Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

The formation of hydroxyectoine in the industrial ectoine producerHalomonas elongata was improved by the heterologous expressionof the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus.The efficient conversion of ectoine to hydroxyectoine was achievedby the concerted regulation of thpD by the H. elongata ectApromoter.

INTRODUCTION

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Introduction

Under hyperosmotic stress, a variety of microorganisms accumulateorganic low-molecular-weight compounds such as polyols, aminoacids, sugars, betaines, and ectoine. These so-called compatiblesolutes enable the organisms to survive under conditions ofhigh osmotic pressure (2). Since it has been demonstrated thatcompatible solutes, especially ectoine, are able to protectenzymes, membranes, and whole cells against stresses causedby exposure to salt, heating, freezing, and desiccation, thereis an increasing interest in the use of compatible solutes forvarious applications in biotechnology (8, 12, 21, 20). Today,ectoine is already used as a moisturizer in cosmetics and skincare products (RonaCare Ectoin; Merck KGaA, Darmstadt, Germany)(12). In addition, ectoine's hydroxyl derivative, hydroxyectoine,has gained attention as a protein-protecting agent which, ina number of applications, has properties superior to those ofectoine (8, 4). Moreover, hydroxyectoine has also been investigatedas a protectant of healthy cells during chemotherapy (1). Thus,the development of hydroxyectoine as a by-product of ectoineis foreseeable.

The biotechnological production of ectoine, a process calledbacterial milking, is carried out with the halophilic gram-negativebacterium Halomonas elongata, which responds to a hypoosmoticshock by the rapid release of the accumulated solutes (15).After growth at 25°C, the typical product solution consistsof more than 93% ectoine, 4% of the ectoine precursor N-acetyl-diaminobutyricacid, and only traces of glutamate, alanine, and hydroxyectoine.However, the relative proportion of hydroxyectoine can be increasedto 50% by using considerably higher salinities or elevated temperatures,factors increasing costs for both the equipment and the process(15). Moreover, the costly separation of ectoine and hydroxyectoineis still necessary. Therefore, the biotechnological productionof hydroxyectoine is carried out with the gram-positive Marinococcussp. strain M52, which produces predominantly hydroxyectoinebut is less amenable to the milking process than H. elongata.

The in vivo formation of hydroxyectoine has been described fora variety of bacteria (7, 10, 18). The in vitro conversion ofectoine to hydroxyectoine by a specific hydroxylase from Streptomyceschrysomallus ATCC 11523 (Fig. 1) has been shown previously (5,6). The corresponding gene (thpD) has been shown to be partof the ectoine-hydroxyectoine gene cluster as shown in Fig.2. Unlike the streptomycetes, the moderately halophilic bacteriaH. elongata and Marinococcus halophilus lack a thpD-like genein the ect gene cluster (3, 9).

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FIG. 1. Reaction catalyzed by ThpD, the ectoine hydroxylase from S. chrysomallus.

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FIG. 2. (A) Map of the ectoine and hydroxyectoine biosynthesis genes of H. elongata (ect) and S. chrysomallus (thp). The thpD gene coding for the S. chrysomallus ectoine hydroxylase, which was transferred to H. elongata, is shown in black. (B) Mobilizable pHE1-derived shuttle vectors for the expression of the S. chrysomallus thpD gene in H. elongata. The ectA promoter region is indicated by small arrows.

For the industrial production of hydroxyectoine, a microorganismwith a broad salt tolerance is favored to perform the bacterialmilking process (15). Since H. elongata is already used forthe industrial production of ectoine, there would be commercialinterest in broadening its industrial application. We thereforeexpressed the ectoine hydroxylase gene of S. chrysomallus, thpD,in H. elongata to improve this industrially used ectoine producerso that it may serve as a hydroxyectoine producer.

Construction of H. elongata expression plasmids.

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Construction of h. elongata...

