Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation.

Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Proteomic insights into the response of Halomonas sp. MNB13 to excess Mn(â ¡) and the role of H2S in Mn(â ¡) resistance.

Halomonas spp. are moderately halophilic bacteria with the ability to tolerate various heavy metals. However, the role of basic cellular metabolism, particularly amino acid metabolism, has not been investigated in Halomonas spp. under excess Mn(Ⅱ). The strain Halomonas sp. MNB13 was isolated from a deep-sea ferromanganese nodule and can tolerate 80 mM Mn(Ⅱ). To comprehensively explore the mechanisms underlying its resistance to excess Mn(Ⅱ), we conducted a comparative proteome analysis. The data revealed that both 10 mM and 50 mM Mn(Ⅱ) significantly up-regulated the expression of proteins involved in Mn(Ⅱ) transport (MntE), oxidative stress response (alkyl hydroperoxide reductase and the Suf system), and amino acid metabolism (arginine, cysteine, methionine, and phenylalanine). We further investigated the role of cysteine metabolism in Mn(Ⅱ) resistance by examining the function of its downstream product, H2S. Consistent with the up-regulation of cysteine desulfurase, we detected an elevated level of H2S in Halomonas sp. MNB13 cells under Mn(Ⅱ) stress, along with increased intracellular levels of H2O2 and O2•-. Upon exogenous addition of H2S, we observed a significant restoration of the growth of Halomonas sp. MNB13. Moreover, we identified decreased intracellular levels of H2O2 and O2•- in MNB13 cells, which coincided with a decreased formation of Mn-oxides during cultivation. In contrast, in cultures containing NaHS, the residual Mn(Ⅱ) levels were higher than in cultures without NaHS. Therefore, H2S improves Mn(Ⅱ) tolerance by eliminating intracellular reactive oxygen species rather than decreasing Mn(Ⅱ) concentration in solution. Our findings indicate that cysteine metabolism, particularly the intermediate H2S, plays a pivotal role in Mn(Ⅱ) resistance by mitigating the damage caused by reactive oxygen species. These findings provide new insights into the amino acid mechanisms associated with Mn(Ⅱ) resistance in bacteria.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Efficient polyhydroxybutyrate production using acetate by engineered Halomonas sp. JJY01 harboring acetyl-CoA acetyltransferase.

Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Construction of a Stable Expression System Based on the Endogenous hbpB/hbpC Toxin-Antitoxin System of Halomonas bluephagenesis.

Halomonas bluephagenesis is a halophilic bacterium capable of efficiently producing polyhydroxyalkanoates and other valuable chemicals through high salinity open fermentation, offering an appealing platform for next-generation industrial biotechnology. Various techniques have been developed to engineer Halomonas bluephagenesis, each with its inherent shortcomings. Genome editing methods often entail complex and time-consuming processes, while flexible expression systems relying on plasmids necessitate the use of antibiotics. In this study, we developed a stable recombinant plasmid vector, pHbPBC, based on a novel hbpB/hbpC toxin-antitoxin system found within the endogenous plasmid of Halomonas bluephagenesis. Remarkably, pHbPBC exhibited exceptional stability during 7 days of continuous subculture, eliminating the need for antibiotics or other selection pressures. This stability even rivaled genomic integration, all while achieving higher levels of heterologous expression. Our research introduces a novel approach for genetically modifying and harnessing nonmodel halophilic bacteria, contributing to the advancement of next-generation industrial biotechnology.

Genome analysis of haloalkaline isolates from the soda saline crater lake of Isabel Island; comparative genomics and potential metabolic analysis within the genus Halomonas.

BACKGROUND: Isabel Island is a Mexican volcanic island primarily composed of basaltic stones. It features a maar known as Laguna Fragatas, which is classified as a meromictic thalassohaline lake. The constant deposition of guano in this maar results in increased levels of phosphorus, nitrogen, and carbon. The aim of this study was to utilize high-quality genomes from the genus Halomonas found in specialized databases as a reference for genome mining of moderately halophilic bacteria isolated from Laguna Fragatas. This research involved genomic comparisons employing phylogenetic, pangenomic, and metabolic-inference approaches. RESULTS: The Halomonas genus exhibited a large open pangenome, but several genes associated with salt metabolism and homeostatic regulation (ectABC and betABC), nitrogen intake through nitrate and nitrite transporters (nasA, and narGI), and phosphorus uptake (pstABCS) were shared among the Halomonas isolates. CONCLUSIONS: The isolated bacteria demonstrate consistent adaptation to high salt concentrations, and their nitrogen and phosphorus uptake mechanisms are highly optimized. This optimization is expected in an extremophile environment characterized by minimal disturbances or abrupt seasonal variations. The primary significance of this study lies in the dearth of genomic information available for this saline and low-disturbance environment. This makes it important for ecosystem conservation and enabling an exploration of its biotechnological potential. Additionally, the study presents the first two draft genomes of H. janggokensis.

