Development of M. alcaliphilum 20Z mutant for efficient genetic engineering by the removal of the native plasmid
The process of the genetic manipulation of methanotrophs has not yet been fully established, and this serves as a critical limitation for bioconversion technologies. Although conjugation methods are commonly used to transform methanotrophs, their transformation efficiency is still very low, and the appearance of transconjugants takes a long time. A previous study reported that the loss of the native plasmid increased the conjugation efficiency in Methylomicrobium buryatense . Methylomicrobium alcaliphilum 20Z also contains several enzymes of the restriction modification system (Type III RM system) and P-type conjugal transfer system, some of which are located on its native 12.8-kb plasmid. Therefore, we constructed a mutant of M. alcaliphilum 20Z to improve the transformation efficiency by eliminating its native plasmid. To achieve this, we knocked out the replication gene repB of the native plasmid locus by pK19mobsacB using the sucrose counter-selection method . The resulting mutant of M. alcaliphilum 20Z was named “M. alcaliphilum 20ZDP,” and was confirmed by colony PCR at three different loci (repB, korB, and trbF) on the native plasmid (Additional file 1: Fig. S1). There was no significant difference in cell growth under normal conditions between the M. alcaliphilum 20ZDP and M. alcaliphilum 20Z wild-type (data not shown), suggesting that the native plasmid does not contain essential genes for growth under normal conditions.
To confirm the increase in the transformation efficiency in the mutant strain, M. alcaliphilum 20ZDP was manipulated to introduce the episomal vector pAWP89 using the conjugation method. Transformation was carried out five times each on both M. alcaliphilum 20Z and M. alcaliphilum 20ZDP, resulting in 16 and 2115 colonies per plate after transformation, respectively (Fig. 2). This result showed an increase in the transformation efficiency by approximately 132-fold on average for M. alcaliphilum 20ZDP compared to M. alcaliphilum 20Z. In addition, there were a number of plates in which no colonies were formed when M. alcaliphilum 20Z was used as a conjugant. This result demonstrates that M. alcaliphilum 20ZDP is able to replicate IncP-based vectors due to the removal of the native plasmid found in M. alcaliphilum 20Z and suggests that there is competition between IncP-based vectors and the native plasmid.
Plasmid-based gene expression systems are useful genetic tools for strain manipulation, and the stability of episomal plasmids in which the desired protein is continuously expressed is very important. To investigate the stability of the episomal plasmid in M. alcaliphilum 20ZDP, we examined the enzyme expression level by the introduction of the episomal vector pAWP89 containing the dTomato reporter gene, and determined the extent to which it is maintained after 10 generations. The fluorescence from dTomato expression in M. alcaliphilum 20ZDP and M. alcaliphilum 20Z was detected using a fluorescence plate reader. As shown in Fig. 3, the dTomato expression levels were similar between M. alcaliphilum 20Z/dt and M. alcaliphilum 20ZDP/dt in the first generation. After the 10th subculture, the expression level in M. alcaliphilum 20ZDP/dt was maintained at 80% of its initial level, while the expression level in M. alcaliphilum 20Z/dt decreased by 50%. Therefore, these results indicate that the loss of the native plasmid not only increases conjugation frequency, but also induces stability during the replication of the episomal plasmids.
Ectoine production of M. alcaliphilum 20ZDP in batch culture
In a previous study, M. alcaliphilum 20Z was assessed for its ability to produce ectoine using methane as the sole carbon source . The full genome sequence containing the genes involved in ectoine biosynthesis has been investigated in M. alcaliphilum 20Z . To evaluate ectoine production by M. alcaliphilum 20ZDP, flask batch fermentation was performed in a Methylomicrobium medium containing 3% NaCl with methane as the sole carbon source for 72 h. Intracellular and extracellular ectoine and hydroxyectoine were measured as described by a previous method . Intracellular ectoine and hydroxyectoine production by M. alcaliphilum 20ZDP was slightly reduced compared to that of the wild-type strain (data not shown). No extracellular ectoine or hydroxyectoine was detected during cultivation under culture conditions. Considering the efficiency of genetic engineering for the improvement of ectoine production, it was decided to use M. alcaliphilum 20ZDP as an ectoine producer in a further study.
