FIG 3.

FIG 3

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Carotenoid composition of various engineering Escherichia coli. (A) Colors of E. coli (engineered) strains harboring different combinations of plasmids. (B) HPLC analysis of the carotenoids produced in E. coli cells harboring different combinations of plasmids. The following are the peak assignments: d, α-carotene; e, all-trans-β-carotene; e1, 9-cis-β-carotene; f, γ-carotene; g, all-trans-lycopene; g1, lycopene isomer1; g2, lycopene isomer2; g3, lycopene isomer3; h, 7,7′,9,9′-tetra-cis-lycopene (prolycopene); h1, prolycopene isomer1; h2, prolycopene isomer2; i, ε-carotene; j, 9,9′-di-cis-ζ-carotene; j1, ζ-carotene isomer1; j2, ζ-carotene isomer2; k, δ-carotene; l, 9,9′,15-tri-cis-ζ-carotene; n, 7,9,9′-tri-cis-neurosporene; n1, neurosporene isomer1.

The carotenoid compositions of ΔPSY, ΔPDS, ΔZISO, ΔZDS, ΔCRTISO, CRTS, and CRT+LYCB strains were detected by high-performance liquid chromatography (HPLC) analysis. The retention time and characteristic absorption spectrum of each carotene are shown in Fig. 3B and in Fig. S1 and Table S2 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. The detected 9,15,9′-tri-cis-ζ-carotene and 9,9′-di-cis-ζ-carotene are consistent with the characteristic peaks in the HPLC results of the E. coli strain containing pAC-ZETAipi (Fig. S2 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf). The liquid chromatogram peak with 9,15,9′-tri-cis-ζ-carotene (l) as the main substance was detected from ΔZISO at a 400-nm band. Likewise, a liquid chromatogram peak with 9,9′-di-cis-ζ-carotene (j) as the main substance was detected in ΔZDS at a 400-nm band for 17.29 min, and a liquid chromatogram peak with 7,7′,9,9′-tetra-cis-lycopene (h) as the main substance was detected in ΔCRTISO at a 450-nm band for 18.06 min. CRTS and CRT+LYCB strains formed all-trans-lycopene and β-carotene at 29.18 and 16.27 min, respectively. Among them, there were more isomers of lycopene, which had a great relationship with 13 carbon-carbon double bonds in lycopene.

Functional catalytic properties of DsLYCB.

Through further analysis of the CRT+LYCB strain HPLC results in Fig. 3B, it was found that in addition to the production of β-carotene, δ-carotene, ε-carotene, γ-carotene, and α-carotene were also generated. Many studies have reported the functional identification and regulatory mechanism of LCYB in D. salina (16, 17). But little research has reported that LYCB can catalyze all-trans-lycopene to produce α-carotene, δ-carotene, and ε-carotene (18).

In the lycopene cyclization process, it may be that C-2 dehydrogenates under the effect of the cofactor NAD, making C-1 exhibit electrophilicity, attacking C-6, and then C-4, C-5, and C-6 form delocalized π bonds, which are conjugated with the C-2 dumbbell electronic cloud to form larger delocalized π bonds (18, 19). A β-ring is formed when C-6 hydrogen is transferred to the C-2 position, and an ε-ring is formed when C-4 hydrogen is transferred to the C-2 position. Under the catalysis of DsLYCB, C-6 hydrogen tends to be transferred to the C-2 position, so most of them form β-carotene (84%) and a small amount of ε-ring (16%). This agreed with our previous report suggesting that DbLCYB is bifunctional in Dunaliella bardawil (20). Interestingly, maize LcyE (ZmLcyE) also showed a low level of β-monocyclase activity (21). But we found that there was another cyclase in Dunaliella (DbLCYE) that only showed ε-cyclase activity (20). It has been reported that a conserved motif (designated the extended motif B) of lycopene cyclases determines the formation of β- and ε-ionone groups (22).

