Pseudomonas mendocina

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Shufang Wang - One of the best experts on this subject based on the ideXlab platform.

  • Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina.
    International journal of biological macromolecules, 2020
    Co-Authors: Xu Fan, Shufang Wang, Fengjie Zhao, Yiting Zhang, Yujie Liu, Yuxin Zhao, Ruihua Liu, Chao Yang
    Abstract:

    Abstract Pseudomonas mendocina NK-01 previously isolated by our lab is able to accumulate medium-chain-length polyhydroxyalkanoate (mcl-PHA) intracellularly and secrete alginate oligosaccharide (AO) to the extracellular milieu. The present study aimed at investigating whether improved production of mcl-PHA and AO by P. mendocina can be accomplished by genome reduction. In this study, 14 large genomic fragments accounting for 7.7% of the genome of P. mendocina NK-01 were sequentially deleted to generate a series of genome-reduced strains by an upp-based markerless knockout method. As a result, the intracellular ATP/ADP ratio of the strain NKU421 with the largest deletion improved by 11 times compared to NK-01. More importantly, the mcl-PHA and AO yields of NKU421 increased by 114.8% and 27.8%, respectively. Enhancing mcl-PHA and AO production by NKU421 may be attributed to improved transcriptional levels of PHA synthase genes and AO secretion-related genes. The present study suggests that rational reduction of bacterial genome is a feasible approach to construct an optimal chassis for enhanced production of bacterial metabolites. In the future, further reduction of the NKU421 genome can be expected to create high-performance chassis for the development of microbial cell factories.

  • Enhanced synthesis of alginate oligosaccharides in Pseudomonas mendocina NK-01 by overexpressing MreB
    3 Biotech, 2019
    Co-Authors: Xu Fan, Shufang Wang, Ting Gong, Fengjie Zhao, Mingqiang Qiao, Chao Yang
    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.

  • morphology engineering for enhanced production of medium chain length polyhydroxyalkanoates in Pseudomonas mendocina nk 01
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Fengjie Zhao, Shufang Wang, Xu Fan, Ting Gong, Xiangsheng Liu, Rui Huang, Weixia Gao, Chao Yang
    Abstract:

    Polyhydroxyalkanoates (PHAs) can be produced by microorganisms from renewable resources and are regarded as promising bioplastics to replace petroleum-based plastics. A medium-chain-length PHAs (mcl-PHA)-producing strain Pseudomonas mendocina NK-01 was isolated previously by our lab and its whole-genome sequence is currently available. Morphology engineering of manipulating cell morphology–related genes has been applied for enhanced accumulation of the intracellular biopolymer short-chain-length PHAs (scl-PHA). However, it has not yet been reported to improve the yield of mcl-PHA by morphology engineering so far. In this work, several well-characterized cell morphology–related genes, including the cell fission ring (Z-ring) location genes minCD, peptidoglycan degradation gene nlpD, actin-like cytoskeleton protein gene mreB, Z-ring formation gene ftsZ, and FtsZ inhibitor gene sulA, were intensively investigated for their impacts on the cell morphology and mcl-PHA accumulation by gene knockout and overexpression in P. mendocina NKU, a upp knockout mutant of P. mendocina NK-01. For a minCD knockout mutant P. mendocina NKU-∆minCD, the average cell length was obviously increased and the mcl-PHA production was improved. However, the nlpD knockout mutant had a shorter cell length and lower mcl-PHA yield compared with P. mendocina NKU. Overexpression of mreB in P. mendocina NKU resulted in spherical cells. When ftsZ was overexpressed in P. mendocina NKU, the cell division was accelerated and the mcl-PHA titer was improved. Furthermore, mreB, ftsZ, or sulA was overexpressed in P. mendocina NKU-∆minCD. Consequently, the mcl-PHA titers were all increased compared with P. mendocina NKU-∆minCD carrying the empty vector. The multiple fission pattern was finally achieved in ftsZ-overexpressing NKU-∆minCD. In this work, improved production of mcl-PHA in P. mendocina NK-01 has been achieved by morphology engineering. This work provides an alternative strategy to enhance mcl-PHA accumulation in mcl-PHA-producing strains.

  • Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01
    Nature Publishing Group, 2019
    Co-Authors: Fengjie Zhao, Shufang Wang, Xu Fan, Yuxin Zhao, Xiangsheng Liu, Weixia Gao, Annie Kong, Chao Yang
    Abstract:

    Abstract Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA

  • enhancement of medium chain length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in Pseudomonas mendocina
    Biotechnology Letters, 2016
    Co-Authors: Yuanyuan Wang, Shufang Wang, Xu Fan, Fengjie Zhao, Cunjiang Song
    Abstract:

    To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

Cunjiang Song - One of the best experts on this subject based on the ideXlab platform.

