Cupriavidus necator

Poly(3-hydroxybutyrate) is a biopolymer produced by numerous bacteria in nature as an intercellular carbon and energy reserve and belongs to the class of poly (hydroxyalkanoate)s (PHAs). In 1925, the French microbiologist Maurice Lemoigne discovered and characterized PHB extracted from Bacillus megaterium. However, it is produced by a various number of microorganisms such as Cupriavidus necator or Ralstonia eutroph. PHAs are biodegradable polyesters with a structure as shown in Fig. 1. 

The residues Cys-319, Asp-480 and His-508 of the class I polyester synthase from Cupriavidus necator are conserved in all PHA synthases and were shown to be essential for covalent catalysis [2,4, 5]. Cys-319 is the proposed catalytic nucleophile that is activated by the general base catalyst His-508. 

Product quality of PHAs is very much dependent on the polyester composition (see 3.2.2.1). In 1987, Byrom found that poly-(3HB-co-3HV) can be produced in large-scale fed-batch culture by supplementing the nutritional medium of a glucose-utilising mutant of Alcaligenes eutrophus (today known as Cupriavidus necator) with propionic acid (precursor for 3HV formation) [50]. Later it was shown that the utilisation of valeric acid instead of propionic acid results in a higher proportion of 3HV units [51]. The improvements in product quality of co- and terpolyesters, however, results in an increase of the production costs of the polymer because of the high price of the precursors. 

I 60 - 73 kDa Cupriavidus necator Sinorhizobium melioti Burkholderia sp. 3HAsci-CoA (-C3-C5) 4HAsci-CoA, 5HAsci-CoA,... 

Bhubalan K, Rathi D-N, Abe H, Iwata T, Sudesh K (2010) Improved synthesis of P(3HB -co-3HV-co-3HHx) terpolymers by mutant Cupriavidus necator using the PHA synthase gene of Chromobacterium sp. USM2 with high affinity towards 3HV. Polym Degrad Stab 95 1436-1442... 

Cavalheiro JMBT, de Almeida MCMD, Grandfils C, da Fonseca MMR (2009) Poly (3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process Biochem 44 509-515... 

Kek Y-K, Lee W-H, Sudesh K (2008) Efficient bioconversion of palm acid oQ and palm kernel acid oil to poly(3-hydroxybutyrate) by Cupriavidus necator. Can J Chem 86 533-539 KekYK, Chang CW, Amirul AA, Sudesh K (2010) Heterologous expression of Cupriavidus sp. USMAA2-4 PHA synthase gene in PHB 4 mutant for the production of poly(3-hydroxybu-tyrate) and its copolymers. World J Microbiol Biotechnol 26 1595-1603 KekYK, Sudesh K (Unpublished)... 

Mifune J, Nakamura S, Eukui T (2008) Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can J Chem 86 621-627... 

Ng KS, Ooi WY, Goh LK, Shenbagarathai R, Sudesh K (2010) Evaluation of Jatropha oil to produce poly(3-hydroxybutyrate) by Cupriavidus necator H16. Polym Degrad Stab 95 1365-1369... 

Rao U, Sridhar R, Sehgal PK (2010) Biosynthesis and biocompatibility of poly(3-hydroxybu-tyrate-co-4-hydroxybutyrate) produced by Cupriavidus necator from spent palm oil. Biochem Eng 149 13-20... 

Wang J, Yue Z-B, Sheng G-P, Yu H-Q (2010) Kinetic analysis on the production of polyhydroxy-alkanoates from volatile fatty acids by Cupriavidus necator with a consideration of substrate inhibition, cell growth, maintenance, and product formation. Biochem Eng J 49 422-428 Wang Y, Yamada S, Asakawa N, Yamane T, Yoshie N, Inoue Y (2001) Comonomer compositional distribution and thermal and morphological characteristics of bacterial poly(3-hydroxybu-tyrate-co-3-hydroxyvalerate)s with high 3-hydroxyvalerate content. Biomacromolecules 2 1315-1323... 

STEM picture of Cupriavidus necator cells harbouring granules of PHA biopolymers (magnification... 

Crude glycerol phase Biodiesel production Glycerol Haloferax mediterranei. Bacillus sphaericus, Methylobacterium rhodesianum Halorcula sp., Methylobacterium extorquens, Novosphingobium capsulatum, Cupriavidus necator. Pseudomonas oleovorans. Pseudomonas corrugata [35, 42 6)... 

