Heterologous production of novel and rare C30-carotenoids using Planococcus carotenoid biosynthesis genes

Background Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4′-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. Results A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1–7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4′-diapophytoene synthase (CrtM) and 4,4′-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4′-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4′-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4′-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds. Conclusions The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-021-01683-3.

In this study, we tried to isolate genes involved in the production of the glucosyl C 30 -carotenoid from P. maritimus strain iso-3, to identify the functions of the isolated genes using E. coli, and to produce novel and rare C 30 -carotenoids with anti-oxidative activity in E. coli by combinations of the obtained genes.

Bacterial strains and growth conditions
Escherichia coli K12 DH5α was used for DNA manipulation. E. coli K12 JM101(DE3) was constructed from strain JM101 using the λDE3 lysogenization kit (Merk, Darmstadt, Germany) and used for expression of the carotenoid biosynthesis genes. These E. coli strains and their transformants were maintained as the frozen stocks including 20% glycerol at − 80 °C, and were grown in 2 × YT medium (16 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl) at 37 °C as needed. Bacillus subtilis strain MI112 was kindly provided from Dr. Atsuhiko Shinmyo (NAIST, Japan). This bacterium was similarly maintained as the frozen stock and routinely grown in LB medium (10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl) at 37 °C.

DNA isolation of Planococcus maritimus strain iso-3
Genome DNA was prepared from Planococcus maritimus strain iso-3 according to a method described by Nishida et al. [24].

Functional cloning experiments
The genome DNA was digested with BamHI, ligated with vector pGETS103, and used to transform Bacillus subtilis strain MI112 as described previously [25]. In generated genome library, one yellow colony appeared, which was found to contain a 7344-bp insert.

Inverse PCR
To isolate the flanking region of the carotenoid biosynthesis gene cluster, inverse PCR was performed. One μg of genomic DNA from the Planococcus was digested with EcoRI. Digested DNA (0.1 μg/μL) was self-ligated with ligation solution (Takara Bio, Ohtsu, Japan). PCR was performed using 50 pg of these circular DNAs as the template in 25 μL of the reaction solution with the gene-specific primers which we designed. The amplified fragments were cloned into the pBC phagemid vector (Agilent, CA, USA), transformed into E. coli (DH5α) and sequenced. The primers used are summarized in Additional file 1: Table S1.

Cloning of the carotenoid biosynthesis genes from P. maritimus strain iso-3
Based on the sequences obtained, the primers containing the restriction sites were designed as shown in Additional file 1: Table S1 and the coding regions of each orf were amplified by PCR of the genomic DNAs.
Then PCR products were cloned into a plasmid vector and sequenced.

Expression of the Planococcus carotenoid biosynthesis genes in Escherichia coli
Plasmids used in this study are summarized in Additional file 1: Fig. S1. Firstly, we constructed the plasmid pAC-HI which contained the Haematococcus pluvialis IDI (isopentenyl diphosphate isomerase) gene between the tac promoter (Ptac) and the rrnB terminator (TrrnB) in the pACYC184 vector. The exogenous expression of the IDI gene in E. coli results in the increase of carotenoid production [26]. Then, the coding region of the Planococcus orf3 was inserted into the plasmid pAC-HI. The resultant plasmid was named as pAC-HIO3. The coding regions of the orf1, orf2, orf4 were cloned into the pAC-HIM which contained IDI and Leuconostoc mesenteroides crtM, independently. These plasmids were designated pAC-HIMO1, pAC-HIMO2, and pAC-HIMO4, respectively. The coding regions of the orf2, orf4 and orf7 were cloned into the pAC-HIMN which contained IDI and L. mesenteroides crtM and crtN, independently. The resultant plasmids were named as pAC-HIMNO2, pAC-HIMNO4, and pAC-HIMNO7, respectively. The orf2 and orf4 were also inserted into the pAC-HIMNF (same as pAC-HIMNO7) and the obtained plasmids were pAC-HIMNFO2 and pAC-HIMNFO4, respectively. The orf5 was further inserted into the pAC-HIMNFNb (same as pAC-HIMNFO4) and this plasmid was named pAC-HIMNFNbO5 (renamed pAC-HIMNFNbG). All plasmids were independently introduced into the wild type E. coli (JM101 (DE3)). Each transformed E. coli was grown in 2YT medium at 37 °C. Next day, this culture was inoculated in a new 2YT medium (100 mL medium in 500 mL Sakaguchi flask) and cultured at 21 °C for 2 days.