Interestingly, analysis of the liquid chromatogram of the ΔCRTISO+LYCB strain in Fig. 4A revealed the production of a new carotenoid not previously reported, which has a distinctly different retention time and characteristic absorption spectrum from that of β-carotene (Fig. 2; Table S2 https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf). It has been reported that neurosporene can be directly cyclized to β-zeacarotene under the catalysis of LYCB (18). Only one end of the ψ-end group with C-7 and C-8 dehydrogenation can be cyclized by LYCB, while the ψ-end group without dehydrogenation cannot be cyclized (18). By comparing the retention time and characteristic absorption spectrum, it was inferred that this new carotenoid was the β-cyclization product of prolycopene, namely, 7,7′,9,9′-tetra-cis-β-carotene. This new carotenoid implies a new way to synthesize β-carotene (Fig. 4B).

FIG 4.

FIG 4

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Functional characteristics of LYCB from D. salina. (A) HPLC scans of carotenoid extracts from ΔCRTISO-LYCB; peaks: m, 7,7′,9,9′-tetra-cis-lycopene; m1, 7,7′,9,9′-tetra-cis-lycopene isomer. (B) CRTISO and LYCB involved in the probable pathway of β-carotene biosynthesis.

All-trans-β-carotene was not detected in the ΔCRTISO+LYCB strain, indicating that the cis structure at C-7 and C-9 was a bond-energy-stable structure, and no isomerization occurred automatically during the cyclization process (Fig. 4B). In addition, the cis structure of C-7 and C-9 has a larger spatial conformation, making it more difficult to enter the catalytic channel, and this location is unlikely to have an isomeric catalytic site (23). Therefore, the catalytic process of the cis structure of C-7 and C-9 is consistent with the cyclization process of all-trans-lycopene; dehydrogenation occurs at the 2 position, and then the 1 position attacks C-6 (Fig. S3 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf).

The traditional view is that LYCB has only trans-ψ-end group catalytic ability (18). This study found that DsLYCB also has cis catalytic ability. This result also explained the reason that the contents of 9-cis-β-carotene and all-trans-β-carotene in D. salina are so close (Fig. 2B). The isomerization of DsCRTISO in the synthesis of β-carotene is not complete, leading to the accumulation of 9-cis-carotene. However, there are different internal environments between E. coli and chloroplasts of Dunaliella. More accurate verification requires the removal of DsLYCB from D. salina by knockout technology.

Functional catalytic properties of DsZISO and DsCRTISO.

Many reports believed that 9,9′,15-tri-cis-ζ-carotene and 7,7′,9,9′-tetra-cis-lycopene can be isomerized into 9,9′-di-cis-ζ-carotene and all-trans-lycopene, provided that they are directly exposed to light (24, 25). A study of Arthrospira platensis showed that light conditions could produce isomeric effects and performed the function of ZISO, but the CrtP of A. platensis itself has the function of ZISO isomerase (26). It is hard to say whether ZISO or CrtP was working.

ΔZISO and ΔCRTISO strains were cultured under light and dark conditions at 28°C, respectively, and we found that light and dark conditions did not change the color of the bacteria (Fig. 5A). HPLC analysis also found that the peaks of ΔZISO and ΔCRTISO strains cultured under dark and light conditions did not change (Fig. 5B). Under light conditions, 9,15,9′-tri-cis-ζ-carotene and 7,7′,9,9′-tetra-cis-lycopene in ΔZISO and ΔCRTISO could not be well isomerized into 9,9′-di-cis-ζ-carotene and all-trans-lycopene to produce red lycopene mycelia. This result indicated that ZISO and CRTISO are necessary for the production of β-carotene by photosynthetic organisms, such as green algae and even plants. This result was consistent with that of Yu et al. (24) but differed from Elio et al. (27) who believed that light restores lycopene biosynthesis in ZISO-silenced fruits.

FIG 5.

