search for




 

Complete genome sequence of Pantoea intestinalis SRCM103226, a microbial C40 carotenoid zeaxanthin producer
Korean J. Microbiol. 2019;55(2):167-170
Published online June 30, 2019
© 2019 The Microbiological Society of Korea.

Jin Won Kim, Gwangsu Ha, Seong-Yeop Jeong, and Do-Youn Jeong*

Microbial Institute for Fermentation Industry (MIFI), Sunchang 56048, Republic of Korea
Correspondence to: E-mail: jdy2534@korea.kr; Tel.: +82-63-653-9579; Fax: +82-63-653-9590
Received March 25, 2019; Revised April 5, 2019; Accepted April 10, 2019.
Abstract

Pantoea intestinalis SRCM103226, isolated from edible insect mealworm overproduces zeaxanthin as a main carotenoid. The complete genome of P. intestinalis SRCM103226 was sequenced using the Pacific Biosciences (PacBio) RS II platform. The genome of P. intestinalis SRCM103226 comprises a 4,784,919 bp circular chromosome (53.41% G+C content), and is devoid of any extrachromosomal plasmids. Annotation using the RAST server reveals 4,332 coding sequences and 107 RNAs (22 rRNA genes, 85 tRNA genes). Genome annotation analysis revealed that it has five genes involved in the carotenoid pathway. The genome information provides fundamental knowledge for comparative genomics studies of the zeaxanthin pathway.

Keywords : Pantoea intestinalis, C40 carotenoid, complete genome sequence, mealworm
Body

Carotenoid, a natural pigment, is an isoprenoid derivative. Until now, more than 700 carotenoid structures in the naturally occurring C30 and C40 carotenoid biosynthetic pathways have been identified (Walter and Strack, 2011; Kim et al., 2014). The carotenoids found in bacteria and yeast have antioxidative functions and play roles in energy transfer, light harvesting and the regulation of membrane fluidity (Holt et al., 2005; Johnson and Schmidt-Dannert, 2008; Kim and Lee, 2012). The bacteria which produce carotenoids have been studied significantly for their potential applicability in terms of microbial sources in the industrial field as feed additives, food and cosmetics ingredients (Lee and Schmidt-Dannert, 2002; Nishino et al., 2009). In the various type of carotenoids, zeaxanthin is a major yellow pigment in the plants such as corn. The zeaxanthin is used for the food of poultry, and it is the base materials for the color of egg yolk, meat, or skin of poultry. In addition to this, the zeaxanthin and lutein which is a one of carotenoid are has the various functionalities to the human health such as involvement in the maintenance of eye health (Krinsky et al., 2003). Some species of carotenoid-producing bacteria belonging to the family Enterobacteriaceae have been isolated (Lee and Schmidt-Dannert, 2002; Sedkova et al., 2005; Albermann, 2011). Here, we describe the complete genome sequence of C40 zeaxanthin- producing P. intestinalis SRCM103226. The gDNA was extracted by employing the Genomic DNA Kit (MGmed). The genome sequence of P. intestinalis SRCM103226 was in single-molecule real-time (SMRT) cells using the PacBio RS II SMRT sequencing (Pacific Biosciences) method. After sub-reads filtering of raw data of PacBio RS II sequencer, 91,717 read and 1,273,390,248 base pairs, with a 158-fold genome coverage, were generated and assembled de novo assembled using Canu v1.3 assembler (Koren et al., 2017). The overlapping regions at both ends of a contig were identified and trimmed to generate a unique stretch on both ends using Circlator (Hunt et al., 2015). Prediction of the open reading frames (ORFs) were the RAST server online (Aziz et al., 2008), Prodigal version 2.6.3 (Hyatt et al., 2010) and Glimmer 3.2 (Delcher et al., 1999). The tRNA and rRNA were predicted by using tRNAscan-SE v1.21 (Lowe and Eddy, 1997) and RNAmmer v1.2 (Lagesen et al., 2007), respectively. Function predictions were based on RPS-BLAST searches (E-value < 10-3) against the non-redundant GenBank protein database (www.ncbi.nlm.nih.gov/protein), the clusters of orthologous groups (COG) database (www.ncbi.nlm.nih.gov/COG). The graphical circular map of the genome was visualized and constructed using Circos v0.67 (Krzywinski et al., 2009). Zeaxanthin was extracted, analyzed using an Agilent 1200 HPLC/MS system equipped with a photodiode array detector according to our previous paper (Kim et al., 2016). The complete genome of P. intestinalis SRCM103226 contains a 4,784,919 bp circular chromosome with a G+C content of 53.41%, and the extrachromosomal plasmid was not included in P. intestinalis SRCM103226. A total of 4,464 coding DNA sequences (CDSs) were predicted with 22 rRNA and 85 tRNA genes. The 3,749 genes were identified and classified to functional categories (Tatusov et al., 2000), and the categorized genes were presented in the circular representation with color codes (Fig. 1). The main carotenoids of strain SRCM103226 were zeaxanthin (Fig. 2), a carotenoid pigment which was reported in other members of the genus Pantoea (Song et al., 2013). Genome annotation analysis identified that P. intestinalis SRCM103226 has carotenoid gene cluster comprising five genes encoding four C40 zeaxanthin pathway genes (Fig. 3): a heterologous copies of gene encoding zeaxanthin glucosyl transferase (crtX: peg 4263), a gene encoding geranylgeranyl pyrophosphate synthase (crtE: peg 404), a gene encoding phytoene synthase (crtB: peg 4266), a gene encoding phytoene desaturase (crtI: peg 4265), a gene encoding lycopene cyclase (crtY: peg 4264). The genome information provides fundamental knowledge for comparative genomics studies of the zeaxanthin pathway.

