Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoine-producer isolated from pulp mill wastewater

Accepted Manuscript Title: Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoine-producer isolated from pulp mill wastewater Authors: Qi Zhao, Yiwei Meng, Shannan Li, Peiwen Lv, Ping Xu, Chunyu Yang PII: DOI: Reference:

S0168-1656(18)30616-3 https://doi.org/10.1016/j.jbiotec.2018.08.017 BIOTEC 8267

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

29-1-2018 20-8-2018 27-8-2018

Please cite this article as: Zhao Q, Meng Y, Li S, Lv P, Xu P, Yang C, Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoineproducer isolated from pulp mill wastewater, Journal of Biotechnology (2018), https://doi.org/10.1016/j.jbiotec.2018.08.017 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.

Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoine-producer isolated from pulp mill wastewater

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Qi Zhao#, Yiwei Meng#, Shannan Li, Peiwen Lv, Ping Xu, Chunyu Yang*

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State Key Laboratory of Microbial Technology, Microbial Technology Institute,

The authors contribute equally to this work

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Shandong University, Qingdao, People’s Republic of China

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*Corresponding author: Dr. Chunyu Yang

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State Key Laboratory of Microbial Technology, Microbial Technology Institute,

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Shandong University, Qingdao, 266237, P. R. China Tel: +86-531-88366231; Fax: +86-531-88366231

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E-mail: [email protected]

Highlights

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The complete genome of Halomonas hydrothermalis Y2 was sequenced by platforms PacBio and Illumina.

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A circular chromosome of 3.9 Mbps contains 3520 protein coding sequences.

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The strain can efficiently produce ectoine under pressures of high salinity and alkalinity. 1

ABSTRACT Halophilic microorganisms have great potentials towards biotechnological applications. Halomonas hydrothermalis Y2 is a halotolerant and alkaliphilic strain that

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isolated from the Na+-rich pulp mill wastewater. The strain is dominant in the bacterial community of pulp mill wastewater and exhibits metabolic diversity in utilizing various

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substrates. Here we present the genome sequence of this strain, which comprises a circular chromosome 3,933,432 bp in size and a GC content of 60.2%. Diverse genes

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that encoding proteins for compatible solutes synthesis and transport were identified

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from the genome. With a complete pathway for ectoine synthesis, the strain could

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produce ectoine from monosodium glutamate and further partially secreted into the

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medium. In addition, around 20% ectoine was increased by deleting the ectoine

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hydroxylase (EctD). The genome sequence we report here will provide genetic information regarding adaptive mechanisms of strain Y2 to its harsh habitat, as well as

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production.

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facilitate exploration of metabolic strategies for diverse compatible solutes, e.g., ectoine

Keywords: Halomonas hydrothermalis Y2; Genome sequence; Compatible solutes;

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Ectoine synthesis

As ideal candidates for the production of many compatible solutes, e.g., ectoine, halophilic hydrolases, biosurfactants/bioemulsifiers, and several other chemicals, 2

halophilic microorganisms have attracted a lot of interests and attentions in various industries (Oren et al., 1999). Halomonas species belong to the moderately halophilic bacteria, which grow best in media containing 3 to 15% NaCl salt concentrations and can even tolerate up to 25% NaCl (Ventosa et al., 1998). Besides diverse chemicals

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produced by these Halomonas strains, unparalleled halophilic properties also make

and continuous fermentation processes (Yin et al., 2015).

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them an ideal and attractive “cell factory”, with the advantages of contamination free

Halomonas species are known as prominent producers for some compatible solutes

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synthesis, especially ectoine or 5-hydroxyectoine. Various isolates including H.

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elongata (Sauer and Galinski, 1998), H. boliviensis (Van-thuoc., 2010), H. salina

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(Zhang et al., 2009) had been reported for their abilities in ectoine synthesis. Previously,

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we have isolated a halotolerant and alkaliphilic strain Y2 from the pulp mill wastewater.

