Complete genome sequence of Stenotrophomonas sp. KACC 91585, an efficient bacterium for unsaturated fatty acid hydration

Accepted Manuscript Title: Complete genome sequence of Stenotrophomonas sp. KACC 91585, an efficient bacterium for unsaturated fatty acid hydration Author: Kyoung-Rok Kim Woo-Ri Kang Deok-Kun Oh PII: DOI: Reference:

S0168-1656(16)31624-8 http://dx.doi.org/doi:10.1016/j.jbiotec.2016.11.024 BIOTEC 7728

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

12-9-2016 22-11-2016 25-11-2016

Please cite this article as: Kim, Kyoung-Rok, Kang, Woo-Ri, Oh, DeokKun, Complete genome sequence of Stenotrophomonas sp.KACC 91585, an efficient bacterium for unsaturated fatty acid hydration.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.11.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Complete genome sequence of Stenotrophomonas sp. KACC 91585, an efficient bacterium for unsaturated fatty acid hydration

Kyoung-Rok Kim, Woo-Ri Kang, and Deok-Kun Oh

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, South Korea

*Corresponding author. Tel: +82-2-454-3118; fax: +82-2-444-5518 E-mail address: [email protected] (D.–K. Oh)

Highlight • We report the complete genome sequence of Stenotrophomonas sp. KACC 91585. • This strain is an efficient hydrating bacterium for unsaturated fatty acids. • A set of genes concerned with the biosynthesis and metabolizing of fatty acids were identified. • Desaturases, thioesterases, and oleate hydratase are involved in hydroxy fatty acid production.

1

Abstract Hydroxy fatty acids (HFAs) such as 10-hydroxystearic acid (10-HSA) and 10-hydroxy-12(Z)octadecenoic acid (10-HOD), which are similar to ricinoleic acid, are important starting materials and intermediates for the industrial manufacture of many commodities. Stenotrophomonas sp. KACC 91585, which was isolated from lake sediment, is an efficient bacterium for unsaturated fatty acid hydration that produces 10-HSA and 10-HOD from oleic acid and linoleic acid, respectively, with high conversion rates. The complete genome of this strain is 4,541,729 bp with 63.83% GC content and devoid of plasmids. Sets of genes involved in the fatty acid biosynthesis and modification as well as modified lipids were identified in the genome, and these genes were concerned with HFA production. This genome sequence provides molecular information and elucidation for HFA production, and will be used as an efficient biocatalyst source for the biotechnological production of HFA.

Keywords: Complete genome sequence; Hydroxy fatty acids; Linoleic acid; Oleic acid; Pacbio; Stenotrophomonas sp. KACC 91585

Stenotrophomonas species are ubiquitously living bacteria, which are found from various environments, including plant, soil, and water. Although Stenotrophomonas maltophilia is notoriously known as an emerging human pathogen, most Stenotrophomonas species are beneficial and industrially valuable. For example, some Stenotrophomonas species promote the growth and development of plant. These species can be applied as biocontrol agents (Hayward et al., 2010). Several Stenotrophomonas species have also been used in biotechnology applications because they exhibit special abilities, including heavy metal 2

tolerance, xenobiotics degradation, and enzymatic biotranformation for fatty acids (Ryan et al., 2009). As a potent hydroxy fatty acid (HFA) producer, Stenotrophomonas sp. KACC 91585 (available from Korea Agricultural Culture Collection), formerly mane as Stenotrophomonas nitritireducens, was isolated from lake sediment (Yu et al., 2008b). Stenotrophomonas sp. KACC 91585 converted oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) to 10-hydroxystearic acid (10-HSA) (Kim et al., 2011), 10-hydroxy-12(Z)-octadecenoic acid (10-HOD) (Jo et al., 2014; Yu et al., 2008a), and 10-hydroxy-12,15(Z,Z)-octadecadienoic acid (Choi et al., 2015), respectively. HFAs are important starting materials for synthesizing carboxylic acid, dicarboxylic acid, nylon, polyamide, and waxes, owing to their superior properties than non-HFAs. Several bacteria have been identified for their ability to produce HFAs from unsaturated fatty acids (UFAs) (Biermann et al., 2011). However, no complete genome information of these HFA-converting bacteria has been provided, and their related metabolism is unknown. Therefore, this strain was subjected to the whole genome sequencing analysis to investigate the genetic background of HFA formation and its related metabolism. Most known HFA-producing strains showed the highest conversion rate toward OA and exhibited significantly lower conversion rate toward polyunsaturated fatty acids (PUFAs), including LA, ALA, and γ-linolenic acid (GLA). However, the conversion rate of HFA from LA by Stenotrophomonas sp. KACC 91585 was almost similar to that from OA. As shown Fig. 1A and 1B, the strain KACC 91585 produced 10-HSA from OA with 95% conversion rate and produced 10-HOD from LA with 81% conversion rate within the same reaction period. The volumetric production rate of Stenotrophomonas sp. KACC 91585 was compared with those of known HFA-producing bacteria, including Enterococcus faecalis, Flavobacterium sp. DS5, Nocardia paraffinae, Nocardia cholesterolicum, and S. maltophilia 3