Fig. 1.

Graphical Circular map of Pantoea intestinalis SRCM103226. From bottom to top: genes on the forward strand (colored by COG categories), genes on the reverse strand (colored by COG categories), RNA genes (tRNA-green, rRNA-red, other RNAs-black), GC content, and GC skew (purple/olive).


Fig. 2.

Carotenoid profile of Pantoea intestinalis SRCM103226. Crude acetone extract of P. intestinalis SRCM10322 was analyzed using HPLC and LC/MS. The left insert shows UV/V is spectrum for peak 1 (λ max = 453 and 480 nm) and the right insert shows the mass spectrum for peak 1 (a molecular ion with [M-H]− = 568.3).


Fig. 3.

C40 carotenoid zeaxanthin biosynthesis gene cluster. Zeaxanthin biosynthesis genes are denoted by colored arrows.


General genomic features of Pantoea intestinalis SRCM103226

Genomic features Chromosome
Genome size (bp) 4,784,919
Number of contigs 1
G+C content (%) 53.41
Total coding DNA sequence (CDS) 4332
rRNA (5S, 16S, 23S) 22
tRNA 85


Nucleotide sequence accession numbers

The complete genome sequence of P. intestinalis SRCM103226 has been deposited in GenBank under accession number CP28271, and at the Korean Culture Center of Microorganisms (KCCM) under accession number KCCM 12194P.

적 요

Pantoea intestinalis SRCM103226은 식용곤충 밀웜으로부터 분리하였으며, zeaxanthin을 메인으로 생산하였다. P. intestinalis SRCM103226의 유전체 분석을 실시하여 4,784,919 bp 크기의 염기서열, GC 비율은 53.41%로 나타났으며, 플라스미드는 존재하지 않는다. RAST server를 이용하여 annotation한 결과 4,332개의 코딩유전자, 22개의 rRNA, 85개의 tRNA 유전자가 확인되었다 지놈분석결과 zeaxanthin 생합성회로 5개 유전자를 가지고 있다. 이러한 유전체 정보는 zeaxanthin 생합성 경로의 분자 진화의 비교 유전체학 연구에 대한 기초 정보를 제공한다.

Acknowledgements

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) and Establishment of Infrastructure for Industrialization of Korean Useful Microbes (R0004073).

