Biogenesis of Microbial PHA Granules:a Platform Technology for the Production of Tailor-made Bioparticles

-PHA: water-insoluble cytoplasmic nano-sized inclusions
- it's spherical shell-core particles composed of polyester core, surrounded by phospholipids and proteins.

Fig. 1 PHA granule

- Key enzyme for polyester biosynthesis and polyester particle formation: polyester synthase (PHA synthase)

• this enzyme catalyze: enantio-selective polymerisation of R-hydroxyacyl-CoA thioesters --> polyesters

- Phasins:

• non-covalently attached proteins (structural proteins) at the particle's surface

Introduction

- microbes are capable to form inclusions
- inclusions are surrounded by phospholipid membrane

Fig. 2 Summarization on types of bacterial inclusions and definition of inclusion

- granules:

• have monolayer of phospholipid
• covalently attached to PHA synthase's surface

- when it's in carbon starvation, PHA acts as reserve polymer and mobilized by intracellular PHA depolymerase (attached at granular's surface)

- PHA synthase:

• during elongation of PHA (PHA will self assembly during the enzymatic reaction): soluble synthase transformed into amphipathic enzymes
• PHA is covalently attached to the enzyme

Fig. 3 Classes of polyester synthase

Polyester synthases are the key enzymes

- there are 4 major classes of polyester synthases:

Class I/II
- comprise of enzyme that has only 1 subunit )phaC)
- MW: 61 - 73 kDa
- Class I (R.eutropha): utilise CoA thioesters of various fatty acid

Table 1: Classes of synthase (click to enlarge)

Genetics of polyester biosynthesis enzyme

- genes biopolyester biosynthesis clustered at bacterial genomes


Polyester synthases

- R.eutropha has: homology to enzymes that is related to superfamily of a/B-hydrolases
- method to identify: CD alignment shows amino acid residues' region exhibited 30 % similarity and 17 % identity to the conserved a/B-hydrolase domain
- mutational analysis shows the factors that affect polyester synthase activity:

• N terminus: the variable non-conserved N terminus (the 1st about 100 amino acid residued) which could be altered by insertion of SmaI restriction sites & deletion of the 1st 100 N terminal amino acid residues without loosing enzyme activity. thus, this is less important for polyester synthase activity

• C terminus: not in the class III polyester synthase, but in class I & II only, essential in enzymatic activity, hydrophobic suggestic that it interacts with hydrophobic core of PHA granules.

Structural Insights Into Biopolymer PHA Granular Phasins: Comparison Analysis & Function Validation

- PHA functions:

• substitute for petroleum derived thermoplastics (petrochemical plastics for 'white pollution', environmentally unfriendly)
• as scaffold/implant biomaterials in tissue engineering

- monomers of PHA depend on:

• type of PHA producing bacterium
• growth conditions

- Typical PHAs:

a) PHB:

• c4 monomer
• SCL
• homopolymer that is synthesized from Wautersia eutropha strain H16

b) poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)

• PHBHHX
• C4 & C6 monomers
• copolymer of SCL-MCL PHA
• heteropolymer from Aeromonas hydrophila strain 4AK4

- Native PHA were accumulated as intracellular granules (inclusion bodies) in vivo
- PHA granules were found enveloped by various granule associated proteins
- Non-enzymatic structural proteins associated to PHA granules are called: PHA granular phasins
- low phasin = low PHA content = PHA number

Fig. 1 Summarized types of PHA envelopes

Fig.2 Summarized phasins' functions

Investigating the Structure-Property Relationship of Bacterial PHA Block Copolymers

- SCLPHA films that have block copolymers = retain high elasticity over time with respect to films of similar random copolymers of comparible composition

- Conclusion I got for types of strength:

Molecular Analysis of the A.eutrophus (PHB) Biosynthetic Operon:Identification of the N Terminus of PHB Synthase &Identification of the Promoter

- P(3HB) = PHB
- This paper is to analyze phbC (by using molecular technique method)
- Analysis:

• Identify translational initiation codon (from cell R.eutrophathat has phbC'-'lac fusion gene)
• the methods: analysis of gel electrophoretic homogeneity by chromatography on DEAE-Sephacel and on aminophenyl-B-D-thiogalacto-pyranosidase-sepharose

- The method of synthesis (Enzymatic reactions):

1. 2 acetyl-CoA (starter compound) --> (condensation) acetoacetyl-CoA
2. acetoacetyl-CoA --> (chiral reduction) D-(-)-3-hydroxy-butyryl-CoA
3. polymerization of D-(-)-3-hydroxybutyrate units

cont... Bacterial and other biological systems for polyester production

Organization of PHA biosynthesis genes

- Genes for PHA biosynthesisand other proteins that involved in metabolism of PHA usually clustered in bacterial genomes
- example:
in R.eutropha, genes for phaC, phaA and phaB contitute the phaCAB operon. a little downstram (4 kbp), a second B-ketothiolase gene was found. it can synthesize 3-ketoaceryl-CoA.


Primary structure of PHA synthase

- Type I & Type II:

constitute of enzyme that is
* has 1 type of subunit (phaC)
* molecular weight: 61-68 kDa

- Type III:

* Preferentially utilise coenzyme A thioester (at least C5)
* enzyme that has 2 subunit:
a) PhaC (MW=40 kDa) : 21 - 28 % simlar to Type I & II
b) PhaE (MW=40 kDa): not similar with PHA synthases
* Both of the enzyme prefer coenzyme A thioester of 3HA SCL

- The mechanism that inveolved 2 thiol groups of PHA synthases during catalytic cycle was proposed by Giebel et. al.

