Enhanced synthesis of alginate oligosaccharides in Pseudomonas mendocina NK-01 by overexpressing MreB

Abstract

This study aimed to investigate the effects of cytoskeleton protein MreB on bacterial cell morphology and the synthesis of alginate oligosaccharides (AO) and polyhydroxyalkanoate (PHA) by Pseudomonas mendocina NK-01. To overexpress the mreB gene, an expression vector encoding MreB-GFP fusion protein was constructed. The scanning electron microscope (SEM) showed that cells expressing MreB were longer than the wild ones, which agrees with MreB’s relationship with the synthesis of peptidoglycan. Cells expressing the MreB-GFP fusion protein emitted green fluorescence under a fluorescence microscope, suggesting that MreB was functionally expressed in strain NK-01. Under a confocal laser scanning microscope, MreB was observed as located around the cell membrane. Furthermore, the recombinant strain could synthesize 0.961 g/L AO, which was 5.86-fold higher than wild-type strain. Through the medium optimization test, we finally selected the addition of 20 g/L glucose as the optimal glycogen addition for AO fermentation based on a high AO yield and high substrate transformation efficiency. The results indicated that overexpression of MreB affected the cell morphology, the activity of AO polymerase, and the efficiency of AO secretion. However, the synthesis of PHA for recombinant strain was slightly reduced. The results suggested that the overexpression of this cytoskeleton protein affected the yield of specific intracellular and extracellular products.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1873-7) contains supplementary material, which is available to authorized users.

Introduction

Pseudomonas mendocina NK-01 can synthesize alginate oligosaccharides (AO) and medium-chain-length polyhydroxyalkanoate (PHA_MCL) from glucose under conditions limiting nitrogen sources (Guo et al. 2012). AO can be obtained by alginate lyase from the degradation of alginate (Zhang et al. 2004) or mollusks in nature (Chaki et al. 2006). It was found that AO has many biological activities, such as antioxidation (Spizzirri et al. 2010), anticoagulation (Lin et al. 2007), and immune regulation (Otterlei et al. 1991). These characteristics lead to the widespread use of AO in the food and pharmaceutical fields. Poly-3-hydroxyalkanoate (PHA) is a family of biopolyesters synthesized by numerous bacteria as intracellular carbon and energy polymers when nitrogen, phosphorus, oxygen, or magnesium is limited in the environment (Anderson and Dawes 1990; Goh and Tan 2012). PHA is used as an environmentally friendly biomedical materials and bioplastics due to its biocompatibility, biodegradability, and thermoprocessibility (Williams and Peoples 1997; Chen and Wu 2005).

In coryneform bacteria, changes in cell morphology may occur throughout the cell cycle. Several genes are involved in maintaining the shape of Escherichia coli and Bacillus subtilis. Mutations in these genes often cause the cells to become round. mreB is the first gene of the murein (mre) gene cluster in the E. coli chromosome (Biondi et al. 2004). mreB is essential in Pseudomonas mendocina NK-01, encoding a 37 kDa protein (Bork et al. 1992). MreB polymerizes to form helical filamentous structures, which are thought to be homologous to eukaryotic actin filaments (Jones et al. 2001; van den Ent et al. 2001). The idea of a helical cytoskeleton within coryneform bacteria has led to MreB being suggested to play key roles in models of not only cell shape determination, but also chromosome segregation (Gitai et al. 2005), cell polarity (Gitai et al. 2004; Shih et al. 2005), motility (Nan and Zusman 2011), and growth (van Teeffelen et al. 2011; Wang et al. 2012). In addition, filamentous recombinant E. coli that can accumulate more PHA has been reported recently (Wang et al. 2014).

To my knowledge, there have been several reports on the relationship between MreB and bacterial metabolism products. The whole 5,434,353 bp genome sequence of P. mendocina NK-01 has been submitted to the GenBank (ID: CP002620). Four cytoskeleton genes, named mreB, ftsA, ftsZ, and minD, have been identified in the strain via nucleotide sequence alignment. The present study aimed to determine whether the overexpression of MreB could affect the synthesis of AO and PHA.

Materials and methods

Strains, plasmids, primers, and culture conditions

The strains, plasmids, and primers used in this study are shown in Table 1. P. mendocina NK-01 was cultured at 30 °C and E. coli was cultured at 37 °C in LB medium, supplemented with appropriate antibiotics: 100 μg/mL of ampicillin (Ap), 170 μg/mL of chloramphenicol (Cm), and 60 μg/mL kanamycin (Km) to promote plasmid retention.