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As a dominant member in the bacterial community of wastewater (Yang et al., 2010a), the strain displays excellent ability in treating the pulp mill wastewater, robust

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resistance to alkaline and saline conditions, as well as metabolic diversities of utilizing

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various organic compounds (Yang et al., 2010b). Special habitats and metabolic diversities of strain Y2 attracted our interests for exploring its application potentials. It was anticipated that the complete genome sequence of H. hydrothemolis Y2 can provide

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information for those biosynthetic gene clusters that encoding various compatible solutes, and thereby creates more possibilities for genetic manipulation and modification.

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Genomic DNA of strain Y2 was isolated using the ChargeSwitch® gDNA Mini Bacteria Kit (Life Technologies) and sequenced by the Illumina Hiseq2000 and Pacbio RSII platforms. After filtered by the SOAPdenovo v2.04 (Li et al., 2010) and SMRT analysis v2.3.0 software (Chin et al., 2013), a high quality data of 18249463

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reads Hiseq clean data, 48253 PacBio reads (C2-chemistry) with an average read length of 6862 bp, was generated. The final assembly generated a circular

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chromosome 3,933,432 bp in size (Fig. 1) with high GC content of 60.2% (Table 1). Phylogenetically, the strain is closely related with H. elongata DSM2581T as shown

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in Fig. 2.

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The assembled genome sequence was annotated using the NCBI Prokaryotic

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Genome Annotation Pipeline version 4.2 (PGAP, released 2013,

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https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and submitted to SwissProt

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(http://uniprot.org), KEGG (http://www.genome.jp/kegg/), and COG (http://www.ncbi.nlm.nih.gov/COG) for functional annotation. Totally 3637 genes

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were predicted, which include 3520 coding sequences (CDS) genes, 61 tRNAs genes,

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18 rRNA genes, 4 ncRNAs genes, and 34 Pseudo genes (Table 1). Among these encoding proteins, five ORFs had been annotated as Na+(Li+, K+)/H+ antiporters and experimentally verified for their contributions to the robust resistance of high

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alkalinity and salinity. We have illustrated that these antiporters in strain Y2 worked in concert upon alkaline and (or) saline environments (Cheng et al., 2016). Moreover, strain Y2 contains gene clusters for ectoine, betaine, and glutamate syntheses, as well

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as the opuABCD gene cluster for osmoprotectant uptake and Na+/proline symporters (Table 2). In its genome, whole pathways for ectoine synthesis and catabolism were identified, with most homologue genes as those in H. elongata DSM2581T

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(Schwibbert et al., 2011) (Table 2). The ability of strain Y2 for ectione synthesis was firstly tested in the 500 ml flask containing 50 ml MG medium (Zhang et al., 2009)

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(pH 8.0). After 48-h incubation, both intracellular and extracellular ectoine were

determined in a Shimadzu HPLC system (Kyoto, Japan). The samples were passed

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through a Venusil XBP NH2 column (4.6 × 250 mm) and monitored by UV detector at

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a wavelength of 204 nm. As shown in Fig. 3, the ectoine peak in spectrum of the

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standard sample (Fig. 3A) was well accordance with that of the intracellular sample

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(Fig. 3B), indicating that ectoine could be accumulated in the cells of strain Y2.

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Differently, only hint ectoine was detected in the culture supernatant (data not shown). Due to the robust tolerance of strain Y2 to high alkalinity and salinity, we further

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compared the ectoine productivity under different NaCl concentrations or pH

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conditions. As shown in Fig. 4A, under lower NaCl concentrations ranging from 20 to 60 g L-1, strain Y2 grew well at all tested pH conditions. However, under high salinity stress (above 80 g L-1), the strain grew better in the media of high alkalinity and the

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best growth was observed at pH 10.0. Contrast to the cell growth, the ectoine synthesis displayed an obviously salinity-dependence as shown in Fig. 4B. More ectoine could be synthesized under lifted salinity, with a maximum ectoine produced in the presence of 80 g L-1 NaCl. This is highly in agreement with some other 5

halotolerant and halophilic ectoine-producers, in which NaCl was determined to be a key factor on the ectoine synthesis (Onraedt et al., 2005). Besides high salinity preference, the synthesis of ectoine also showed a highly pH-dependence, with a maximum ectoine content observed at pH 9.0 while a minimal production at pH 8.0.