FIG 5

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Functional identification of ZISO, CRTISO, and ZDS. (A) Colors of ΔZISO and ΔCRTISO E. coli strains under light and dark culture conditions, respectively. (B) HPLC scans of carotenoid extracts showing no differences between light and dark culture of ΔZISO and ΔCRTISO strains; peaks: g, all-trans-lycopene; h, 7,7′,9,9′-tetra-cis-lycopene (prolycopene); h1, prolycopene isomer1; h2, prolycopene isomer2; l, 9,9′,15-tri-cis-ζ-carotene. (C) Putative γ-carotene biosynthesis pathway catalyzed by ZDS and CRTISO.

Due to the influence of the cell wall and stroma, light energy is greatly weakened in cells, resulting in 7,7′,9, 9′-tetra-cis-lycopene not being well isomerized. It should be noted that a small amount of all-trans-lycopene was detected in the ΔCRTISO strain cultured under light and dark conditions. It can be speculated that if CRTISO is removed, the green algae or plants may still survive. This may be the reason why some of the CRTISO-mutant plants can continue to survive despite being cultured in the dark (24, 27).

Functional catalytic properties of DsZDS.

In plants, ZDS is a well-studied enzyme (28–31). ZDS catalyzed the production of prolycopene from 9,9′-di-cis-ζ-carotene, but liquid chromatogram analysis of the CRTS strain revealed the presence of a small amount of γ-carotene, which was not reported previously. It is speculated that this phenomenon may occur during DsZDS-mediated dehydrogenation at C-7 and C-9, and its catalytic process is shown in Fig. 5C. Dehydrogenation occurs first at C-7, followed by conjugation at C-5, C-6, and C-7. It is known that the 6-membered ring structure is more stable than other ring structures, so C-6 attacks C-1, and the hydrogen of the 5 position shifts to the 2 position to form the β-ring structure (32, 33). Because of the presence of DsCRTISO, the C-7 and C-9 positions were isomerized into trans structures, forming γ-carotene (Fig. 5C).

Phylogenetic distribution of crts.

To study the phylogenetic distribution of crts, a genome-wide survey of phototrophic organisms of crts was investigated. It was found that crts homologs are widely distributed in the genomes of photosynthetic organisms, such as plants, algae, and cyanobacteria. Because DsGGPS, DsPSY, DsPDS, DsZDS, and DsLYCB are common carotenoid synthases, they will not be discussed here. Only DsZISO and DsCRTISO with relatively few reports were analyzed.

By analyzing the evolutionary tree of ZISO, it was found that DsZISO and ZISO of green algae form a family cluster. Interestingly, ZISO of cyanobacteria had closer homology with higher plants (Fig. S4A at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf), indicating that prokaryotes produced branches during their evolution into plants and green algae. DsCRTISO did not have high homology with the CRTISO of plants and CrtH of cyanobacteria and was far apart in classification (Fig. S4B at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf). The two homologous isomerases of DsCRTISO were closer to those of plants and cyanobacteria; in particular, DsCRTISO-homo1 was close to that of the cyanobacteria Nostoc piscinale. However, our functional validation found that DsCRTISO-homo1 and DsCRTISO-homo2 did not have isomerase activity; they could not isomerize prolycopene to lycopene. This is an important reason why CRTISO isomerase had not been validated in green algae.

Phytoene desaturases of different biological origins show functional diversity. In most cyanobacteria, algae, and higher plants, carotenoid biosynthetic pathways consist of multiple enzymes involved in lycopene formation, whereas in most microorganisms, only one enzyme, CrtI-type, is involved in the dehydrogenation of phytoene, such as in Blakeslea trispora, Xanthophyllomyces dendrorhous, and Rhodosporidium diobovatum, while Neurospora crassa CrtI catalyzes the five-step dehydrogenation of phytoene to all-trans-3,4-didehydrolycopene.

