Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production guided by genome analysis.


 Background

The species in family Elizabethkingia meningoseptica are interesting strain for investigating Vitamin K2 metabolic analysis. However, their genomic sequence, metabolic pathway, potential abilities, and evolutionary status are still unknown.
Results

This study therefore aimed to perform a genome sequencing of Elizabethkingia meningoseptica sp. F2 and further accomplished comparative analysis with other Vitamin K2 strains reveals overall identifying its unique/shared metabolic genes across genomes. The 3,874,794–base pair sequence of Elizabethkingia meningoseptica sp. F2 is presented. Of 3,539 genes annotation was applied. Results of synteny block demonstrated Elizabethkingia meningoseptica sp. F2 shares high levels of synteny with Elizabethkingia meningoseptica ATCC 13253 and Elizabethkingia meningoseptica NBRC 12535. Identification of Vitamin K2 metabolic pathway in Elizabethkingia meningoseptica sp. F2 were also accomplished. In addition, Elizabethkingia meningoseptica sp. F2 was resistant to gentamicin, streptomycin, ampicillin and caramycin, consistent with the presence of multiple genes encoding diverse multidrug efflux pump protein in the genome. Furthermore, By co-overexpression experiments of MenA and MenG, we showed that Vitamin K2 content was enhanced by 37% compared with control strain.
Conclusions

The genome analysis of Elizabethkingia meningoseptica sp. F2 in conjunction with the comparative metabolic pathways analysis among the E.coli, Bacillus subtilis and Streptomyces provided a useful information on the Vitamin K2 biosynthetic pathway and other related pathways at systems level.


Abstract Background
The species in family Elizabethkingia meningoseptica are interesting strain for investigating Vitamin K2 metabolic analysis. However, their genomic sequence, metabolic pathway, potential abilities, and evolutionary status are still unknown.

Results
This study therefore aimed to perform a genome sequencing of Elizabethkingia meningoseptica sp. F2 and further accomplished comparative analysis with other Vitamin K2 strains reveals overall identifying its unique/shared metabolic genes across genomes. The  accomplished. In addition, Elizabethkingia meningoseptica sp. F2 was resistant to gentamicin, streptomycin, ampicillin and caramycin, consistent with the presence of multiple genes encoding diverse multidrug efflux pump protein in the genome. Furthermore, By co-overexpression experiments of MenA and MenG, we showed that Vitamin K2 content was enhanced by 37% compared with control strain.

Conclusions
The genome analysis of Elizabethkingia meningoseptica sp. F2 in conjunction with the comparative metabolic pathways analysis among the E.coli, Bacillus subtilis and Streptomyces provided a useful information on the Vitamin K2 biosynthetic pathway and other related pathways at systems level.