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This is different from the weaker alkaline conditions for many other ectoine producers (medium pH 7.5), while is consistent with the alkaliphilic property of strain Y2 (Yang

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et al., 2010b). Furthermore, we have deleted the ectoine hydroxylase (EctD) and

obtained a modestly improved ectoine production. In a 1-liter bioreactor containing

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500 ml of modified MG medium (80 g L-1 NaCl, pH 9.0), a maximum intracellular

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ectoine reached to its plateau of 3.4 g L-1 (114.0 mg g DCW-1) by the △ectD mutant

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after 24 h fermentation. As for the wild-type strain, only 2.9 g L-1 (96.7 mg g DCW-1)

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was produced. Different from the flask cultivations, high contents ectoine (2.8 and 2.2

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g L-1) was detected in the 24-h fermentation media of both strains, implying that ectoine could be secreted into the medium by the strains. In agreement with the

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identified ectoine catabolic pathway (Table 2, designated as Doe pathway in H.

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elongata), further cultivation resulted in a drastically decrease in both strains, with less than half ectoine retained after 48 h incubation (data not shown). This suggests that ectoine could be consumed by strain Y2 and its catabolic pathway is functional in

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the strain.

In conclusion, the complete genome sequence of strain H. hydrothermalis Y2 revealed that the strain can adopt many adaptive strategies for coping with saline and alkaline environments. Robust saline and alkaline resistance, metabolic diversity, and 6

rapidly reproduce of this strain provide great potentials to explore more desired chemicals from it, with reference of the genome disclosed in this study, e.g., the genome sequence provides molecular basis for the genetic modification and metabolic

Strain and nucleotide sequence accession numbers

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engineering works of ectoine production.

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H. hydrothermalis strain Y2 has been deposited at China Center for Type Culture Collection under the deposition CCTCC M 208188. The genome sequence has been

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deposited at GenBank under the accession CP023656.1.

Acknowledgements

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The authors declare no conflicts of interest.

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Conflicts of interest

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This work was supported by the National Natural Science Foundations of China

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(Grant nos. 31670109 and 31370153).

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References Cheng, B., Meng, Y.W., Cui, Y.B., Li, C.F., Tao, F., Yin, H.J., Yang, C.Y., Xu, P., 2016. Alkaline response of a halotolerant alkaliphilic Halomonas strain and

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functional diversity of its Na+(K+)/H+ antiporters. J. Biol. Chem. 291, 26056– 26065.

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Chin, C.S., Alexander, D.H., Marks, P., Klammer, A.A., Drake, J., Heiner, C., Clum, A., Copeland, A., Huddleston, J., Eichler, E.E., Turner, S.W., Korlach, J., 2013.

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sequencing data. Nat. Methods 10, 563–569.

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Nonhybrid, finished microbial genome assemblies from long-read SMRT

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Li, R., Zhu, H., Ruan, J., Qian,W., Fang. X.D., Shi, Z.B., Li, Y.R., Li, S.T., Shan, G.,

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Kristiansen, K., Li, S., Yang, H., Wang, J., Wang, J., 2010. De novo assembly of

265–272.

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human genomes with massively parallel short read sequencing. Genome Res. 20,

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Oren, A., 1999. Bioenergetic aspects of halophilism. Microbiol. Mol. Biol. R.

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63, 334–348.

Sauer, T., Galinski, E.A., 1998. Bacterial milking: A novel bioprocess for production of compatible solutes. Biotechnol. Bioeng. 57, 306–313.

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Schwibbert, K., Marin-Sanguino, A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S.C., Klenk, H.P., Pfeiffer, F., Oesterhelt, D., Kunte, H.J., 2011. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environ. Microbiol. 13, 1973–1994. 8

Van-thuoc, D., Guzmán, H., Thihang, M., Hattikaul, R., 2010. Ectoine production by Halomonas boliviensis: optimization using response surface methodology. Mar. Biotechnol. 12, 586–593. Ventosa, A., Nieto, J.J., Oren, A., 1998. Biology of moderately halophilic aerobic

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bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544. Yang C.Y., Niu Y., Su H.J., Wang Z., Tao F., Wang X., Tang, H.Z., Ma, C.Q., Xu, P.,

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2010a. A novel microbial habitat of alkaline black liquor with very high pollution load: Microbial diversity and the key members in application potentials.

