Genomics of PHA Synthesizing Bacteria

Authored by: Parveen K. Sharma , Jilagamazhi Fu , Nisha Mohanan , David B. Levin

The Handbook of Polyhydroxyalkanoates

Print publication date:  November  2020
Online publication date:  November  2020

Print ISBN: 9780367275594
eBook ISBN: 9780429296611
Adobe ISBN:

10.1201/9780429296611-4

 

Abstract

Polyhydroxyalkanoates (PHA) have gained popularity as biodegradable polymers that could be used to displace some petroleum-derived plastics. PHA are polymerized by PHA synthase enzymes, which produce polymers that consist of either short-chain-length (scl-) or medium-chain-length (mcl-) subunits. PHA synthesis has been detected in heterotrophic, autotrophic, halophilic, and methylotrophic bacteria. Some bacteria, such as Cupriavidus necator and Pseudomonas spp., have revolutionized PHA production on the industrial scale. However, other bacterial species, such as Halomonas sp. and Paracoccus sp., have also been investigated as PHA producers. The availability of complete genomes for PHA producers has led to improved PHA production via genetic manipulation. Halomonas, with its ability to grow in high salt concentrations, has enabled low-cost PHA production processes in non-sterilized conditions. Paracoccus, a methylotrophic, denitrifying autotroph present in marine microbial mats, is also a producer of a high quantity of poly(3-hydroxybutyrtate) (PHB) under natural conditions. The enzymes that polymerize PHA in C. necator, Halomonas, and Paracoccus belong to the class I PHA synthases. These species synthesize and accumulate only short-chain-length PHA (scl-PHA) from different substrates. The enzymes that polymerize PHA in Pseudomonas species are grouped as class II PHA synthases and result in the synthesis and accumulation of medium-chain-length PHA with different monomer compositions depending on the carbon substrate used by the bacteria. Multiple PHA synthases (2–3) were identified in the respective genomes. All these PHA synthases have a lipase box and a catalytic domain possessing conserved active site residues Cys, His, and Asp. Genome analysis of PHA producers will identify new genes for manipulating and advancing PHA production.

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Genomics of PHA Synthesizing Bacteria

3.1  Introduction

Polyhydroxyalkanoates (PHA) are a family of bacterial polyesters synthesized as intracellular energy storage molecules that allow cells to survive in conditions of nutritional stress [1]. To date, over 150 types of PHA monomers have been identified that endow PHA polymers with a wide range of properties that may be tailored for various potential applications [2]. The PHA monomers can be generally divided into two groups according to the number of carbon atoms in their side-chains. Short-chain-length polyhydroxyalkanoates (scl-PHA) contain 3–5 carbon atoms, for example, polyhydroxybutyrate [PHB a.k.a. P(3HB)] consists of subunits with four carbon atoms. Medium-chain-length PHA (mcl-PHA) contain 3-hydroxyalkanoate subunits that contain 6–14 carbon atoms and are more structurally diverse than scl-PHA [3].

A wide range of eubacteria, archaea, and even eukaryotes, such as some yeast species, are known to store PHA as a carbon and energy source, but the majority of microorganisms that synthesize PHA are found in eubacteria. Attempts have been made to correlate various bacteria taxonomies with their genome content associated with PHA biosynthesis, although the machinery for PHA biosynthesis has various degrees of similarity. In this chapter, we review the literature on different bacteria that synthesize PHA polymers, with special emphasis on the genomics of the metabolic pathways used for PHA synthesis, and the genes and gene products involved.

3.2  Scl-PHA Producing Bacteria

Cupriavidus necator H16, previously known as Alcaligenes eutrophus and Ralstonia eutropha H16, is the most well-studied scl-PHA producing bacterium [4]. C. necator is well known for its ability to synthesize large quantities of intracellular PHB, which accumulates as much as 72% to 76% of the cell dry mass (CDM) when cultured in media containing soybean oil [5]. C. necator can also synthesize and accumulate up to 75 wt.-% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV) a.k.a. PHBV] in CDM copolymers when grown in cultures containing glucose and propionate [6]. Alcaligenes latus, unlike C. necator, does not require nutrient limitation to stimulate the synthesis of PHB from various sugars. However, increased PHB accumulation was observed under nitrogen-limiting conditions, resulting in an increase in the accumulation of PHB from 50% to 78% CDM [7].

Halomonas strains were isolated from deep‐sea hydrothermal sites characterized by high pressure, high temperature gradients, and high concentrations in toxic elements (sulfides, heavy metals). Their ability to grow under conditions of high salt concentration and high pH has opened commercial applications in biotechnology. The presence of 3‐hydroxyalkanoic acids in sediments recovered from the deep‐sea hydrothermal vents suggests the synthesis of PHA by bacteria associated with these deep-sea vents [8].

A number of Halomonas spp. have been characterized for PHA production [9,10]. PHA production in cells of Halomonas spp. allows them to survive under high osmotic pressure. PHA production appears to be a universal phenomenon among halophiles and protects bacteria from the harmful effects of high salt concentration [11,12]. Genome sequences of 120 Halomonas species are available on the Integrated Microbial Genome (IMG) platform. Recently, Halomonas sp. SF2003 has been studied in detail for the presence of genes typically involved in PHA biosynthesis, such as phaA, phaB, and phaC, which has enabled preliminary analysis of their organization and characteristics [13].

Paracoccus species also show great potential for accumulating PHA. These Gram-negative methylotrophic bacteria are well known for possessing denitrification abilities and thus are considered very useful in the biotreatment of wastewater [14]. Paracoccus denitrificans and Paracoccus pantotrophus are the two most widely studied bacteria that can metabolize various carbon sources, including glycerol, methanol, n-pentanol, and carbon dioxide (CO2) to PHA [15,16]. They display both autotrophic as well as heterotrophic physiology to produce PHA on simple substrates, as well as mixtures of other compounds that are mostly present in industrial effluent, wastewater, and lignocellulosic biomass hydrolysates [17,18]. Interestingly, PHA production in these bacteria has been observed to be both growth-associated as well as growth-limited and associated with carotenoid production [19]. However, large-scale production of PHA using Paracoccus spp. has not yet been demonstrated. The complete genome sequences of 59 Paracoccus species are available on IMG but have not been studied in detail.

3.3  Mcl-PHA Producing Bacteria

To date, Pseudomonas species have been extensively investigated for their ability to synthesize mcl-PHA; the species tested include P. aeruginosa, P. entomophila, P. fluorescens, P. oleovorance, P. putida, P. mendocina, and P. stutzeri (and many more). Pseudomonas sp. can use substrates unrelated to the 3-hydroxyalkanoate structure of PHA polymer side-chains (glucose and glycerol), as well as substrates related to PHA side-chain structures (fatty acids, oils) for PHA production.

The carbon source used to grow the bacterial species that synthesize mcl-PHA has a significant effect on the subunit composition of the polymer produced (see Table 3.1). Pseudomonas species can use a wide range of carbon sources for synthesizing mcl-PHA producing versatile, functional groups in their side-chains, such as branched alkyl groups [35], halogens [36], epoxy moieties [37], alkyl esters [38], unsaturated aliphatic groups [39], and aromatic groups [40]. These functional groups can greatly change the physical properties of the mcl-PHA polymers, imparting excellent modifications for different applications.

Table 3.1   PHA Production and Monomer Composition of Polymers Produced by Pseudomonas Species

Strain

Substrate

Concentration (g/L)

C/N ratio

PHA

(wt.-%)

C6

C8

C10

C12

C12:1

C14

Ref.