(Fig. 1C), and the strain showed the highest volumetric production rate among the known HFA-producing bacteria. The volumetric production rates of the strain KACC 91585 for 10HSA and 10-HOD were 1.6- and 4.4-fold higher, respectively, than those of S. maltophilia. The substrate preference of the strain KACC 91585 for UFAs showed that highest toward OA. However, the relative specific activities for LA, ALA, and GLA to OA were not low as 87%, 61%, and 59%, respectively (Fig. 1D). This unique property of the strain indicated that it could be used to efficiently convert UFAs, including OA, LA, ALA, and GLA, to 10-HFAs. Thus, Stenotrophomonas sp. KACC 91585 can be used as an effective biocatalyst for producing 10-HFAs from oils because they contained OA, LA, ALA and/or GLA as major components. A phylogenic tree based on 16S rRNA sequences was constructed by MEGA (version 6.0) and neighbor-joining method (Fig. 2). The tree showed a branch of Stenotrophomonas sp. KACC 91585 with S. terrae R-32768T and distinguishing with other Stenotrophomonas strains. In the previous report, the strain KACC91585 was named as S. nitritireducens. However, in the detailed comparison of 16S rRNA, the strain KACC 91585 showed 99.9% and 99.3% identities with S. terrae R-32768T and S. maltophilia IAM 12672, respectively. In contrast, the isolated strain exhibited 99.2% identity with the type strain S. nitritireducens L2 (DSM 12575) (Table S1). Furthermore, ANI (analyzed in EzGenome) and DDH (analyzed in GGDC; http://ggdc.dsmz.de/distcalc2.php) values of the strain KACC 91585 to the strain L2 were lower than those to the strain R-32768T (Table S2). These results indicating that the strain KACC 915825 did not belong to S. nitritireducens. Therefore, we proposed the strain as Stenotrophomonas sp. To obtain the detailed genetic information involved in HFA production, we sequenced whole genome of the strain KACC 91585. The genomic DNA was purified using a GenElute 4

Bacterial Genomic DNA Kit (Sigma-Aldrich) for constructing library, and the validity of quality and quantity of the genomic DNA were analyzed by Aglient 2100 Bioanalyzer. A 20kb insert SMRTbell library was constructed using a SMRTbell template prep kit (VarelaÁlvarez et al., 2006), and whole genome sequencing of the strain KACC 91585 was performed using a combined strategy of PacBio RSII sequencing (Pacific Biosciences) and Illumina paired-end sequencing technology using an illumina Hiseq model 2000. The complete genome was assembled using hierarchical genome assembly process (HGAP) Version 2.3. Around 582.1 Mb data were obtained with 128-fold average coverage. Coding DNA sequences (CDSs) were identified using Glimmer v3.02 (Delcher et al., 2007), and open reading frames (ORFs) were obtained. Gene annotation was performed using Blastall alignment against the NCBI non-redundant (nr) protein database for all species. Gene ontology (GO) annotation was assigned to each of ORFs by Blast2GO software analyzing the best hits of the BLAST results (Conesa et al., 2005). Additionally, ribosomal RNAs and transfer RNAs were predicted using RNAmmer 1.2 (Lagesen et al., 2007) and tRNAscan-SE 1.4 (Lowe and Eddy, 1997). CRISPR analysis was used in the online tool (http://crispr.i2bc.paris-saclay.fr/Server/). The complete genome features of Stenotrophomonas sp. KACC 91585 are summarized in Table 1. The complete genome of this strain consisted of a single circular chromosome without plasmids. The size of the chromosome was 4,541,729 bp. The GC content of the chromosomal DNA was 63.8% with 3,975 protein-coding genes, 14 rRNA genes, 67 tRNA genes, 91 pseudogenes, and 9 clustered regularly interspaced short palindromic repeats (CRISPR) were identified in the genome. To access more detailed classification of identified genes, we performed clusters of orthologous genes (COG) analysis (Tatusov et al., 2000) in webMGA (http://weizhonglab.ucsd.edu/metagenomic-analysis). The genomes of three model 5