Extraction and HPLC analysis of carotenoids from E. coli cells
Extraction of carotenoids from the recombinant E. coli was performed by the method described by Fraser et al. [27]. E. coli cultures were centrifuged and cell pellets were extracted with methanol (MeOH) using mixer for 5 min. Tris-HCl (50 mM, pH 7.5) and 1 M NaCl were added and mixed. Then chloroform was added to the mixture and mixed for 5 min. After centrifugation, the chloroform phase was collected and dried by centrifugal evaporation. Dried residues were re-suspended with ethyl acetate (EtOAc), and applied to HPLC with a Waters Alliance 2695-2996 (PDA) system (Waters, Milford, MA, USA). HPLC was carried out according to the method described [28] using TSKgel ODS-80Ts (4.6 × 150 mm, 5 μm; Tosoh, Tokyo, Japan). Briefly, the extract was eluted at a flow rate of 1.0 mL/min at 25 °C with solvent A [water (H 2 O)-MeOH, 5:95] for 5 min, followed by a linear gradient from solvent A to solvent B (tetrahydrofuran-MeOH, 3:7) for 5 min, and solvent B alone for 8 min. The produced carotenoids were identified by comparing both retention times and absorption spectra with those of our authentic standards. When the produced carotenoids are not compounds in our authentic standards as described in the following sections, we isolated the produced carotenoids and determined their structures using HRESI-MS (high resolution electrospray ionization-mass spectrometry) and NMR (nuclear magnetic resonance) analyses.

Singlet oxygen-quenching activity
For the measurement of singlet oxygen-quenching activity, 80 μL of 25 μM methylene blue and 100 μL of 0.24 M linoleic acid, with or without 40 μL of carotenoid (final concentration, 1-100 μM; each dissolved in ethanol), were added to 5 mL glass test tubes. The tubes were mixed well and were illuminated at 7000 lx at 22 °C for 3 h in a styrofoam box. Then, 50 μL of the reaction mixture was removed and diluted to 1.5 mL with ethanol, and OD 235 was measured to estimate the formation of conjugated dienes [29]. The OD 235 in the absence of carotenoids was measured as negative control [no singlet oxygen ( 1 O 2 )-quenching activity], and the 1 O 2 -quenching activity of carotenoids was calculated from OD 235 in the presence of carotenoids relative to this reference value. The activity was indicated as the IC 50 value, which represents the concentration at which 50% inhibition was observed.

Isolation of the carotenoid biosynthesis gene cluster from P. maritimus strain iso-3
A yellow colony including a 7.3-kb genomic insert from P. maritimus strain iso-3 was obtained by functional cloning experiments using Bacillus subtilis as the host. Sequence analysis of this insert showed the presence of seven open reading frames (orfs), named orfs 2-8 ( Fig. 1). Six orfs, orf2-7, were predicted to be involved in carotenoid biosynthesis, and the remaining orf, orf8, was unlikely to be involved in it. When the 7.3-kb (7344-bp) DNA fragment was expressed in E. coli, the recombinant cells did not become yellow. This DNA fragment contains the 4,4′-diapophytoene synthase (crtM) gene, but it does not include the 4,4′-diapophytoene desaturase (crtNa) gene, as shown later. It is thus likely that the recombinant E. coli produced only the colorless 4,4′-diapophytoene. On the other hand, the B. subtilis used as the host retained 4,4′-diapophytoene desaturase activity; therefore, the recombinant Bacillus cells turned yellow.
The complete genome sequences of several Planococcus, such as P. plakortidis (NZ_CP016539.2), P. faecalis (NZ_CP019401.1), and P. halocrytophilus (NZ_CP016537.2), have been determined. Each of the sequences contains a similar carotenoid biosynthesis gene cluster that includes one more carotenoid biosynthesis gene other than the orfs2-8. Then, we performed inverse PCR to isolate the flanking region of orfs2-8. As a result, the 2.1-kb fragment adjacent to orf2 was isolated and the additional orf, named orf1, was found. In total, a 9.4-kb genomic DNA was analyzed to find out that eight orfs existed (Fig. 1). The accession no. of the sequences of this genomic DNA is LC620265.

Sequence analysis of the carotenoid biosynthesis gene candidates
Next, we performed a sequence analysis of orf1-8. Homology searches suggested that seven orfs, orf1-7, were involved in carotenoid biosynthesis, but not orf8, which was homologous to the genes encoding the MurR/ RpiR family of transcriptional regulators. The predicted amino acid sequences of Orf1, Orf2, and Orf4 were homologous to that of diapophytoene desaturase. Orf1 was most homologous, displaying approximately 61% identity to the H. halophilus CrtNa. Whereas Orf2 and Orf4 were homologous to the H. halophilus CrtNc and CrtNb, displaying about 50 and 52% identity, respectively ( Fig. 2A). On the other hand, Orf3 showed homology to diapophytoene synthase (CrtM) (Fig. 2B). Meanwhile, Orf5 and Orf6 were similar to glycosyltransferase and acyltransferase, respectively. Lastly, Orf7 was homologous to the genes that were annotated as carotenoid biosynthesis genes but with unknown functions. However, we found that the Orf7 was slightly similar, at 22% identity, to CruF (1′-hydroxylase).

