75NISR Research GRANT
2005 Young Investigator Research Grant

Regulation of Molecular Weight of Biopolyesters Produced byBacteria
Takeharu TSUGE
Department of Innovative and Engineering Materials, Tokyo Institute of Technology

Research aims
A variety of bacteria can synthesize biopolyester, polyhydroxyalkanoate (PHA), as carbon and energy storage materials in the cells. Unlike petroleum-based polymers, PHA
can be produced from renewable resources such as biomass
and carbon dioxide and be used as biodegradable thermoplastic material. Natural PHA-producing bacteria produce
PHA with the weight-average molecular weight ranging
from 500,000 to 1,000,000. Molecular weight of polymer is
one of the important factors for material properties and themolecular weight-regulated polymers are practically useful.
However, as for PHA, regulation of the molecular weight inbiosynthesis method has not been established. This study
aims to develop a method for regulating molecular weightof PHA by use of PHA synthase mutants in recombinantbacteria.
Methods
PHA synthase gene (phaCAc) from Aeromonas caviae
was used in this study. The A. caviae PHA synthase mutants, substituting an alanine at position 505 (A505) withthe other 19 amino acids, were generated by site-specificmutagenesis. As a host for PHA accumulation, PHA negative mutant of Ralstonia eutropha H16, strain PHB4, wasused. Recombinant plasmids for functional expression ofphaCAc genes in R. eutropha PHB4 were constructedusing a broad-host-range vector and introduced into thehost cells by a transconjugation method.
Recombinant strains of R. eutropha PHB4 were cultivated on a reciprocal shaker in 500-ml flasks containing
100 ml mineral salt (MS) medium supplemented with 2%(wt./vol.) fructose at 30°C for 72 h. After cultivation, collected cells were washed with water to remove the remaining carbon source, and then lyophilized.
The PHA content in dried cells was determined by gas
chromatography (GC) after methanolysis of lyophilizedcells. The polymers accumulated in the cells were extractedwith chloroform at room temperature and purified by reprecipitation with methanol. Molecular weight data were obtained by gel permeation chromatography (GPC).
Results
Sequence alignment analysis of PHA synthases
At present, approximately 60 different PHA synthases
have been found and characterized. Based on their substratespecificity and subunit composition, PHA synthases havebeen classified into four types, type I to IV. PHA synthasefrom Ralstonia eutropha is the well-known enzyme belonging to type I group. Previously, we reported that an aminoacid substitution at position 510 (A510) in R. eutropha enzyme, a highly conserved position in type I enzymes andlocated two residues downstream from active-site histidine(H508), is beneficial to change the molecular weight ofPHA1). Also, similar effect was observed for type II mutant
enzyme, in which the corresponding amino acid residue(Q481) was substituted with other amino acids2).
PHA synthase from Aeromonas caviae belongs to type I
group; this enzyme shows broader substrate specificity thantypical type I enzymes. Thus, it has been recognized as animportant tool for biosynthesis of high performance PHA.
Based on the sequence alignment analysis of PHA synthases, we identified that alanine at position 505 (A505) inA. caviae enzyme is the effective residue to change in molecular weight of PHA by introducing amino acid substitution.
PHA synthesis by use of mutant enzymes
R. eutropha PHB4 expressing PHA synthase mutants
A505X from A. caviae were cultivated and all strains
showed comparable growth (2.8–4.1 g/l of dry cell concentration). To determine the monomer composition of PHA
and the content in the cells, the cells were subjected to GC
analysis. The results showed that all strains produced homopolymer of (R)-3-hydroxybutyrate, P(3HB). As for PHA
accumulation, high content was observed for the strain expressing the wild-type enzyme (79 wt%), followed by
A505M (78 wt%) and A505T (77 wt%). The low PHA content was observed for three mutants of A505P, A505R andA505K (66 wt%). All mutant enzymes, however, produced
relatively large amounts of PHA in the cells, suggestingthat amino acid substitution at position 505 does not affectprimary polymerization function of the enzyme.
Molecular weight analysis
All PHA samples isolated from each strain were subjected to GPC analysis. The weight-average molecularweight (Mw) of PHA synthesized by the wild-type enzyme
was 650,000, whereas those by mutant enzymes were in therange of 280,000–1,070,000. These mutants did not synthesize PHA with significantly higher molecular weight than
the wild-type. However, A505M (Mw280,000) andA505I (Mw400,000) synthesized low-molecular weight
PHA with high reproducibility. The polydispersity index of

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PHA synthesized by the wild-type enzyme, A505M andA505I were 2.6, 2.5 and 3.3, respectively. Taking PHA content and polydispersity index into consideration, A505M
mutant is a possible candidate for synthesis of PHA withthe molecular weight regulated to half of that synthesized
by the wild-type enzyme.
Conclusion
The molecular weights of PHA synthesized by mutantsof PHA synthase from A. caviae were characterized. Aminoacid substitution at position 505 was found to be effective
for changing molecular weight of PHA without reducing
polyester productivity. Particularly, A505M mutant is a possible candidate for synthesis of low-molecular weight PHA.

This study demonstrates the feasibility of synthesizing
PHA with regulated-molecular weight by use of PHA synthase mutants from A. caviae.
References1) Tsuge, T., Saito, Y., Narike, M., Muneta, K., Yahaya, N. M.,
Kikkawa, Y., Hiraishi, T., Doi, Y.: Mutation effects of a conserved
alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis; Macromol. Biosci., 4,963–970 (2004)
2) Tsuge, T., Yano, K., Imazu, S., Numata, K., Kikkawa, Y., Abe, H.,
Taguchi, S., Doi, