Medium chain length polyhydroxyalkanoates, promising new biomedical materials for the future

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.

Download Full-text

Metabolic Engineering for Microbial Production and Applications of Copolyesters Consisting of 3-Hydroxybutyrate and Medium-Chain-Length 3-Hydroxyalkanoates

Macromolecular Bioscience ◽

10.1002/mabi.200600186 ◽

2007 ◽

Vol 7 (2) ◽

pp. 174-182 ◽

Cited By ~ 16

Author(s):

Xiang Hui Zou ◽

Guo-Qiang Chen

Keyword(s):

Metabolic Engineering ◽

Chain Length ◽

Microbial Production ◽

Medium Chain ◽

Medium Chain Length

Download Full-text

Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46

Canadian Journal of Microbiology ◽

10.1139/w2012-074 ◽

2012 ◽

Vol 58 (8) ◽

pp. 982-989 ◽

Cited By ~ 24

Author(s):

Parveen K. Sharma ◽

Jilagamazhi Fu ◽

Nazim Cicek ◽

Richard Sparling ◽

David B. Levin

Keyword(s):

Pseudomonas Putida ◽

Chain Length ◽

Saturated Fatty Acids ◽

Dry Mass ◽

Nucleotide Sequence Analysis ◽

Glucose Medium ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Production ◽

Nitrogen Excess

Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P. putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P. putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12 h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48 h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C6–C14 saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20 mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1–68.8 mol%) in P. putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88 mol%).

Download Full-text

Effects of propionate and carnitine on the hepatic oxidation of short- and medium-chain-length fatty acids

Biochemical Journal ◽

10.1042/bj2500819 ◽

1988 ◽

Vol 250 (3) ◽

pp. 819-825 ◽

Cited By ~ 52

Author(s):

E P Brass ◽

R A Beyerinck

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Organic Acid ◽

Chain Length ◽

Ketone Body ◽

Acetyl Coa ◽

Body Formation ◽

Medium Chain ◽

Medium Chain Length ◽

Hepatic Oxidation

Accumulation of propionate, or its metabolic product propionyl-CoA, can disrupt normal cellular metabolism. The present study examined the effects of propionate, or propionyl-CoA generated during the oxidation of odd-chain-length fatty acids, on hepatic oxidation of short- and medium-chain-length fatty acids. In isolated hepatocytes, ketone-body formation from odd-chain-length fatty acids was slow as compared with even-chain-length fatty acid substrates, and increased as the carbon chain length was increased from five to seven to nine. In contrast, rates of ketogenesis from butyrate, hexonoate and octanoate were all approximately equal. Propionate (10 mM) inhibited ketogenesis from butyrate, hexanoate and octanoate by 81%, 53% and 18% respectively. Addition of carnitine had no effect on ketogenesis from the even-chain-length fatty acids, but increased the rate of ketone-body formation from pentanoate (by 53%), heptanoate (by 28%) and from butyrate or hexanoate in the presence of propionate. The inhibitory effect of propionate could not be explained by shunting acetyl-CoA into the tricarboxylic acid cycle, as CO2 formation from butyrate was also decreased by propionate. Examination of the hepatocyte CoA pool during oxidation of butyrate demonstrated that addition of propionate decreased acetyl-CoA and CoA as propionyl-CoA accumulated. Addition of carnitine decreased propionyl-CoA by 50% (associated with production of propionylcarnitine) and increased acetyl-CoA and CoA. Similar changes in the CoA pool were seen during the oxidation of pentanoate. These results demonstrate that accumulation of propionyl-CoA results in inhibition of short-chain fatty acid oxidation. Carnitine can partially reverse this inhibition. Changes in the hepatocyte CoA pool are consistent with carnitine acting by generating propionylcarnitine, thereby decreasing propionyl-CoA and increasing availability of free CoA. The data provide further evidence of the potential cellular toxicity from organic acid accretion, and supports the concept that carnitine's interaction with the cellular CoA pool can have a beneficial effect on cellular metabolism and function under conditions of unusual organic acid accumulation.

Download Full-text

Draft Genome Sequence of Medium-Chain-Length Polyhydroxyalkanoate-Producing Pseudomonas putida Strain LS46

Genome Announcements ◽

10.1128/genomea.00151-13 ◽

2013 ◽

Vol 1 (2) ◽

Cited By ~ 6

Author(s):

P. K. Sharma ◽

J. Fu ◽

X. Zhang ◽

B. W. Fristensky ◽

K. Davenport ◽

...

Keyword(s):

Pseudomonas Putida ◽

Chain Length ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Medium Chain ◽

Medium Chain Length

Download Full-text

Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans : economic considerations

Applied Microbiology and Biotechnology ◽

10.1007/s002530051100 ◽

1997 ◽

Vol 48 (5) ◽

pp. 588-596 ◽

Cited By ~ 35

Author(s):

W. Hazenberg ◽

B. Witholt

Keyword(s):

Chain Length ◽

Efficient Production ◽

Pseudomonas Oleovorans ◽

Medium Chain ◽

Medium Chain Length ◽

Economic Considerations

Download Full-text

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.185.18.5391-5397.2003 ◽

2003 ◽

Vol 185 (18) ◽

pp. 5391-5397 ◽

Cited By ~ 64

Author(s):

Si Jae Park ◽

Sang Yup Lee

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Chain Length ◽

Biosynthetic Pathway ◽

Pha Synthase ◽

Enzymatic Analysis ◽

E Coli ◽

Medium Chain ◽

Medium Chain Length ◽

Enoyl Coa Hydratase

ABSTRACT The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

Download Full-text