P. putida LS46

Glucose

10

22.4

22

2.2

15.7

76.1

5.6

[19]

Octanoic acid

3.2

11.7

56

6.9

89.3

3.4

Decanoic acid

2.8

10.7

34

4.6

47.5

46.6

1.3

Biodiesel glycerol

16.3

2.78

37.4

62.3

0.9

0.5

P. putida KT2440

Sodium octanoate

12

39

52

18.8

81.2

[20]

Glucose

18.5

41

25

5

11.8

70.3

4.7

9.4

0.8

[21]

Glycerol

3.7

40

19

P. entomophila L48

Dodecanoic acid

15

50.4

6.4

44.5

38.6

10.6

[22]

P. putida CA3

Phenylacetic acid

2

24

28

[23]

P. aeruginosa PAO1

Sodium gluconate

15

48

20

14

21

61

4

[24]

P. aeruginosa L2-1

Waste fryer oil

2

43

37.5

42.1

13.2

2.2

2.1

[25]

P. aeruginosa IFO3924

Palm oil

7

27

36

4

38

43

12

[26]

P. aeruginosa P14

Decanoic acid

5.2

30

16.9

8.2

55

36.8

[27]

P. mendocina ymp

Gluconate

15

54

13

3

15

68

13.8

[28]

Octanoic acid

10

65

30

9.1

90.9

[29]

P. fulva TY16

Glucose

10

39

27

2.1

17.7

60.1

8.5

11.7

[30]

Gluconic acid

10

36

34

2.4

20.5

63.5

7.1

6.5

Octanoic acid

4

26

55

9.6

79.1

11.3

3.7

P. chlororaphis PA23-63-1

Glucose

20

11.2

30.56

5.7

46.7

36.3

12.4

1.6

[31]

Octanoic acid

3.2

11.7

32.5

10.0

83.2

3.6

Glycerol bottom

20

24.37

7.2

40.1

36.2

12.8

2.2

0.4

Glycerol bottom + Valeric acid#

20

30.8

(27) 5.2

33.8

24.0

6.2

This study

Glycerol bottom + Hexanoic acid#

20

36.5

(7.4) 38.3

26.3

20.9

5.4

1.6

P. fluorescens GK13

Glucose

15

58

38

1.6

9.4

68.5

19.3

[32]

Octanoic acid

10

65

31

4.2

95.8

P. fluorescens BM07

Gluconate

12.6

42

8.2

5.2

35.5

19.3

32.5

7.4

[33]

Octanoate

5.8

32

23.3

11.8

84.4

1.8

0.4

0.4

0.4

P. stutzeri 13167

Glucose

10

45

58

2.4

21.3

63.2

4.2

6.1

1

[34]

Soybean oil

10

63

8.2

63.4

2.6

#3.2 g/L hexanoic acid, 5.0 g/L valeric acid.

Species such as P. chlororaphis, P. fluorescens, and P. fulva synthesize mcl-PHA with “uncommon” monomer compositions, and polymers synthesized by these bacteria may contain subunits with significant amounts of unsaturated or longer carbon chain length side-chains. P. mendocina CH50 is the only wild-type strain producing C8 homo-polymer from octanoate that has been reported to date [41]. A number of reviews on the genome organization and regulation of PHA synthesis are available for Pseudomonas sp. [42–45].

3.4  Scl-co-mcl-Copolymer Producers

Mcl-PHA polymers containing medium length 3-hydroxyalkanoate side-chains are very amorphous, with low to no crystallinity, which makes them tacky at room temperature and unsuitable for structural or fiber applications. Scl-PHA consists of 100% 3-hydroxybutanoate side-chains and has high tensile strength, but low elasticity, which makes them very brittle. Scl-PHA polymers consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have slightly lower tensile strength, but greater elasticity, and scl-co-mcl copolymers composed of mostly C4 monomer and small amounts of C6 monomer have been shown to have properties most similar to polypropylene, with the added advantage that they are biodegradable [46,47]. The PHA synthases derived from copolymer producing strains, such as Pseudomonas sp. 61-3 and Thiocapsa pfennigii, have significant amino acid sequence identities and many highly conserved amino acid residues with the class I–IV PHA synthases [48,49].

3.5  Genomics of Mcl-PHA Producing Bacteria

Pseudomonas putida KT2440 is the most extensively studied mcl-PHA synthesizing bacterium. Omic studies of P. putida KT2440 have led to the development of new strategies for PHA production. In our laboratory (Department of Biosystems Engineering, University of Manitoba), we have focused on P. putida LS46, a recently isolated strain that is closely related to P. putida KT2440. We have compared the genome of P. putida LS46, which was isolated specifically for its ability to produce mcl-PHA, with 13 other mcl-PHA producing Pseudomonas species, which were isolated from diverse environments and not for their ability to produce PHA (see Table 3.2).

Table 3.2   Pseudomonas Genomes Used in this Study

Genome name

Isolated from

Genome size (Mb)

ANI with

P. putida ls46 (%)

Gene count

GC ratio

Horizontally transferred gene count

P. putida KT2440

Rhizosphere soil

6.18

97.34

5481

0.62

173

P. fluorescens SBW25

Leaf surface

7.14

78.67

6492

0.6

40

P. fluorescens Pf0-1

Agricultural soil

6.43

78.80

5857

0.61

256

P. entomophila L48

Fruit fly

5.88

85.41

5293

0.64

369

P. putida F1

Polluted creek

5.95

98.38

5422

0.62

133

P. aeruginosa PAO1

Infected wound

6.05

77.63

6401

0.66

2

P. mendocina ymp

Sediment surface

5.07

78.30

4730

0.65

231

P. chlororaphis PA23

Soybean roots

7.12

79.88

6264

0.65

45

P. monteilii SB3078

Contaminated soil

5.86

90.00

5525

0.63

2

P. putida BIRD-1

Rhizosphere soil

5.73

97.18

5046

0.62

112

P. putida LS46

Wastewater

5.87

100

5346

0.62

41

P. stutzeri A1501

Rice roots

4.56

76.91

4237

0.64

133

P. pseudoalcaligenes NBRC 14167

Sinus drainage

4.68

78.15

4713

0.62

68

Some of the P. putida strains (KT2440, BIRD-1, as well as P. fluorescens Pf0-1), were isolated from rhizosphere soil, while P. chlororaphis PA23, P. stutzeri A1501, and P. fluorescens SBW25 were isolated from plant roots or leaf surfaces. P. putida LS46, P. putida F1, P. monteilii SB3078, and P. mendocina ymp were isolated from polluted environments. P. aeruginosa PAO1 and P. pseudoalcaligenes NBRC14167 were isolated from wounds and human sinus drainage. Originally, none of these strains were isolated as PHA producers, but later it was observed the all these strains have the genetic machinery for mcl-PHA synthesis, and some of these strains were developed as industrial strains for PHA production. The genome sizes of these bacteria range from 4.56 to 7.14 Mb, with gene counts of 4,237 to 6,401. These bacteria showed average nucleotide identities of 76.91% to 98.38%.

3.5.1  Evolutionary Relationship among Pseudomonas Species

The most widely used method to study evolutionary relationships among bacteria is 16S rRNA gene sequence analysis. Protein-encoding genes are known to provide higher levels of taxonomic resolution than non-protein-encoding genes, like 16S rRNA [50]. Therefore, a protein-coding gene, cpn60 (also known as hsp60 or GroEL), was used for phylogenetic analysis of Pseudomonas species and strains [51]. Sequences were aligned using ClustalW, and a neighbor-joining tree was constructed using MEGA7 [52,53]. Phylogenetic relationships based on Cpn60 protein amino acid sequences separated these 14 strains into 4 clades. P. putida LS46 formed a clade along with ten other strains. In this clade, BIRD-1, F1, KT2440, P. monteilii SB3078, and P. entomophila L48 formed a subclade, which was separate from the clade consisting of P. aeruginosa PAO1, P. stutzeri A1501, P. mendocina ymp, P. oleovorans, and P. pseudoalcaligenes NBRC14167. P. fluorescens SBW25 and P. fluorescens Pf01 were different from all the other strains and related in a separate subclade. P. chlororaphis PA23 was different from all other P. chlororaphis strains and formed an out-group clade in the phylogenetic tree based on Cpn60 (Figure 3.1).

Phylogenetic tree depicting the relationships among

Figure 3.1   Phylogenetic tree depicting the relationships among Pseudomonas species. The tree is based on Cpn60 amino acid sequences, which were aligned by ClustalW; a neighbor-joining tree was generated using MEGA7 program. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distance used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT (Jones, Taylor, Thornton) method. The bootstrap method was used as a test of the phylogeny with 500 replications. Bootstrap values are indicated at the nodes.

3.5.2  Shared Genes among Pseudomonas Strains

P. putida strains have 68,811 genes, of which 44,900 (65.25%) were present among the 14 strains analyzed. A large number of the genes (87.2% to 90.5%) were conserved among the 14 P. putida strains analyzed (see Table 3.3). All P. putida strains are closely related and have highly conserved fatty acid metabolism and PHA synthesis genes. P. entomophila L48 and P. monteilii SB3078 are closely related to P. putida strains and share 76% and 84% of the P. putida genes, respectively. In contrast, the 14 P. putida strains shared only 58.0% and 59.7% of the conserved genes with P. stutzeri and P. pseudoalcaligenes.

