Metabolic engineering of Pseudomonas mendocina NK-01 for enhanced production of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer

doi:10.1016/j.ijbiomac.2019.11.044

International Journal of Biological Macromolecules

Volume 154, 1 July 2020, Pages 1596-1605

https://doi.org/10.1016/j.ijbiomac.2019.11.044 Get rights and content

Highlights

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The mcl-PHA titer was improved by inhibition of the β-oxidation pathway.
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Knockout of phaZ improved the contents of the dominant monomer of mcl-PHA.
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The physical properties of mcl-PHA were improved.
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The cell morphology obviously changed for the β-oxidation pathway inhibition strains.

Abstract

In this study, six genes involved in β-oxidation pathway of P. mendocina NK-01 were deleted to construct mutant strains NKU-∆β1 and NKU-∆β5. Compared with the wild strain NKU, the mcl-PHA titers of NKU-∆β5 were respectively increased by 5.58- and 4.85-fold for culturing with sodium octanoate and sodium decanoate. And the mcl-PHA titers of NKU-∆β1 was increased by 10.02-fold for culturing with dodecanoic acid. The contents of dominant monomers 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD) of the mcl-PHA synthesized by NKU-∆β5 were obviously increased to 90.01 and 58.60 mol%, respectively. Further deletion of genes phaG and phaZ, the 3HD and 3HDD contents were further improved to 94.71 and 68.67 mol%, respectively. The highest molecular weight of mcl-PHA obtained in this study was 80.79 × 10⁴ Da, which was higher than the previously reported mcl-PHA. With the increase of dominant monomer contents, the synthesized mcl-PHA showed better thermal properties, mechanical properties and crystallization properties. Interestingly, the cell size of NKU-∆β5 was larger than that of NKU due to the accumulation of more PHA granules. This study indicated that a systematically metabolic engineering approach for P. mendocina NK-01 could significantly improve the mcl-PHA titer, dominant monomer contents and physical properties of mcl-PHA.

Graphical abstract

Introduction

Polyhydroxyalkanoates (PHA) are a kind of intracellular biopolyesters which are accumulated by many bacteria under unbalanced growth conditions [[1], [2], [3]]. Microbial PHA can be used for packaging and biomedicine materials due to their unique biodegradability, biocompatibility, and excellent thermal and mechanical properties [[4], [5], [6], [7]]. The R-3-hydroxyalkanoate methyl ester which was obtained by acid catalyzed hydrolysis of PHA can be used as biofuels [5,8]. PHA have been used as potential alternatives to petroleum-based chemicals. PHA are traditionally classified into two major types based on monomer carbon chain length, i.e., short-chain-length PHA (scl-PHA) with monomer units of C3–C5 and medium-chain-length PHA (mcl-PHA) with monomer units of C6–C14 [[9], [10], [11]].

scl-PHA commonly consist of monomers of 3-hydroxypropionate (3HP), 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3 HV), such as polyhydroxybutyrate (PHB), while the mcl-PHA normally contain monomer of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD). PHB is brittle and stiff due to its high crystallization (melting temperatures, T_m > 170 °C) [3,12,13]. While mcl-PHA are elastomeric and amorphous which have low T_m, weak tensile strength and longer elongation rate [3,14]. Thus, mcl-PHA can be used as biodegradable elastomers, adhesives or blending materials to improve the physical properties of PHA [15,16].

