Ectoine Biosynthesis Research Articles

Scalable Electro‐Biosynthesis of Ectoine from Greenhouse Gases

Converting greenhouse gases into valuable products has become a promising approach for achieving a carbon‐neutral economy and sustainable development. However, the conversion efficiency depends on the energy yield of the substrate. In this study, we developed an electro‐biocatalytic system by integrating electrochemical and microbial processes to upcycle CO2 into a valuable product (ectoine) using renewable energy. This system initiates the electrocatalytic reduction of CO2 to methane, an energy‐dense molecule, which then serves as an electrofuel to energize the growth of an engineered methanotrophic cell factory for ectoine biosynthesis. The scalability of this system was demonstrated using an array of ten 25 cm2 electrochemical cells equipped with a high‐performance carbon‐supported isolated copper catalyst. The system consistently generated methane at the cathode under a total partial current of approximately −37 A (~175 mmolCH4 h–1) and O2 at the anode under a total partial current of approximately 62 A (~583 mmolO2 h–1). This output met the requirements of a 3‐L bioreactor, even at maximum CH4 and O2 consumption, resulting in the high‐yield conversion of CO2 to ectoine (1146.9 mg L–1). This work underscores the potential of electrifying the biosynthesis of valuable products from CO2, providing a sustainable avenue for biomanufacturing and energy storage.

Scalable Electro-Biosynthesis of Ectoine from Greenhouse Gases.

Converting greenhouse gases into valuable products has become a promising approach for achieving a carbon-neutral economy and sustainable development. However, the conversion efficiency depends on the energy yield of the substrate. In this study, we developed an electro-biocatalyticsystem by integrating electrochemical and microbial processes to upcycle CO2 into a valuable product (ectoine) using renewable energy. This system initiates the electrocatalytic reduction of CO2 to methane, an energy-dense molecule, which then serves as an electrofuel to energize the growth of an engineered methanotrophic cell factory for ectoine biosynthesis. The scalability of this system was demonstrated using an array of ten 25 cm2 electrochemical cells equipped with a high-performance carbon-supported isolated copper catalyst. The system consistently generated methane at the cathode under a total partial current of approximately -37 A (~175 mmolCH4 h-1) and O2 at the anode under a total partial current of approximately 62 A (~583 mmolO2 h-1). This output met the requirements of a 3-L bioreactor, even at maximum CH4 and O2 consumption, resulting in the high-yield conversion of CO2 to ectoine (1146.9 mg L-1). This work underscores the potential of electrifying the biosynthesis of valuable products from CO2, providing a sustainable avenue for biomanufacturing and energy storage.

Genome-Scale Metabolic Modeling of Halomonas elongata 153B Explains Polyhydroxyalkanoate and Ectoine Biosynthesis in Hypersaline Environments.

Halomonas elongata thrives in hypersaline environments producing polyhydroxyalkanoates (PHAs) and osmoprotectants such as ectoine. Despite its biotechnological importance, several aspects of the dynamics of its metabolism remain elusive. Here, we construct and validate a genome-scale metabolic network model for H. elongata 153B. Then, we investigate the flux distribution dynamics during optimal growth, ectoine, and PHA biosynthesis using statistical methods, and a pipeline based on shadow prices. Lastly, we use optimization algorithms to uncover novel engineering targets to increase PHA production. The resulting model (iEB1239) includes 1534 metabolites, 2314 reactions, and 1239 genes. iEB1239 can reproduce growth on several carbon sources and predict growth on previously unreported ones. It also reproduces biochemical phenotypes related to Oad and Ppc gene functions in ectoine biosynthesis. A flux distribution analysis during optimal ectoine and PHA biosynthesis shows decreased energy production through oxidative phosphorylation. Furthermore, our analysis unveils a diverse spectrum of metabolic alterations that extend beyond mere flux changes to encompass heightened precursor production for ectoine and PHA synthesis. Crucially, these findings capture other metabolic changes linked to adaptation in hypersaline environments. Bottlenecks in the glycolysis and fatty acid metabolism pathways are identified, in addition to PhaC, which has been shown to increase PHA production when overexpressed. Overall, our pipeline demonstrates the potential of genome-scale metabolic models in combination with statistical approaches to obtain insights into the metabolism of H. elongata. Our platform can be exploited for researching environmental adaptation, and for designing and optimizing metabolic engineering strategies for bioproduct synthesis.

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass

BackgroundEctoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization.ResultsIn this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively.ConclusionsThis study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method

Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method

The tropical marine actinomycete Nocardiopsis dassonvillei NCIM 5124 as novel source of ectoine: Genomic and transcriptomic insights

Since ectoine is a high-value product, overviewing strategies for identifying novel microbial sources becomes relevant. In the current study, by following a genome mining approach, the ectoine biosynthetic cluster in a tropical marine strain of Nocardiopsis dassonvillei (NCIM 5124) was located and compared with related organisms. Transcriptome analysis of Control and Test samples (with 0 and 5% NaCl, respectively) was carried out to understand salt induced stress response at the molecular level. There were 4950 differentially expressed genes with 25 transcripts being significantly upregulated in Test samples. NaCl induced upregulation of the ectoine biosynthesis cluster and some other genes (stress response, chaperone/Clp protease, cytoplasm, ribonucleoprotein and protein biosynthesis). The production of ectoine as a stress response molecule was experimentally validated via LCMS analysis. The investigation sheds light on the responses exhibited by this actinomycete in coping up with salt stress and provides a foundation for understanding salt induced molecular interactions.

Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered Halomonas elongata.

In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB-encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated.

Microbial inoculum effects on the rumen epithelial transcriptome and rumen epimural metatranscriptome in calves

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions.

Efficiently manufacturing ectoine via metabolic engineering and protein engineering of L-2,4-diaminobutyrate transaminase

Ectoine, so-called tetrahydropyrimidine, is an important osmotic adjustment solute and widely applied in cosmetics and protein protectant. Some attempts have been made to improve the ectoine productivity. However, the strains with both high ectoine production capacity and high glucose conversion were still absent so far. Aim to construct a strain for efficiently producing ectoine, ectoine synthetic gene cluster ectABC from Pseudomonas stutzeri was overexpressed in E. coli BL21 (DE3). The ection production was improved by 382 % (ectoine titer increased from 1.73 g/L to 8.33 g/L) after the rational design of rate-limiting enzyme L-2,4-diaminobutyrate transaminase EctBps (protein engineering) combined with the metabolic engineering that focused on the enrichment and conversion of precursors. The final strain YW20 was applied to overproduce ectoine in fed-batch fermentation and yield 68.9 g/L of ectoine with 0.88 g/L/h of space-time yield and the highest glucose conversion reported [34 % (g/g)]. From the fermentation broth, ectoine was purified with 99.7 % purity and 79.8 % yield. This study successfully provided an engineered strain as well as an efficient method for the industrial bio-synthesis and preparation of ectoine.

Advances in ectoine biosynthesis and biochemical characteristics of key enzymes

Compatible solutes are highly water-soluble organic osmolytes produced by microorganisms to adapt to extreme environments, such as high salinity and osmotic pressure. Among these, ectoine plays a crucial role in repairing and protecting nucleic acids, protein, biofilms, and cells. As a result, it has found widespread applications in cosmetics, biological agents, the enzyme industry, medicine, and other fields. Currently, the market value of ectoine is around US$ 1 000/kg, with a global demand reaching 15 000 tons per year. Although halophilic bacteria serve as the natural source of ectoine synthesis, its production in high-salinity media presents challenges such as equipment corrosion and high cost for industrial production. Advancements in functional genomics, systems biology, and synthetic biology have paved the way for the development of high-yielding cell factories through metabolic engineering, leading to significant progress. For example, engineered Escherichia coli achieved a maximum ectoine titer of 131.8 g/L, with a productivity of 1.37 g/(L·h). This review aims to explore the biosynthetic pathway, biochemical characteristics of key enzymes, and the biosynthesis of ectoine, shedding light on current research status and offering insights for industrial-scale ectoine production.

Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

Metabolic Engineering of Corynebacterium glutamicum for Highly Efficient Production of Ectoine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

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

BackgroundThe 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.ResultsThis 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.ConclusionsThis 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.

Growth pattern, Protein quantification, and Antibiotic responses of four Halophilic Bacterial Strains in different salt concentration

Abstract: Background: Halophilic bacteria are studied extensively to explore molecular basis of adaptation, enzymatic potential, drug resistance and their other biotechnological implications. Their ability to survive in hypersaline condition is often linked to the potential of biosynthesizing a well-known compatible solute, namely ectoine. Aim: In this study, four bacterial strains, which were previously identified from hypersaline environment were studied for their growth pattern and ectoine biosynthesis proteins expression under varying NaCl concentration. In addition, antibiotic susceptibility of the strains was also investigated. Methods: The growth pattern of the bacterial strains were studied in LB broth supplemented with different NaCl concentration. Bradford method was employed for protein estimation, and optical densities of the samples were quantified through UV-Vis spectrophotometer. Moreover, expression of the ectoine proteins were detected through SDS-PAGE, and disc diffusion method was used for profiling the antibiotic resistance of the strains. Results: The results indicated distinct growth patterns, with each strain demonstrating preferences for specific salinity levels. Notably, Staphylococcus cohnii strain F exhibited optimal growth at 10% NaCl, while Jeotgalicoccus huakuii strain 3.7 thrived at 15% NaCl. Total protein analysis revealed a consistent trend of decreasing protein content with rising NaCl concentrations, elucidating a potential adaptive strategy. Staphylococcus cohnii strain F stood out, maintaining higher protein levels at 20% NaCl, indicative of unique adaptive mechanisms. Antibiotic susceptibility testing uncovered diverse resistance profiles, emphasizing the strain-specific nature of responses. The expression of ectoine biosynthesis genes in Staphylococcus cohnii strain F underlines its adaptive advantage in high salinity. Conclusion: The findings contribute not only to basic microbial physiology knowledge but also hold implications for biotechnological applications and clinical practices, emphasizing the importance of tailored approaches in antibiotic therapy and highlighting the strain-specific nature of bacterial adaptations.