Orf3 acts as a diapophytoene synthase
To investigate the function of orf3 which was homologous to diapophytoene synthase, we made the plasmid pAC-HIO3 which included H. pluvialis IDI and orf3, and introduced it into the E. coli (JM101(DE3)). Wild type E. coli cannot produce diapophytoene from farnesyl pyrophosphate (FPP). On the other hand, a new peak (named 1) was observed in the E. coli expressing pAC-HIO3 with IDI and orf3 (Fig. 3A). Then, the produced compound 1 was purified from the cells via acetone-MeOH extraction, n-hexane/90% MeOH partition, and silica gel column chromatography and analyzed by ESI-MS (+), 1 H, Fig. 1 Gene organization of the DNA fragments obtained from the P. maritimus strain iso-3 and 13 C NMR spectra. From these spectra, the produced compound was identified as 15-cis-4,4′-diapophytoene (carotenoid 1) (Fig. 4) [30]. Thus, orf3 was confirmed to encode a 15-cis-4,4′-diapophytoene synthase (CrtM).

Orf1 acts as a diapophytoene desaturase
Because orf1, orf2, and orf4 were found to be homologous to phytoene desaturase, the catalytic activities of these Orfs were examined by constructing the plasmids pAC-HIMO1, pAC-HIMO2, and pAC-HIMO4 containing H. pluvialis IDI, Leuconostoc mesenteroides crtM, and each orf. The recombinant E. coli expressing pAC-HIMO1 generated a new carotenoid product, as indicated by the new peak (2) (Fig. 3B). On the other hand, the introductions of pAC-HIMO2 and pAC-HIOM4 did not affect the carotenoid profiles (Fig. 3B) We  Table S2. Orf1 and orf4 belonged to the crtNa and crtNb groups, respectively. B Phylogenetic tree of the crtM-related genes. The accession numbers of these sequences are shown in Additional file 1: Table S3. Orf3 belonged to the crtM group cultured the E. coli expressing pAC-HIMO1 and purified the carotenoid 2 using the same process described in the previous section. The ESI-MS (+), 1 H, and 13 C NMR spectral analyses indicated carotenoid 2 as 4,4′-diaponeurosporene (Fig. 4) [31]. These results suggest that the orf1, but not orf2 or orf4, acts as a diapophytoene desaturase (CrtNa). However, the activity of Orf1 was low in E. coli; therefore, we use L. mesenteroides crtN for further experiments.

Orf7 acts as a diaponeurosporene hydratase
The synthetic pathway from FPP to diaponeurosporene is thought to be general, but the reaction after diaponeurosporene varies. So, we investigated the activities of Orf2, Orf4, and Orf7 by constructing the plasmids, pAC-HIMNO2, pAC-HIMNO4, and pAC-HIMNO7, which contained H. pluvialis IDI, L. mesenteroides crtM, L. mesenteroides crtN and each orf. When pAC-HIMNO2 or pAC-HIMNO4 was introduced into the E. coli, it only  (Fig. 3C). In the E. coli expressing pAC-HIMNO7 (orf7), a new peak (3) was detected, which could not be identified using our standards, in addition to 4,4′-diaponeurosporene (Fig. 3C). Thus, we cultured this recombinant E. coli and purified the new carotenoid 3 (13.9 mg) as orange powder as  (Fig. 4). Carotenoid 3 was a new one according to the CAS database. The assigned 1 H and 13 C signals in 3 were listed in Table 1.
These results indicate that orf7 encodes a diaponeurosporene hydratase; thus, we named orf7 as cruF, encoding a γ-carotene 1′ hydratase involved in myxoxanthophyll biosynthesis in Synechococcus. We also renamed the plasmid pAC-HIMNO7 to pAC-HIMNF.

