Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z”

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles.     

Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis

Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, l-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the l-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

Production of ectoine through a combined process that uses both growing and resting cells of <Emphasis Type="Italic">Halomonas salina</Emphasis> DSM 5928<Superscript>T</Superscript>

Using ectoine-excreting strain Halomonas salina DSM 5928^T, we developed a new process for high-efficiency production of ectoine, which involved a combined process of batch fermentation by growing cells and production by resting cells. In the first stage, batch fermentation was carried out using growing cells under optimal fermentation conditions. The second stage was the production phase, in which ectoine was synthesized and excreted by phosphate-limited resting cells. Optimal conditions for synthesis and excretion of ectoine during batch fermentation in a 10 l fermentor were 0.5 mol l⁻¹ NaCl and an initial monosodium glutamate concentration of 80 g l⁻¹ respectively. The pH was adjusted to 7.0 and the temperature was maintained at 33°C. In phosphate-limited resting cells medium, monosodium glutamate and NaCl concentration was 200 g l⁻¹ and 0.5 mol l⁻¹, respectively, as well as pH was 7.0. The total concentration of ectoine produced was 14.86 g l⁻¹, the productivity and yield of ectoine was 7.75 g l⁻¹ day⁻¹ and 0.14 g g⁻¹, respectively, and the percentage of ectoine excreted was 79%. These levels of ectoine production and excretion are the highest reported to date.     

Biotechnological Coproduction of Compatible Solutes and Polyhydroxyalkanoates using the Genus Halomonas

An increased usage of poly‐β‐hydroxyalkanoates (PHA), for instance as bulk biodegradable and biocompatible plastics, will require a cheaper production and downstream processing. If the synthesis of this intracellular biopolyester could be combined with the production of another valuable intracellular product, the economic balance of the process could be improved. It was found that the moderately halophilic bacterium Halomonas elongata simultaneously synthesizes PHA and a protector molecule, called ectoine. Whereas the synthesis of PHA is a response to the shortage of nutrients, the production of ectoine counteracts osmotic imbalances. This behavior is in so far surprising as the conditions of a bi‐factorial stress initiate the fast simultaneous synthesis of ectoine and PHA. In the presence of 100 g/L NaCl, Halomonas elongata accumulated up to 50 % w/w PHA and up to 14 % ectoine within 2–3 days under so far non‐optimized conditions. Furthermore, it was found that other Halomonas species (e.g. Halomonas halodenitrificans and own isolates of Halomonas halodeneurihalina and Halomonas salina) were able to produce both ectoine and PHA.     

转录组学分析盐单胞菌四氢嘧啶合成代谢相关的表达差异基因与qRT-PCR验证

[目的]探究盐适应条件下坎帕尼亚盐单胞菌(Halomonas campaniensis)的差异基因表达水平,挖掘四氢嘧啶(ectoine)合成代谢相关联的差异基因.[方法]设置无盐组NS(0 mol/LNaCl)、中盐组 MS(1.5 mol/L NaCl)和高盐组 HS(2.5 mol/L NaCl),培养H.cam...     

A process for the production of ectoine and poly(3-hydroxybutyrate) by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH₄⁺, and PO₄³⁻. In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.     

The role of trehalose as a substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris)

The halophilic phototrophic bacterium Ectothiorhodospira halochloris is able to synthesize both nitrogen-containing (betaine, ectoine) and nitrogen-free (trehalose) compatible solutes. In the absence of external ammonium and under nitrogen-limited growth conditions ectoine was metabolized and trehalose partly replaced betaine. The cytoplasmic trehalose concentration did not exceeded 0.5 mol/kg water (approx. 30% of total compatible solutes). A decreasing content of betaine in cells growing under nitrogen limitation is a result of decreased biosynthesis. Apparently, the betaine pool cannot be used as a nitrogen source, not even in a situation of total nitrogen depletion.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

<Emphasis Type="Italic">Methylophaga muralis</Emphasis> Bur 1, a haloalkaliphilic methylotroph isolated from the Khilganta soda lake (Southern Transbaikalia,Buryat Republic)