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Bioresour. Technol. 101, 1737–1744.

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Yang, C.Y., Wang, Z., Li, Y., Niu, Y., Du, M.F., He, X.F., Ma, C.Q., Tang, H.Z., Xu,

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P., 2010b. Metabolic versatility of halotolerant and alkaliphilic strains of

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Halomonas isolated from alkaline black liquor. Bioresour. Technol. 101, 6778–

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6784.

Yin, J., Chen, J.C., Wu, Q., Chen, G.Q., 2015. Halophiles, coming stars for industrial

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biotechnology. Biotechnol. Adv. 33, 1433–1442.

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Zhang, L.H., Lang, Y.J., Nagata, S., 2009. Efficient production of ectoine using

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ectoine-excreting strain. Extremophiles 13, 717–724.

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Figure legends

Fig. 1. Classical circular genome maps of the H. hydrothermalis Y2. From the outermost circle to the inner, each circle represents that (1) Genome size (0.5

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Mb/scale), (2) Forward CDS, (3) Reverse CDS, (4) rRNA/tRNA, (5) GC content, (6)

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GC skew.

Fig. 2. Phylogenetic tree of H. hydrothermalis Y2 based on the neighbor-joining (NJ) method, among publically available reference genomes from some alkaliphilc or

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halotolerant strains. The tree was constructed with MEGA 7.0 after homologous analysis and multiple sequence alignment.

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Fig. 3. High performance liquid chromatography (HPLC) analysis for ectoine

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production by H. hydrothermalis Y2. A, spectrum of the standard ectoine; B,

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spectrum of the intracellular sample of H. hydrothermalis Y2.

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Fig. 4. Ectoine production in the media containing various concentrations of NaCl, at

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different pH conditions. A, cell growth determined at 600 nm; B, ectoine productivity.

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Table 1 Genome features of Halomonas hydrothermalis Y2. Chromosome

Molecular shape

circular

Genome size (bp)

3,933,432

Total number of the genes

3637

CDS (total)

3554

CDS (coding)

3520

GC content in genome (%)

60.2

rRNAs (5S, 16S, 23S)

18

tRNAs

61

ncRNAs

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Pseudo genes

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GenBank accession number

CP023656.1

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Features

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Table 2 Compatible solutes encoding genes in the genome of Halomonas hydrothermalis Y2. Protein

ORF Number

lysC

aspartate kinase

orf01948

Asd

aspartic semialdehyde dehydrogenase

orf02775

ectA

L-2,4-diaminobutyric acid acetyltransferase

orf02990

ectB

diaminobutyrate-2-oxoglutarate transaminase

orf02989

ectC

L-ectoine synthase

orf02988

ectD

ectoine hydroxylase

orf00558

doeA

ectoine hydrolase

orf00349

doeB

N-alpha-acetyl diaminobutyric acid deacetylase

orf00348

doeC

NAD-dependent succinate-semialdehyde dehydrogenase

orf00345

doeD

unknown

orf00344

doeX

AsnC family transcriptional regulator

Beta

choline dehydrogenase

betB

betaine-aldehyde dehydrogenase

betI

BetI family transcriptional regulator

betT/betS

choline transporter

TC.BCT

BCCT family transporter

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Gene

orf00347

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orf03145 orf03144 orf03143 orf00375 orf00267 orf00516

opuA

ABC transporter

orf02899

opuBD

osmoprotectant uptake system permease

orf02900

opuBD

osmoprotectant uptake system substrate-binding protein

orf02898

opuC

osmoprotectant uptake system substrate-binding protein

orf02897

TC.SSS

sodium:proline symporter

orf02587

TC.SSS

sodium:proline symporter

orf01527

glutamate dehydrogenase

orf03519

glutamate dehydrogenase

orf00761

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gdhA

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gdhA

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ABC transporter glycine/betaine ABC transporter permease

NAD-glutamate dehydrogenase

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GDH2

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orf02335