A phylogenetic tree was constructed based on protein sequence homology searches of phytoene desaturase from different sources, and the evolutionary pathway of the enzyme was analyzed (Table 1; Fig. 6A and B). The CrtI-type monoenzyme of a bacterial source may be the “common ancestor” of phytoene desaturase (Fig. 6B), which evolved into the Haloarchaea CrtI-type phytoene desaturase and the fungal CrtI-type phytoene desaturase (34). In addition, during the evolution of bacterial phytoene desaturase, substrate-specific alterations were exhibited, such as Staphylococcus aureus CrtN catalyzing C30 diapophytoene to generate C30 diapolycopene (35). The high homology of Nostoc CrtP and CrtQa with bacterial-derived CrtI-type phytoene desaturase suggests that Nostoc CrtP and CrtQa evolved from bacterial-derived CrtI-type to form the CrtP/CrtQa double enzyme system. Nostoc CrtQa evolved into the cyanobacterial cis-trans isomerases CrtH and CrtQb (36), resulting in the cyanobacterial CrtP/CrtQb/CrtH-type tri-enzyme system. Finally, The CrtP/CrtQb/CrtH-type tri-enzyme system has evolved into the more complex plant-derived PDS/ZDS/ZISO/CRTISO-type quadruple-enzyme system. It is noteworthy that the discovery of ZISO in cyanobacteria has recently been reported and has been shown to be widespread (26), but its presence is nonessential. In contrast, it has not been found in Chlorobiaceae, which is more evidence that the plant four-enzyme system evolved from cyanobacteria.

TABLE 1.

Functional diversity of CrtI-type phytoene dehydrogenase

Type Organism Phytoene ζ-Carotene Neurosporene Lycopene Reference
One enzyme Rhodosporidium diobovatum CrtI (FAD dependent) 46
Rhodobacter azotoformans 47
Erwinia uredovora 38
Neurospora crassa Al-1 (NAD dependent) 48
Double enzyme Myxococcus xanthus CrtIa CrtIb 49
Triple enzyme Chlorobaculum tepidum CrtP CrtQb CrtH 50
Chloracidobacterium 26
Quadruple enzyme Cyanobacteria CrtP CrtQa 36
ZISO CrtQb CrtH 26
Algal PDS ZISO ZDS CRTISO In this study
Plant 27
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FIG 6.

FIG 6

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Evolutionary analysis of phytoene desaturase. (A) Phylogenetic tree analysis of phytoene dehydrogenases based on the amino acid sequences. The phylogenetic tree was constructed using the maximum-likelihood method of MEGA7 software with the JTT substitution model. Numbers associated with the branches were the bootstrap values (n = 1,000). Accession numbers of phytoene dehydrogenases protein sequences are shown in Table S5 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. (B) Evolutionary pathway analysis of phytoene dehydrogenases.

Comparison between algal and bacterial carotenoid synthesis pathways.

The content of all-trans-β-carotene in the CRT+LYCB strain constructed in this study was 3.3 mg/g (cell dry weight) without optimization, while that in the engineered E. coli W07 strain constructed with the Erwinia gene was 2.75 mg/g (Fig. 7A and B). The content of all-trans-lycopene in CRTS strain was 3.8 mg/g, which was more than 1.6 times of that in Erwinia genetic engineered E. coli strain W05 (2.36 mg/g) (Fig. 7A and B). We note that many lycopene isomers were generated in the W05 strain, whereas our constructed CRTS strain mainly generated all-trans-lycopene (Fig. 7A and C). This indicates a higher specificity of the carotenoid synthesis pathway in algae.

FIG 7.

FIG 7

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Comparison of carotenoid composition of algal and bacterial carotene synthesis pathway engineering Escherichia coli. (A) HPLC analysis of the carotenoids produced in E. coli cells harboring different pathways of plasmids; peaks: d, α-carotene; e, all-trans-β-carotene; e1, 9-cis-β-carotene; f, γ-carotene; g, all-trans-lycopene; g1, lycopene isomer1; g2, lycopene isomer2; g3, lycopene isomer3; g4, lycopene isomer4; g5, lycopene isomer5; g6, lycopene isomer6; g7, lycopene isomer7. (B) Carotenoid content in W05, CRTS, W07, and CRT+LYCB strains. (C) Relative content of lycopene isomers in W05 and CRTS strains.