Saturated biodiesel fraction Biodiesel production from animal lipids Methyl esters of fatty acids Cupriavidus necator [47,48]... 

In the case of 5C/-PHAs, the monomers consist of three to five carbon atoms, and mainly constitute R-configured chiral 3-hydroxyalkanoates. jcZ-PHAs mainly feature properties of classical thermoplasts hence, on the market, they compete with poly(ethylene) (PE) or poly(propylene) (PP), and, to a certain extent, also with bio-based poly(Z,-lactic acid). The strain Cupriavidus necator, a member of the Burkholderiaceae family, can be regarded as the best investigated bacterial jcZ-PHA producer. 

The type of PHA produced biotechnologically is highly dependent on the microbial production strain. Cupriavidus necator can only polymerize 3HAs consisting of 3-5 C-atoms... 

Biomer Germany 1993 Cupriavidus necator Glucose from corn starch PHB Biomer 500t-1000t... 

Imperial Chemical Industries (ICI) (Later Zeneca, Monsanto) UK 1976-1998 Alcaligenes eutrophus (today Cupriavidus necator) Glucose from carbohydrate feedstocks PHB, later also PHBHV BIOPOL 8001 (later phase under Monsanto)... 

Tianjin Green Bioscience DSM Tianan PR China PR China 2004- ongoing n.r. Cupriavidus necator P(3HB-co-4HB) PHBHV Green Bio Enmat 10 0001 lOOt-IOOOt... 

PHB Industrial/ Copersucar (PHBISA) Brazil 1995-ongoing Alcaligenes eutrophus (today Cupriavidus necator) Burkholderia sacchari Cane sugar PHB and PHBHV BIOCYCLE 1001 (capacity 5000 t)... 

Atlic, A., Koller, M., Scherzer, D. et al. (2011) Continuous production of poly([(R]-3-hydroxybutyrate) by Cupriavidus necator in a multistage bioreactor cascade. Applied Microbiology and Biotechnology, 91, 295-304. 

Park and Kim [15] demonstrated the production of homopolymer P(3HB) from soybean oil as the sole carbon source through batch and fed-batch cultures of Cupriavidus necator with 15-32 g/1 of CDW, PHA content of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. Copolymer poly(3HB-co-4HB) with a 4HB composition ranging from 6-10 mol% was also produced by feeding soybean oil and y-butyrolactone together. The copolymers with various 4HB fractions were produced with a CDW of 10-21 g/1 and PHA yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50 g-PHA/g-carbon sources). Marjadi and Dharaiya [16] have also reported the production of homopolymer P(3HB) with the highest CDW of 13.1 g/1 and PHA content of 87 wt% from soybean oil (5 g/1) by Bacillus subtilis. The soybean oil used was comprised of 61% polyunsaturated fats and 24% monounsaturated fats which is comparable to the total unsaturated fat content of other vegetable oils (-85%). 

There are four classes of PhaC reported so far. PhaC belonging to the first class utilise (R)-3-hydroxy fatty acids with 3-5 carbon atoms and produce short-chain-length (sc/)-monomers this type of PhaC can be found in Cupriavidus necator [2, 7]. Class II PhaC can be found in Pseudomonas aeruginosa, catalyse medium-chain-length... 

Slater and co-workers [38] reported that poly[3HB-co-3-hydroxyvalerate (3HV)] could be synthesised by a special mutant (atoC fadR) strain of Escherichia coli LS5218 harbouring the PHA biosynthesis genes of Cupriavidus necator, which enabled the constitutive expression of the enzymes responsible for the utilisation of short-chain fatty acids however, Escherichia coli LS5218 did not produce a high cell density culture. Next, Choi and Lee (1999)... 

In another study by Dennis and co-workers [41], the PHA accumulation of several microorganisms Escherichia coli, Klebsiella aerogenes and PHA-negative mutants of Cupriavidus necator and Pseudomonas putida) that expressed the phaC and acetoacetyl-coenzyme A reductase phaB) of Cupriavidus necator were analysed. This results in a construct that puts phaC and phaB under the control of the original Cupriavidus necator promoter. It was found that wild-type Cupriavidus necator was able to produce sc/-PHA, however the recombinant Cupriavidus necator expressing its own phaC and phaB without P-ketothiolase phaA) were able to accumulate poly(3HB-co-3HHx) when cultivated with even number chain fatty acids. The same trend was reported for Klebsiella aerogenes and Pseudomonas putida. 

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