Table 3.3   Shared Genes in Different PHA Producing Pseudomonas Strains

Strain

LS46

KT2440

BIRD-1

F1

PAO-1

SBW25

Pf0-1

L48

ymp

SB3078

PA23

A1501

NBRC14167

LS46

100

KT2440

87.2

100

BIRD-1

88.5

86.0

100

F1

90.5

87.0

89.9

100

PAO-1

63.6

66.7

64.5

63.7

100

SBW25

64.1

62.7

64.4

64.6

61.2

100

Pf0-1

70.3

68.7

71.3

71.1

66.8

71.2

100

L48

76.5

74.5

77.5

76.2

64.0

65.2

73.4

100

ymp

62.4

61.3

63.4

62.3

67.1

57.5

62.2

61.4

100

SB3078

83.9

82.7

83.5

85.4

63.8

63.9

70.5

74.3

61.5

100

PA23

67.8

66.5

67.9

68.1

68.1

70.4

77.2

70.1

60.2

67.9

100

A1501

59.7

56.5

58.6

58.0

60.0

51.3

56.2

55.7

66.9

57.3

54.8

100

NBRC14167

58.4

57.7

59.1

58.7

60.2

53.2

57.3

56.7

75.2

59.0

55.5

64.7

100

3.5.3  The PHA Synthesis Operon in Pseudomonas Species

The enzymes involved in mcl-PHA polymer synthesis are encoded by six genes that are organized into a PHA synthesis operon, which is identical in all the Pseudomonas strains analyzed. The six genes are phaC1, phaZ, phaC2, phaD, phaF, and phaI. The mcl-PHA biosynthesis cluster forms two putative transcriptional units: phaC1-phaZ-phaC2-phaD and phaF-phaI, which are under the regulation of a transcriptional regulator, PhaD [54]. The regulator was thought to be activated by an intermediate from the fatty acid β-oxidation pathway, resulting in higher transcriptional levels of mcl-PHA synthesis genes when the cell is grown -on fatty acid substrates (such as octanoic acid) versus glucose (Figure 3.3). The phaZ encodes a PHA depolymerase, which hydrolyzes the PHA monomers when they are required as a carbon source and are catabolized via a central metabolism for growth [55,56]. PhaF and phaI encode phasin proteins involved in PHA granule structure and size [57].

3.5.4  Mcl-PHA Synthesis Genes

Pseudomonas species code for class II PHA synthases and the PHA synthesis operon of most Pseudomonas species have two PHA synthases: PhaC1 and PhaC2. These are highly conserved among Pseudomonas species, with up to 90% amino acid sequence identity, but within P. putida, the phaC1 and phaC2 genes share only 71% nucleotide identity and 55% amino acid sequence identity. PHA synthases catalyze the stereoselective conversion of (R)-3-hydroxyacyl-CoA substrates to PHA with the concomitant release of CoA [58]. Recognition of the (R)-3-hydroxyacyl-CoA intermediate is highly dependent on the substrate specificity of the PHA synthase itself. Mcl-PHA synthases are preferentially active toward CoA thioesters of various mcl-3-HA comprising 6–14 carbon atoms.

PHA synthases have different structures and subunit compositions among class I, II, III, and IV synthases. Class I and class II PHA synthases have only one subunit: PhaC for class I; PhaC1 and/or PhaC2 for class II. Class III and class IV PHA synthases are heterodimers: PhaC and PhaE for class III; PhaC and PhaR for class IV [59,60]. However, polymerases within and between PHA synthase classes are highly conserved in all four classes containing six conserved blocks, with eight conserved amino acid residues, and a lipase box [GX (S/C) XG]. With respect to specific catalytic activity and substrate specificity, the conserved Cys-319, Asp-480, and His-508 of the class I PHA synthases, and the conserved Asp-452 and His-453 of the class II PHA synthases, are crucial catalytic residues [61].

3.5.5  Evolutionary Relationship among PhaC1 and PhaC2 Proteins

The PhaC1 and PhaC2 of different Pseudomonas species formed different clusters in neighbor-joining trees. The PhaC1 and PhaC2 in Pseudomonas strains were highly conserved among Pseudomonas species (Figure 3.2). However, P. stutzeri A1501 had only one PhaC, while P. pseudoalcaligenes NBRC 14167 encodes three PhaC proteins. PhaC from P. stutzeri A1501 and P. pseudoalcaligenes formed an out-group. PhaC1 and PhaC2 of P. aeruginosa PAO1 formed a separate group, while the rest of the PhaC1 and PhaC2 proteins are in another group. P. putida PhaC1 proteins are closely related to P. entomophila and P. monteilii, while P. fluorescens PhaC1 and PhaC2 are clustered with P. chlororaphis, P. mendocina, and P. pseudoalcaligenes. The PhaC1 proteins have a significant effect on the subunit composition of PHA polymers [34,48].

Phylogenetic tree depicting the relationship among PHA synthase proteins (PhaC1 and PhaC2) of

Figure 3.2   Phylogenetic tree depicting the relationship among PHA synthase proteins (PhaC1 and PhaC2) of Pseudomonas species. Amino acid sequences were aligned by ClustalW, and a neighbor-joining tree was generated using the MEGA7 program. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT method. The bootstrap method was used as a test of the phylogeny with 500 replications. Bootstrap values are indicated at the nodes.

3.6  The Genomics of Mcl-PHA Metabolism

3.6.1  Mcl-PHA Synthesis via the Fatty Acids De Novo Synthesis Pathway

Carbon metabolism pathways are highly conserved among 14 Pseudomonas genomes, with 75% to 100% amino acid sequence homology in glycerol metabolism enzymes. The fatty acid de novo route for mcl-PHA biosynthesis of P. putida refers to mcl-PHA synthesis from sugars, glycerol, and other carbon sources that are metabolized by the fatty acid de novo synthesis pathway to provide the (R)-3-hydroxyacyl-ACP (acyl carrier protein) intermediates that are polymerized to form mcl-PHA (see Figure 3.3) [62].

Proposed metabolic pathways for mcl-PHA biosynthesis in P. putida LS46.

The mechanisms of initial carbon uptake and metabolism (such as glucose vs. glycerol) are virtually identical in Pseudomonas species. A glycerol uptake cluster is specifically required for glycerol metabolism in Pseudomonas [63], while the transportation of glucose into the cytoplasm requires an ABC (ATP binding cassette)-type glucose transporter. Glycerol uptake clusters and ABC transporters for glucose are present in all Pseudomonas species. Simultaneously, enzymes, such as glucose dehydrogenase and gluconate dehydrogenase, carry out oxidation reactions in the periplasmic space, converting glucose into gluconate and keto-gluconate, respectively [64]. These are the enzymes of the Entner–Duodoroff (ED) pathway.

Glycerol uptake in Pseudomonas species is mediated by glycerol “uptake facilitators,” which are integral membrane proteins (glpF, locus tag PPUTLS46_022196 in the P. putida LS46 genome), catalyzing the rapid equilibration of glycerol concentration gradients across the cytoplasmic membrane [65]. Glycerol is converted to glycerol-3-phosphate (G3P) by phosphorylation of glycerol by the ATP-dependent glycerol kinase (glpK, locus tag PPUTLS46_022201 in P. putida LS46), followed by the dehydrogenation of G3P into dihydroxyacetone phosphate (DHAP) by three glyceraldehyde-3-phosphate dehydrogenase (GPDHs: locus tags PPUTLS46_022211, PPUTLS46_005991, and PPUTLS46_012690 in P. putida LS46). Glycerol is not a preferential carbon source for PHA production, and about two-thirds of the glycerol added to the medium as the sole carbon source for mcl-PHA production remained unused by P. putida LS46 after 72 h [66]. Phosphorylation of glycerol by GK is the rate-limiting step. GK, along with glycerol-3-phosphate dehydrogenase (GlpD), is induced by the presence of glycerol under aerobic conditions.