microorganisms, S. maltophilia K279a (CP012122) (Crossman et al., 2008), DDT-6 (LEKR00000000) (Pan et al., 2016), and Stenotrophomonas rhizophila DSM14405 (CP007597) (Alavi et al., 2014), were compared with that of the strain KACC 91585 (Table S1). Genome analysis showed that Stenotrophomonas sp. KACC 91585 had many functions although its genome size was relatively small. These results seem to be associated with residing environment of strain KACC 91585, which was isolated from lake sediment. To adapt its competitive environment for survival, the strain may have many functions to maintain versatile metabolism and catalysis. The numbers of genes involved in de novo biosynthesis, chain elongation, and degradation of fatty acid in Stenotrophomonas sp. KACC 91585 were 12, 4, and 9, respectively. Twelve ORFs for fatty acid biosynthesis were accA (acetyl-CoA carboxylase carboxyl transferase subunit alpha), accB (acetyl-CoA carboxylase biotin carboxyl carrier protein), accC (acetyl-CoA carboxylase), accD (acetyl-CoA carboxylase carboxyl transferase subunit beta), fabA (3-hydroxyacyl-acyl carrier protein (ACP) dehydratase/isomerase), fabB (3-oxoacyl-ACP synthase I), fabD (ACP S-malonyltransferase), fabF (3-oxoacylACPsynthase II), fabH (3-oxoacyl-ACP synthase III), fabG (3-oxoacyl-ACP reductase), fabY (enoyl ACP reductase), and fabZ (3-hydroxyacyl-ACP dehydratase) synthase III (Table 2). Notably, S. nitritireducens KACC 91585 genome contained more than one copy of certain genes that were involved in fatty acid biosynthesis, such as fabA (A1~4), fabB (B1~3), fabF (F1~3), and fabG (G1~4). These multiplicated genes were seemed to overexpress and significantly increase fatty acid production, comparing with the duplicated fabH, fabG and fabD in other Stenotrophomonas species such as S. maltophilia K279a. Four genes encoding hdaH (3-hydroxyacyl-CoA dehydrogenase), ech1 (enoyl-CoA hydratase), mer1 (trans-2enoyl-CoA reductase), and tes1 (palmitoyl-protein thioesterase) were involved in fatty acid 6

chain elongation. Nine genes involved in fatty acid degradation were fadA (acetyl-CoA acyltransferase), fadE (acyl-CoA dehydrogenase/enoyl-CoA hydratase), fadJ (fatty-acid oxidation protein subunit alpha), fadN (enoyl-CoA hydratase), gcdH (glutaryl-CoA dehydrogenase), frmA (S-(hydroxymethyl)glutathione dehydrogenase/alcohol dehydrogenase), aldH (aldehyde dehydrogenase), and acaT1 and acaT2 (acetyl-CoA acetytransferases 1 and 2). Desaturase is important for the formation of UFAs such as OA, LA, ALA, and GLA. Two bacterial-type desaturases, stereaoyl-CoA delta-9 desaturase (SCD) and linoleoyl-CoA delta-6 desaturase (LCD), have been reported to catalyze the conversion of stearic acid and OA to OA and GLA, respectively, with the aid of two acyl-CoA thioesterases (ACOTs). In Stenotrophomonas sp. KACC 91585 genome, two desaturases scd1 and scd2 genes; and one lcd gene were identified (Table 2). The genome of S. maltophilia strains, including D457 and K279a, also had scd1, scd2, and lcd genes. However, S. acidaminiphila (CP012900.1) genome lacked lcd gene, and S. rhizophila DSM14405 genome lacked lcd and scd2 genes. These results indicate that S. nitritireducens KACC 91585 and S. maltophilia strains possessed well-developed metabolisms for UFAs. The one gene encoding a fatty acid double-bond hydratase involved in 10-HSA or 10HOD formation from OA or LA, respectively, was identified. The fatty acid hydratase of S. nitritireducens KACC 91585 preferred OA over LA, indicating that the enzyme was oleate hydratase (OhyA), which was responsible in the HFA production and catalyzed the conversion of oleic acid to 10-HAS with the highest catalytic efficiency. Notably, the oleate hydratase genes (ohyAs) of Stenotrophomonas strains are highly conserved (Fig. S1). Since only one ohyA gene of S. maltophilia KCTC 1773 was functionally analyzed, we compared the activity of S. nitritireducens KACC 91585 OhyA with that of S. maltophilia KCTC 1773 7

