Question

Ectoine is a compatible solute produced by halophilic bacterium as an osmoregulatory compound in a harsh environment. Its attractive properties such as UV-radiation reduction and anti-inflammation. To recover intracellular ectoine, cell disruption needs to be conducted. Provide one mechanical disruption method and one chemical disruption method that is used to recover intracellular ectoine, covering the aspects of working principles, advantages and disadvantages. Recommend a relatively more suitable method to recover the intracellular ectoine at industrial scale with providing scientific justifications.

Answer 1

In the microorganisms, ectoine and 5-hydroxyectoine are not only accumulated as excellent stress protectants but can also serve as valuable nutrients for cell growth . Similar gene clusters involving in ectoine catabolism were disclosed in strain Sinorhizobium meliloti[21], Ruegeria pomeroyi DSS-3 [22], and H. elongata DSM 2581^T . In H. elongata DSM 2581^T, the gene cluster comprising of doeA (ectoine hydrolase), doeB (Na-acetyl-L-2,4-diaminobutyric acid deacetylase), doeC (aspartate-semialdehyde dehydrogenase), and doeD(diaminobutyric acid transaminase) was verified by in-frame deletion. Recently, more detailed catabolic pathway, as well as its regulatory system was identified in strain R. pomeroyi DSS-3 .These genetic circuit(s) suggest avenues for the genetic controlling of ectoine accumulation and are valuable for ectoine production.

Since hyper-osmolarity pressure is required for compatible solutes accumulation, fermentation medium with high salinity is generally used for their enrichments and raise big challenges for the fermenter antirust and wastewater treatment. Therefore, low-salinity medium is desirable for large-scale ectoine production, as well as for some other compatible solutes. H. hydrothermalis Y2 was isolated from an artificial alkaline environment of pulp mill wastewater. As a halotolerant extremophile, the strain grows well in a broad range salinity that from 0 to 180 g L⁻¹ NaCl [26]. As we previously observed, four Na⁺/H⁺ antiporters work in a labor division way to deal with saline and alkaline environments, in which NhaD2 and Mrp play a notable physiological role in pH and osmotic homeostasis .In the present study, based on a double mutant that lacking doeA and ectD genes, Mrp and (or) NhaD2 were in-frame deleted and their effluence to the ectoine productivity was investigated. recovery of intracellular ectoine using the PPG/salt ABS. In this study, ectoine exhibited a salt-rich bottom phase preference in the PPG/salt ABS because of the hydrophilic surface properties of ectoine. The effects of concentrations of PPG 425 and salts, crude load, pH and concentrations of neutral salts (NaCl and KCl) on the recovery of ectoine were investigated. Optimum recovery of intracellular ectoine was obtained with ABS (pH 5.5) composed of 30% (w/w) PPG 425 and 20% (w/w) sulfate; 15% (w/w) crude load; and 1.5% (w/w) of KCl. High yield of 94.7% of intracellular ectoine was successfully recovered from H. salina cells with an enrichment factor (E_F) of 1.7 and a purity of 87.03%.

Ectoine can be synthesized by a large number of aerobic, heterotrophic bacteria, such as genus Halorhodospira, genera Halomonas, Chromohalobacter, Vibrio, Pseudomonas of the class γ-proteobacteria, and even archaea Nitrosopumilus maritimus or methanotroph Methylomicrobium alcaliphilum strain . Commercially, it is produced by the moderate halophilic bacterium Halomonas elongata, which has an established biosynthetic pathway for ectoine metabolism. Starting from precursor L-aspartate-β-semialdehyde (ASA), ectoine is synthesized by the catalytic combination of a diaminobutyric acid transaminase (EctB), a diaminobutyric acid acetyltransferase (EctA), and an ectoine synthase (EctC) Under certain stress conditions (e.g. elevated temperatures), it is demonstrated that some ectoine could be converted to 5-hydroxyectoine by ectoine hydroxylase (EctD)

In the microorganisms, ectoine and 5-hydroxyectoine are not only accumulated as excellent stress protectants but can also serve as valuable nutrients for cell growth .Similar gene clusters involving in ectoine catabolism were disclosed in strain Sinorhizobium meliloti Ruegeria pomeroyi DSS-3 and H. elongata DSM 2581^T [23]. In H. elongata DSM 2581^T, the gene cluster comprising of doeA (ectoine hydrolase), doeB (Na-acetyl-L-2,4-diaminobutyric acid deacetylase), doeC (aspartate-semialdehyde dehydrogenase), and doeD(diaminobutyric acid transaminase) was verified by in-frame deletion. Recently, more detailed catabolic pathway, as well as its regulatory system was identified in strain R. pomeroyi DSS-3 These genetic circuit(s) suggest avenues for the genetic controlling of ectoine accumulation and are valuable for ectoine production.

Since hyper-osmolarity pressure is required for compatible solutes accumulation, fermentation medium with high salinity is generally used for their enrichments and raise big challenges for the fermenter antirust and wastewater treatment. Therefore, low-salinity medium is desirable for large-scale ectoine production, as well as for some other compatible solutes. H. hydrothermalis Y2 was isolated from an artificial alkaline environment of pulp mill wastewater. As a halotolerant extremophile, the strain grows well in a broad range salinity that from 0 to 180 g L⁻¹ NaCl [26]. As we previously observed, four Na⁺/H⁺ antiporters work in a labor division way to deal with saline and alkaline environments, in which NhaD2 and Mrp play a notable physiological role in pH and osmotic homeostasis .In the present study, based on a double mutant that lacking doeA and ectD genes, Mrp and (or) NhaD2 were in-frame deleted and their effluence to the ectoine productivity was investigated.