Mcl-PHA biosynthesis by P. putida is regulated at the substrate transportation step. A repressor protein (coded by glpR) located in the glycerol uptake cluster was knocked out to reduce the lag phase of P. putida KT2440 grown on glycerol [67]. Biodiesel-derived waste glycerol, despite the presence of various impurities, was as good a carbon source as pure glycerol for PHA production. Heavy metal contamination in biodiesel glycerol led to the induction of transcripts for Cu, Cd, Ni, and Hg stress response genes, and proteomic analyses detected a number of proteins encoded by genes scattered across the P. putida LS46 genome, which were putatively involved in sensing, transporting, and the efflux of (heavy) metal ions, such as iron, cobalt/zinc/cadmium, and nickel [68].

In contrast, the glucose uptake pathway has been modified by knocking-out glucose dehydrogenase in P. putida KT2440, leading to increased mcl-PHA production from glucose [22]. Glucose is metabolized by the ED pathway because the Embden–Meyerhof–Parnas (EMP) pathway is not functional in P. putida due to the absence of the key glycolytic enzyme, 6-phosphofructo-1-kinase (Pfk). The biomass yield could be maximized by constructing a functional EMP pathway in P. putida. Sánchez et al. [69] designed two modules to construct a functional EMP pathway in P. putida KT2440. In engineered P. putida, 95% of the pyruvate was generated by the engineered EMP pathway, compared with 93% pyruvate generated by the ED pathway in wild-type P. putida. The authors did not test mcl-PHA production in the engineered strain but reported an increase in the carotenoid yield due to the production of higher pyruvate concentrations.

During de novo fatty acid synthesis in P. putida LS46, acetyl-CoA was used by the pathway enzymes to generate 3-hydroxyacyl-ACPs, the key precursor molecule for mcl-PHA biosynthesis. “Omics” data has suggested a few gene products were up-regulated during active mcl-PHA synthesis at the steps that provide the key pathway intermediates for polymer synthesis. The protein levels of a putative ketoacyl-ACP reductase (fabG, encoded by PPUTLS46_023353 in P. putida LS46), is one of eight homologs identified in the P. putida LS46 genome that provide various 3-hydroxyacyl-ACP intermediates. FabG was highly up-regulated in the stationary phase of waste glycerol cultures when there was active mcl-PHA synthesis [62]. RNA sequencing (RNAseq) analysis, however, indicated down-regulation of the fabG gene at the transcriptional level. Two isoforms of 3-hydroxyacyl-ACP dehydratases (FabA and FabZ) identified in the genome of P. putida LS46 carry out dehydration reactions to produce trans-2-acyl-ACP, and FabA also carries out an isomerization reaction leading to the biosynthesis of unsaturated fatty acids [70], which could also be a critical point for unsaturated mcl-PHA production from fatty acid de novo synthesis.

In the next step, intermediates are converted into acetyl-CoA via partial glycolysis through the ED pathway, which is then fluxed to fatty acid biosynthesis (see Figure 3.3). (R)-3-hydroxyalkanoates-acyl carrier proteins are used as the intermediate for mcl-PHA biosynthesis. However, (R)-3-hydroxyalkanoates-acyl carrier proteins must be converted into their CoA derivatives, which is carried out by PhaG, a 3-hydroxyacyl-ACP-CoA transacylase. PhaG (also known as an mcl-PHA monomer supplying protein) is identified biochemically as a unique protein that is required for mcl-PHA synthesis from fatty acid de novo synthesis in Pseudomonas. Its expression was highly up-regulated in P. putida grown in either glycerol or glucose cultures under mcl-PHA producing conditions [71].

3.6.2  Mcl-PHA Synthesis via the Fatty Acid β-Oxidation Pathway

The fatty acid β-oxidation pathway is used for mcl-PHA biosynthesis when fatty acids are used as a sole carbon source. The key precursor for mcl-PHA synthase, (R)-3-hydroxyalkanoate-CoA, is derived from the conversion of trans-2-enoyl-CoA, the intermediate of fatty acid β-oxidation. The key enzyme that carries out this reaction is an (R)-specific enoyl-CoA hydratase coded by phaJ in Pseudomonas species. There are four phaJ homologs identified in P. aeruginosa, and their gene products expressed in recombinant Escherichia coli were demonstrated to provide monomer for mcl-PHA biosynthesis from fatty acids [72]. When expressed in E. coli, PhaJ1 of P. aeruginosa showed substrate specificity toward enoyl-CoAs with acyl chain lengths from C4 to C6 (scl to mcl), while PhaJ2, PhaJ3, and PhaJ4 exhibited substrate specificities toward enoyl-CoAs with acyl chain lengths from C6 to C12 (mcl). Only two phaJ genes (phaJ1 and phaJ4, based on the sequence similarity to those of P. aeruginosa) are present in P. putida, and the product of the phaJ4 ortholog was shown to act as the primary mcl-PHA monomer supplying enzyme in P. putida [73]. Another putative monomer supplying enzyme for mcl-PHA synthesis from fatty acid β-oxidation is a 3-ketoacyl-CoA reductase coded by fabG. This enzyme is an NADPH-dependent 3-ketoacyl reductase and identified in P. aeruginosa as an mcl-PHA monomer supplying protein by reducing mcl-3-ketoacyl-CoAs to mcl-3-hydroxyacyl-CoA from fatty acid β-oxidation [74]. Yet, no evidence has shown that the protein (FadG) played a role as a monomer supplying enzyme for mcl-PHA biosynthesis from fatty acid β-oxidation in P. putida.

Pseudomonas spp. are known to produce other secondary metabolites like rhamnolipids and antimicrobial substances (phenazines). These secondary metabolites have common precursors and compete with each other for the partitioning of carbon substrates and reducing power. A defect in phenazine or rhamnolipid production led to higher PHA production in P. chlororaphis PA23 and P. aeruginosa [28,32]. Furthermore, the regulatory pathways for phenazine production and rhamnolipid production are very similar and regulated by gacA/gacS, rpoS stationary phase sigma, and stringent response (relA/spoT) [75].

3.7  Mcl-PHA Synthesis from Vegetable Oils and Fats

Genome analyses of 13 Pseudomonas species indicated the presence of genes encoding triacylglycerol lipase/esterase (COG 1075), acetyl esterase (COG0657), and lipase chaperone (COG5380) proteins. Many Pseudomonas species, like P. putida KT2440, P. putida LS46, P. entomophila L48, P. fluorescens Pf01, and P. corrugata 388, can use long-chain fatty acids (LCFAs) derived from vegetable oils as sole carbon sources, but are unable to use vegetable oils directly for growth and PHA production because they lack genes encoding lipase/esterase enzymes that can be secreted extracellularly. The extracellular lipase/esterase enzymes cleave the ester bonds between the LCFAs and glycerol, making the LCFAs available for metabolism via the β-oxidation pathway. Evidence in support of this statement was provided by experiments in which recombinants of P. corrugata 388 were constructed by cloning and expressing a gene encoding a secreted lipase enzyme, which enabled the recombinant strains to grow on animal fat (lard) and synthesize PHA [76]. Plasmid-containing Pseudomonas lipase genes were procured from the American Type Culture Collection.

In contrast, a number of Pseudomonas species, including P. chlororaphis PA23, P. stutzeri A1501, P. aeruginosa PAO1, P. mendocina ymp, P. monteilii SB3078, and P. pseudoalcaligenes NBRC14167, can use vegetable oils directly as a carbon source for growth and PHA synthesis [32]. Analyses of the genomes of P. stutzeri A1501, P. aeruginosa PAO1, and P. mendocina ymp revealed the presence of genes encoding triacylglycerol lipases with signal peptides, which suggests that they are secreted and function extracellularly. These three species also encode lipase chaperone proteins, which ensure proper folding of the lipase proteins that are excreted from the cell [32]. The lipase chaperone gene is absent from the genomes of P. putida KT2440, P. putida LS46, P. putida BIRD-1, and P. putida F1. Strains that can use vegetable oils directly for growth and PHA synthesis, such as P. chlororaphis PA23, P. monteilii SB3078, and P. pseudoalcaligenes NBRC14167, also encode genes for a protease/lipase (EYO4_16115) ABC transporter.