References
  1. Albermann C. 2011. High versus low level expression of the lycopene biosynthesis genes from Pantoea ananatis in Escherichia coli. Biotechnol. Lett. 33, 313-319.
    Pubmed CrossRef
  2. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, and Kubal M. 2008. The RAST server:rapid annotations using subsystems technology. BMC Genomics. 9, 75.
    Pubmed KoreaMed CrossRef
  3. Delcher AL, Harmon D, Kasif S, White O, and Salzberg SL. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27, 4636-4641.
    Pubmed KoreaMed CrossRef
  4. Holt NE, Zigmantas D, Valkunas L, Li XP, Niyogi KK, and Fleming GR. 2005. Carotenoid cation formation and the regulation of photosynthetic light harvesting. Science. 307, 433-436.
    Pubmed CrossRef
  5. Hunt M, Silva ND, Otto TD, Parkhill J, Keane JA, and Harris SR. 2015. Circlator:automated circularization of genome assemblies using long sequencing reads. Genome Biol. 16, 294.
    Pubmed KoreaMed CrossRef
  6. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, and Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 11, 119.
    Pubmed KoreaMed CrossRef
  7. Johnson ET, and Schmidt-Dannert C. 2008. Light-energy conversion in engineered microorganisms. Trends Biotechnol. 26, 682-689.
    Pubmed CrossRef
  8. Kim SH, Kim JH, Lee BY, and Lee PC. 2014. The astaxanthin dideoxyglycoside biosynthesis pathway in Sphingomonas sp. PB304. Appl. Microbiol. Biotechnol. 98, 9993-10003.
    Pubmed CrossRef
  9. Kim SH, Kim MS, Lee BY, and Lee PC. 2016. Generation of structurally novel short carotenoids and study of their biological activity. Sci. Rep. 6, 21987.
    Pubmed KoreaMed CrossRef
  10. Kim SH, and Lee PC. 2012. Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli. J. Biol. Chem. 287, 21575-21583.
    Pubmed KoreaMed CrossRef
  11. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, and Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722-736.
    Pubmed KoreaMed CrossRef
  12. Krinsky NI, Landrum JT, and Bone RA. 2003. Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu. Rev. Nutr. 23, 171-201.
    Pubmed CrossRef
  13. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, and Marra MA. 2009. Circos:an information aesthetic for comparative genomics. Genome Res. 19, 1639-1645.
    Pubmed KoreaMed CrossRef
  14. Lagesen K, Hallin P, Rødland EA, Stærfeldt HH, Rognes T, and Ussery DW. 2007. RNAmmer:consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100-3108.
    Pubmed KoreaMed CrossRef
  15. Lee P, and Schmidt-Dannert C. 2002. Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl. Microbiol. Biotechnol. 60, 1-11.
    Pubmed CrossRef
  16. Lowe TM, and Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955-964.
    Pubmed KoreaMed CrossRef
  17. Nishino H, Murakoshi M, Tokuda H, and Satomi Y. 2009. Cancer prevention by carotenoids. Arch. Biochem. Biophys. 483, 165-168.
    Pubmed CrossRef
  18. Sedkova N, Tao L, Rouvière PE, and Cheng Q. 2005. Diversity of carotenoid synthesis gene clusters from environmental Enterobacteriaceae strains. Appl. Environ. Microbiol. 71, 8141-8146.
    Pubmed KoreaMed CrossRef
  19. Song GH, Kim SH, Choi BH, Han SJ, and Lee PC. 2013. Heterologous carotenoid-biosynthetic enzymes:functional complementation and effects on carotenoid profiles in Escherichia coli. Appl. Environ. Microbiol. 79, 610-618.
    Pubmed KoreaMed CrossRef
  20. Tatusov RL, Galperin MY, Natale DA, and Koonin EV. 2000. The COG database:a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33-36.
    Pubmed KoreaMed CrossRef
  21. Walter MH, and Strack D. 2011. Carotenoids and their cleavage products:biosynthesis and functions. Nat. Prod. Rep. 28, 663-692.
    Pubmed CrossRef


September 2019, 55 (3)
Full Text(PDF) Free

Social Network Service
Services

Author ORCID Information