Phenotypic and genomic characterization of the first alkaliphilic aceticlastic methanogens and proposal of a novel genus Methanocrinis gen.nov. within the family Methanotrichaceae.

Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7-10.2 (optimum 9.3-9.5) in the presence of optimally 0.2-0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3-98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.

Complete genome sequence of Marinobacter shengliensis D49 harboring ectABC genes for ectoine synthesis.

A complete genome sequence of Marinobacter shengliensis D49 in the class Gamma-proteobacteria was isolated from activated sludge treating landfill leachate. The genome encodes the functional genes for the biosynthesis of ectoine (ectABC), a compatible solute for cosmetics. Deciphering the genome helps pave the way for ectoine production by the isolate.

Growing in Saltwater: Biotechnological Potential of Novel Methylotuvimicrobium- and Methylomarinum-like Methanotrophic Bacteria.

Methanotrophic bacteria that possess a unique ability of using methane as a sole source of carbon and energy have attracted considerable attention as potential producers of a single-cell protein. So far, this biotechnology implied using freshwater methanotrophs, although many regions of the world have limited freshwater resources. This study aimed at searching for novel methanotrophs capable of fast growth in saltwater comparable in composition with seawater. A methane-oxidizing microbial consortium containing Methylomarinum- and Methylotuvimicrobium-like methanotrophs was enriched from sediment from the river Chernavka (water pH 7.5, total salt content 30 g L-1), a tributary river of the hypersaline Lake Elton, southern Russia. This microbial consortium, designated Ch1, demonstrated stable growth on natural gas in a bioreactor in media with a total salt content of 23 to 35.9 g L-1 at a dilution rate of 0.19-0.21 h-1. The highest biomass yield of 5.8 g cell dry weight (CDW)/L with a protein content of 63% was obtained during continuous cultivation of the consortium Ch1 in a medium with a total salt content of 29 g L-1. Isolation attempts resulted in obtaining a pure culture of methanotrophic bacteria, strain Ch1-1. The 16S rRNA gene sequence of strain Ch1-1 displayed 97.09-97.24% similarity to the corresponding gene fragments of characterized representatives of Methylomarinum vadi, methanotrophs isolated from marine habitats. The genome of strain Ch1-1 was 4.8 Mb in size and encoded 3 rRNA operons, and about 4400 proteins. The genome contained the gene cluster coding for ectoine biosynthesis, which explains the ability of strain Ch1-1 to tolerate high salt concentration.

Effect of Exogenous and Endogenous Ectoine on Monascus Development, Metabolism, and Pigment Stability.

Monascus, a key player in fermented food production, is known for generating Monascus pigments (MPs) and monacolin K (MK), possessing bioactive properties. However, the limited stability of MPs and mycotoxin citrinin (CTN) constrain the Monascus industry. Extremolytes like ectoine, derived from bacteria, exhibit cytoprotective potential. Here, we investigated the impact of ectoine on Monascus purpureus ATCC 16365, emphasizing development and secondary metabolism. Exogenous 5 mM ectoine supplementation substantially increased the yields of MPs and MK (105%-150%) and reduced CTN production. Ectoine influenced mycelial growth, spore development, and gene expression in Monascus. Remarkably, ectoine biosynthesis was achieved in Monascus, showing comparable effects to exogenous addition. Notably, endogenous ectoine effectively enhanced the stability of MPs under diverse stress conditions. Our findings propose an innovative strategy for augmenting the production and stability of bioactive compounds while reducing CTN levels, advancing the Monascus industry.

NhaR, LeuO, and H-NS Are Part of an Expanded Regulatory Network for Ectoine Biosynthesis Expression.

Bacteria accumulate compatible solutes to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, among others, 3 regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed in exponential and stationary phase cells. PectA-gfp expression was significantly repressed in the ΔleuO mutant and significantly induced in the ΔnhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the Δhns mutant, PectA-gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a ΔleuO/Δhns mutant, PectA-gfp showed reduced expression, but significantly more than ΔleuO, suggesting H-NS and LeuO interact to regulate ectoine expression. However, ΔnhaR/Δhns had no additional effect compared to ΔnhaR, suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a PleuO-gfp reporter analysis was examined that showed significantly increased expression in the ΔleuO, Δhns, and ΔleuO/Δhns mutants compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance outside of regulating ectoine biosynthesis gene expression. IMPORTANCE Ectoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and showed that, similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.