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production / El análisis genómico comparativo de cepas mutadas por radiación ultravioleta y de tipo salvaje de Halomonas campaniensis revela diferencias genómicas asociadas con una mayor producción de ectoína

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 (CCTCCM2019776) 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 G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) 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 G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 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 G8-52 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 G8-52 is suitable for large-scale fermentation applications.(AU)

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution.

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Valorization of lignin-derived compounds into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by engineered Halomonas sp. Y3.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biopolyester with great potential, but its high production cost via the propionate-dependent pathway has hindered its development. Herein, we engineer Halomonas sp. Y3 to achieve efficient conversion of various LDCs into PHBV without propionate supplement. Initially, we successfully achieve PHBV production without propionate supplement by overexpressing threonine synthesis. The resulting biopolyester exhibits a 3 HV proportion of up to 7.89 mol%, comparable to commercial PHBV (8 mol%) available from Sigma Aldrich (403105). To further enhance PHBV production, we rationally design the reconstruction of aromatic compound catabolism. The engineered strain Y3_18 efficiently assimilates all LDCs containing syringyl (S), guaiacyl (G), and p-hydroxyphenyl-type (H) units. From 1 g/L of S-, G-, and H-type LDCs, Y3_18 produces PHBV at levels of 449 mg/L, 488 mg/L, and 716 mg/L, respectively, with yields of 44.9 % (g/g), 48.8 % (g/g), and 71.6 % (g/g). Moreover, to improve PHBV yield from lignin, we integrate laccase-secretion and PHBV production modules. This integration leads to the accumulation of 425.84 mg/L of PHBV with a yield of 21.29 % (g/g) and a 3 HV proportion of 6.38 mol%. By harnessing the capabilities of Halomonas sp. Y3, we demonstrate an efficient and sustainable approach for PHBV production from a variety of LDCs.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrification-aerobic denitrification bacterium Halomonas sp. DN3.

Recently, the functional microorganisms capable of eliminating nitrogenous waste have been applied in mariculture systems. As a potential candidate for treating mariculture wastewater, strain DN3 eliminated 100% of ammonia and nitrate and 96.61%-100% of nitrite within 72 h, when single nitrogen sources at concentrations of 0-50 mg/L. Strain DN3 also exhibited the efficient removal performance of mixed-form nitrogen (ammonia, nitrate, and nitrite) at salinity 30 ‰, C/N ratio 20, and 180 rpm. The nitrogen assimilation pathway dominated inorganic nitrogen metabolism, with less nitrogen (14.23%-25.02% of TN) lost into the air via nitrification and denitrification, based on nitrogen balance analysis. Moreover, the bacterial nitrification pathway was explored by enzymatic assays and inhibition assays. These complex nitrogen assimilation and dissimilation processes were further revealed by bacterial genome analysis. These results provide important insight into nitrogen metabolism of Halomonas sp. and theoretical support for treating mariculture wastewater with strain DN3.

Machine learning methods for predicting the key metabolic parameters of Halomonas elongata DSM 2581 T.

Ectoine is generally produced by the fermentation process of Halomonas elongata DSM 2581 T, which is one of the primary industrial ectoine production techniques. To effectively monitor and control the fermentation process, the important parameters require accurate real-time measurement. However, for ectoine fermentation, three critical parameters (cell optical density, glucose, and product concentration) cannot be measured conveniently in real-time due to time variation, strong coupling, and other constraints. As a result, our work effectively created a series of hybrid models to predict the values of these three parameters incorporating both fermentation kinetics and machine learning approaches. Compared with the traditional machine learning models, our models solve the problem of insufficient data which is common in fermentation. In addition, a simple kinetic modeling is only applicable to specific physical conditions, so different physical conditions require refitting the function, which is tedious to operate. However, our models also overcome this limitation. In this work, we compared different hybrid models based on 5 feature engineering methods, 11 machine-learning approaches, and 2 kinetic models. The best models for predicting three key parameters, respectively, are as follows: CORR-Ensemble (R2: 0.983 ± 0.0, RMSE: 0.086 ± 0.0, MAE: 0.07 ± 0.0), SBE-Ensemble (R2: 0.972 ± 0.0, RMSE: 0.127 ± 0.0, MAE: 0.078 ± 0.0), and SBE-Ensemble (R2:0.98 ± 0.0, RMSE: 0.023 ± 0.001, MAE: 0.018 ± 0.001). To verify the universality and stability of constructed models, we have done an experimental verification, and its results showed that our proposed models have excellent performance. KEY POINTS: • Using the kinetic models for producing simulated data • Through different feature engineering methods for dimension reduction • Creating a series of hybrid models to predict the values of three parameters in the fermentation process of Halomonas elongata DSM 2581 T.