Background
Vitamin K is a group name for a family of related compounds, generally subdivided into phylloquinone K1(vitamin K1) and menaquinone K2 (Vitamin K2) [1]. Vitamin K2 is an essential lipid-soluble vitamin that play important roles in a number of vital physiolog-ical processes including haemostasis, calcium and bone metabolism, as well as cell growth regulation [2,3]. In addition, it can also functions as an electron carrier in prokaryotes [4]. Therefore, Vitamin K2 is receiving increasing attention in the domain of nutritional supplements for humans. Vitamin K2 refers to a group of menaquinones (MKs) varying in side chain length. Different forms of MKs are written as MK-n (Fig. S1), where n stands for the number of isoprene units in the side chain [5][6][7]. Till date, some of the most widely examined Vitamin K2 production microorganisms include Bacillus subtilis natto, Bacillus subtilis168, Elizabethkingia meningoseptica, Escherichia coli, and others.
Isolates of the genus Chryseobacterium, formerly Flavobacterium, named for its yellow pigment, are non-motile, non-fastidious, Gram-negative bacilli [8].The genus Elizabethkingia was established in 2005 based on an analysis of 16S rRNA gene sequences from strains within the family Flavobacteriaceae [9]. E. meningoseptica are most frequently isolated from soil, saltwater and freshwater and from dry and municipal water supplies including those which have been adequately chlorinated [10]. It has also been reported that E. meningoseptica is resistant to multiple antibiotics, such as extended-spectrum β-lactam agents and aminoglycosides [11,12] .
Mutational improvement of cells has contributed to the development of microbial processes for the MK production of Elizabethkingia meningoseptica [13][14][15][16]. Modular pathway engineering is another effective method for improving the biosynthesis of specific chemicals [17,18]. Nevertheless, the genome and Vitamin K2 relevant metabolic pathway of Elizabethkingia meningoseptica sp. F2 remain unclear. Despite the recent publication of several complete genomes of Elizabethkingia meningoseptica at NCBI database, no studies have yet used whole genome information more extensively in order to understand the Vitamin K2 biosynthetic pathway of Elizabethkingia meningoseptica. Genome sequencing and comparative analysis can help identify shared and speciesspecific Vitamin K2 in closely-related species.
In this study, We firstly studied Elizabethkingia meningoseptica F 2 using whole-genome sequencing (WGS) and compared it with other available strain genomes(microorganisms can be used to biosynthesize Vitamin K2). Specifically, the orthologous genes involved in the Vitamin K2 metabolic pathway were identified. Subsequently, the identification of Vitamin K2 metabolic pathway in Elizabethkingia meningoseptica sp. F2 were also accomplished. Moreover, the recombinant expression of the synthetic menaquinone pathway operons of Elizabethkingia meningoseptica F 2 increased the production of Vitamin K2 in our engineered strains. The information and materials presented in this study will be of great use in improving and modifying Elizabethkingia meningoseptica and other strains for the production of Vitamin K2.