A haloalkaliphilic restricted facultative methylotroph, strain Bur 1, which used methanol, methylated amines, and fructose as carbon and energy sources, was isolated from the soda Lake Khilganta (Buryat Republic, Russia). The cells were gram-negative non-spore-forming, motile rods reproducing by binary fission. The organism was aerobic, reduced nitrates to nitrites. Growth occurred at temperatures from 4 to 37°C (optimum at 25–29°C), pH 7.5–10.5 (optimum at 8.5–9.5), and NaCl concentration in the medium from 0.05 to 10.0% NaCl (optimum at 3–4%). Ectoine, glutamate, and sucrose were accumulated as osmoprotectants. Activity of the enzymes of de novo ectoine biosynthesis were detected. The organism utilized C₁ compounds via the KDPG variant of the ribulose monophosphate pathway. The DNA G + C content was 44.67 mol %. Based on the similarity of the 16S rRNA gene sequences (94.7–99.1%) and the results of DNA–DNA hybridization (24–74%) with type strains of the neutrophilic and alkaliphilic Methylophaga species, the isolate was identified as Methylophaga muralis Bur 1 (VKM B-3046 = DSM 103617). The genome of M. muralis Bur 1 contained 2585 protein-encoding genes; 634 proteins with unidentified functions were predicted. Three rRNAs (5S, 16S, and 23S) and 38 tRNAs were identified. Apart from the mxaFJGIRSACKLDEH classical cluster of methanol oxidation genes, the xoxF gene was found. Methylamine was oxidized to formaldehyde by methylamine dehydrogenase and via the N-methylglutamate pathway. Orthologs of type III glutamine synthetases were revealed in the genome. The operons of ectoine and sucrose biosynthesis, ectRABC-ask and sps-spp-fruK-ams, were found. The genomes of M. muralis Bur 1 and M. lonarensis MPL^T, unlike that of M. nitratireducenticrescens JAM1^T, were found to contain the genes encoding the proteins of bicarbonate transport.     

Comparison of ectoine synthesis regulation in secreting and non-secreting strains of Halomonas

Ectoine is an osmotic pressure compatible solute. It is synthesized by Halomonas and other microorganisms in a hypertonic environment. As a stabilizing agent of cells proteins, nucleic acids and other biological products, ectoine has wide applications. Therefore, an efficient production method for ectoine is in great demand. Ectoine is overproduced by Halomonas salina DSM 5928, an ectoine-secreting strain, in which the synthesis of ectoine is not limited by its intracellular threshold concentration. In order to explain the mechanism of secretion of ectoine, the response to NaCl stress, and the release and uptake kinetics of ectoine were compared between H. salina DSM 5928 and Halomonas elongata DSM 2581, a non-ectoine-secreting strain. Moreover, the ectoine binding protein TeaA from each of these two strains was cloned and expressed, and binding abilities were examined in vitro. The results indicated that H. salina DSM 5928 and H. elongata DSM 2581 respond to NaCl in the medium in different ways. Compared with H. elongata DSM 2581, the amount of ectoine released was higher and the uptake of ectoine under NaCl stress was lower in H. salina DSM 5928. In addition, the binding ability of TeaA to ectoine in H. salina DSM 5928 was also lower. These results reveal the secretion mechanism of ectoine as well as critical regulation and control factors involved in ectoine synthesis.     

新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

研究旨在克隆新的四氢嘧啶合成基因簇,并对其功能进行鉴定,为应用于四氢嘧啶的生产奠定基础。从新喀里多尼亚弧菌CGJ02-2中克隆获得四氢嘧啶合成基因簇ectABC,ectABC与表达载体pBAD连接后转化至大肠杆菌BW25113中,通过L-阿拉伯糖诱导表达。采用SDS-PAGE和液质联用鉴定重组表达蛋白,利用全细胞催化合成四氢嘧啶,通过高分辨质谱鉴定四氢嘧啶,并从天冬氨酸浓度、KCl浓度、温度和pH 4个方面优化催化条件。结果表明,来自新喀里多尼亚弧菌CGJ02-2基因组的ectABC大小为2 235 bp。SDS-PAGE显示表达产物中有3个重组蛋白产生,液质联用鉴定表明其分子量分别与ectA、ectB、ectC的理论分子量一致。高分辨质谱分析发现全细胞催化上清中有四氢嘧啶产生。优化后的最适全细胞催化条件为：天冬氨酸浓度100 mmol·L^-1,KCl浓度100 mmol·L^-1,温度30℃,pH 7.0,最优条件下产量为1.11 mg·mL^-1。研究从弧菌中克隆了四氢嘧啶合成基因簇ectABC,并在大肠杆菌BW2511...