3.8  Genome Analysis of Halomonas Species

As indicated in the Introduction, Halomonas strains isolated from deep‐sea hydrothermal sites have been shown to produce PHA polymers. Three major classes of PHA were identified in Halomonas species: poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) [77]. Under non-sterile conditions, H. elongata, H. halophila, H. nitroreducens, and H. boliviensis produced 40% to 69% PHA in the presence of 6% to 10% salinity [78,79]. Halomonas species can accumulate PHA up to 80% of CDM [80]. In addition to PHA production, H. smyrnensis AAD6T and other Halomonas strains can also produce extracellular polymeric substances (EPS) and levan [81–83]. Halophiles with osmo-adaptations and metabolic diversity have not yet been exploited for industrial applications to produce bioplastics, EPS, and/or halophilic enzymes.

A large number (120) of Halomonas sp. genome sequences (draft, permanent, and finished) are available on the IMG platform (https://img.jgi.doe.gov). Complete genome sequences of Halomonas sp. SF2003 and H. smyrnensis AAD6T have been recently published [13,81]. We selected 15 genomes of Halomonas species for further study. The genome size of Halomonas species varies from 2.87 to 5.33 Mb, with gene contents between 2,636 to 4,807. The H. halocynthiae DSM 14573 genome is 2.87 Mb, while the H. titanicae BH1 genome is 5.33 Mb (see Table 3.4). The % GC ratio is in the range of 0.52–0.62. A maximum of 1,002 genes was horizontally transferred into H. anticariensis DSM 16096. The 15 genomes of Halomonas spp. had up to 77.4% average nucleotide identity with Halomonas anticariensis SDSM16096. Analyses of genome size, gene counts, and % average nucleotide identity (ANI) indicate that there is wide diversity among the genomes of Halomonas species.

Table 3.4   Halomonas Genomes Used in the Present Study

Genome name

Genome size

Gene count

% GC

16S rRNA count

Horizontally transferred genes

% ANI

H. smyrnensis AAD6

3561919

3326

0.68

1

311

76.10

H. meridiana R1t3

3507875

3525

0.57

8

130

72.39

H. anticariensis DSM 16096

5019539

4807

0.59

1

1002

74.79

H. illicicola DSM 19980

3960292

3746

0.63

5

413

77.40

H. lutea DSM 23508

4533090

4368

0.59

1

617

100.00

H. zincidurans B6

3554760

3292

0.64

2

209

78.34

H. elongata DSM 2581

4061296

3556

0.64

4

743

75.55

H. stevensii S18214

3693745

3523

0.6

4

154

73.62

H. campaniensis LS21

4074048

3631

0.53

6

76

70.74

H. zhejiangensis DSM 21076

4060520

3739

0.55

3

330

71.75

H. titanicae BH1

5339792

2908

0.55

1

138

71.86

H. halocynthiae DSM 14573

2878444

2773

0.54

2

316

71.07

H. alkaliantarctica FS-N4

3797897

3484

0.52

1

125

70.63

H. boliviensis LC1

4136366

3914

0.55

4

245

71.79

H. halodenitrificans DSM 735

3466026

3256

0.64

1

119

74.99

3.8.1  Carbon Metabolism in Halomonas Species

The H. smyrnensis AAD6T genome has genes for glycolysis via the EMP and ED pathways, as well as for gluconeogenesis, the pentose phosphate pathway, and all de novo amino acid biosynthesis pathways. Genes for the complete tricarboxylic acid (TCA) cycle and glyoxylate shunt were also present. However, the absence of phosphofructokinases (PFKs) indicated the absence of a functional EMP pathway [81]. Halomonas species can use a wide variety of carbon substrates for PHA production. Halomonas elongate 2FF was reported to produce PHB from sucrose, glucose, galactose, pyruvic acid, and acetic acid. H. campisalis and H. boliviensis used xylose to produce PHB [82–86]. H. halophila produced PHA in high salt conditions (20–100 g/L NaCl) and was able to use chemical-grade sugars like glucose, fructose, sucrose, cellubiose, mannose, rhamnose, and arabinose, as well as carbohydrates derived from complex compounds, such as hydrolyzed cheese whey, hydrolyzed lignocellulose, and molasses [87], as carbon sources for growth and PHA production.

The TCA cycle provides energy and intermediates for the synthesis of many important biological compounds. H. bluephagenesis TD01 is unable to synthesize PHBV because the precursor propionyl-CoA is not synthesized. A recombinant strain with increased metabolic flux toward PHBV synthesis, H. bluephagenesis TD08AB, was constructed by deleting two genes in the TCA cycle: the succinate dehydrogenase gene, sdhE, and the isocitrate lyase gene, icl. The addition of α-ketoglutarate, citrate, or succinate to the culture media did not increase the cell mass production or the total PHBV content of the cells. It did, however, increase the intracellular concentrations of acetyl-CoA and propionyl-CoA, which are precursors for PHB and PHBV, and increased the molar ratio of 3-hydroxyvalerate in the PHBV to 90% [88].

The general pathway for synthesis of scl-PHA is very similar in both Halomonas and C. necator. There are three main steps: (i) formation of 3-ketoacyl-CoA from acyl-CoA and acetyl-CoA, a reaction catalyzed by 3-ketothiolase (PhaA); (ii) conversion of 3-ketoacyl-CoA to (R)-3-hydroxyacyl-CoA by NADP-dependent 3-ketoacyl-CoA reductase (PhaB); and (iii) polymerization by stereoselective conversion of (R)-3-hydroxyacyl-CoA to scl-PHA polymers by PHA synthase (PhaC). Halomonas spp. carries the genes for supplying fatty acyl-CoA precursors for PHA synthesis as well as the gene for degradation of stored PHA.

3.8.2  Evolutionary Relationship among Halomonas

The phylogenetic relationships of 15 Halomonas genomes (permanent or finished) were determined using Cpn60 amino acid sequences. This analysis indicated a wide diversity among Halomonas species (see Figure 3.4). The 15 strains were clustered in two clades: nine strains were present in clade I and six strains were present in clade II. Clade I was further split into three subclades, and clade II was split into two subclades. Diversity among Halomonas strains was identified using 16S rRNA genes and Halomonas spp. SF2003 and Halomonas halodurans are closely related to Chromohalobacter salarius and Cobetia species [13].

Phylogenetic tree depicting the relationships among

Figure 3.4   Phylogenetic tree depicting the relationships among Halomonas species. The tree is based on Cpn60 amino acid sequences, which were aligned by ClustalW, and a neighbor-joining tree was generated using MEGA7. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distance used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT method. The bootstrap method was used as a test of the phylogeny with 500 replications. Bootstrap values are indicated at the nodes.

3.8.3  PHA Synthase in Halomonas Species

Genome analyses of the 15 Halomonas strains indicated wide variation in PhaC proteins (see Figure 3.5). Encoding of multiple phaC genes in Halomonas species is very common among the genomes examined. The genomes of three Halomonas spp. each encoded more than one phaC gene (see Figure 3.5). H. boliviensis LC1 and H. anticariensis DSM16096 encoded genes for two PHA synthases. H. illicicola DSM 19980 is unique and encodes genes for five PHA synthases. Two PHA synthases of H. illicicola are identical and present in the same cluster, while three other PHA synthases are divergent and present in different clusters. Of the 15 genomes studied, 14 contained multiple phaC genes with homologs that were different and present in different gene clusters of the neighbor-joining tree. Two phaC of H. boliviensis LC1 are present in different clusters. Homologs of the five PHA synthases of H. illicicola are present in both clades (see Figure 3.5). Two PHA synthases of H. illicicola are identical, while three other PHA synthases are diverse. How these variations in PHA synthases in Halomonas species control their specificity is still not known.

Phylogenetic tree depicting the relationships among PHA synthase proteins (PhaC) of

Figure 3.5   Phylogenetic tree depicting the relationships among PHA synthase proteins (PhaC) of Halomonas species. Amino acid sequences were aligned by ClustalW, and a neighbor-joining tree was generated using MEGA7. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT method. The bootstrap method was used as a test of the phylogeny with 500 replications. Bootstrap values are indicated at the nodes.

3.8.4  PHA Synthesis in Halomonas Species

Halomonas species can metabolize glucose, glycerol, sucrose, maltose, volatile fatty acids (VFAs), lignocellulosic waste (sawdust, corn stover), and industrial effluent to produce PHA (see Table 3.5). Batch and fed-batch, or one-step or two-step processes have been tried for PHA production. Accumulation of PHA was between 23% and 88% of cell weight under different conditions, with 10% to 60% salt concentrations. H. boliviensis, H. venusta, Halomonas KM-1, and Halomonas TD-1 were the best producers and accumulated PHA to 80% to 88% CDM [90–92]. The addition of propionate, pyruvate, or valerate to the culture medium led to the production of copolymers of PHB and PHV with 15% to 35% 3-hydroxyvalerate (3HV) [100].