  • enhancement of medium chain length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in Pseudomonas mendocina
    Biotechnology Letters, 2016
    Co-Authors: Yuanyuan Wang, Shufang Wang, Xu Fan, Fengjie Zhao, Cunjiang Song
    Abstract:

    To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

  • an upp based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina nk 01 and Pseudomonas putida kt2440
    Journal of Microbiological Methods, 2015
    Co-Authors: Yuanyuan Wang, Xu Fan, Ting Gong, Fengjie Zhao, Chao Yang, Chi Zhang, Zhenqiang Zuo, Cunjiang Song
    Abstract:

    Abstract A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA MCL )-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P . mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA MCL depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P . mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA MCL polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P . putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp -based counterselection system could be further adapted for P . mendocina NK-01 and P . putida KT2440 and used for genome reduction and metabolic pathway engineering.

  • comparison of medium chain length polyhydroxyalkanoates synthases from Pseudomonas mendocina nk 01 with the same substrate specificity
    Microbiological Research, 2013
    Co-Authors: Wenbin Guo, Shufang Wang, Weitao Geng, Jun Feng, Jingjing Duan, Cunjiang Song
    Abstract:

    The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.

  • augmented production of alginate oligosaccharides by the Pseudomonas mendocina nk 01 mutant
    Carbohydrate Research, 2012
    Co-Authors: Wenbin Guo, Yuanyuan Wang, Cunjiang Song, Weitao Geng, Jun Feng, Ning Chen, Shufang Wang
    Abstract:

    Abstract Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHA MCL ) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHA MCL synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30 L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight ( M W ). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHA MCL and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.

  • complete genome of Pseudomonas mendocina nk 01 which synthesizes medium chain length polyhydroxyalkanoates and alginate oligosaccharides
    Journal of Bacteriology, 2011
    Co-Authors: Wenbin Guo, Chao Yang, Yuanyuan Wang, Cunjiang Song, Xiumei Sun, Dongfang Song, Xiaojuan Yang, Shufang Wang
    Abstract:

    Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHAMCL) and alginate oligosaccharides (AO) simultaneously from glucose under conditions of limited nitrogen. Here, we report the complete sequence of the 5.4-Mbp genome of Pseudomonas mendocina NK-01, which was isolated from farmland soil in Tianjin, China.

Wenbin Guo - One of the best experts on this subject based on the ideXlab platform.

  • comparison of medium chain length polyhydroxyalkanoates synthases from Pseudomonas mendocina nk 01 with the same substrate specificity
    Microbiological Research, 2013
    Co-Authors: Wenbin Guo, Shufang Wang, Weitao Geng, Jun Feng, Jingjing Duan, Cunjiang Song
    Abstract:

    The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.

  • augmented production of alginate oligosaccharides by the Pseudomonas mendocina nk 01 mutant
    Carbohydrate Research, 2012
    Co-Authors: Wenbin Guo, Yuanyuan Wang, Cunjiang Song, Weitao Geng, Jun Feng, Ning Chen, Shufang Wang
    Abstract:

    Abstract Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHA MCL ) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHA MCL synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30 L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight ( M W ). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHA MCL and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.

  • complete genome of Pseudomonas mendocina nk 01 which synthesizes medium chain length polyhydroxyalkanoates and alginate oligosaccharides
    Journal of Bacteriology, 2011
    Co-Authors: Wenbin Guo, Chao Yang, Yuanyuan Wang, Cunjiang Song, Xiumei Sun, Dongfang Song, Xiaojuan Yang, Shufang Wang
    Abstract:

    Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHAMCL) and alginate oligosaccharides (AO) simultaneously from glucose under conditions of limited nitrogen. Here, we report the complete sequence of the 5.4-Mbp genome of Pseudomonas mendocina NK-01, which was isolated from farmland soil in Tianjin, China.

  • simultaneous production and characterization of medium chain length polyhydroxyalkanoates and alginate oligosaccharides by Pseudomonas mendocina nk 01
    Applied Microbiology and Biotechnology, 2011
    Co-Authors: Wenbin Guo, Yuanyuan Wang, Cunjiang Song, Weitao Geng, Meimei Kong, Shufang Wang
    Abstract:

    When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L−1 medium-chain-length polyhydroxyalkanoate (PHAMCL) and 0.57 g L−1 alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHAMCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T m and T g values for the PHAMCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was 700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3.

