Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

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Role of phaD in Accumulation of Medium-Chain-Length Poly(3-Hydroxyalkanoates) inPseudomonas oleovorans

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3705-3710.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3705-3710 ◽

Cited By ~ 43

Author(s):

Stefan Klinke ◽

Guy de Roo ◽

Bernard Witholt ◽

Birgit Kessler

Keyword(s):

Chain Length ◽

Wild Type ◽

Knockout Mutant ◽

Medium Chain ◽

Medium Chain Length ◽

Associated Proteins ◽

Intracellular Storage ◽

Mutant Complementation ◽

Mutant Cells

ABSTRACT Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated aphaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.

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Role of carboxyl pendant groups of medium chain length poly(3-hydroxyalkanoate)s in biomedical temporary applications

Journal of Applied Polymer Science ◽

10.1002/app.32041 ◽

2010 ◽

Vol 117 (4) ◽

pp. 1888-1896 ◽

Cited By ~ 11

Author(s):

E. Renard ◽

L. Timbart ◽

G. Vergnol ◽

V. Langlois

Keyword(s):

Chain Length ◽

Medium Chain ◽

Medium Chain Length

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Production of medium chain length polyhydroxyalkanoate from acetate by engineered Pseudomonas putida KT2440

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-019-02159-5 ◽

2019 ◽

Vol 46 (6) ◽

pp. 793-800 ◽

Cited By ~ 9

Author(s):

Songyuan Yang ◽

Suhang Li ◽

Xiaoqiang Jia

Keyword(s):

Pseudomonas Putida ◽

Chain Length ◽

Pseudomonas Putida Kt2440 ◽

Medium Chain ◽

Medium Chain Length

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Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

Applied and Environmental Microbiology ◽

10.1128/aem.00564-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3813-3821 ◽

Cited By ~ 24

Author(s):

Jo-Ann Chuah ◽

Satoshi Tomizawa ◽

Miwa Yamada ◽

Takeharu Tsuge ◽

Yoshiharu Doi ◽

...

Keyword(s):

Chain Length ◽

Fold Increase ◽

Short Chain ◽

Pha Synthase ◽

Wild Type ◽

Short Chain Length ◽

Medium Chain ◽

Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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