Table 3.5   PHA Production by Halomonas Species

Strain

NaCl [%]

Carbon source

CDM [g/L]

PHB [%]

Condition

Ref.

H. elongata 2FF

10.0

Glucose (1%)

2.5

40.0

Batch

[78]

H. halophila CCM 3662

10.0

60.0

Glucose (2%)

Glucose (2%)

3.2

5.1

48.0

72.0

Batch

[87]

H. boliviensis

4.5

Butyrate & Acetate 0.8%

2.0

88.0

Batch

[89]

Halomonas sp. KM-1

1.94

Glycerol (10%)

0.67

24.5

Batch

[90]

H. venusta KT832796

1.5

Glucose

37.9

88.1

Fed-batch

[91]

H. boliviensis

Glucose + VFA

44.0

79.5

[92]

Halomonas TD01

1.94

Glucose

83.0

78.0

[93]

Halomonas KM-1

1.94

Glucose

72.1

83.0

Batch

[90]

Halomonas sp. SF2003

1.1

Glucose + VA$ 10 mol%

Glucose + VA 46 mol%

1.4

2.0

64.0 (15)*

56.0 (27)

Two steps

Two steps

[94]

#IE

IE + VA

4.2

6.2

31.0

23 (35)

One step

One step

H. smyrnensis AAD6

13.72

Glucose

1.34

45.8

Batch

[95]

13.72

Sucrose (5%)

0.5

26.9

Batch

H. campaniensis LS21

2.62

Glucose

8.0

75.0

Fed-batch

[96]

H. nitroreducens JCCO25.8

25% @

Glucose

33.0

Batch

[97]

H. campisalis MCM B-1027

4.5

Maltose

1.3

81.0

Batch

[98,99]

3.0

Banana peel extract (1%)

0.7

21.5

Batch

$ Valeric acid, *3hydroxyvalerate %, # IE industrial effluent, @ seawater.

A number of biotechnology techniques have been used to enhance PHA production in halophilic bacteria. These include increasing substrate specificity, modifying the β-oxidation pathway, and constructing new metabolic pathways [101,102]. An open and continuous process using seawater with mixed substrates containing cellulose, starch, lipids, and proteins was used to produce PHB by recombinant H. companiensis LS21. It produced 70% PHB after 65 days in the presence of 27 g/L NaCl [80]. Deletion of phasin genes like phbP1 and phaP2 in H. bluephagenesis resulted in greater PHB granule size. Further deletion of minC and minD genes, which regulate cell fission, increased the PHB granule size to 10 µm [103].

3.9  Genome Analysis of Paracoccus Species

The genus Paracoccus (alphaproteobacteria) currently comprises 40 recognized and validly named species. Paracoccus spp. are Gram-negative methylotrophs and are known to inhabit a wide range of environments, like contaminated soil, rhizospheric soil of leguminous plants, and marine sediment. Some Paracoccus spp. are opportunistic human pathogens. Paracoccus spp. have applications in bioremediation due to their ability to carry out denitrification and degrade toxic compounds, such as herbicides (chlorpyrifos), acetone, dichloromethane, formamide, N,N-dimethylformamide (DMF), and methylamine [104,105]. The metabolic flexibility of Paracoccus is based on its ability to use a variety of nitrogen sources, including nitrate, nitrite, nitrous oxide, and nitric oxide, and C1-compounds, like methane, methanol, methylated amines, halogenated methanes, and methylated sulfur species, as alternative electron acceptors. In addition, Paracoccus spp. have a great ability to accumulate PHA polymers using glycerol, methanol, n-pentanol, and CO2 as carbon sources. PHA is synthesized under autotrophic as well as heterotrophic growth conditions. Microbial mats are excellent starting materials for isolating PHA producing bacteria, and a number of Paracoccus species have been isolated as PHA producers from marine mats [106]. Paracoccus sp. LL1 has been reported to produce carotenoids along with PHA production [19].

In the present study, we selected 22 Paracoccus genomes from the IMG platform, which were draft, permanent draft, or finished genomes (Table 3.5). These bacteria were isolated from different environments and geographic locations. The genome size of the Paracoccus species analyzed ranged between 3.03 and 5.62 Mb, and the gene contents ranged between 3,258 and 5,522. The genomes of Paracoccus spp. have multiple replicons of chromosomes as well as plasmids. Paracoccus denitrificans PD1222 is composed of two chromosomes (ChI = 2.9 Mb and ChII = 1.7 Mb), plus a single mega-plasmid (Plasmid 1) of 653 kb [107]. P. aminophilus JCM 7686 carries a single circular chromosome plus eight plasmids (pAMI1 to pAMI8), ranging in size from 5.6 to approximately 440 kb. Plasmid pAMI2 carries genes for DMF degradation [108]. The genome of another strain of P. aminophilus, JCM 7685, is composed of four circular DNA molecules [109]. P. yeei is an opportunistic human pathogen, which has a circular chromosome and eight extrachromosomal replicons [109]. P. yeei has PHA production machinery like other Paracoccus strains. The other genomes, like P. versutus DSM 532 and P. bengalensis DSM 17099, have genome sizes comparable to those of P. aminophilus JCM 7686 and Paracoccus denitrificans PD1222, which are finished genomes.

Eighteen plasmids have been identified and sequenced in different Paracoccus species. It has been speculated that the bacteria of genus Paracoccus usually carry at least one mega-plasmid of more than 100 kb [110]. Finished genomes have reported one or more plasmids, but draft or permanent draft genomes have not identified plasmids in Paracoccus species. It is possible that draft genomes, when finished, will also show other replicons and plasmids. Some of these plasmids are associated with carotenoid production [111]. P. denitrificans DSM 15148, P. denitrificans DSM 415, P. versutus DSM 582, P. bengalensis DSM 17099, and P. aminophilus JCM 7686 genomes each have four 16S rRNA genes, while the genomes of other species have fewer 16S rRNA genes. Paracoccus species acquired up to 21% of their genes from other bacteria by horizontal gene transfer (see Table 3.6). The average nucleotide identity is 76.95% to 78.52%.

Table 3.6   Paracoccus Genomes Used in the Present Study

Genome name

Genome size

Gene count

% GC

16S rRNA count

% ANI

Horizontally transferred gene count

P. sanguinis DSM 29303

3588279

3457

0.71

1

76.95

139

P. alkenifer DSM 11593

3191900

3099

0.67

3

77.36

323

P. halophilus CGMCC 1.6117

4008709

3912

0.65

2

77.44

335

P. versutus DSM 582

5627664

5522

0.68

4

78.52

487

P. denitrificans DSM 15418

4767709

4677

0.67

4

78.20

156

P. saliphilus DSM 18447

4570660

4442

0.61

1

77.77

399

P. chinensis CGMCC 1.7655

3632870

3608

0.68

1

77.09

390

P. bengalensis DSM 17099

4989582

5009

0.67

4

78.43

188

P. aminovorans DSM 8537

3946204

3832

0.68

2

78.17

191

P. isoporae DSM 22220

3523473

3411

0.66

1

75.94

179

P. denitrificans PD1222

5236194

5158

0.67

3

78.04

976

P. solventivorans DSM 6637

3377602

3258

0.69

1

77.22

147

P. contaminans RKI 16-01929

3033494

2937

0.69

2

76.09

74

P. halophilus JCM 14014

3998285

3884

0.65

1

77.45

4

P. pantotrophus J46

4658858

4675

0.67

1

78.33

511

P. homiensis DSM 17862

3868263

3898

0.64

2

78.70

129

P. denitrificans DSM 415

5192961

5177

0.67

4

78.03

19

P. seriniphilus DSM 14827

4195486

4050

0.62

1

77.41

125

P. yeei ATCC BAA-599

4428895

4280

0.67

2

78.30

546

P. sediminis DSM 26170

3645751

3597

0.66

1

100.00

179

P. aminophilus JCM 7686

4917798

4642

0.63

4

75.31

987

P. zhejiangensis J6

4637247

4512

0.66

2

78.30

176

3.9.1  Evolutionary Relationship among Paracoccus

The evolutionary relationship among the 22 Paracoccus isolates, belonging to 17 species, was studied using Cpn60 proteins. Generally, most eubacteria encode a single copy of the cpn60 gene. However, all of the Paracoccus species studied have multiple copies of the cpn60 gene, all of which are identical. The genome of P. pantotrophus J46 encoded four copies of the cpn60 gene, while P. nitrificans DSM 415 encoded three cpn60 genes. The genomes of P. aminophilus JCM 7686, P. versutus DSM 582, and P. bengalensis contain two copies of the cpn60 gene (see Figure 3.6). P. aminophilus JCM 7686 forms an out-group, while the rest of the Paracoccus species are present in a single clade. The 16 Paracoccus species further clustered into two subclades. The amino acid sequences of Cpn60s from three strains of P. denitrificans are highly conserved and form a subclade. Likewise, two strains, P. halophilus JCM 14040 and P. halophilus CGMCC 1.6117, are very closely related and clustered together with P. yeei ATCC-BBA 599. P. denitrificans is related to P. pantotrophus, P. versutus, and P. bengalensis, and forms another subclade. P. isoporae, P. saliphilus, P. homiensis, and P. seriniphilus form another group. Paracoccus strains isolated from different environments have close phylogenetic relationships.