Some strains of Pseudomonas spp. have an ability to accumulate mcl-PHA via either fatty acid de novo biosynthesis pathway from unrelated carbon source (e.g. glucose) or β-oxidation pathway from related carbon source (e.g. fatty acids). Although metabolic engineering approaches, such as the increase of NADPH availability and the reduction of by-products accumulation, could improve the mcl-PHA production using glucose as carbon source through fatty acid de novo biosynthesis [17,18]. The monomers composition of mcl-PHA accumulated from this biosynthetic pathway is not easy to control. For fatty acids as the carbon source, β-oxidation pathway plays an important role in providing intermediates for PHA synthesis. When fatty acids enter β-oxidation pathway, two carbon atoms are lost after each cycle and the remaining acyl-CoA enter into the next cycle. The intermediate 2-tran-enoyl-CoA can be catalyzed to R-3-hydroxyacyl-CoA by an R-enoyl-CoA hydratase (PhaJ) and then R-3-hydroxyacyl-CoA are polymerized into mcl-PHA by PHA synthetase (PhaC). 3-ketoacyl-CoA thiolase (FadA) and 3-hydroxyacyl-CoA dehydrogenase (FadB) play important roles in catalyzing the last two steps of β-oxidation cycle [14,[19], [20], [21], [22]]. Many researches have focused on the two key steps to construct β-oxidation pathway inhibited strains to increase mcl-PHA accumulation and synthesize novel functionalized mcl-PHA.

It has been shown that mutated in the fadA and fadB genes on β-oxidation pathway elicited a strong intracellular accumulation of biopolymers for Pseudomonas putida. An enhanced accumulation of functionalized mcl-PHA which contained side groups has been obtained through this strategy in P. putida [20,23]. Ouyang et al. [14] found that the mutant strain KTOY06 with fadA and fadB knocked out in P. putida KT2442 accumulated 84 wt% mcl-PHA, much higher than 50 wt% in the wild type when cultured with dodecanoate as the carbon source. In addition, the HDD fraction in mcl-PHA produced by KTOY06 was 41 mol% along with increased crystallinity and tensile strength, much higher than 7.5 mol% in mcl-PHA synthesized by KT2442 [14]. Ma et al. [10] further inhibited the β-oxidation pathway to construct mutant strain P. putida KT2047A which could synthesize copolymer P(3HD-co-56 mol% 3HDD) with a higher T_m in the presence of dodecanoate. Combined β-oxidation pathway inhibition and phaG gene knockout, the mutant strain P. putida KTQQ20 could synthesized homopolymer poly-3-hydroxydecanoate (PHD) and copolymer P(3HD-co-84 mol% 3HDD), when grown on decanoic acid and dodecanoic acid, respectively [24]. However, the mcl-PHA titers (wt%) were all lower than 15% for the above copolymer and homopolymer. Pseudomonas entomophila L48 which showed a close relationship to Pseudomonas putida was deleted some of the genes encoding key enzymes in β-oxidation cycle. The mutant strain P. entomophila LAC26 accumulated mcl-PHA over 90 wt% with a high 3HDD fraction of 99 mol% compared with 50 wt% and 11 mol% 3HDD for wild type in the presence of dodecanoic acid [22]. When simultaneously cultured with two fatty acids as the carbon source, random and block PHA could be synthesized by β-oxidation pathway deficient pseudomonas, which could be a platform for the production of PHA with adjustable monomer contents and compositions [[25], [26], [27], [28]].

Sodium acrylate which was a β-oxidation pathway inhibitor was also used to block the β-oxidation cycle. It was reported that Ralstonia eutropha could accumulate scl/mcl polyhydroxyalkanoate copolymers in the presence of sodium acrylate when cultured with sodium octanoate [29]. For P. putida KT2440, the monomer contents of 3HN, 3HO and 3HD were all obviously increased in the mcl-PHA when cultured with the relevant carbon source and sodium acrylate [30,31].

Pseudomonas mendocina NK-01, which was isolated by our lab from farmland soil, can synthesize mcl-PHA and alginate oligosaccharides (AO) simultaneously from glucose [32]. The synthesized mcl-PHA possessed special monomer compositions with a small number of longer chain length monomers and also showed superior physical properties [33]. To date, whole-genome sequencing of P. mendocina NK-01 has been completed and the biosynthetic pathway of mcl-PHA has been elucidated in this strain [32,33]. A genome editing system has also been developed for P. mendocina NK-01 [34]. We have done many works to enhance the mcl-PHA accumulation of NK-01 when cultured with glucose as the carbon source, such as metabolic engineering, promoter engineering and morphology engineering [[35], [36], [37]].