In our previous study, we observed that the growth of M. alcaliphilum 20Z in a batch culture, with methane as the sole carbon source, was stimulated by the addition of tungsten . To investigate the effect of tungsten on the increase in biomass and thereby on ectoine production, tungsten (0.05 μM) was added to the Methylomicrobium medium containing 3% NaCl, and cell growth and ectoine production were measured. The time-course cultivation profiles of M. alcaliphilum 20ZDP in tungsten-added and tungsten-free media are shown in Fig. 4. As expected, the maximum dry cell weight (DCW) in the tungsten-added medium was greater than that in the tungsten-free medium (2.29 g/L vs 0.69 g/L at 96 h) (Fig. 4A), and the maximum production of ectoine remarkably increased up to 2.3-fold in the tungsten-added medium compared with the tungsten-free medium (31.43 mg/L at 48 h vs. 13.85 mg/L at 72 h) (Fig. 4B). Ectoine yield (mg/DCW g) was also increased up to 1.38-fold in the tungsten-added medium until 48 h before ectoine production rapidly decreased in the tungsten-added medium. These results suggest that tungsten addition plays a positive role in both biomass production and ectoine production in M. alcaliphilum 20ZDP. Thus, tungsten (0.05 μM) was added to the medium in all subsequent batch cultures. Interestingly, ectoine accumulated slowly until 48 h and was maintained or decreased slightly in the tungsten-free medium, whereas ectoine production increased rapidly until 48 h and then decreased remarkably in the tungsten-added medium. It is assumed that this is because the ectoine might be reused as a cell component for cell growth in tungsten-added medium.
Construction of the ectD deletion mutant and batch cultivation of the mutant using methane
Although M. alcaliphilum 20ZDP could produce ectoine using methane as a carbon source, the formation of the by-products needed to be decreased for efficient ectoine production. It was confirmed that the production ratio of hydroxyectoine to ectoine accounts for approximately 4–8% by M. alcaliphilum 20ZDP cultivated in Methylomicrobium medium containing 3% NaCl. To reduce the formation of hydroxyectoine, the ectD gene encoding ectoine hydroxylase, which is responsible for the synthesis of hydroxyectoine from ectoine, was deleted in the chromosome using the sucrose counter-selection method. PCR and nucleotide sequencing data (data not shown) confirmed that the ectD gene of M. alcaliphilum 20ZDP was successfully deleted (Additional file 1: Fig. S2), and this mutant strain was named M. alcaliphilum 20ZDP1 (Table 1). When batch flask fermentation was conducted with the M. alcaliphilum 20ZDP1 strain in Methylomicrobium medium containing 3% NaCl, ectD deletion had a positive effect on ectoine production compared to M. alcaliphilum 20ZDP without the inhibition of cell growth (Fig. 5). Furthermore, the deletion of the ectD gene in M. alcaliphilum 20ZDP1 resulted in abolished hydroxyectoine formation, compared with that observed in M. alcaliphilum 20ZDP (2.2 mg/L at 72 h). Methylomicrobium alcaliphilum 20ZDP1 showed more ectoine production than M. alcaliphilum 20ZDP (34.5 mg/L vs. 27.3 mg/L). This demonstrates that the disruption of ectD not only completely inhibited hydroxyectoine production, but also induced more ectoine production.
In previous study, deletion of genes encoding ectoine hydroxylase (EctD) and ectoine hydrolase (DoeA) that responsible for ectoine catabolism were resulted in an obviously enhanced productivity of ectoine in H. hydrothermalis Y2 . A significant increase in ectoine production was observed by simultaneous deletion of ectD and doeA rather than deletion of ectD alone in H. hydrothermalis Y2. Since it was also found that ectoine was degraded in M. alcaliphilum 20Z, it is expected that ectoine production can be increased by deletion of gene that responsible for ectoine catabolism in further study.
Construction of ectD and ectR double deletion mutant and batch cultivation of the double mutant using methane
The genes encoding key enzymes of the ectoine synthesis pathway in M. alcaliphilum 20Z were organized in the ectABC operon. Upstream of this gene cluster, the ectR gene encoding a MarR-like transcription regulator (EctR) was identified in M. alcaliphilum 20Z, and was indicated to be a negative regulator of the ectABC operon . To investigate the effect of EctR on ectoine biosynthesis in M. alcaliphilum 20Z, we created the double deletion mutant strain M. alcaliphilum 20ZDP ΔectDΔectR which was named M. alcaliphilum 20ZDP2 (Table 1). This was done by removing the ectR gene from the ΔectD mutant (M. alcaliphilum 20ZDP1). Successful deletion of ectR was confirmed by PCR amplification (Additional file 1: Fig. S2) and nucleotide sequencing data (data not shown).
To confirm the effect of the deletion of ectD and ectR on ectoine production, batch cultures in flasks were conducted with methane as the carbon source using M. alcaliphilum 20ZDP2 (Fig. 6). Batch fermentation was performed for 96 h, and cell growth and intracellular ectoine and hydroxyectoine were measured every 24 h. The maximum ectoine concentration reached 45.58 mg/L at 48 h without hydroxyectoine production, and then rapidly decreased. As shown in Figs. 5 and 6, ectoine production increased up to 1.3-fold compared to that of M. alcaliphilum 20ZDP1 (45.58 mg/L vs. 34.53 mg/L). These results clearly indicate that the deletion of ectD and ectR had a beneficial effect on ectoine production without the inhibition of cell growth. More importantly, the disruption of ectD resulted in the inhibition of hydroxyectoine production, and the deletion of ectD and ectR further induced ectoine production, resulting in a remarkable increase in ectoine yield, which was up to 1.6-fold of that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L).
Enhanced production of ectoine from methane by M. alcaliphilum 20ZDP2 in batch cultivation
To improve ectoine production by M. alcaliphilum 20ZDP2, some process parameters such as agitation speed and temperature were examined. When the agitation speed was adjusted to 150, 230, and 300 rpm, there was no significant change in the cell growth and ectoine production by M. alcaliphilum 20ZDP2 (Additional file 1: Fig. S3). However, when the temperature was adjusted to 25 °C, 30 °C, and 35 °C, the cell growth and ectoine production were the best at 30 °C (Additional file 1: Fig. S4) by using M. alcaliphilum 20ZDP2. Therefore process parameters for ectoine production were determined to be 230 rpm and 30 °C.
NaCl concentration is a major factor in the production of intracellular ectoine in M. alcaliphilum 20Z . To examine the salinity adaptation range and the optimal concentration of NaCl for intracellular ectoine production in M. alcaliphilum 20ZDP2, media of varying salinity (3%, 6%, and 9% NaCl) were used and cell growth and intracellular ectoine production were comparatively evaluated. It was observed that the growth was severely inhibited in a medium containing 9% NaCl (data not shown). As shown in Figs. 6 and 7, it was confirmed that the higher the salinity of the medium, the lower the cell growth rate. However, when M. alcaliphilum 20ZDP2 was cultivated in a medium containing 6% NaCl, DCW increased up to 120 h and finally reached 1.81 g/L. Meanwhile, maximum ectoine production was 3.1-fold higher (142.32 mg/L at 96 h) than that obtained with 3% NaCl-containing medium (45.58 mg/L at 48 h) (Figs. 6 and 7). No hydroxyectoine was detected in any of the experiments using M. alcaliphilum 20ZDP2. Moreover, the maximum ectoine yield (mg/DCW g) was 2.1-fold higher in the medium containing 6% NaCl than in the medium containing 3% NaCl. As seen in Figs. 4 and 7, 0.05 μM tungsten- and 6% NaCl-containing medium was the most effective for ectoine production by M. alcaliphilum 20ZDP2. Comparing Figs. 6 and 7, it can be observed that ectoine production dramatically increased with increasing salinity, even when the ectR gene was deleted. These results demonstrated that the ectABC-ask operon was activated in response to the high osmolarity of the growth medium, but ectR1 is most likely not essential for the activation of the ectABC operon in response to elevated salinity. Thus, EctR-mediated control of the ectABC-ask operon is not a single mechanism, and an alternative uncharacterized regulatory system of ectoine production might exist in M. alcaliphilum 20Z.