Phylogenetic tree depicting the relationships among

Figure 3.6   Phylogenetic tree depicting the relationships among Paracoccus species. The tree is based on Cpn60 amino acid sequences, which were aligned by ClustalW, and a neighbor-joining tree was generated using MEGA7. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using the JTT method. The bootstrap method was used as a test of the phylogeny with 500 replications. Bootstrap values are indicated at the nodes.

3.9.2  Carbon Metabolism for PHA Production by Paracoccus Species

Some species of Paracoccus are methylotrophs, use C1 compounds, and have a role in carbon, nitrogen, and sulfur recycling. Genes for carbon metabolism have been identified on one of the four replicons in P. aminophilus JCM 7685. These genes are for l-arabinose use, C4-dicarboxylates transport, the glyoxylate shunt for acetate use, methylcitrate cycle genes for propionate use, methylotrophy (oxidation of methylated amines and the serine cycle for assimilation of C1 units), and stachydrine or D-amino acid use [109]. Strains of Paracoccus are known to produce PHB and PHBV copolymers. P. pantotrophus, P. seriniphilus, P. denitrificans, and Paracoccus sp. LL1 have been reported to produce PHB from glucose, glycerol, pentanol, corn stover hydrolysates, and CO2 (10% in gas mixture). The maximum accumulation of up to 72% has been reported under batch culture conditions of glycerol and corn stover hydrolysate [19].

3.9.3  PHA Synthase Proteins in Paracoccus Species

Analysis of the P. denitrificans genome identified phaA, B, C, P, R, and Z genes related to PHA biosynthesis, which are present in two different clusters. The phaA (3-ketothiolase) and phaB (acetoacetyl-CoA reductase) are present in one cluster. The other cluster carries phaC (PHA synthase), phaP (PHA granule-associated phasin protein), and phaR (putative regulator of PHA synthesis) genes [112]. PHA production is higher under nitrogen-deficient conditions. P. denitrificans degrades PHA under carbon starvation conditions, and a PHA depolymerase gene (phaZ) was identified [113]. The analysis of 22 genomes of Paracoccus identified phaC genes encoding 49 PHA synthase proteins (Figure 3.7). PHA synthase proteins of Paracoccus belong to class I PHA synthases and produce scl-PHA. Genomic analyses revealed multiple copies of phaC genes in 15 Paracoccus strains, and 1–3 phaC genes are present in the genomes of different species. Ten Paracoccus strains have three phaC genes, five strains have two phaC genes, and the rest have a single phaC gene.

Phylogenetic tree depicting the relationship among PhaC of

Figure 3.7   Phylogenetic tree depicting the relationship among PhaC of Paracoccus species. Amino acid sequences were aligned by ClustalW, and a neighbor-joining tree was generated using MEGA7 program. The tree is drawn to scale with branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were calculated using JTT method. Bootstrap method was used as test of phylogeny with 500 replications. Bootstrap values are mentioned at the nodes.

While P. denitrificans PD1222 has three phaC genes, one copy is present on chromosome I, chromosome II, and on the mega-plasmid. P. denitrificans DSM415 and P. denitrificans DSM 15148 carry three phaC genes, and the PhaC proteins encoded by them are not identical. The PHA synthase of P. denitrificans PD1222 present on chromosome I (Pden_0958) has a 33% amino acid sequence identity with the PHA synthase (Pden_4207) carried on chromosome II, and a 35% amino acid sequence identity with the PHA synthase carried on the mega-plasmid (Pden_5103). The PHA synthase (Pden_4207) present on chromosome II and the PHA synthase present on the mega-plasmid (Pden_5103) share 65% amino acid sequence identity. The PHA synthase on chromosome I (Pden_0958) has 173 additional amino acids at its N-terminus compared with the PHA synthase carried on chromosome II (Pden_4207), and 184 additional amino acids at its N-terminus compared with the PHA synthase on the mega-plasmid (Pden_5103). The genomes of P. versutus DSM 582, P. alkenifer DSM 11593, P. halophilus DSM 14040, P. halophilus CGMMC 1.6117, and P. aminovorans JCM 7686 encode two phaC genes. One of the phaC genes in P. aminovorans JCM 7686 is present on the chromosome, while the other is encoded on the mega-plasmid.

Phylogenetic analyses of PHA synthases identified three subclades. The first subclade has 25 PHA synthase proteins, the second clade has 23 PHA synthase proteins, and the third has one PHA synthase. The first clade is divided into four subclades. The first subclade is divided into two further subclades; P. seriniphilus DSM 14827 forms an out-group, while the rest of the 24 proteins are present in different groups.

Three P. denitrificans strains have nine PHA synthase proteins, which are identical and form three clusters in different groups. The first group is closely associated with PHA synthases from P. versutus DSM 582 and P. pantotrophus J46. The second and third PHA synthase proteins are also related to PHA synthases from P. versutus DSM 582 and P. pantophila J46. Likewise, the three PHA synthases from P. halophilus CGMCC 1.6117 and P. halophilus JCM 14014 are identical, but present in different groups. The first copy of PHA synthases from P. halophilus CGMCC 1.6117 and P. halophilus JCM 14014 are closely related to P. yeei ATCC-BBA 599. The second copy is related to P. alkenifer DSM 11593 and P. solventivorans DSM 6637. The third copy of the PHA synthase from P. halophilus JCM 14014 has high homology with the PHA synthases of P. denitrificans, P. aminophilus, P. pantophilus, P. versutus, and P. bengalensis.

3.9.4  PHA Production in Paracoccus Species

Earlier studies reported PHA production in Paracoccus species in batch and shake-flask cultures using glucose, n-pentanol, glycerol, corn stover hydrolysate, and CO2. The maximum production was 72.4% of CDM in Paracoccus sp. LL1, while P. pantotrophus, P. denitrificans NBRC13301, P. denitrificans PD01, and P. seriniphilus E71 are able to accumulate PHA only up to 50% of CDM [19].

3.10  The PHA Production Machinery in Pseudomonas Putida, Cupriavidus necator, Halomonas spp., and Paracoccus spp.

The genes and proteins involved in PHA synthesis in P. putida and C. necator are well studied and have been discussed in previous review articles [43,59,60,114–116]. The genome of Halomonas lutea encodes only one PHA synthase gene (F568DRAFT_04081). Genes encoding two PHA synthases are encoded by the genomes of P. putida (PhaC1, PPUTLS46_005621, and PhaC2, PPUTLS46_005611); P. denitrificans has three PHA synthases (Pden_0957, Pden_4615, and Pden_5103). In contrast, C. necator encodes genes for two PHA synthases (H16_A1437 and H16_A2003) [117].

While the genes involved in PHA synthesis in P. putida and C. necator are clustered in operons, the organization of genes involved in PHA synthesis in P. denitrificans and Halomonas lutea is different; they are scattered all over the genomes. However, homologs of most of the PHA genes present in C. necator are present in H. lutea and P. denitrificans [112] (see Table 3.7). The P. denitrificans genome encodes three phaC genes (encoding PHA synthase), two phaZ genes (encoding PHA depolymerase), and one phaR gene (encoding a PHA repressor). Likewise, H. lutea encodes one phaC gene, one phaZ gene, and one phaR gene. The genomes of both P. denitrificans and H. lutea carry genes for multiple 3-ketothiolases (PhaA) and β-ketoacyl reductase (PhaB) enzymes.