Materials And Methods
Bacterial strains, plasmids, media and cultivation.
All microorganisms and plasmids used in this study are listed in Table S1. E. coli strain DH5α was used for plasmids construction and propagation. Elizabethkingia meningoseptica sp. F2 (CCTCC AB2011070) were as industrial strain for Vitamin K2 production. Plasmids pMBIS and pTric-hisA were used for pathway construction. Luria-Bertani (LB) media containing 10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract was used for plasmid propagation. The media and cultivation of Elizabethkingia meningoseptica sp. F2 were performed as previously described [19].
For extraction from bacterial biomass, bacteria were harvested by centrifugation at 12000 rpm for 15 min. The supernatant was discarded. For Vitamin K2 extraction from was extracted from the fermentation media, the method was adapted from Wei et al [20]. Vitamin K2 analysis was performed by HPLC(Shimadzu, Kyoto, Japan) using eclipse plus a C18 column (Shimadzu, 250 mm x 4.6 mm ID), and aliquots of 20 µL were injected manually using a loop injection valve (Shimadzu). In addition, the Resistance to tolerance test of Elizabethkingia meningoseptica sp. F2.
Previous studies revealed that the resistome of E. meningoseptica contain resistance genes and is resistant to most antimicrobial agents commonly [22][23][24]. Elizabethkingia meningoseptica sp. F2 were tested for antibiotic susceptibility refering to the Kirby-Bauer disk diffusion method as previously described [25]. To determine antimicrobial resistance concentrations, the different concentrations of six antimicrobial agents were assessed. The ranges of antimicrobial agents were as follow: Ampicillin General DNA manipulation was conducted according to standard protocols. DNA purification and plasmid isolation kits were purchased from Sangon Biotech (Shanghai, China). DNA manipulation enzymes and restriction enzymes were provided by Thermo Inc(ThermoScientific, USA). Primer sequences are listed in Table.S2. The structures of the plasmids are shown in Fig. S2. Identification of metabolic bottlenecks in the quinonoid ring modification pathway were done. The MenA ~ MenG gene was amplified from Elizabethkingia meningoseptica sp. F2. genome using primers as described in Table S2. And then PCR product was digested by Xhol and SacI and then inserted into the corresponded sites of pMBIS, formed pMBISMenA ~ pMBISMenG. The combinatorial overexpression of homologous genes were expressed under control of the strong induced promoter Ptrc in plasmid pTrc-hisA. All the plasmids were transferred into Elizabethkingia meningoseptica sp. F2 by heat shock transformation (Table S1). Then the constructs were confirmed by Sanger sequencing and all the strains were validated by colony PCR.  . 1). Our phylogenomic tree also showed a distinct separation of strains of into there different clusters. Moreover, this tree obviously indicated that Elizabethkingia meningoseptica strains are genetically distinct from the outgroup Elizabethkingia anophelis and Elizabethkingia miricola. The morphological appearance of cells was also studied using scanning electron microscopy as our previously described [19]. Similar results were observed that the cells are Gram-negative, non-motile, (0.5 × 1.0-2.0 µm, Fig.S3)non-spore-forming rods [26].