OhyA. The specific activity of S. maltophilia KCTC 1773 OhyA toward OA was 1.6-fold higher than that of S. nitritireducens KACC 91585, whereas the specific activities toward LA, ALA and GLA were 1.8-, 3.9- and 2.7-fold higher than those of S. maltophilia KCTC 1773 (Fig. S2). The gene encoding Stenotrophomonas sp. KACC 91585 OhyA contained 654 amino acids, whereas the gene encoding S. maltophilia KCTC 1773 OhyA consisted of 555 amino acids. Thus, the gene encoding the strain KACC 91585 OhyA may be differently evolved with the gene encoding S. maltophilia KCTC 1773 OhyA, and the strain KACC 91585 OhyA has more effective for the production of PUFAs, including LA, ALA and GLA; and oils into HFAs than S. maltophilia KCTC 1773 OhyA. The genome data of the strain KACC 91585 showed a full set of genes involved in the metabolism of long-chain UFAs. In silico analyses predicted that 10-HFAs could be formed by subsequent reactions of two SCDs, single LCD, two ACOTs, and OhyA. Transport protein such as long chain fatty acid tranporter (FadL) involved in the transfer of fatty acid and free HFA was identified. These results may provide clues to explain the high-level production of HFA in S. nitritireducens KACC 91585. Furthermore, several ORFs involved in the biosynthesis of metabolites, including lipoic acid, phospholipid, and cardiolipin derived from fatty acids and lipids were also identified. Therefore, S. nitritireducens KACC 91585 is a potential biotransformation factory for producing various metabolites originated from fatty acid and lipid resources, although future studies are needed to validate whether these pathways are functional. In conclusion, the complete genome sequence of Stenotrophomonas sp. KACC 91585 will enhance our understanding and application for not only the production of HFAs from UFAs but also formation of modified lipids in this bacterium. Our data implies that the strain

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KACC 91585 and its genes can be utilized as efficient biocatalyst sources for the biotechnological production of HFA.

Nucleotide sequence accession number This whole-genome project has been deposited at GenBank under the accession number CP016756.

Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2014R1A1A2057370).

References

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research. Bioinformatics 21, 3674-3676. Crossman, L.C., Gould, V.C., Dow, J.M., Vernikos, G.S., Okazaki, A., Sebaihia, M., Saunders, D., Arrowsmith, C., Carver, T., Peters, N., Adlem, E., Kerhornou, A., Lord, A., Murphy, L., Seeger, K., Squares, R., Rutter, S., Quail, M.A., Rajandream, M.-A., Harris, D., Churcher, C., Bentley, S.D., Parkhill, J., Thomson, N.R., Avison, M.B., 2008. The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia eveals an organism heavily shielded by drug resistance determinants. Genome Biol. 9, 1-13. Delcher, A.L., Bratke, K.A., Powers, E.C., Salzberg, S.L., 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23, 673-679. Hayward, A.C., Fegan, N., Fegan, M., Stirling, G.R., 2010. Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology. J. Appl. Microbiol. 108, 756-770. Jo, Y.S., An, J.U., Oh, D.K., 2014. gamma-Dodecelactone production from safflower oil via 10-hydroxy-12(Z)-octadecenoic acid intermediate by whole cells of Candida boidinii and Stenotrophomonas nitritireducens. J. Agric. Food Chem. 62, 6736-6745. Kim, B.N., Yeom, S.J., Oh, D.K., 2011. Conversion of oleic acid to 10-hydroxystearic acid by whole cells of Stenotrophomonas nitritireducens. Biotechnol. Lett. 33, 993-997. Lagesen, K., Hallin, P., Rødland, E.A., Stærfeldt, H.-H., Rognes, T., Ussery, D.W., 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35, 3100-3108. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955-964. Pan, X., Lin, D., Zheng, Y., Zhang, Q., Yin, Y., Fang, H., Yu, Y., 2016. Biodegradation of 10

