Structural and functional analysis of the carotenoid biosynthesis genes of a Pseudomonas strain isolated from the excrement of Autumn Darter

Figures & data

Table 1. Primers used in this study.

Primer	Sequence
16S rRNA
27F	5′- AGAGTTTGATCMTGGCTCAG −3´
1492R	5′- TACGGYTACCTTGTTACGACTT −3´
idi
PAidi F1	5′-GCTGCAGATGAACAAAAGCCCGTTG-3´
PAidi R1	5′-CGGATCCTCATTTGGGGTCGAGCTG-3´
crtE
PAcrtE F1	5′-CGAATTCATGACGACCATCGCCCCC-3´
PAcrtE R1	5′-CGCGGCCGCTTAGACGTGATGCATGAC-3´
crtB
PAcrtB F1	5′-CGGATCCATGACCGCACCGTCCCAC-3´
PAcrtB R1	5′-CGCGGCCGCTATCGAGCGGCTGCGCC-3´
crtI
PAcrtI F1	5′-CGAATTCATGAGCAACGAACGATCC-3´
PAcrtI R1	5′-CGCGGCCGCTCATGCGCCCAGGTCCTC-3´
crtY
PAcrtY F1	5′-CGAATTCATGCCCTATGACCTGATC-3´
PAcrtY R1	5′-CGCGGCCGCTCATGGATAGTCCTTGAA-3´
crtZ
PAcrtZ F1	5′-CGAATTCATGCTGTTCAATATCCTC-3´
PAcrtZ R1	5′-CGCGGCCGCTCAGAGAGCAGAGCCCCT-3´
PAcrtZ R2	5′-CGCGGCCGCTCAGTGACCGGCGTCCC-3´
crtX
PAcrtX F1	5′-CGAATTCATGAGCCACTTCGCCCTG-3´
PAcrtX R1	5′-CGCGGCCGCTCAGGTCATAGGGCACGG-3´
Inverse PCR
PACrtBF3	5′-GCGTCATCGCGCAGCCGTCG-3'
PACrtBR3	5'-GCCAGGTCGTCTGCACTGAG-3'
PACrtEF3	5'-TTGTTCTCGGCCTTGCTGGG-3'
PACrtER3	5'-CCCCGGTCTTGAGTTGATTG-3'
PACrtEF4	5'-AACAGTGCCTGACCAGCCTG-3'
PACrtER4	5'-ACGCTCTGCAAATGGGCTTC-3'
PACrtZF3	5'-CCTGCTGGGCACACTGGTGG-3'
PACttZR3	5'-ACGAGGATATTGAACAGCAT-3'
PACrtZF4	5'-CATCGCTCGCACCACGAACC-3'
PACrtZR4	5'-ACCAGCCCCAGCCATGCATG-3'

The underlined sequences were added for the cloning.

Figure 1. Schematic representation of plasmids used for E. coli expression system. crtE, GGPP synthase gene from Pantoea ananatis; crtB, phytoene synthase gene from P. ananatis; crtI, phytoene desaturase gene from P. ananatis; crtY, lycopene β-cyclase gene from P. ananatis; crtZ, β-carotene hydroxylase gene from P. ananatis; crtX, zeaxanthin glucosyltransferase from P. ananatis; Cm^r, Chloramphenicol resistance gene.

Table 2. Retention time and absorbing spectra of the authentic standards.

	Retention time (min.)	Absorbing spectra(nm)
phytoene	16.4	286
lycopene	14	446 473 503
β-carotene	14.7	452 479
β-cryptoxanthin	11.4	452 478
zeaxanthin	9.1	450 477

Figure 2. Analysis of carotenoids extracted from Pseudomonas sp. strain Akiakane. (a) HPLC chromatogram of carotenoids extracted from Pseudomonas sp. strain Akiakane. 1; β-cryptoxanthin, 2; β-carotene. (b) Absorbing spectra of the peak 1. (c) Absorbing spectra of the peak 2.

Figure 3. Phylogenetic tree based on the 16S rRNA sequences of Pseudomonas. A rooted neighbor-joining tree was constructed. Numbers adjacent to branches are bootstrap values supporting the presented final tree. Each accession number is shown in parentheses.

Figure 4. Presumed carotenoid biosynthesis pathway of Pseudomonas sp. strain Akiakane. The gene indicated by gray letters has not been isolataed. From the extract of Akiakane we detected β-cryptoxanthin and β-carotene.

Figure 5. HPLC analysis of carotenoids produced by the recombinant E. coli. HPLC chromatograms of the extracts from E. coli that carried (a) pACCAR25ΔcrtEX plus pETD vector; (b) ACCAR25ΔcrtEX plus pETD-crtE; (c) pACCAR25ΔcrtB plus pETD vector; (d) pACCAR25ΔcrtB plus pETD-crtB. 1; zeaxanthin, 2; β-cryptoxanthin.

Table 3. Carotenoids produced by recombinant E. coli.

	pETD	pETD-crtE	t test
zeaxanthin (μg/gFW)	0.448 ± 0.026	9.768 ± 0.133	***
β-cryptoxanthin (μg/gFW)	ND	0.324 ± 0.008

Results are the mean ± SD of the three experiments.

*** p < 0.01.

Figure 6. HPLC analysis of carotenoids produced by the recombinant E. coli. HPLC chromatograms of the extracts from from E. coli that carried (a) pACCRT-EB plus pETD vector; (b) pACCRT-EB plus pETD-crtI; (c) pACCRT-EIB plus pETD vector; (d) pACCRT-EIB plus pETD-crtY. Individual peak of 1; phytoene, 2; lycopene, 3; β-carotene.

Figure 7. Lycopene contents of the recombinant E. coli that carried pACCRT-EIB plus pRK404 or pRK-idi. Results are the mean ± SD of the three experiments. ***; p < 0.01.

Figure 8. The carotenoid gene clusters of carotenoid-producing bacteria.