Sequencing and gene annotation of the Elizabethkingia meningoseptica sp. F2 genome.  Figure S4. According to the analysis of the sequencing samples, Circos [27] software was used to display the genome (Fig. 2), ncRNA, repetitive sequences, annotation information, methylation, GC content, GC skew and other information on the genome of sequencing strains. We compared the genomes of Eme.ATCC13253, Eme.NBRC12535, B.subtilis.natto, E.coli.K-12 to that of Elizabethkingia meningoseptica sp. F2 (All of microorganisms can be used to biosynthesize Vitamin K2) to identify any differences in their genomic content. Structure variation analysis could detect the evolution of homology genomes, for example, the location variations of gene clusters with similar function. Nucleic acid level synteny of those five strains (Fig. 3, S5). The level of vertical colour filling in Eme.ATCC13253 and Eme.NBRC12535 was the highest, which revealed the close relative of them and the absence of any species-specific regions. This result also supports the close relationship between these three strains. While whole-genome alignments of B.subtilis.natto and E.coli.K-12 to that of Elizabethkingia meningoseptica sp. F2 revealed without any levels of synteny. Similar results are observed from the distribution of protein identity of Eme.ATCC13253, Eme.NBRC1253, B.subtilis.natto, E.coli.K-12 with Eme.F 2 (Fig. S6). The clustering calculated with the COG function classification data in dispensable gene heat map (Fig. 4)   UbiE (Q-8/MK-8 biosynthesis methyltransferase) were found. Our data also support the fact of absent coenzyme Q in Elizabethkingia meningoseptica sp. F2 (Fig.S7, S8).  (Table 2). Our results also show that has some resistance genes are found in Elizabethkingia meningoseptica sp. F2 genomes( Figure S9), including those encode TolC family protein, multidrug ABC transporter, MFS transporter, multidrug SMR transporter, RND family efflux transporter MFP subunit, bleomycin resistance protein, outer membrane efflux protein, multidrug transporter, hydrophobic/ amphiphilic exporters etc. This observation is consistent with resistance to antibiotics in our tests.  Increasing expression of the menaquinone pathway.
As discussed in the genome-scale the Vitamin K2 metabolic network analysis section, overexpression of Men genes to increase precursor supply may be a useful strategy to improve Vitamin K2 production. To increase the intracellular concentration of precursor supplied to synthetise the Vitamin K2, a series of constitutive plasmids carrying different Men enzymes, pMBIS_MenA, pMBIS_MenB, pMBIS_MenC, pMBIS_MenD, pMBIS_MenE, pMBIS_MenF and pMBIS_MenG, were constructed for preliminary screening of key enzymes. To test the functionality of the homologous over expression pathway, Vitamin K2 production by recombinant strains was evaluated. The results (Table 3)indicate that constitutive overexpression of Men genes could strengthen metabolic flux flow into target product and slightly promote the accumulation of biomass compared with the control strain.
Elizabethkingia meningoseptica sp. F2 harboring pMBIS_MenC exhibited a slightly decrease in Vitamin K2 production due to the depression of cell mass. These modifications in pMBIS_MenA, pMBIS_MenG resulted in 19.13%, 22% respectively increase in Vitamin K2 production compared with control strain.
The improvements resulting from overexpression of the Men operon motivated us to examine the use of strong and inducible promoters and construct the MenA-MenG operon. And then the menA gene and menG gene was co-expressed in the plasmid pTrc-hisA to synthsis the final product Vitamin K2 from 1,4-Dihydroxy-2-naphthoate (DHNA), the Vitamin K2 production had further improved to 27.8 ± 0.91 mg/L (increased by 37% in comparison to control strain). However, the overexpression of these genes had an unobvious effect on the cell growth. Based on these results, enhancing metabolic flux from DHNA to menaquinone could be more effective for improving Vitamin K2 yield. Nevertheless, Engineering Elizabethkingia meningoseptica sp. F2 for Vitamin K2 production still have a lot of work to do. Therefore, the combination between overexpression key genes of menaquinone pathway(Module II) and modification of the mevalonate pathway(Module III) will also be explored in subsequent experiments.