DDT by Stenotrophomonas sp. DDT-1: Characterization and genome functional analysis. Sci. Rep. 6, 21332. Ryan, R.P., Monchy, S., Cardinale, M., Taghavi, S., Crossman, L., Avison, M.B., Berg, G., van der Lelie, D., Dow, J.M., 2009. The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat. Rev. Microbiol. 7, 514-525. Tatusov, R.L., Galperin, M.Y., Natale, D.A., Koonin, E.V., 2000. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33-36. Varela-Álvarez, E., Andreakis, N., Lago-Lestón, A., Pearson, G.A., Serrão, E.A., Procaccini, G., Duarte, C.M., Marbá, N., 2006. Genomic DNA isolation from green and brown Algea (Caulerpales and Fucales) for microsatellite library construction. J. Phycol. 42, 741-745. Yu, I.S., Kim, H.J., Oh, D.K., 2008a. Conversion of linoleic acid into 10-Hydroxy-12(Z)octadecenoic acid by whole cells of Stenotrophomonas nitritireducens. Biotechnol. Prog. 24, 182-186. Yu, I.S., Yeom, S.J., Kim, H.J., Lee, J.K., Kim, Y.H., Oh, D.K., 2008b. Substrate specificity of Stenotrophomonas nitritireducens in the hydroxylation of unsaturated fatty acid. Appl. Microbiol. Biotechnol. 78, 157-163.

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Legend of Figures Fig. 1. Ability of Stenotrophomonas sp. KACC 91585 for HFA production. 10-HSA from OA (A) and 10-HOD from LA (B). HFA formation was detected by GC. Palmitic acid (C16:0) was used as an internal standard. Volumetric production rates of 10-HSA and 10-HOD by HFA-producing bacteria (C). Cells were cultivatied in Luria-Bertani (LB) medium for 10 h. After incubation, cells were harvested. The reactions of 10-HSA or 10-HOD using harvested whole cells were conducted at 35 °C in 50 mM citrate/phosphate buffer (pH 7.0) containing 10 g l−1 cells and 20 g l−1 oleic acid or linoleic acid, and 0.05% (w/v) Tween 40 for 6 h, respectively. Volumetric production rates were calculated from the produced amounts of 10HSA and 10-HOD. Relative substrate preference of Stenotrophomonas sp. KACC 91585 for UFAs (D).

Fig. 2. Phylogenetic tree of Stenotrophomonas sp. KACC 91585 within Stenotrophomonas strains. The tree was constructed using MEGA 6.0 by neighbour-joining method with Pdistance based on 16S rDNA gene sequences with 1,000 replications in bootstrap test.

Fig. 3. Circular genome map of Stenotrophomonas sp. KACC 91585. Circular genome-map was drawn using dnaplotter (http://www.sanger.ac.uk/science/tools/dnaplotter). From the innermost circles, circle (1) illustrates the GC skew (G-C/G+C). The value is plotted as the deviation from the average GC skew of the entire sequence. Circle (2) GC content, the GC content is plotted using a sliding window, as the deviation from the average GC content of the entire sequence. Circle (3) denotes tRNA (orange yellow)/rRNA (green). Circle (4, 5) 12

indicates the CDSs, colored according to GO2Blast functional categories, 4 is backward strand (red), and 5 is forward strand (blue).

13

(A)

(B)

Fig. 1-continued

14

(C)

Volumetric production rate (g/L/h)

9 10-HSA 10-HOD

8 7 6 5 4 3 2 1 0

i lia um alis p. DS5 nae ens affi terolic altoph reduc aec i E. f rium s N. par t s m i e itr S. hol acte S. n N. c vob

Fla

(D)

Relative specific activity (%)

120

100

80

60

40

20

0

OA

LA

ALA

GLA

Unsaturated fatty acids

Fig. 1

15

Fig. 2

16

Fig. 3

17

Table 1 Genome features of Stenotrophomonas nitritireducens KACC 91585

Features

Characteristics

Length (bp)

4,541,729

G + C contents (%)

63.83

CDS

3,975

rRNA

14

tRNA

67

GenBank accession no.

CP016756

18

Table 2. Genes involved UFAs, HFAs, and related pathway in S. nitritireducens KACC 91585 and other Stenotrophomonas strains.

Strain

Biosynthesis

UFAs and HFAs

FabB

FabD

FabE

FabF

FabG

FabH

FabI

FabK

FabP

FabQ

FabR

Tes1

Tes2

Scd1

Scd2

Lcd1

OhyA

FadL

Mfs1

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

■

■

■

■

■

■

S. maltophilia D457

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

■

■

■

■

■

■

S. maltophilia K279a

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

■

■

■

■

■

■

S. acidaminiphila

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

■

■

■

■

■

S. rhizophila DSM14405

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

■

■

■

■

19

FabL

FabA S. nitritireducens KACC 91585