Table 3.7   Amino Acid Sequence Identities Among Class I PHA Synthases of Cupriavidus necator H16 and Halomonas Lutea DSM 23508

Species

Locus tag

1

2

3

4

5

6

1) Cupriavidus necator H16

H16_A2003

100

43.4

46.4

28.1

31.8

31.7

2) Paracoccus denitrificans PD1222

Pden_4207

100

63.1

29.0

34.5

34.8

3) Paracoccus denitrificans PD1222

Pden_5103

100

29.3

35.0

34.4

4) Paracoccus denitrificans PD1222

Pden_0958

100

37.5

37.4

5) Halomonas lutea DSM 23508

F568DRAFT_01350

100

40.3

6) Cupriavidus necator H16

H16_A1437

100

Phylogenetic analyses of the P. putida LS46, C. necator H16, and Halomonas lutea DSM 23508 PHA synthase enzymes revealed some interesting relationships (Figure 3.8). PHA synthases of H. lutea show 40.35% and 31.84% homology to C. necator PHA synthases (H16_A1437 and H16_A2003, respectively). The three PHA synthases of P. denitrificans (Pden_4207, Pden_5103, and Pden_0958) share 43.46%, 46.40%, and 28.17% amino acid sequence identity with the C. necator PHA synthase (H16_A2003), respectively. The amino acid sequence identities of the P. denitrificans PHA synthases (Pden_4207, Pden_5103, and Pden_0958) with the C. necator PHA synthase (H16_A1437) are low and range from 34.40% to 37.43%. The PHA synthase of P. denitrificans PD1222 encoded by Pden_0958 is closely related to the other PHA synthase (H16_A1437) of C. necator, indicating that this could be the main PHA synthase in P. denitrificans. Likewise, the PHA synthase protein of H. lutea (F568DRAFT_04081) shares 40.35% and 31.84% amino acid sequence identity with the two C. necator PHA synthases (H16_A1437 and H16_A2003), respectively.

Phylogenetic tree depicting the relationships among PHA synthase proteins (PhaC) of

Figure 3.8   Phylogenetic tree depicting the relationships among PHA synthase proteins (PhaC) of Pseudomonas putida LS46, C. necator (Ralstonia eutropha) H16, and Halomonas lutea DSM 23508. Amino acid sequences were aligned by ClustalW, and a neighbor-joining tree was generated using MEGA7.

3.11  Domain Organization and Structural Comparison of PhaC from Cupriavidus necator, Halomonas lutea, and Paracoccus denitrificans

PHA synthases consist of two domains, the N-terminal domain, which plays an important role in stabilizing the PhaC dimer, and the C-terminal catalytic domain that possesses the conserved active site amino acid residues Cys, His, and Asp [118]. The catalytic His activates the nucleophilic Cys once the substrate acyl-CoA enters the catalytic pocket. The activated Cys then attacks the thiol group of the acyl-CoA, and the catalytic Asp is postulated to activate the 3-hydroxyl group of the substrate/acyl-CoA and attack the second incoming substrate for the elongation process [119,120]

The domain organization of PhaCs from C. necator, H. lutea, and P. denitrificans indicates a conserved α/β hydrolase-fold region, characterized as the α/β core subdomain (Figure 3.9), featuring a central mixed β-sheet flanked by α-helices on both sides. This domain contains a catalytic pocket comprising a catalytic triad (Cys-Asp-His) at its core. A flexible catabolite activator protein (CAP) subdomain covers the α/β core subdomain from the top.

Schematic representation of PhaC domains from

Figure 3.9   Schematic representation of PhaC domains from Cupriavidus necator (H16_A2003, H16_A1437), Paracoccus denitrificans (Pden_4207, Pden_5103, Pden_0958), and Halomonas lutea (F568_01350).

The C. necator PhaC enzyme was found to exist in equilibrium with the monomer and dimer in solution, with the dimer being the more catalytically active form [121–123]. The structural conformation of the CAP subdomain (closed/open) is the key indicator of the enzyme’s active status. The closed form prevents the substrates from entering the catalytic pocket by covering the active site within the CAP subdomain, particularly, a short stretch of amino acid residues termed the LID region. The LID region in the partially opened PhaC of C. necator undergoes structural changes to allow substrate entry, as revealed by X-ray crystallography of its catalytic domain (residues 201–368 and 378–589; residues 369–377 are disordered) [124,125]. This contrasts with the closed configuration of the catalytic domain reported for the PhaC of Chromobacterium sp. [126]. The structure of C. necator PhaC represents the dimeric organization with the active site of each monomer separated by 33 Å across a dimeric interface, indicating that the polyhydroxybutyrate biosynthesis occurs at a single active site [124]. Thus, the CAP subdomain changes its conformation during catalysis, which involves rearrangement of the dimer to facilitate substrate entry as well as product formation [127].

Multiple amino acid sequence alignments of PHA synthases from C. necator, H. lutea, and P. denitrificans revealed the potential conserved amino acid residues involved in catalysis, substrate specificity, and possible dimerization and/or positioning of catalytic residues (Figure 3.10). The putative residues involved in altering the enzyme function are located near the catalytic triad, but not inside the catalytic pocket [127]. For instance, the alanine residue near the catalytic His of PhaC (Ala510 of C. necator H16_A1437) and the corresponding alanine residues in others (Ala514 of C. necator H16_A2003; Ala505, Ala516, and Ala516 of P. denitrificans Pden_4207, Pden_5103, and Pden_0958, respectively, and Ala521 of H. lutea F568_01350) are involved in the open-close regulation and have an important role in substrate specificity and activity [127]. Moreover, the residues corresponding to Ala510 of C. necator PhaC are conserved in the respective PHA synthase classes: Ala in class I, Gln in class II, Gly in class III, and Ser in class IV, which confirms its role in the substrate specificity of PhaC.

Sequence alignment of PhaC from

Figure 3.10   Sequence alignment of PhaC from Cupriavidus necator (H16_A2003, H16_A1437), Paracoccus denitrificans (Pden_4207, Pden_5103, Pden_0958), and Halomonas lutea (F568_01350). Catalytic amino acid residues (Cys-Asp-His) are highlighted in black. Phenylalanine and the conserved tryptophan residues involved in dimerization are marked in dark gray. The conserved alanine residues involved in regulating substrate specificity are shown in light gray.

A conserved Phe420 of the C. necator PhaC is one of the residues (corresponding to Phe608 of P. denitrificans Pden_0958 and Phe431 of H. lutea F568_01350) involved in dimerization; its mutation to serine greatly reduces the lag phase. The mutation of another conserved residue, Trp425 of C. necator H16_A1437, showed reduced dimerization of PhaC [128,129]. The corresponding residues in others are Trp430 of C. necator H16_A2003, Trp421, Trp432, and Trp610 of P. denitrificans, Pden_4207, Pden_5103, and Pden_0958, respectively, and Trp436 of H. lutea F568_01350. Thr348 of C. necator H16_A1437 interacts with catalytic Cys319 at a distance of 3.6 Å. It contributes to stability enhancement of catalytic Cys through stronger hydrogen bond interaction between the main chain of Cys and the side chain of Thr.

Efforts were made to widen the substrate specificities of PhaCs to integrate both scl and mcl monomers into synthesized copolymers with better characteristics. Studies on the evolutionary engineering approach of class I PhaC from C. necator can be traced back to 2001 [130]. For instance, the fusion enzyme with the N-terminal portion (26%) of PhaC from Aeromonas caviae and the C-terminal portion (74%) of C. necator PhaC exhibit complete enzyme activity with broad substrate specificity [131]. The transfer of a phaC1 gene from a metagenomic clone to P. putida LS46 broadens its substrate specificity and produces copolymers with different combinations of scl- and mcl-monomers, depending on the carbon source used to culture the recombinant bacterium [132].

Limited information on the three-dimensional structure of PHA synthases has limited our understanding of the molecular basis of the substrate specificity and the mechanisms involved in polymerization as well as chain length. The structural information of the intermediate complex of PHA synthases and its substrates will be extremely beneficial in identifying its substrate recognition and/or substrate specificity.

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