Table 1.

Strains, plasmids, and primers used in this study

Strain/plasmid	Relevant characteristics	Source or reference
E. coli
DH5α	△(lacZYA-argF)U169, recA1, endA1, phoA, supE44, thi-1, relA1	Takara
S17-1	recA; harbors the tra genes of plasmid RP4 in the chromosome; proA, thi-1	Guo et al. (2011)
P. mendocina NK-01
Wild type	PHAMCL and AO producing strain; CmR	Guo et al. (2011)
NK-01(pBBR1MCS-2)	NK-01 derivative containing pBBR1MCS-2; CmR; AmpR; KmR	This study
NK-01(pBBR-mreB-gfp)	NK-01 derivative containing pBBR-mreB-gfp; CmR; AmpR; KmR	This study
Plasmids
pMD19-T Simple Vector	T easy vector for gene cloning; AmpR	Takara
T-gfp	T cloning vector containing gfp; AmpR	Takara
pMD19-mreB-gfp	pMD19-T simple vector derivative consists of mreB-gfp and a constitutive promoter of NK-01	This study
pBBR1MCS-2	Cloning vector; KmR	This study
pBBR-mreB-gfp	pBBR1MCS-2 derivative containing mreB gene from P. mendocina NK-01 genome and gfp from T-gfp; KmR	This study
Primers
P1-XbaI	5′-GGGTCTAGAAGATCGGCGGACATGATGAG-3′	This study
P2	5′-TCTTGAACATGGGCTGGGATCTCCAACGGG-3′	This study
P3	5′-TTCTCCTTTACTCATCTCGGTGGAGAGCAGGTCCA-3′	This study
P4	5′-CTGCTCTCCACCGAGATGAGTAAAGGAGAAGAACT-3′	This study
P5	5′-ATCCCAGCCCATGTTCAAGAAACTGCGTGG-3′	This study
P6-SacI	5′-GGGGAGCTCTCACTCGGTGGAGAGCAGGT-3′	This study

Vector construction and genetic manipulation of P. mendocina NK-01

The nucleotide sequences of the conserved promoter and the mreB gene in strain NK-01 were PCR amplified from the chromosomal DNA of strain NK-01 using two pairs of primers P1/P2 and P3/P4, respectively. The gfp gene was amplified from the plasmid T-gfp using primers P5 and P6. The promoter-mreB-gfp fusion construct was amplified from the above three products using primers P1 and P6, digested with XbaI/SacI, and subcloned into XbaI/SacI-digested pBBR1MCS-2, a Pseudomonas expression vector, to produce pBBR-mreB-gfp. All the constructed plasmids were verified by restriction enzyme digestion and DNA sequencing analysis. Subsequently, the plasmid was transferred into P. mendocina NK-01 using Escherichia coli S17-1 as a donor strain as described above (Guo et al. 2012).

Scanning electron microscopy

The cells were grown on LB agar plates at 30 °C. One centimeter square plugs containing the bacterial colonies were excised. The surface mucus of the colonies was removed by isotonic phosphate buffer (pH 7.0–7.2). The samples were fixed with glutaraldehyde overnight in a 5-mL plastic tube, and then, glutaraldehyde was gently poured off. The residual glutaraldehyde was removed by dialyzation against isotonic phosphate buffer five times. The samples were then dialyzed using a linear gradient of ethanol (v/v) (30, 50, 70, 80, 90, and 100%). The samples were lyophilized in a vacuum freezing dryer after twice more treatments with 100% ethanol. Gold dust was sprayed on the samples. To investigate the effects of MreB overexpression on the cell shape of NK-01, cells were observed using a scanning electron microscopy (SEM) (FEI, Hillsboro, USA) with an accelerating voltage (EHT) of 15 kV and a magnification factor of 20,000.

The lengths of long and short axes were then measured using the image processing software (Adobe Photoshop CS3) as previously described (Shiomi et al. 2008). At least 50 cells were measured in each group. All experiments were repeated at least twice.

Production of AO and PHA_MCL by P. mendocina NK-01

The recombinant or wild-type P. mendocina NK-01 was incubated in LB medium with or without 60-μg/mL kanamycin in a shaker at 30 °C and 180 rpm for 12 h. Then, a 1% (v/v) inoculum was added to a 500-mL flask containing 200 mL of fermentation medium (Guo et al. 2011) with or without 60 μg/mL kanamycin and incubated in a shaker at 30 °C and 180 rpm. After 48 h of incubation, cells were pelleted by centrifugation at 5000g for 5 min and lyophilized in a vacuum freezing dryer. The PHA_MCL was extracted with chloroform from the lyophilized cell, and the AO was obtained from the supernatant by repeated precipitation with cold ethanol. The detailed procedures for PHA_MCL and AO extraction are described in Guo et al. (2011).