For expression of the thpD gene, we assembled an autonomousreplicating plasmid for H. elongata by combining the Escherichiacoli pUC derivative pSP72 (Promega) with the H. elongata plasmidpHE1 (17) by the insertion of a spectinomycin resistance cassettefor selection in Halomonas and an oriT gene for plasmid transfer.First, oriT of Pseudomonas aeruginosa was PCR amplified as an0.8-kb PstI cassette from plasmid pHM10a (11) with oligonucleotidesprim1a (5'-ATT ACT GCA GTC GGT CTT GCC TTG CTC GTC GG-3') andprim1b (5'-TTT CTG CAG TGC ATA ACC CTG CTT CGG GG-3'), subclonedinto plasmid pLITMUS28 (New England Biolabs) cleaved with NsiI,reisolated as a BglII-XhoI fragment, and cloned into pSP72 cleavedwith BamHI and XhoI. The next step was insertion of the spectinomycincassette, which was obtained as an EcoRI fragment from plasmidpHP45omega (14), and insertion of the BglII-linearized plasmidpHE1 into the respective sites of the pSP72 backbone. The resultantplasmid, pJP-0, harbors a single PstI site located in the pHE1region, which was used for inserting two different thpD genecassettes (Fig. 2B). A promoterless thpD gene cassette was generatedby PCR from a genomic thpD clone (plasmid pAF1sub10) (unpublisheddata) with oligonucleotide prim2a (5'-GCC GAA TTC CAT ATG ACCACC GAA GTA CGC GCC GAT-3'), introducing an NdeI site encompassingthe ATG translation start codon of the thpD stop codon, andwith oligonucleotide prim2b (5'-AGC GAA TTC CCC TGC AGG GGCCGG GAC GGC GTA CCC GTC CCG G-3'), introducing a PstI and EcoRIsite about 50 bp downstream of the thpD gene. The NdeI-EcoRIfragment was subcloned into pSL1180 (Amersham Biosciences) cleavedwith NdeI and EcoRI, thus maintaining untouched the PCR-introducedPstI site downstream of thpD and generating a second PstI siteupstream of thpD located in the pSL1180 polylinker. We alsogenerated a promoter-driven variant of the thpD gene cassetteby inserting the ectA promoter of H. elongata DSM 3043 as a250-bp NotI-NdeI fragment immediately upstream of thpD by usinga single NotI site of the pSL1180 polylinker. The ectA promoterwas PCR amplified from genomic DNA with oligonucleotides prim3a(5'-CGG GGA TCC GCG CCG ACG AGC GCT CGA TCG-3') and prim3b (5'-AGCGAA TTC CCC TGC AGG GGC CGG GAC GGC GTA CCC GT-3') to generaterestriction sites. All PCR-generated fragments were sequencedto confirm the entire DNA sequence. Insertion of these thpDgene cassettes into the PstI site of plasmid pJP-0 generatedplasmids pJP-2 (thpD with ectA promoter) and pJP-1 (promoterlessthpD gene) (Fig. 2B). In both plasmids, the orientation of theinserted thpD gene was chosen in such a way that a known pHE1promoter region (16) providing basal gene expression was alwayslocated upstream of the thpD gene cassette. Thus, we also expectedthe promoterless thpD gene in pJP-1 to be expressed to someextent.

Plasmids were transferred from E. coli S17-1 to H. elongataby conjugation. To eliminate E. coli cells after plasmid transfer,a spontaneous rifampin-resistant mutant of H. elongata DSM 304was isolated by serial plating on solid medium saline (SWYE)containing rifampin (25 µl/ml) (17, 13). Comparison ofthe resulting rifampin-resistant strain H. elongata AD-98 withthe wild-type strain H. elongata DSM 3043 did not reveal anydissimilarities regarding the production of ectoine or hydroxyectoine,even in the presence of plasmid pJP-D-0 (data not shown). Wetherefore routinely used H. elongata AD-98 for studying theeffect of the introduction of thpD on the formation of hydroxyectoinein that strain.

ThpD-dependent hydroxyectoine production in H. elongata.

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Thpd-dependent hydroxyectoine...

To determine the effect of the thpD gene on the formation ofhydroxyectoine, H. elongata/pJP-2 (thpD with ectA promoter)and H. elongata/pJP-1 (promoterless thpD) were cultivated at30°C in 10 ml of SWYE medium in 100-ml flasks, with shakingat 200 rpm, to an optical density at 600 nm of 2.4 to 2.6. Bacterialmilking was carried out by lowering the salt concentration from10 to 2% (wt/vol) with distilled water. An aliquot of the cell-freesupernatant was incubated at 95°C for 15 min, cleared bycentrifugation, and subjected to high-pressure liquid chromatography(HPLC) analysis (LiChroCART 250-4, LiChrospher 100 NH₂; 5-µmparticle size with 70% acetonitrile as the eluent). Ectoineand hydroxyectoine were quantified by using commercial ectoineand hydroxyectoine standards (Biomol). At 30°C and with10% (wt/vol) salts, H. elongata/pJP-2 converted 76% of the ectoineinto hydroxyectoine with a yield of 340 nmol of hydroxyectoineper ml (Fig. 3B, left chromatogram). In contrast, the controlstrain H. elongata/pJP-0 (plasmid without insert) did not convertsignificant amounts of ectoine into hydroxyectoine under theseconditions (Fig. 3A, left chromatogram). Also, in the case ofH. elongata/pJP-1, none or only traces (<3%) of the ectoinewere converted into hydroxyectoine. These results show thatthe thpD gene of S. chrysomallus was actively expressed in H.elongata in the presence of a suitable promoter.