Orf5 acts as a glycosyltransferase
A novel carotenoid previously isolated from Planococcus iso-3, methyl 5-glucosyl-5,6-dihydro-4,4′diapolycopenate, was glycosylated. Because orf5 showed homology to glycosyltransferase, we investigated the activity of Orf5. The introduction of the plasmid pAC-HIMNFNbO5 into E. coli resulted in the detection of a new peak (5), which could not be identified using the standards (Fig. 3E) (5) and the attachment of a hexose at C-5 of carotenoid 4. The hexose in carotenoid 5 was shown to be β-glucose because all vicinal coupling constants among H-1″ to H-5″ were large (7.9-9.1 Hz) in the 1 H NMR spectrum of acetylated carotenoid 5 in CDCl 3 (Additional file 1: Fig. S3). From these observations, carotenoid 5 was determined to be 5-glucosyl-5,6-dihydro-4,4′-diapolycopene (Fig. 4). Carotenoid 5 was a new compound according to the CAS database. The assigned 1 H and 13 C signals in 5 were listed in Table 1. These results indicate that the orf5 encodes a glycosyltransferase and we renamed pAC-HIMNFNbO5 as pAC-HIMNFNbG.
The whole-genome sequences of several kinds of Planococcus were available in the genome database (https:// www. ncbi. nlm. nih. gov/ genome/). All of them, including P. faecalis AJ003 T , had identical carotenoid biosynthesis gene clusters to that found in this study [19,23]. Similar gene clusters have also been reported in Halobacillus halophilus, Staphylococcus aureus, Bacillus indicus, and Bacillus firmus, which all belong to the order of Bacillales (Fig. 5) [14,20]. In particular, H. halophilus has nearly the same gene organization as Planococcus regarding gene order and transcriptional direction, whereas they fall into distinct families, the Bacillaceae and Planococcaceae families, respectively [18]. On the other hand, the gene organization is much different between B. indicus and B. firmus [20]. These results suggested that the ancestor of Bacillales retained the same carotenoid biosynthesis gene cluster, and during evolution, the gene organizations have been rearranged independently.
Functional analysis of the genes isolated from P. maritimus indicated that the crtM and crtNa genes encoded proteins with enzymatic activities as predicted from the sequence homology analysis. On the other hand, the enzyme encoded by crtNb was found to generate a product different from those by other crtNb gene products. For example, Steiger et al. [20] demonstrated that the proteins encoded by B. indicus and B. firmus crtNb functioned as aldehyde synthases for utilizing 4,4′-diapolycopene or 4,4′-diaponeurosporene. In contrast, the functions of the crtNb genes in H. halophilus and P. fecalis sp. Nov have not been revealed [18,19,32]. To discuss the evolution of crtNb gene, it will be required to characterize the function of various crtNb genes.
We also found a carotenoid 1,2-hydratase gene that showed a slight similarity to cruF, initially found in Deinococcus [33]. CruF catalyzes the reaction from γ-carotene to 1′-OH-γ-carotene and is required to produce deinoxanthin in Deinococcus. The homologous genes with this were present not only in the Planococcus genomes but also in the Halobacillus genomes at the same position, whereas their functions have not been elucidated. As for other-type hydratase genes, crtC has been reported in Rubivivax gelatinosus [34,35]. However, the Orf7 did not show any similarity to the CrtC protein. Here, the entire carotenoid biosynthetic pathway was almost elucidated using complementation analysis with E. coli (Fig. 4). After 15-cis-4,4′-diapophytoene (1) is synthesized, it is desaturated to 4,4′-diaponeurosporene (2), hydrated to produce 5-hydroxy-5,6-dihydro-4,4′diaponeurosporene (3), and then desaturated to produce 5-hydroxy-5,6-dihydro-4,4′-diapolycopene (4). Finally, 5-glucosyl-5,6-dihydro-4,4′-diapolycopene (5) is produced by the glycosylation of a terminal group of 5-hydroxy-5,6-dihydro-4,4′-diapolycopene (4). However, according to previous reports, the final product is methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate, indicating that more reactions should occur. We found orf6, homologous to acyltransferase in the carotenoid gene cluster and speculated that it catalyzed the esterification. On the other hand, the genes involved in generating carotenoid carboxylic acid were not identified in the gene cluster. Indeed, the introduction of orf2 into the E. coli carrying the plasmid pAC-HIMNFNb or pAC-HIM-NFNbO5 did not affect the carotenoid profile (Additional file 1: Fig. S4). Lee et al. [19] reported that crtP and aldH genes coded for 4,4-diaponeurosporene oxidase and aldehyde dehydrogenase, respectively; that these genes were located away from the carotenoid gene cluster in P. faecalis. Thus, it is necessary to analyze the genes outside of the carotenoid synthesis gene cluster in the Planococcus iso-3 genome.
We evaluated the 1 O 2 -quenching activities of carotenoids 1-5, and compared their activities with 5-glucosyl-5,6-dihydro-4,4′-diapolycopenoate. It is very interesting that the compound produced through more advanced biosynthetic pathway shows more potent 1 O 2 -quenching activity. P. maritimus strain iso-3 may have completed this biosynthetic pathway to obtain a strong singlet oxygen scavenger.