In the step-by-step construction from DsGGPS to DsLYCB, the products were efficiently synthesized to the end products and their isomers of the cutoff step, while the contents of intermediate products and original substrates were few, such as lycopene, indicating that the catalytic efficiency of these enzymes was extremely high, which was probably one of the important reasons for the high accumulation of β-carotene in D. salina. This provides us with a more efficient pathway than the bacterial-derived carotenoid synthesis pathway (37, 38).

Conclusion.

Here, the DsGGPS, DsPSY, DsPDS, DsZISO, DsZDS, DsCRTISO, and DsLYCB genes from D. salina were successfully constructed, and highly expressed β-carotene engineering bacteria CRT+LYCB were obtained, confirming the β-carotene synthesis pathway of D. salina. Carotenoid synthesis of D. salina is highly similar to that of plants but has some differences. The functions of some enzymes have been redefined; in addition to the dehydrogenation function, DsZDS also has a cyclization function. DsLYCB also has catalytic activity for 7,9-cis-type lycopene, and is different from some plants, DsZISO and DsCRTISO cannot be replaced by photoisomerism. A new carotenoid 7,7′,9,9′-tetra-cis-β-carotene was discovered. These findings provided new ideas for the development of a high-yield carotenoid series of engineering bacteria (Fig. 8).

FIG 8.

FIG 8

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The β-carotene biosynthesis pathway in Dunaliella; blue area, new pathway to synthesize γ-carotene; orange area, new pathway to synthesize β-carotene.

MATERIALS AND METHODS

Chemicals and reagents.

Carotenoid standards (β-carotene, all-trans-lycopene, α-carotene, γ-carotene, ε-carotene, δ-carotene, zeaxanthin, and lutein) were purchased from Sigma-Aldrich (USA) with purity ranging from 90% to 99%. 9,15,9′-Tri-cis-ζ-carotene and 9,9′-di-cis-ζ-carotene are carotenoid extracts from an engineered E. coli strain constructed from plasmids in the Addgene database pAC-ZETAipi (Addgene 53287) (39). The strain carrying the plasmids pACCRT-EIB and pACCAR16ΔcrtX, generously provided by Norihiko Misawa from Japan Ishikawa Prefectural University, can produce lycopene and β-carotene, respectively (40).

RNA extraction and cDNA template preparation.

D. salina CCAP 19/18 cells were cultivated in a defined medium containing 1.5 M NaCl at 26°C and 8,000 lx provided under a 16-h light/8-h dark cycle with shaking at 96 rpm for about 14 days (exponential phase) (31). Total RNA was obtained from D. salina using the TRIzol reagent system (Invitrogen). First-strand cDNA was synthesized by PCR reverse transcription using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific) with oligonucleotide (dT)18 primers and total RNA as a template. The 5′- and 3′-end sequences of the CDS were validated using SMARTer RACE cDNA amplification kit (Clontech).

Sequence alignment and phylogenetic analyses.

Sequence alignment was performed using the MUSCLE algorithm of MEGA7 (41, 42). Based on the Jones-Taylor-Thornton (JTT) matrix-based model, evolutionary history was inferred using the maximum-likelihood method (43). Bootstrap values were obtained using 1,000 repeated calculations. All phylogenetic analyses were performed using MEGA7.

Cloning of carotene synthesis genes from cDNA.

High-fidelity KOD DNA polymerase (TOYOBO) was used for PCR amplification using the cDNA of D. salina as a template. The amplified gene was cloned into the target vector using Gibson assembly (44). Restriction endonucleases were purchased from Thermo Fisher Scientific, and Gibson assembly reagents were purchased from New England BioLabs (NEB). Oligonucleotide primers were purchased from Sangon Biotech. Table S3 (available at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf) lists the primers used for cloning.