Quantitative real-time PCR

Total RNA was purified using Trizol, according to the protocol provided by Cwbiotech. The RNA was then reverse transcribed to obtain cDNA using a PrimeScript RT reagent kit (Takara, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was performed and analyzed using the SYBR Premix Ex Taq and an Eppendorf qRT-PCR System. mRNA was purified from three independent clones for each strain. The transcription levels of these genes were normalized to the levels of 16S rRNA. The PCR reaction was performed with the following cycling profile: initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 30 s, and extension at 72 °C for 20 s. Each sample was analyzed in three independent experiments. The results were standardized as fold changes compared to the wild-type strain.

Fluorescence microscope and confocal microscope

Cells carrying pBBR or pBBR-mreB-gfp were grown to mid-exponential phase (OD₆₀₀ = 0.6) at 30 °C, washed with PBS buffer (pH 7.4) twice, and then resuspended in PBS. Cells were observed by a fluorescence microscope (Zeiss, Germany) with blue exciting light at 480–500 nm. The expression level of the MreB-GFP was reliably predicted from the fluorescence intensity.

The cells carrying pBBR-mreB-gfp were treated in the same manner as above when observed by confocal microscopy, except that the cells were treated with 10 µM FM4-64/L for 15 min. Cells were observed with a Leica TCS SP5 confocal microscope, fitted with a Leica 100 × 10 numerical aperture objective lens, using parameters appropriate for the fluorescence excitation. The whole process of cell division was observed.

Results

Construction of the recombinant strain overexpressing mreB-gfp fusion protein

The successful construction of the recombinant strain harboring pBBR-mreB-gfp was verified by PCR using the primers P1 and P6 listed in Table 1. Genomic DNA of wild-type NK-01 was used as a negative PCR control. PCR positive colonies were selected and used in the subsequent experiments. The characteristics of the expression vector pBBR-mreB-gfp are shown in Fig. S1.

Measurement of the cell size by SEM

The cells of the wild-type and recombinant strains were observed by SEM. As shown in Fig. 1, the average length of the recombinant cells bearing pBBR-mreB-gfp was longer than that of the wild-type cells and recombinant cells carrying the empty vector pBBR1MCS-2. We randomly selected 50 cells and calculated their length and width. The statistical analysis is shown in Fig. 2. The recombinant cells harboring pBBR-mreB-gfp (triangle) were distributed around the top right corner, which indicated their fatter cell shape. In contrast, the majority of the wild-type cells (circle) and recombinant cells harboring pBBR1MCS-2 (crossing) were gathered in the middle of Fig. 2, which represented shorter and thinner cells.

Fig. 1.

Scanning electron micrographs of the wild-type and recombinant P. mendocina NK-01. Cells were observed using a scanning electron microscope after 24 h of cultivation on the LB agar plates. Scale bar is 2 μm

Fig. 2.

Measurement of the lengths of the long and short axes. Fifty cells of the three kinds of strains (NK-01 indicated by circles, NK-01/pBBR1MCS-2 indicated by crosses, and NK-01/pBBR-mreB-gfp indicated by triangles) were observed under SEM and measured. The graph shows the values of the lengths of long and short axes

As shown in Table 2, the average length of 2.051 µm of the recombinant cells overexpressing MreB was 1.27- and 1.23-fold higher, respectively, than that of the wild-type cells and recombinant cells harboring pBBR1MCS-2. The average width of 0.565 µm of the recombinant cells overexpressing MreB was 1.49- and 1.51-fold higher, respectively, than that of the wild-type cells and recombinant cells harboring pBBR1MCS-2. The recombinant cells overexpressing MreB had a smaller length–width ratio of 3.66 compared to the wild-type cells and recombinant cells harboring pBBR1MCS-2 with a length–width ratio of 4.35 and 4.53, respectively. These results demonstrated that the recombinant strain NK-01 (pBBR-mreB-gfp) was longer and wider than the wild-type cells and recombinant cells harboring pBBR1MCS-2.

Table 2.

Average cell length and width of the wild and recombinant P. mendocina NK-01

	Average length (µm)	Average width (µm)	Length–width ratio
NK-01	1.610 ± 0.173	0.380 ± 0.072	4.355 ± 0.784
NK-01 (pBBR1MCS-2)	1.667 ± 0.303	0.373 ± 0.064	4.538 ± 0.844
NK-01 (pBBR-mreB-gfp)	2.051 ± 0.412	0.565 ± 0.058	3.663 ± 0.785

Transcription levels of the AO and PHA synthase genes

As shown in Fig. 3, the transcription level of mreB was enhanced in the recombinant strain compared to the wild-type strain, while the transcription levels of the other genes (the AO synthase gene algA and PHA synthase genes phaC1 and phaC2) were slightly decreased. Thus, the overexpression of MreB might have a detrimental effect on the transcription of the AO synthase gene and PHA synthase genes.

Fig. 3.

Quantification of the mRNA transcription levels of mreB, algA, phaC1, and phaC2 in the wild-type NK-01 and recombinant NK-01 bearing pBBR-mreB-gfp by qRT-PCR using total RNA samples obtained from exponentially growing cells (OD₆₀₀ = 0.6). The transcription level of the mRNAs in the wild-type NK-01 was set as 1. The data are mean values ± standard deviations from three independent experiments

Bacterial growth and product analysis

As shown in Fig. 4, the growth of the overexpressing strain was slightly slower than the wild-type strain during the first 16 h; however, the plateau of growth occurred between 18 and 40 h in both the wild-type and recombinant strains. These results suggested that overexpression of MreB did not affect the growth of the strain significantly.

After 48 h of fermentation, PHA_MCL and AO were recovered from the cell and supernatant, respectively. We found that the extracellular AO produced by the recombinant strain was about 5.86 times higher than the wild-type strain (Fig. 5). In contrast, the yield of the intracellular PHA slightly declined for the recombinant strain, which might be explained by the competition for carbon sources between AO and PHA.

Fig. 5.

Effects of the overexpression of MreB on the production of PHA and AO. The yields (g/L) of PHA (dark bars) and AO (light bars) in the wild-type NK-01 and recombinant NK-01 harboring pBBR-mreB-gfp are shown

Optimize AO fermentation medium by changing the amount of glucose added

Since the yield of AO is significantly increased after overexpression of mreB, we consider optimizing the fermentation medium to further increase the yield. On the basis of adding 20 g/L of glucose to the original fermentation medium, we chose to add 10 g/L, 20 g/L, 30 g/L, 40 g/L, and 50 g/L glucose to the medium, respectively. It is desirable to obtain a glycogen addition amount which is more advantageous for AO fermentation. Three experiments were performed in each group.

We monitored the growth of the cells in the medium for different amounts of glycogen added. Sampling was performed every 4 h with three parallels for each medium. As shown in Fig. 6, in most of the growth cycles, the cells of any one of the experimental groups did not have a significant growth advantage or disadvantage compared to the control group. This indicates that the change in the amount of glycogen does not have a significant effect on the growth of the cells under the premise that the glycogen can be fully utilized.

Fig. 6.

The cell growth curve in AO fermentation medium with 10 g/L (circles), 20 g/L (squares), 30 g/L (triangles), 40 g/L (forks), and 50 g/L (diamonds) glucose, respectively. The growth of cell was determined by measuring the optical density at 600 nm (OD₆₀₀) using a UV spectrophotometer (UV-1800, SHIMADZU)

After the 48-h fermentation experiment, we measured the AO yield, the dry weight of the bacteria, and the AO produced by the dry weight per unit of the bacteria in each experimental group. As shown in Fig. 7, with the increase of glucose addition, the yield of AO showed an upward trend, but the dry weight of the cells and the dry weight of the unit cells did not increase significantly. Compared with 20 g/L glucose, the dry weight of the cells was significantly reduced and the AO yield was slightly decreased in the medium supplemented with 10 g/L glucose. In contrast, in the medium supplemented with 40 g/L and 50 g/L glucose, the dry weight of the cells and the AO yield were both increased. In the medium supplemented with 50 g/L glucose, the AO produced by the dry weight per unit cell was also slightly increased.

Fig. 7.

