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!