The correlation between ectoine production and the growth phase in M. alcaliphilum 20Z has been previously reported in batch cultivation . Intracellular ectoine production was the highest in the mid-exponential growth phase of M. alcaliphilum 20Z, and decreased rapidly during the stationary growth phase, suggesting that accumulated ectoine might be used for the synthesis of cell constituents [24, 34]. The fact that extracellular ectoine was not detected at all when intracellular ectoine rapidly decreased indicates that ectoine was not excreted outside of cells at that point but was degraded inside. Therefore, maximum ectoine production can be achieved by identifying the appropriate time to stop the culture before the rapid degradation of ectoine. In order to determine an appropriate growth phase for the maximum production of ectoine, M. alcaliphilum 20ZDP2 was cultured in a medium containing 0.05 μM tungsten and 6% NaCl, along with methane as the sole carbon source, and sampled each time the OD600 value increased (OD 1–9) while the accumulated ectoine was compared according to the growth phase. As shown in Fig. 8, the accumulation of ectoine increased with cell growth, and when the cell optical density was 6, the cells could synthesize the most ectoine up to 138.44 ± 9.93 mg/L. After reaching the highest ectoine synthesis, it decreased dramatically with cell growth.
Reshetnikov et al.  showed that M. alcaliphilum 20Z possesses the doeBDAC gene cluster, which is responsible for ectoine degradation. This gene cluster codes for putative ectoine hydrolase (DoeA), Nα-acetyl-l-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD), and aspartate semialdehyde dehydrogenase (DoeC). These four enzymes catalyze ectoine hydrolysis, producing N-acetyl-DAB, and further deacetylate it to diaminobutyrate (DAB) and acetate (Fig. 1) . DAB can either flow off to aspartate or re-enter the ectoine synthesis pathway. Therefore, it is expected that the genetic manipulation of genes involved in ectoine degradation can further increase ectoine production by preventing the rapid degradation of the accumulated ectoine.
Table 2 compares ectoine production from glucose and methane from previous reports and this study. The main process for ectoine production was focused on sugar fermentation using Halomonas species and metabolically engineered E. coli and C. glutamicum. Until now, Gießelmann et al.  reported the highest ectoine production (65.3 g/L) from glucose and molasses by metabolically engineered C. glutamicum. Zhao et al.  obtained 10.5 g/L of ectoine with the yield of 0.22 g/DCW g by H. hydrothermalis Y2 from glucose and sodium glutamate. However, this process is costly due to the need for high quality carbon source such as glucose, yeast, and sodium glutamate . M. alcaliphilum 20Z was able to accumulate ectoine up to 70 mg/g DCW during the treatment of dilute methane emission under continuous mode . Additionally, a recent study demonstrated that M. alcaliphilum 20Z and a mixed haloalkaliphilic consortium were able to produce ectoine up to 109 mg/g DCW using upgraded biogas under continuous mode . So far, previous studies on ectoine production from methane have been conducted using parental strain due to limitations in genetic manipulation of methanotrophs, and it was focused on bio-milking process to enhance ectoine production. In this study, we constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of episomal plasmid by removal of native plasmid to facilitate genetic manipulation of the strain. And then, the enhanced ectoine production from methane was fulfilled by deletion of ectD and ectR in M. alcaliphilum 20ZDP in a medium containing 6% of NaCl and 0.05 μM of tungsten. Overall, a new record of the highest ectoine concentration from methane (142.32 mg/L) as the sole carbon source was achieved without hydroxyectoine production.
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