Ectoine bioconversion was also performed with higher density cells (OD_{600 nm} = 20) in a fermentor When the reaction proceeded for 12 h, the aspartate and glycerol were all consumed and feeding solution was added into the reaction system with a flow speed of 25 mL/h. During the reaction phase, a significant quantity of ectoine was synthesized and excreted at a rate of 1046 mg/L/h. After a reaction time of 24 h, the ectoine concentration reached 25.1 g/L in the medium with a productive yield of 4048 mg/g DCW, which was significantly higher than that in flask reactions with low density cells (OD_{600 nm} = 5). The ectoine bioconversion was successfully amplified in a fermentor, the yield of ectoine increased at least nine times compared with that in flasks.Ectoine production in fermentor. An initial reaction mixture containing 200 mM sodium aspartate, 200 mM glycerol, 100 mM KCl, 100 mM sodium phosphate buffer (pH7.0), and recombinant E. colicells of 20 OD/mL was used. (A) Time profiles of aspartate consumption and ectoine excretion in fermentor. Legend: excreted ectoine (black squares), aspartate (white squares). After 12 h of bioconversion the feeding solution was added (↓) with a flow speed of 25 mL/h. (B) Repeated use of cells to synthesize ectoine in fermentor. Error bars respresent standard deviations from triplicate biological replicates.

Production of ectione by multiple rounds of whole-cell biocatalysis

To test whether the cells in the whole-cell biocatalysis could be used by multiple rounds, the process of whole-cell biocatalysis using aspartate and glycerol as substrates was repeated for another two cycles with the same batch of E. colicells. The results demonstrated that the extracellular ectoine concentrations of second and third round were 21.1 g/L and 17.2 g/L, respectively. In other words, 84% and 69% of yields were achieved for the second and the third round of catalysis as compared to the first round, respectively . Thus, multiple rounds of whole-cell biocatalysis are cost effective to further improve the production of ectione.

In recent decades, whole-cell biocatalysis has been widely applied as an alternative to chemical methods for large-scale synthesis of chemicals because it is more environmentally friendly. Whole-cell biocatalysis allows cascades of enzymatic reactions that involve multiple enzymes, cofactors, and substrates; it can also help stabilise enzymes with the protective nature of cell envelopes and makes cofactor regeneration much easier [24]. E. coli K12 is often used as a suitable cell factory in bioconversion because it is a nonpathogenic strain with a well-studied genetic background and a powerful genetic tool system for metabolic engineering .With a relatively well-developed fermentation, E. coli K12 is also easy to grow because of its short generation time and ability to synthesize everything needed for generating a completely new cell from low raw materials. Most advantageously, the E. coli genome sequence lacks an ectoine catabolic pathway, eliminating product degradation due to the reuse of ectoine as a carbon or nitrogen source. The biosynthesis of ectoine from L-aspartate is a complex process containing multiple enzymatic reactions in microorganisms. Using the above whole-cell biocatalytic method for the production of ectoine by recombinant E. coli has the potential to become the most promising method for commercial production.

As an industrial strain for ectoine production, the ectoine synthesis gene cluster of H. elongata has been cloned for more than 10 years and the characteristics of ectoine biosynthetic enzymes have been described in detail .

. However, there were still no reports describing heterologous ectoine synthesis in E. coli using the ectABC genes from this industrial strain. In our experiment, the ectABC gene cluster was first introduced into E. coli K strain BW25113 using the expression plasmid pBAD/HisA. Under the control of the arapromoter, all three enzymes corresponding to ectoine synthesis achieved soluble expression. Whole-cell biocatalysis was introduced into ectoine synthesis using aspartate and glycerol as substrates. The bioconversion was performed in shaking flasks and in a fermentor with cell concentrations of 5 OD/mL and 20 OD/mL, respectively. In the fermentor the extracellular ectoine concentration reached 25.1 g/L with a productive yield of 4048 mg/g DCW, which is markedly higher than the yield in the flask reaction of 1748 mg/g DCW. By controlling the pH and substrate feeding, the cell catalysis efficiency can be significantly improved in fermentor. The highest yield in history was achieved in C. salexigens (540 and 400 mg/g DCW for ectoine and hydroxyectoine respectively) [15], which is significantly lower than our ectoine yield (4048 mg/g DCW). After the first round of bioconversion, the cells were reused two more times, and a total yield of 63.4 g ectoine was obtained using one litre cells.

Ectoine is synthesized from aspartate-semialdehyde, the central intermediate in the synthesis of amino acids belonging to the aspartate family. Therefore, in recombinant E. coli, the synthesis rate of aspartate-semialdehyde determines the efficiency of ectoine conversion . In the ectoine bioconversion system, sodium aspartate was added to the reaction mixture, and under the catalysis of E. coliendogenous Ask and Asd, aspartate could be converted to aspartate-semialdehyde more effectively than other substrates. In addition, under catalysis by glutamate-aspartate aminotransferase the amino group of aspartate could also be transferred to glutamate, which could provide the amino group for the transamination catalysed by EctB, the first reaction of the specific route for ectoine synthesis. The 2-oxoglutarate converted from glutamate by EctB could further supply substrate for the glutamate-aspartate transamination reaction to form glutamate. In summary, aspartate should be an ideal substrate for ectoine bioconversion because it can be used as both a synthetic precursor and an amino donor.