Engineering the b -Carotene Metabolic Pathway of Microalgae Dunaliella To Con ﬁ rm Its Carotenoid Synthesis Pattern in Comparison To Bacteria and Plants

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The aim of this study was to clone all the key enzyme genes of the b-carotene metabolic pathway in D. salina, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), PDS, ZISO, ZDS, CRTISO, and lycopene b-cyclase (LYCB), and construct these genes into Escherichia coli to express them completely. By observing the expression efficiency of b-carotene and various intermediate carotenoids upstream, the application potential of b-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 b-carotene will accumulate, making the cells yellowish red (Fig. 2).
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 59 rapid amplification of cDNA ends (RACE) PCR, and the termination codon of each gene was verified by 39 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.
Construction and expression of the D. salina b-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-b-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 DZDS strain produced beige bacteria, the DCRTISO strain formed golden yellow bacteria, the CRTS strain formed red bacteria, and the CRT1LYCB 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 b-carotene.
Engineering the b-Carotene Metabolic Pathway of Dunaliella Microbiology Spectrum strains formed all-trans-lycopene and b-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 CRT1LYCB strain HPLC results in Fig. 3B, it was found that in addition to the production of b-carotene, d -carotene, « -carotene, g -carotene, and a-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-translycopene to produce a-carotene, d -carotene, and « -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 p bonds, which are conjugated with the C-2 dumbbell electronic cloud to form larger delocalized p bonds (18,19). A b-ring is formed when C-6 hydrogen is transferred to the C-2 position, and an « -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 b-carotene (84%) and a small amount of « -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 b-monocyclase activity (21). But we found that there was another cyclase in Dunaliella (DbLCYE) that only showed « -cyclase activity (20). It has been reported that a conserved motif (designated the extended motif B) of lycopene cyclases determines the formation of band « -ionone groups (22).
Interestingly, analysis of the liquid chromatogram of the DCRTISO1LYCB 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 b-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 b-zeacarotene under the catalysis of LYCB (18). Only one end of the c -end group with C-7 and C-8 dehydrogenation can be cyclized by LYCB, while the c -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 b-cyclization product of prolycopene, namely, 7,79,9,99-tetra-cis-b-carotene. This new carotenoid implies a new way to synthesize b-carotene (Fig. 4B).
All-trans-b-carotene was not detected in the DCRTISO1LYCB 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-c -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-b-carotene and all-trans-b-carotene in D. salina are so close (Fig. 2B). The isomerization of DsCRTISO in the synthesis of b-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 9,99,15-tri-cis-z -carotene and 7,79,9,99-tetra-cis-lycopene can be isomerized into 9,99-di-cis-z -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.
DZISO and DCRTISO 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 DZISO and DCRTISO strains cultured under dark and light conditions did not change (Fig. 5B). Under light conditions, 9,15,99-tri-cis-z -carotene and 7,79,9,99-tetra-cis-lycopene in DZISO and DCRTISO could not be well isomerized into 9,99-di-cis-z -carotene and all-trans-lycopene to produce red lycopene mycelia. This result indicated that ZISO and CRTISO are necessary for the production of b-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.
Due to the influence of the cell wall and stroma, light energy is greatly weakened in cells, resulting in 7,79,9, 99-tetra-cis-lycopene not being well isomerized. It should be noted that a small amount of all-trans-lycopene was detected in the DCRTISO 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)(29)(30)(31). ZDS catalyzed the production of prolycopene from 9,99-di-cis-z -carotene, but liquid chromatogram analysis of the CRTS strain revealed the presence of a small amount of g -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 Microbiology Spectrum 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 b-ring structure (32,33). Because of the presence of DsCRTISO, the C-7 and C-9 positions were isomerized into trans structures, forming g -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 Microbiology Spectrum 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 alltrans-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 trienzyme 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 fourenzyme system evolved from cyanobacteria.
Comparison between algal and bacterial carotenoid synthesis pathways. The content of all-trans-b-carotene in the CRT1LYCB 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.
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 b-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 b-carotene Engineering the b-Carotene Metabolic Pathway of Dunaliella Microbiology Spectrum engineering bacteria CRT1LYCB were obtained, confirming the b-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 7,79,9,99-tetra-cis-b-carotene was discovered. These findings provided new ideas for the development of a high-yield carotenoid series of engineering bacteria (Fig. 8).
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 59-and 39-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   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 (OD 600 ) of 0.8 was reached. Expression was then induced with 1.0 mM isopropyl b-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 mL of acetone.
Engineering the b-Carotene Metabolic Pathway of Dunaliella Microbiology Spectrum