In this work, we deleted six key genes which encode FadA and FadB to inhibit the β-oxidation pathway of P. mendocina NK-01. The mcl-PHA titers were obviously improved through inhibition of the β-oxidation pathway when cultured with fatty acids as the carbon source. The dominant monomer (3HD, 3HDD) contents for the mcl-PHA synthesized by β-oxidation pathway inhibition strain were greatly increased compared with that of the wild strain. Then, deletion of genes phaG and phaZ further increased the 3HD and 3HDD contents. With the increase of dominant monomer contents, the physical properties of mcl-PHA were also improved. The cell morphology obviously changed for the β-oxidation pathway inhibition strain.

Section snippets

Bacterial strains, plasmids, and growth conditions

E. coli S17–1 was used for plasmid construction and conjugal transfer. P. mendocina NK-01, which was deposited in China Center for Type Culture Collection (CCTCC, no. CCTCC M 208005), is resistant to chloramphenicol and can synthesize mcl-PHA [32]. P. mendocina NKU, an upp knockout mutant of P. mendocina NK-01 [34], was used as the starting strain. A suicide plasmid pEX18Tc-upp [34] was used for gene knockout. All plasmids and strains used in this study are listed in Table 1.

Luria-Bertani (LB)

Construction of mutant strains of P. mendocina NKU

Firstly, the key genes which encode 3-ketoacyl-CoA thiolase (FadA) and 3-hydroxyacyl-CoA dehydrogenase (FadB) of β-oxidation pathway were deleted in P. mendocina NKU to inhibit the shortening of the carbon chain for fatty acids as the carbon source. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG), MDS_1633 and MDS_1634 which are located together in a gene cluster are fadB gene and fadA gene, respectively. MDS_3526 and MDS_2865 encode the same product as gene fadA. MDS_2116 and MDS_2754

Discussion

In this study, enhanced production of mcl-PHA with high dominant monomer contents (3HO, 3HD and 3HDD) were obtained in P. mendocina NK-01 by inhibition of β-oxidation pathway, when cultured with fatty acids as the carbon source. Compared with the wild strain, the mcl-PHA accumulation of β-oxidation mutant strains were greatly increased at most 10-fold (Fig. 1), which were more significant than the P. putida [24]. These results indicate that weakened of β-oxidation pathway can promote the fatty

Conclusion

In this study, we improved the production of mcl-PHA with enriched content of the dominant monomer in P. mendocina NKU through a systematically metabolic engineering approach when using fatty acids as the carbon source. The contents of the dominant monomers 3HD and 3HDD were significantly increased by inhibition of the β-oxidation pathway, followed by deletion of phaG and phaZ when using C10 and C12 as the carbon source. Moreover, the physical properties including the molecular weight, thermal

Acknowledgments

This work was supported by the National Natural Science Fund of China (Grant Nos. 31570035 and 31670093), and the Tianjin Natural Science Foundation (Grant Nos. 17JCZDJC32100 and 18JCYBJC24500).

Declaration of competing interests

The authors declare that they have no competing interests.

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Metabolic engineering of Pseudomonas mendocina NK-01 for enhanced production of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Bacterial strains, plasmids, and growth conditions

Construction of mutant strains of P. mendocina NKU

Discussion

Conclusion

Acknowledgments

Declaration of competing interests

Bioresour. Technol.

Bioresour. Technol.

Curr. Opin. Biotechnol.

Biomaterials

Bioresour. Technol.

Metab. Eng.

Thermochim. Acta

Bioresour. Technol.

Metab. Eng.

Metab. Eng.

Bioresour. Technol.

J. Microbiol. Methods

Microbiol. Res.

Microb. Cell Factories

Metab. Eng.

Metab. Eng.

J. Biol. Chem.

Occurrence, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates

Microbial. Rev.

Plastics derived from biological sources: present and future: a technical and environmental review

Chem. Rev.

A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry

Chem. Soc. Rev.

Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels

Biotechnol. Biofuels

Polyester synthases: natural catalysts for plastics

Biochem. J.

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Biomacromolecules

Preparation and properties of a novel class of polyhydroxyalkanoate copolymers

Biomacromolecules