The AO fermentation results with different amounts of glucose added. a The AO production per liter of fermentation medium after 48 h of fermentation. b The dry weight of cells per liter of fermentation medium after 48 h of fermentation. c The AO production per gram of dry cell weight after 48 h of fermentation. Three of each experimental group were parallel in all experiments

After the fermentation, we also measured the amount of residual glucose. As shown in Fig. 8, with the continuous increase of the initial glucose addition, although the AO yield has a slow upward trend, the glucose at the end of the fermentation has a more significant increase. At the same time, the utilization rate of glucose has also dropped significantly. Although the medium supplemented with 40 g/L and 50 g/L glucose, the AO yield, the dry weight of the cells, and the amount of AO produced per unit of dry weight of the cells were increased compared to the medium supplemented with 20 g/L glucose, but glucose utilization rates were only 31.25% and 32.64%, respectively, which was much lower than 51.5% when 20 g/L glucose was added. On the other hand, although the glucose utilization rate reached 78.4% in the medium supplemented with 10 g/L glucose, both the AO yield and the dry weight of the cells were reduced. Based on the results of the above fermentation experiments, we believe that the addition of 20 g/L glucose is more conducive to the fermentation of AO.

Fig. 8.

The glucose residue (a) and glucose utilization rate (b) after 48 h of fermentation in medium with different glucose concentrations. Three of each experimental group were parallel in all experiments

Localization of MreB by GFP using a confocal microscope

The expression vector pBBR-mreB-gfp encoding the MreB-GFP fusion protein was constructed for overexpressing MreB and demonstrating localization of MreB. The cell morphology of the recombinant NK-01 overexpressing MreB was observed under a light microscope (63×) or fluorescence microscope (63×) under the same vision. The green fluorescence was observed on the cells when exited by blue light under a fluorescence microscope (Fig. 9). However, no fluorescence was observed on the control cells carrying pBBR1MCS-2 (data not shown). These results indicated that the MreB-GFP fusion protein was successfully expressed in strain NK-01. We used a native constitutive promoter to drive the expression of the MreB-GFP fusion protein; therefore, no inducer was required.

Fig. 9.

Observation of the cell morphology of the recombinant NK-01 harboring pBBR-mreB-gfp using a light microscope (63×) or fluorescence microscope (63×) in the same field of vision

MreB could be tracked for its cellular localization when fused to GFP. The whole process of cell division was observed by a confocal microscope. As shown in Fig. 10, MreB in the cells to be split is first moved to the cell pole to provide traction. However, MreB was evenly distributed within the whole cell after the cell separated completely. We estimate that more MreB expression may contribute to the secretion of bacterial products during this process.

Fig. 10.

Observation of the recombinant NK-01 carrying pBBR-mreB-gfp by a confocal microscope. a Green fluorescence on the cell for the localization of MreB. b Outline of the cell membrane stained with FM4-64. c Merged image of (a) and (b). Diamond (♦) indicates that MreB is located at poles of cells that are about to divide. Triangle (▲) indicates septum of cells that have just finished cell division

Discussion

Recently, most studies have focused on improving output of metabolites by modifying relevant metabolic pathways rather than exploring the effects of the cytoskeleton on cellular metabolism. P. mendocina NK-01 can simultaneously synthesize extracellular biomacromolecule AO and intracellular biomacromolecule PHA. Both the alginate and the PHA_MCL synthetic pathways in Pseudomonas species have been clearly elucidated (Rehm and Steinbüchel 1999). The biosynthetic pathways of PHA_MCL and AO have been initially proposed in P. mendocina NK-01, suggesting that acetyl-CoA may be a co-precursor for the synthesis of AO and PHA_MCL. PHA polymerases (PhaC1 and PhaC2) are the key enzymes in PHA biosynthesis, while the AO polymerase gene is algA. In addition, there are many other gene products that participate in AO secretion.

It has been proposed that E. coli MreB also has an important function in the regulation of the short axis of the cell (Kruse et al. 2003). Indeed, our data support an elementary system that controls the lengths of the long and short axes in NK-01 (Fig. 2 and Table 2). Compared to the wild-type cell, the long axis of the recombinant cell overexpressing MreB was increased by 1.27 times and its short axis was increased by 1.49 times.

MreB is involved in peptidoglycan synthesis (Kruse and Gerdes 2005; van den Ent et al. 2006). Fatter cells were generated under the context of MreB overexpression, suggesting that cell division was moderately inhibited. MreB was marked by green fluorescence at both poles and pulled in two directions when the cell intended to divide. After cell division, MreB tended to be diffused throughout the whole intracellular space (Fig. 10). The cytoskeletal MreB also participates in transportation inside cell (Varma and Young 2009). All proteins involved in AO biosynthesis are located on the cell membranes; therefore, overexpression of MreB might supply more transport pathways and then influence secretion of AO into the extracellular space (Remminghorst and Rehm 2006).

