Polyhydroxyalkanoates production

Genes coding for key enzymes of PHA biosynthesis (PhaA, PhaB and PhaC) are frequently organized in one operon, such as Cupriavidus necator (formerly Ralstonia eutropha) ¹⁷ or Pseudomonas sp. (Luengo et al., 2003; Babel and Ackerman, 2001). Organization of the biosynthesis operons depends on bacterial species and also on the PHA synthase class ⁵² as represented in Figure 4. Four classes of PHA synthases are described: Synthase class I and class II are both composed of one phaC gene but class I synthases specifically use substrates composed of 3 to 5 carbon atoms and class II substrates with 6 to 14 carbon atoms. Class I synthases are responsible for the production of scl-PHA and can be found in species like C. necator while class II synthases produce medium chain length PHA (mcl-PHA) and are described in Pseudomonas species. Class III PHA synthases is composed of two genes, phaC and phaE, and possess specificity for substrates with 3 to 5 carbon atoms like class I but can also synthetize PHA with 6 to 8 carbon atoms. Finally, class IV PHA synthases are also composed of two genes: phaC and phaR but synthesized mainly scl-PHA and are found in bacteria of the Bacillus genus 17,29,51,53. Genome annotation, using the platforms MaGe and NCBI, enabled the identification of different genes involved in PHA metabolism such as phaA, phaB, phaC1/phaC2 and phaR coding for acetyl-CoA acetyltransferase (PhaA), acetoacetyl-CoA reductase (PhaB)

,

(PhaC1 and PhaC2) and polyhydroxyalkanoate synthesis repressor (PhaR) respectively. All the data are shown in Table 4.

These results orientate classification of the PHA synthase of Halomonas sp. SF2003 to class I.

The strain belongs to the Halomonas genus, produces scl-PHA and does not have gene phaE, so the PHA synthase clearly does not belong to class II and III. Results also show that genes coding for the three key enzymes of the PHA synthesis pathway are not clustered in one operon but are clearly distant from each other. To our knowledge, this kind of organization has never been reported elsewhere.

Classically, these genes are organized in one operon, or not, with a different order or orientation depending on the bacterial species genes related to PHA biosynthesis, as represented in Figure 4 ⁵⁴.

Figure 4: Schematic representation of a PHA cluster for different representative bacterial species.

Based on data from previous papers ^51–53,55.

Studies of PhaC1 and PhaC2 proteins were carried out, using server Rapid Annotation and Subsystem Technology (RAST). MaGe, NCBI and UniProt databases provided additional information.

Protein PhaC1, being the size of 71.5 kDa, is involved in butanoate and PHA metabolisms. Sequence alignments showed the presence of functional domains of PHA synthase and identity, of about 60-70%, with PHA synthases of other bacterial species like Cobetia marina, Halomonas boliviensis LC1, Halomonas sp. R57-5, Sulfitobacter guttiformis KCTC 32187, Puniceibacterium sediminis or Halocynthiibacter arcticus. These data are in accordance with first results of the bioinformatics analysis and production tests. Like PhaC1, the study of PhaC2 revealed its implication in PHA metabolism and the presence of a functional domain of PHA synthase (in N-term) with an identity, ranging from 65% to 96%, with other bacterial species such as Cobetia amphilecti, Cobetia marina, Halomonas anticariensis

DSM 16096, Halomonas denitrificans or Halomonas boliviensis LC1. Results of sequence alignments, exposing conserved domains of proteins PhaC1 and PhaC2, are available in Figure 5. However, bioinformatics analysis also revealed atypical characteristics of protein PhaC2: the protein being the size of 101.7 kDa and is potentially involved in the metabolism of aromatic compounds. Usually, PhaC proteins have a molecular mass of between 40 to 70 kDa, depending on PHA synthase classes ^29,53. The implication of PHA synthase in aromatic compounds metabolism has, to our knowledge, never been described. This characteristic of protein PhaC2 can be linked to the adaptability of Halomonas sp. SF 2003 to polycyclic aromatic compounds, (see below in the stress-related proteins section).

Figure 5: Conserved domains of PHA synthases PhaC1 and PhaC2 of Halomonas sp. SF2003 using NCBI database.

First results of the automatic annotation of the Halomonas sp. SF2003 genome also included fabG in the pathway of PHA production (MaGe Database). This gene codes as for a 3-oxoacyl-ACP reductase involved in fatty acid metabolism and especially in their biosynthesis, as described in Figure 1 ^56,57. These results suggested that PHA and fatty acid metabolism are closely related. More relevant research was thus conducted to identify potential genes involved in fatty acid metabolism to determine if Halomonas sp. SF2003 can deviate the β-oxidation cycle for PHA production. Genome analysis revealed the presence of characteristic genes encoding enzymes of fatty acid metabolisms: accB (acetyl-CoA carboxylase), fabI (enoyl-ACP reductase), yngI/fadD (acyl-(acetyl-CoA synthetase), acdA/fadE (acyl-CoA dehydrogenase). A selection of identified genes is summarized in Table 4. These data are in accordance with results of preliminary experiments of PHA production using fatty acids, oleic and undecenoic acids which enabled the production of 0.082 and 0.152g/L of PHA, respectively (unpublished data).

Table 4: Genes from Halomonas sp. SF2003 predicted to be involved in PHA and fatty acid biosynthesis and in fatty acid

PhaA / Acetyl-CoA C-acyltransferase C8233_08335 42.4 PhaA / Acetyl-CoA C-acyltransferase C8233_10360 40.6 PhaB / Beta-ketoacyl-ACP reductase C8233_12530 27.0 PhaC1 / Poly-beta-hydroxybutyrate

polymerase C8233_00780 71.5

PhaC2 / Poly-beta-hydroxybutyrate

polymerase (modular protein) C8233_06040 101.8 PhaR PhaR Polyhydroxyalkanoate

synthesis repressor C8233_09450 19.9

Fatty acid biosynthesis

YngI Putative acyl-CoA synthetase COBI473_v1_3625

(MaGe) 64.0 AccC AccC Acetyl-coa carboxylase biotin

carboxylase subunit C8233_07040 48.6 FabG FabG 3-oxoacyl-ACP reductase C8233_15110 27.5 FabG 3-oxoacyl-ACP reductase C8233_06625 25.9 FabI / Enoyl-[acyl-carrier-protein]

reductase C8233_00775 29.1

Fatty acid degradation (β-oxidation)

FadA FadA Acetyl-coa C-acyltransferase C8233_08120 41.1 FadB FadB Fatty acid oxidation complex

subunit alpha C8233_08125 77.4

FadD / Long-chain fatty acid--coa

ligase C8233_02555 61.4

CaiD / Enoyl-CoA hydratase C8233_00770 52.1

/ Enoyl-CoA hydratase C8233_16045 30.3

/ Enoyl-CoA hydratase C8233_16135 29.1

/ Enoyl-CoA hydratase C8233_16140 43.4

/ Crotonase/enoyl-coa

hydratase family protein C8233_13655 29.2 /

Enoyl-coa hydratase/isomerase family

protein C8233_03930 31.3