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production.

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 (CCTCCM2019776) 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 G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) 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 G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 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 G8-52 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 G8-52 is suitable for large-scale fermentation applications.

Non-sterilized conversion of whole lignocellulosic components into polyhydroxybutyrate by Halomonas sp. Y3 with a dual anti-microbial contamination system.

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is challenging due to the need for whole components and energy-effective conversion. Herein, Halomonas sp. Y3, a ligninolytic bacterium with the capacity to produce PHB from lignin and cellulose- and hemicellulose-derived sugars, is employed to explore its feasibility. This strain shows high sugar tolerance up to 200 g/L of glucose and 120 g/L of xylose. A dual anti-microbial contamination system (DACS) containing alkali-halophilic system (AHS) and phosphite-urea system (PUS) is presented, successfully achieving a completely aseptic effect and resulting in a total of 8.2 g of PHB production from 100 g bamboo biomass. We further develop a stage-fed-batch fermentation to promote the complete utilization of xylose. Approximately 69.99 g of dry cell weight (DCW) and 46.45 g of PHB with 66.35 % are obtained from a total of 296.58 g of sugars and 5.70 g of lignin, showing a significant advancement for LCB bioconversion. We then delete the native phosphate transporters, rendering the strain unable to grow on phosphate-loaded media, effectively improving the strain biosafety without compromising its ability to produce PHB. Overall, our findings demonstrate the potential of Y3 as a classic bacterium strain for PHB production with potential uses in industry.

Exploring the potential of Halomonas levan and its derivatives as active ingredients in cosmeceutical and skin regenerating formulations.

Demand on natural products that contain biological ingredients mimicking growth factors and cytokines made natural polysaccharides popular in pharmaceutical and cosmetic industries. Levan is the ß-(2-6) linked, nontoxic, biocompatible, water-soluble, film former fructan polymer that has diverse applications in pharmacy and cosmeceutical industries with its moisturizing, whitening, anti-irritant, anti-aging and slimming activities. Driven by the limited reports on few structurally similar levan polymers, this study presents the first systematic investigation on the effects of structurally different extremophilic Halomonas levan polysaccharides on human skin epidermis cells. In-vitro experiments with microbially produced linear Halomonas levan (HL), its hydrolyzed, (hHL) and sulfonated (ShHL) derivatives as well as enzymatically produced branched levan (EL) revealed increased keratinocyte and fibroblast proliferation (113-118 %), improved skin barrier function through induced expressions of involucrin (2.0 and 6.43 fold changes for HL and EL) and filaggrin (1.74 and 3.89 fold changes for hHL and ShHL) genes and increased type I collagen (2.63 for ShHL) and hyaluronan synthase 3 (1.41 for HL) gene expressions together with fast wound healing ability within 24 h (100 %, HL) on 2D wound models clearly showed that HL and its derivatives have high potential to be used as natural active ingredients in cosmeceutical and skin regenerating formulations.

PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene.

Food wastes can be hydrolyzed into soluble microbial substrates, contributing to sustainability. Halomonas spp.-based Next Generation Industrial Biotechnology (NGIB) allows open, unsterile fermentation, eliminating the need for sterilization to avoid the Maillard reaction that negatively affects cell growth. This is especially important for food waste hydrolysates, which have a high nutrient content but are unstable due to batch, sources, or storage conditions. These make them unsuitable for polyhydroxyalkanoate (PHA) production, which usually requires limitation on either nitrogen, phosphorous, or sulfur. In this study, H. bluephagenesis was constructed by overexpressing the PHA synthesis operon phaCABCn (cloned from Cupriavidus necator) controlled by the essential gene ompW (encoding outer membrane protein W) promoter and the constitutive porin promoter that are continuously expressed at high levels throughout the cell growth process, allowing poly(3-hydroxybutyrate) (PHB) production to proceed in nutrient-rich (also nitrogen-rich) food waste hydrolysates of various sources. The recombinant H. bluephagenesis termed WZY278 generated 22 g L-1 cell dry weight (CDW) containing 80 wt% PHB when cultured in food waste hydrolysates in shake flasks, and it was grown to 70 g L-1 CDW containing 80 wt% PHB in a 7-L bioreactor via fed-batch cultivation. Thus, unsterilizable food waste hydrolysates can become nutrient-rich substrates for PHB production by H. bluephagenesis able to be grown contamination-free under open conditions.