In addition to altering the cell shape, overexpression of MreB affected cell growth rate. While the surface area of a cell remained unchanged, cell volume was increased by about 1.5 times. One of the important parameters that characterize prokaryotes is the surface-to-volume ratio. A decrease in the surface-to-volume ratio might cause impairment of the transport of nutrients. Nutrients and metabolites can be easily transported among all parts of the cell when bacteria have a large surface-to-volume ratio. This suggested that a rod shape would be advantageous for cell growth of relatively larger bacteria (Shiomi and Niki 2013). These observations are consistent with our results that the recombinant strain, with a smaller length–width ratio and a larger inner space, grew slightly slower than the wild type during the logarithmic phase. The recombinant NK-01 overexpressing MreB requires more energy for physical growth rather than synthesis of products, which could explain the low transcription level of algA, phaC1, and phaC2 in the recombinant strain (Fig. 3).

Fig. 4.

Cell growth of the wild-type NK-01 (circles) and recombinant NK-01 carrying pBBR-mreB-gfp (triangles). The wild-type NK-01 and recombinant NK-01 were cultured, respectively, in LB and LB supplemented with 60 μg/mL kanamycin at 30 °C. The growth of cell was determined by measuring the optical density at 600 nm (OD₆₀₀) using a UV spectrophotometer (UV-1800, SHIMADZU)

In addition, a mutation of the rodZ gene, which is also related with cytoskeletal formation, leads to metabolic change in the ability of the cells to utilize certain carbon sources and other metabolites (Ito et al. 2005). Another cell division ring protein, FtsZ, is an important protein of cell division and its inactivation leads to cell elongation from a rod shape to a filamentary shape. This elongated cell accumulates more intracellular products such as sulfur, polyphosphates, magnetosomes, and carboxyl bodies, and these intracellular products are associated with increased intracellular volume (Wang et al. 2014). When the cytoskeleton protein MreB was overexpressed in strain NK-01, cells of larger size were obtained and AO production was enhanced.

Glycolysis, which occurs in the cytosol, was accelerated in the recombinants instead of oxidative phosphorylation, which is critically depending on the membrane. This may explain why carbon metabolism flux becomes more prone to AO production than PHA after overexpressing MreB. In addition, overexpressing MreB may reduce the cell number and prevent cell growth (Fenton and Gerdes 2013), which might explain why the recombinant cells overexpressing MreB grew a little slower than the wild-type cells. Thus, the change in cell shape not only affected the structure of the cell membrane, but also affected various activities inside the cell associated with cell proliferation (Shiomi et al. 2009).

Overexpression of mreB leads to an increase in cell volume and an increase in cell membrane secretion capacity, resulting in a significant increase in AO production. On this basis, we optimized the carbon source content of AO fermentation medium, hoping to find more suitable fermentation conditions for overexpressing strains to further increase AO yield. The results showed that although the cell volume and secretion capacity of the overexpressing strain were more advantageous, the glycogen utilization ability did not change significantly. This was mainly because the overexpression of mreB did not change the main metabolic capacity of the cell. As an extracellular product, AO, the increase in the amount obtained in the overexpressing strain is mainly due to the increase in cell membrane area and secretion capacity, but not to the production capacity of the cell itself.

Taking all these factors into consideration, we concluded that overexpression of MreB in P. mendocina NK-01 could increase extracellular AO production. Currently, we have focused on elucidating the mechanism of MreB-induced AO increase, which will be helpful for increasing our understanding of the roles of MreB in cellular metabolism.

Conclusion

Pseudomonas mendocina NK-01 can synthesize two products, alginic acid oligosaccharide (AO), and medium-chain long polyhydroxyalkanoate (PHA_MCL). At the same time, cytoskeletal proteins play an important role in cell morphology and cell size. Therefore, this experiment hopes to study the relationship between bacterial cell morphology and bacterial product yield, that is, the amount of AO and PHA synthesis, by overexpressing the cytoskeletal protein MreB. In this experiment, the mreB gene was overexpressed by constructing an expression vector encoding the MreB-GFP fusion protein, and introduced into the strain NK-01. The results showed that overexpression of MreB increased cell size and cell length, increased AO polymerase activity and secretion efficiency, and increased AO yield by 5.86 times, although the PHA synthesis by recombinant strain was slightly reduced. In addition, 20 g/L glucose is the best addition amount of glycogen when AO is fermented. The results showed that the overexpression of cytoskeletal protein had certain effects on the production of intracellular and extracellular products, and had positive significance for increasing the yield of specific products through cell morphology transformation.

Electronic supplementary material

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Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.