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FIG. 3. HPLC detection of ectoine (E) and hydroxyectoine (HE) produced by H. elongata strains containing plasmid pJP-0 (A) and pJP-2 (B). After growth of H. elongata at the indicated temperatures, ectoine and hydroxyectoine were released by hypoosmotic shock and subjected to HPLC analysis.

Increased yields of hydroxyectoine have been described for H.elongata at elevated temperatures and higher salinities (19).We therefore measured the hydroxyectoine formation in the transformedstrains at 37°C (instead of the 30°C described above)and 10% (wt/vol) salts. As expected, the control strain H. elongata/pJP-0produced significantly higher amounts of hydroxyectoine (37%)but still less than the ectoine produced (63%) (Fig. 3A, rightchromatogram). In contrast, strain H. elongata/pJP-2 (thpD withectA promoter) produced exclusively hydroxyectoine (100%) (Fig.3B, right chromatogram). No ectoine could be detected underthese growth conditions. Remarkably, the introduction of thepromoterless thpD gene (by plasmid pJP-1), driven only by anintrinsic pHE1 promoter (16), also resulted in an enhanced formationof hydroxyectoine (80%). Ectoine (20%), however, was still presentunder these conditions (Table 1). Complete conversion of ectoineto hydroxyectoine was also observed when the P_ectA-thpD cassettewas in the opposite orientation (pJP-2R) (Table 1). This clearlyindicates that the P_ectA-thpD cassette alone is well suitedfor the industrial production of hydroxyectoine.

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TABLE 1. Hydroxyectoine formation by H. elongata strains at 37°C

The economic benefit of the bacterial milking process, comparedto fed-batch cultures, implies that at least two cycles of compatiblesolute extraction have to be applied (15). We therefore studiedthe formation of hydroxyectoine in the absence of the selectionmarker streptomycin at 37°C. Cultures were subjected tohypoosmotic shocks and used as seed culture every 24 h overa period of 9 days. After three cycles of bacterial milking,the amount of hydroxyectoine dropped from 100 to 96%. Prolongedcultivation led to wild-type levels of hydroxyectoine (36%)after nine cycles. In parallel, aliquots of cultures were usedto assay the amount of plasmid still present. The results ofthe plasmid quantification indicate that pJP-2 is not stableunder nonselective conditions. However, since the milking processcan be repeated at least two times without the detectable appearanceof ectoine, the genetically engineered H. elongata strain maybe valuable for future biotechnological applications.

In this study, we showed the functional expression of the ectoinehydroxylase gene of the gram-positive bacterium S. chrysomallus,thpD, in the gram-negative host H. elongata. Introduction ofthe P_ectA-thpD gene cassette greatly stimulates production ofhydroxyectoine from ectoine at 30°C and completely convertsall ectoine to hydroxyectoine at 37°C. The efficient conversionof ectoine to hydroxyectoine is achieved by the concerted regulationof thpD via the H. elongata ectA promoter. Although the thpDexpression plasmid is not stable under nonselective conditions,the results suggest that the bacterial milking process may beapplicable for the industrial production of hydroxyectoine.To this end, we plan to transfer the thpD expression cassettestably into the genome of H. elongata.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The nucleotide sequence of the thp genes from S. chrysomallus(ATCC 11523) has been deposited in the GenBank nucleotide sequencedatabase under accession number AY524544.

FOOTNOTES

* Corresponding author. Mailing address: Institut für Chemie, Fachgebiet Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, 10587 Berlin, Germany. Phone: 49-30-31425653. Fax: 49-30-31424783. E-mail: ullrich.keller{at}tu-berlin.de.

REFERENCES

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