  • synthesis and characterization of alginate oligosaccharides produced by Pseudomonas mendocina nk 01
    Chinese Journal of Biotechnology, 2009
    Co-Authors: Wenbin Guo, Shufang Wang, Mingfeng Cao, Weitao Geng, Cunjiang Song
    Abstract:

    In this study, we found that when Pseudomonas mendocina NK-01 accumulated intracellular carbon reserve, medium chain length poly (3-hydroxyalkanoates), it also synthesized extracellular saccharides, alginate oligosaccharides. The high carbon nitrogen ratio of culture medium facilitated alginate oligosaccharides production. We analyzed the structure of alginate oligosaccharide by Ultraviolet-Visible Spectrophotometry, Fourier Transform Infrared Spectroscopy, 1H and 13C of Nuclear Magnetic Resonance, and found that it was compounded in line from beta-D-mannuronic acids and alpha-L-gluronic acids via beta-(1-->4)/ alpha-(1-->4) bonds, which acetylated partly on the 2- and/or 3-hydroxy. In addition, we determined the weight-average molecular weight of alginate oligosaccharides by gel permeation chromatography to be 2054.

Chao Yang - One of the best experts on this subject based on the ideXlab platform.

  • Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina.
    International journal of biological macromolecules, 2020
    Co-Authors: Xu Fan, Shufang Wang, Fengjie Zhao, Yiting Zhang, Yujie Liu, Yuxin Zhao, Ruihua Liu, Chao Yang
    Abstract:

    Abstract Pseudomonas mendocina NK-01 previously isolated by our lab is able to accumulate medium-chain-length polyhydroxyalkanoate (mcl-PHA) intracellularly and secrete alginate oligosaccharide (AO) to the extracellular milieu. The present study aimed at investigating whether improved production of mcl-PHA and AO by P. mendocina can be accomplished by genome reduction. In this study, 14 large genomic fragments accounting for 7.7% of the genome of P. mendocina NK-01 were sequentially deleted to generate a series of genome-reduced strains by an upp-based markerless knockout method. As a result, the intracellular ATP/ADP ratio of the strain NKU421 with the largest deletion improved by 11 times compared to NK-01. More importantly, the mcl-PHA and AO yields of NKU421 increased by 114.8% and 27.8%, respectively. Enhancing mcl-PHA and AO production by NKU421 may be attributed to improved transcriptional levels of PHA synthase genes and AO secretion-related genes. The present study suggests that rational reduction of bacterial genome is a feasible approach to construct an optimal chassis for enhanced production of bacterial metabolites. In the future, further reduction of the NKU421 genome can be expected to create high-performance chassis for the development of microbial cell factories.

  • Enhanced synthesis of alginate oligosaccharides in Pseudomonas mendocina NK-01 by overexpressing MreB
    3 Biotech, 2019
    Co-Authors: Xu Fan, Shufang Wang, Ting Gong, Fengjie Zhao, Mingqiang Qiao, Chao Yang
    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.

  • morphology engineering for enhanced production of medium chain length polyhydroxyalkanoates in Pseudomonas mendocina nk 01
    Applied Microbiology and Biotechnology, 2019
    Co-Authors: Fengjie Zhao, Shufang Wang, Xu Fan, Ting Gong, Xiangsheng Liu, Rui Huang, Weixia Gao, Chao Yang
    Abstract:

    Polyhydroxyalkanoates (PHAs) can be produced by microorganisms from renewable resources and are regarded as promising bioplastics to replace petroleum-based plastics. A medium-chain-length PHAs (mcl-PHA)-producing strain Pseudomonas mendocina NK-01 was isolated previously by our lab and its whole-genome sequence is currently available. Morphology engineering of manipulating cell morphology–related genes has been applied for enhanced accumulation of the intracellular biopolymer short-chain-length PHAs (scl-PHA). However, it has not yet been reported to improve the yield of mcl-PHA by morphology engineering so far. In this work, several well-characterized cell morphology–related genes, including the cell fission ring (Z-ring) location genes minCD, peptidoglycan degradation gene nlpD, actin-like cytoskeleton protein gene mreB, Z-ring formation gene ftsZ, and FtsZ inhibitor gene sulA, were intensively investigated for their impacts on the cell morphology and mcl-PHA accumulation by gene knockout and overexpression in P. mendocina NKU, a upp knockout mutant of P. mendocina NK-01. For a minCD knockout mutant P. mendocina NKU-∆minCD, the average cell length was obviously increased and the mcl-PHA production was improved. However, the nlpD knockout mutant had a shorter cell length and lower mcl-PHA yield compared with P. mendocina NKU. Overexpression of mreB in P. mendocina NKU resulted in spherical cells. When ftsZ was overexpressed in P. mendocina NKU, the cell division was accelerated and the mcl-PHA titer was improved. Furthermore, mreB, ftsZ, or sulA was overexpressed in P. mendocina NKU-∆minCD. Consequently, the mcl-PHA titers were all increased compared with P. mendocina NKU-∆minCD carrying the empty vector. The multiple fission pattern was finally achieved in ftsZ-overexpressing NKU-∆minCD. In this work, improved production of mcl-PHA in P. mendocina NK-01 has been achieved by morphology engineering. This work provides an alternative strategy to enhance mcl-PHA accumulation in mcl-PHA-producing strains.

  • Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01
    Nature Publishing Group, 2019
    Co-Authors: Fengjie Zhao, Shufang Wang, Xu Fan, Yuxin Zhao, Xiangsheng Liu, Weixia Gao, Annie Kong, Chao Yang
    Abstract:

    Abstract Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA

  • an upp based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina nk 01 and Pseudomonas putida kt2440
    Journal of Microbiological Methods, 2015
    Co-Authors: Yuanyuan Wang, Xu Fan, Ting Gong, Fengjie Zhao, Chao Yang, Chi Zhang, Zhenqiang Zuo, Cunjiang Song
    Abstract:

    Abstract A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA MCL )-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P . mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA MCL depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P . mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA MCL polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P . putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp -based counterselection system could be further adapted for P . mendocina NK-01 and P . putida KT2440 and used for genome reduction and metabolic pathway engineering.

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  • enhancement of medium chain length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in Pseudomonas mendocina
    Biotechnology Letters, 2016
    Co-Authors: Yuanyuan Wang, Shufang Wang, Xu Fan, Fengjie Zhao, Cunjiang Song
    Abstract:

    To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

  • an upp based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina nk 01 and Pseudomonas putida kt2440
    Journal of Microbiological Methods, 2015
    Co-Authors: Yuanyuan Wang, Xu Fan, Ting Gong, Fengjie Zhao, Chao Yang, Chi Zhang, Zhenqiang Zuo, Cunjiang Song
    Abstract:

    Abstract A markerless gene replacement method was adapted by combining a suicide plasmid, pEX18Tc, with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the medium-chain length polyhydroxyalkanoates (PHA MCL )-producing strain Pseudomonas mendocina NK-01. An NK-01 5-fluorouracil (5-FU) resistant background strain was first constructed by deleting the chromosomal upp gene. The suicide plasmid pEX18Tc, carrying a functional allele of the upp gene of P . mendocina NK-01, was used to construct the vectors to delete the algA (encoding mannose-1-phosphate guanylyltransferase) and phaZ (encoding PHA MCL depolymerase) genes, and a 30 kb chromosomal fragment in the 5-FU resistant background host. The genes were removed efficiently from the genome of P . mendocina NK-01 and left a markerless chromosomal mutant. In addition, two exogenous genes were inserted into the phaC1 (PHA MCL polymerase) loci of Pseudomonas putida KT-∆UPP simultaneously. Thus, we constructed a genetically stable and marker-free P . putida KT2440 mutant with integrated mpd (encoding methyl parathion hydrolase (MPH)) and pytH (encoding a pyrethroid-hydrolyzing carboxylesterase (PytH)) gene on the chromosome. The upp -based counterselection system could be further adapted for P . mendocina NK-01 and P . putida KT2440 and used for genome reduction and metabolic pathway engineering.

  • augmented production of alginate oligosaccharides by the Pseudomonas mendocina nk 01 mutant
    Carbohydrate Research, 2012
    Co-Authors: Wenbin Guo, Yuanyuan Wang, Cunjiang Song, Weitao Geng, Jun Feng, Ning Chen, Shufang Wang
    Abstract:

    Abstract Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHA MCL ) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHA MCL synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30 L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight ( M W ). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHA MCL and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.

  • complete genome of Pseudomonas mendocina nk 01 which synthesizes medium chain length polyhydroxyalkanoates and alginate oligosaccharides
    Journal of Bacteriology, 2011
    Co-Authors: Wenbin Guo, Chao Yang, Yuanyuan Wang, Cunjiang Song, Xiumei Sun, Dongfang Song, Xiaojuan Yang, Shufang Wang
    Abstract:

    Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHAMCL) and alginate oligosaccharides (AO) simultaneously from glucose under conditions of limited nitrogen. Here, we report the complete sequence of the 5.4-Mbp genome of Pseudomonas mendocina NK-01, which was isolated from farmland soil in Tianjin, China.

  • simultaneous production and characterization of medium chain length polyhydroxyalkanoates and alginate oligosaccharides by Pseudomonas mendocina nk 01
    Applied Microbiology and Biotechnology, 2011
    Co-Authors: Wenbin Guo, Yuanyuan Wang, Cunjiang Song, Weitao Geng, Meimei Kong, Shufang Wang
    Abstract:

    When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L−1 medium-chain-length polyhydroxyalkanoate (PHAMCL) and 0.57 g L−1 alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHAMCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T m and T g values for the PHAMCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was 700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3.

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