Discussion
The 16S rRNA gene has been used for the classification of microorganism species [28,29]. In this context, Our strain can certainly be placed among Elizabethkingia meningoseptica species. In this study, a high-quality assembled genome of an industrially relevant Elizabethkingia meningoseptica sp. F2 used for Vitamin K2 production. This was achieved by a hybrid sequencing approach which  (Table S3), indicating their diverse genome evolution under differential selection [30]. Additionally, our results also show that Elizabethkingia meningoseptica sp.
F2 has some virulence factors and resistance genes (Table 1).
We were mostly interested in Vitamin K2 pathway related genes. Hence, Mining of the orthologous genes(KEGG database) involved in Vitamin K2 biosynthesis of other six species was also performed (Fig. 7), a comparative Vitamin K2 metabolic pathway approach was used in this study, in order to understand the gene cluster composition of different strains genome. The results strongly suggest that the mevalonate pathway of Elizabethkingia meningoseptica sp. F2 is unique. On account of previously metabolic pathway analysis demonstrated that the mevalonate pathway is mainly found in eukaryotes, whereas the MEP pathway is normally found in prokaryotes [31]. Interestingly, The gene menI was identified in the genome of Elizabethkingia meningoseptica and E. coli, whereas absent in other five strains. Unfortunately, PMK gene was not identified in the genome (Figure.7a). On the other hand, we also found that same emzyme GGPPS(geranylgeranyl diphosphate synthase) catalyze the synthesis of Geranylgeranyl diphosphate from IPP or DMAPP by three reactions. In the past, the MK-4, MK-5 and MK-6 of Elizabethkingia meningoseptica were identified as our described [20,32]. However, only geranylgeranyl diphosphate synthase and octaprenyl diphosphate synthase were identified, the hexaprenyl diphosphate synthase(catalyze farnesyl diphosphate to hexaprenyl diphosphate) was not found. As shown in Figure S10a, the mechanism of how to synthetize pentenyl diphosphate is remain unclear in this pathway.
Similar results were observed from the overexpression of 1,4-dihydroxy-2-naphthoate octaprenyltransferase(MenA) in B. subtilis 168 and E. coli increased the Vitamin K2 content, in which imply that the reaction of converting 1,4-Dihydroxy-2-naphthoate to 2-Demethylmenaquinone plays the most important role in increasing Vitamin K2 production [33][34][35]. Therefore, the enzyme MenA was listed as rate-limiting enzymes of the Menaquinone pathway.
It has been reported that Escherichia coli could synthesizes a naphthoquinone-type menaquinone-8(MK-8) under anaerobic conditions [35,36]. Corresponding to this result is IspB(octaprenyldiphosphate synthase, convert Famesyl-PP to Octaprenyl-PP) was identified ( Figure.7b). The 2Cmethyl-d-erythritol-4-phosphate (MEP) pathway consists of eight enzyme-catalyzed reactions in Escherichia coli [37]. Further details about MEP biosynthetic pathway can be found in FigureS10b. MEP pathway are not sufficient for the high level production of terpenoids in E. coli [38]. The components of the heterologous MVA pathway have been introduced into E. coli genomes for increase the supply of IPP and DMAPP [39]. Till date, B. subtilis168 and Bacillus subtilis natto has been identified as attractive hosts for the production of Vitamin K2( Figure.7c, g). De novo biosynthesis of Menaquinone-7 from glycerol in the metabolic engineered Bacillus subtilis 168, the MK-7 production had insignificant increase to 69.5 ± 2.8 mg/L upon 144 h fermentation in a 2 L baffled flask [34]. In addition, Berenjian et al. further increased menaquinone-7 concentration of 226 mg/L at 1,000 rpm, 5 vvm, 40 degrees C after 5 days of fermentation [40]. MK7 can exist as geometric isomers that can occur in the cis, trans, and cis/trans forms; however, only the all-trans from is biologically significant [41]. Those result is in accord with the fact of heptaprenyl diphosphate synthase(convert Famesyl-PP to Heptaprenyl-PP) were identified in Bacillus subtilis genomic DNA.
It is important to mention that Streptomyces possessed the futalosine pathway for menaquinone biosynthesis (Fig. 7d, S10c), encompassing seven enzymes encoded by the Mqn gene cluster [42]. Our Figure also show that the geranylgeranyl diphosphate synthase(four isoprene units) and heptaprenyl diphosphate synthase(seven isoprene units) are found in Streptomyces genomes, which is not consistent with previous studies have shown that Streptomyce possess MK-9 as the major menaquinone component [43]. It was believed that Streptomyces possessed a mevalonate pathway or MEP pathways in different species [44][45][46][47]. This issues of Nocard's bacillus and Mycobacterium tuberculosis were few previously described in detail. In Mycobacterium strains, the predominant menaquinones had nine isoprenoid units with one double bond hydrogenated (dihydromenaquinones), abbreviated as MK-9 while Nocardia strains predominantly contained tetrahydromenaquinones MK-8 [48]. Interestingly, it must be noted that Nocard's bacillus possessed MVA pathway and MEP pathway at same time in one strain(Figure.7e). Table 4, the analysis of MKn among different strains reveal that almost every strain produces multiple types of MK and a certain type of MK accounts for the predominant proportion [20,43,[49][50][51][52]. In addition, a confusing phenomenon were observed in view of the above analysis: theoretical types of MK n were not consistent with experimental values. Hence, the relationship between the length of side chain and the type of diphosphate synthase still need to be explored in detail.

Availability of data and materials
All data supporting the conclusions of this study are included in this article and the additional file.

Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication
Not applicable. Figure 1 Phylogenetic relationship among the Elizabethkingia species strains based on 16S rRNA gene sequences. The Phylogenetic tree was made with NCBI BLAST pairwise alignments using the Fast Minimum Evolution method. Strand ncRNA 6. Repeat 7. GC 8. GC-SKEW synteny.Yellow box stands for forward chain and blue box stands for reverse chain within the upper and following sequence region. In the box of sequence, the yellow region stands for the nucleic acid sequence in the forward chain of this genome sequence and the blue region stands for the nucleic acid sequence in the reverse chain of this genome sequence.

Figures
In the middle region of two sequences, the yellow line stands for forward alignment and the blue line stands for reverse complementary alignment.   Venn graph of Pan gene.Each ellipse represent one strain, the number in the ellipse means the only cluster number. One cluster have the genes that more than 50 percent identity and less than 0.3 length diversity.