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Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCC M 2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G 8 -52 genomes. The mutant strain G 8 -52 (CCTCC M 2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L −1 (0.65 g g −1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L −1 ) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G 8 -52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G 8 -52 produced a relatively stable ectoine yield (1.50 g L −1 ) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 ( lipA ) of the wild-type strain mutated to davT and gabD in strain G 8 -52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G 8 -52 is suitable for large-scale fermentation applications.

Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis , which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC , lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L −1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L −1 ectoine and 32 g L −1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine. Halomonas bluephagenesis is a halophilic platform bacterium for next generation industrial biotechnology. Here, the authors employ a stimulus response-based flux-tuning method for coproduction of bioplastic PHB and ectoine under open unsterile and continuous growth conditions.

Background Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. Results Here , we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia , reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C.   glutamicum Ptuf ectD PST achieved a titre of 74 g L −1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABC opt , was successfully achieved without intermediate purification. Conclusions C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.

Background Halomonas species have recently emerged as promising chassis organisms for next-generation industrial biotechnology, due to their ability to thrive under high-salt conditions, where most microorganisms cannot survive. This feature minimizes contamination risks, thus enabling cultivation under open, unsterile conditions. In addition, many Halomonas species naturally produce large amounts of the bioplastic polyhydroxybutyrate and the high-value osmolyte ectoine. Main text This review explores the development of genetic manipulation tools and their pivotal role in establishing the genus Halomonas as an industrial chassis. Key additions to the synthetic biology toolbox, including cloning vectors, genetic parts, and genome editing systems are highlighted, along with challenges faced for their adoption, such as difficulties in transformation. In addition, we showcase how these tools have been employed for the development of more robust, high-producing strains through metabolic engineering, as well as for expanding the portfolio of target metabolites produced by Halomonas . Conclusion Recent developments in synthetic biology tools and metabolic engineering highlighted in this review underscore the potential of Halomonas for large scale metabolite production and provide a promising outlook towards their role as a microbial chassis in industrial biotechnology.

Osmotic adaptation and accumulation of compatible solutes is a key process for life at high osmotic pressure and elevated salt concentrations. Most important solutes that can protect cell structures and metabolic processes at high salt concentrations are glycine betaine and ectoine. The genome analysis of more than 130 phototrophic bacteria shows that biosynthesis of glycine betaine is common among marine and halophilic phototrophic Proteobacteria and their chemotrophic relatives, as well as in representatives of Pirellulaceae and Actinobacteria, but are also found in halophilic Cyanobacteria and Chloroherpeton thalassium. This ability correlates well with the successful toleration of extreme salt concentrations. Freshwater bacteria in general lack the possibilities to synthesize and often also to take up these compounds. The biosynthesis of ectoine is found in the phylogenetic lines of phototrophic Alpha- and Gammaproteobacteria, most prominent in the Halorhodospira species and a number of Rhodobacteraceae. It is also common among Streptomycetes and Bacilli. The phylogeny of glycine-sarcosine methyltransferase (GMT) and diaminobutyrate-pyruvate aminotransferase (EctB) sequences correlate well with otherwise established phylogenetic groups. Most significantly, GMT sequences of cyanobacteria form two major phylogenetic branches and the branch of Halorhodospira species is distinct from all other Ectothiorhodospiraceae. A variety of transport systems for osmolytes are present in the studied bacteria.

A novel Gram-stain-negative, aerobic rod-shaped bacterial strain, which was catalase- and oxidase-positive, designated as DMV24BSW_D T , was isolated from the estuarine waters of the Bhavnagar (India) coast of the Arabian Sea. Its 16S rRNA gene exhibited 99.52% similarity with Neopusillimonas maritima 17-4A T , followed by 97.95% similarity with the Pusillimonas caeni strain EBR-8–1 and 97.4% similarity with the P. noertemannii strain BN9 T . Phylogenomic analysis using BPGA (14,332 aa) and UBCG (90,261 bp) tools revealed a unique phylogenetic position within the genus Neopusillimonas . The genome exhibited a G + C content of 53.25%. In comparison with N. maritima 17-4A T , the strain demonstrated an average nucleotide identity (ANIb) of 94.47% and a digital DNA-DNA hybridization (dDDH) value of 60.1%, indicating distinct genomic divergence. The genome of DMV24BSW_D T contains several unique metabolic genes that facilitate efficient electron transfer during aerobic respiration. Additionally, it harbours one intact prophage and four defective prophages, indicating ongoing viral interactions. The genome encodes a complete pathway for ectoine biosynthesis and transportation. Strain DMV24BSW_D T tested positive for gelatin hydrolysis and demonstrated the ability to utilize a wide range of carbohydrates, including α-D-glucose, D-melibiose, D-fructose, L-rhamnose, and various organic acids, such as methyl pyruvate and propionic acid, along with tolerance to fluctuating pH (5 to 10) and salinity (0–4% NaCl). The major polar lipids included phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine, while fatty acid analysis revealed C 12:0 , C 16:0 , C 17:0 cyclo, and summed feature 2 (C 12:0 aldehyde/unknown) as major components. The respiratory quinones identified were MK-7 and MK-8. These comprehensive phenotypic, chemotaxonomic, and genomic characteristics support the unique taxonomic position of DMV24BSW_D T within the genus Neopusillimonas and the proposal of a novel species of the genus Neopusillimonas, for which the name Neopusillimonas aestuarii sp. nov. (Type strain DMV24BSW_D T  = MCC 2506  T  = KCTC 72453  T  = JCM 34508  T ) is proposed.

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a “bacterial milking” process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina . However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

Computational investigations into the structure and function of metalloenzymes with transition metal cofactors require proper preparation of the model, which requires obtaining reliable force field parameters for the cofactor. Here, we present a test case where several methods were used to derive amber force field parameters for a bonded model of the Fe(II) cofactor of ectoine synthase. Moreover, the spin of the ground state of the cofactor was probed by DFT and post-HF methods, which consistently indicated the quintet state is lowest in energy and well separated from triplet and singlet. The performance of the obtained force field parameter sets, derived for the quintet spin state, was scrutinized and compared taking into account metrics focused on geometric features of the models as well as their energetics. The main conclusion of this study is that Hessian-based methods yield parameters which represent the geometry around the metal ion, but poorly reproduce energy variance with geometrical changes. On the other hand, the energy-based method yields parameters accurately reproducing energy-structure relationships, but with bad performance in geometry optimization. Preliminary tests show that admixing geometrical criteria to energy-based methods may allow to derive parameters with acceptable performance for both energy and geometry.

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients.

Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.