Reconstitution of carotene biosynthesis in E. coli.

The plasmid construction schemes and maps of carotenoid engineering E. coli are shown in Fig. S5 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. The crts genes were inserted into pACYDuet-1, pETDuet-1, and pCDFDuet-1 (Novagen), respectively, using the Gibson assembly system. Primers for plasmid construction are shown in Table S3 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. The constructed vectors were respectively transformed into E. coil BL21(DE3) in the form of cotransformation to form a series of carotenoid engineered bacteria. The cotransformation scheme was shown in Table S4 at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf. The engineered E. coli single colonies were cultured in 50 mL of Luria-Bertani (LB) medium containing the corresponding antibiotics and shaken at 37°C at 200 rpm overnight. Overnight cultures were transferred to 250 mL of LB medium with corresponding antibiotics for expanded culture until an optical density at 600 nm (OD600) of 0.8 was reached. Expression was then induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 48 h in a light or dark environment at 28°C and 100 rpm.

Carotenoids extraction from D. salina and E. coli.

Carotenoids are easily degraded and must be extracted under weak light conditions. Carotenoids in E. coli were extracted with cold acetone using a homogenizer for 20 min. After centrifugation, the carotenoid supernatant was collected. For the extraction of carotenoids from D. salina, cold acetone was added to the algal cells and dispersed on a homogenizer. After centrifugation, the supernatant was collected, and KOH solution was added until the final concentration reached 6%. After centrifugation at 10,000 rpm and 4°C, chlorophylls were extracted into the aqueous phase, and carotenoids were retained in the acetone phase. The extracted carotenoid solution of E. coli and D. salina was filtered with a 0.22-nm filter membrane and dried under a nitrogen evaporator. Then, the dried residue was redissolved in 200 μL of acetone.

HPLC analysis of carotenoids.

Carotenoid analysis was performed using an Agilent 1260 HPLC system with a reversed-phase C30 YMC carotenoid column (5 μm, 250 × 4.6 mm) protected by a C30 guard column (5 μm, 10 × 4 mm) (YMC Co., Ltd., Japan) (20, 45). The solvent systems were A, consisting of methanol (MeOH)/water (97/3 [vol/vol]), 0.05 M ammonium acetate, and 0.1% butylated hydroxytoluene (BHT), and B, consisting of tert-butyl-methyl ether with 0.1% BHT. The following gradient elution was used: 0 min 90% A and 10% B, 0 to 10 min 60% A and 40% B, 10 to 20 min 50% A and 50% B, 20 to 25 min 10% A and 90% B, 25 to 29 min 10% and 90% B, 29 to 29.5 min 90% A and 10%, and 29.5 to 40 min 90% A and 10% B. The flow rate was 1 mL/min, and the injection volume was 10 μL. The detection of analytes was performed by UV absorbance at 450 nm and three-dimensional scanning from 200 to 700 nm.

ACKNOWLEDGMENTS

We acknowledge the contribution of X. B. Guo regarding carotenoid structure, analysis, and characterization. This project was supported by the National Natural Science Foundation of China (31871778, 31801468, and 32072201), Guangdong Basic and Applied Basic Research Foundation (2019A1515010656 and 2022A1515010926), Guangzhou Basic Research Program (202102020109), Fundamental Research Funds for the Central Universities (2018MS89), and the Open Research Fund Program of Guangxi Key Lab of Mangrove Conservation and Utilization (GKLMC-202003).

H.-H.C. was the main author of this work. H.-H.C., M.-H.L., Z.-W.Y., and Y.-H.Z. conducted laboratory experiments for this work. H.-H.C. and J.-G.J. wrote the paper. J.-G.J. provided an overall assessment of this work.

We declare that there are no conflicts of interest.

Contributor Information

Jian-Guo Jiang, Email: jgjiang@scut.edu.cn.

Cesar de la Fuente-Nunez, University of Pennsylvania.

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