* But, in the multiple alighnment of PHA synthases, only cysteine residue (cys-319) is able to be conserved
* So, scientist are trying to find another 1 thiol group.

- cys-319: determintaion of its role in reaction mechanism was obtained from site-specific mutagenesis

- cis-459:
* supposely involved in catalytic cycle that could make the second thiol grioup to be discovered.
* but, the site-specific mutagenesis shows that this amino acid residue is not essential for catalytic activity
* proven when we look at the alignment of PHA synthase sequence.

Secondary and Quarternary Structures of PHA Synthases

- secondary structure (prediction)
* based on multiple alignment of PHA synthases (accuracy 72 %)
* mainly composed of variable loop (49.7 %), a-helical (39.9 %), B-sheet (10.4 %)

Factors determining the MW of PHAs

- Type I: synthesize high MW (500 000 - millions)
- Type II: synthesize 50000 - 500 000
- Type III: synthesize MW between Type I and Type II

Biochemical and Genetic Analysis of PHA synthases and other proteins required for PHA synthesis

Introduction

• most prokaryotes synthesize Poly(3HB) and other PHAs
• PHA is:

1. high molecular weight
2. thermoplastic
3. has elastomeric features

• many prokaryote and eukaryote can produce low MW Poly(3HB) molecules.
• Poly(3HB) will complexed with other biomolecules. this happens when the concentration is less 3 to 4 orders of magnitude than the storage PHAs in the cells

Terminology

• short chain length (SCL): PHA that has 3-5 C atoms
• medium chain length (MCL): PHA that has 6-14 C atoms

SCL/MCL are applied also to the CoA thioesters or substrate range of PHA synthases in the process to produce PHA.

to use the SCL/MCL terms: put on a subcript (eg. PHASCL, HAMCL-CoA thioesters, PHAMCL synthase etc)

• Name/designations for genes that involved in PHA biosynthesis, auxiliary structural proteins bound to PHA granules and PHA degrading enzyme:

a) Genes coding for protein biosynthesis PHA follow alphabetical order:
- phaA (B-ketothiolase)
- phaB (acetoacetyl-CoA reductase)
- phaC (PHA synthase)
- phaG (3-hydroxy-acyl carrier protein-coenzyme A transferase)
- phaJ (enoyl-CoA hydratase)
- etc

b) Genes coding for degradation follow reverse alphabetical order:
- phaZ (PHA depolymerase)
- phaY
- phaX
- phaW
- etc

c) Method to use:
- name of genes + subscript of Genus(capitalised) and sp(small letter) of the organism
- eg. phaCRe = phaC for R.eutropha


Strategies for cloning of PHA biosynthesis genes

• there 8 strategies
• these methods are differs in quality and elegance
• what differentiate them are the time and required to execute the process

1. Strategy A
- 'enzymatic analysis'
- screened clone of the expression of active enzymes involved in the PHA biosynthesis

2. Strategy B
- the homologous gene probes obtained after transposon mutagenesis are used to identify the respective intact gene of the same genome

3. Strategy C
- heterologous gene probes are used to identify corresponding genes in genomic libraries prepared from other bacteria

4. Strategy D
- 'consensus oligonucleotides (hybridization or PCR technique)'
- uses short oligonucleotides that has been designed according to short, highly conserved stretches of PHA synthases

5. Strategy E
- 'oligonucleotides designed according to N-terminal amino acid sequence of enzyme'
- PHA synthase protein purified --> oligonucleotide designed from N-terminal amino acid sequence --> identify the corresponding gene in genomic library

6. Strategy F
- 'opaque colonies in PHA-negative host after heterologous expression'
- the genomic libraries are screen for:
a) to find phenotypic complementation of a PHA-negative mutant
b) confering the ability to synthesize and accumulate PHA to a PHA-negative wild type

7. Strategy G
- 'growth after detoxification of media due to removal of fatty acids'

8. Strategy H
- 'analysis of genome sequence and application of PCR technique'


p.s- dr., i still didn't finish reading this journal, i'll update again. thank you!

Bacterial and other biological systems for polyester production

Introduction

• most bacteria and family Halobactericeae of Archae can synthesize PHA
• function of PHA in cells: storage compound (it is polyester)
• in a form of: insoluble inclusions in cytoplasm
• it is synthesized when:
  

1. abundant in carbon source
2. low of other nutrient (eg. N, P, S, O)

• uses in the industry:

1. source for synthesis of enantiomerically pure chemicals
2. raw materials for production of paint

Biosynthetic Pathways

• key enzyme: PHA synthase (also known as PhaC or PhaEC)
  
• for Ralstonia eutropha:
  

acetyl-CoA is utilise to produce PHB via -R(-)-3-hydroxybutyryl-CoA. it uses 3-step pathway with B-ketothiolase (it is NADPH-dependent acetoacetyl-CoA reductase) & PHA synthase

• acetyl-CoA is the central intermediate in metabolism of any organism. thus, it can be formed from any carbon sources (eg. carbohydrate & fatty acid)

PHA synthase

• PHA synthase in R.eutropha is in class I
• the classes are based on:

1. the number of different subunits that constitute the active PHA-synthase protein
2. substrate range

• site-directed mutagenesis: a method used to identify & confirm the extended homologies that are

1. shared by all PHA synthases & presumed
2. presumed important to the catalytic cycle of PHA synthases

• in R.eutropha: cys-319 in CXCXGG motif is needed for:

1. PHA synthase activity
2. provide 1 of the 2 thiols groups thought to be important for the proposed catalytic cycle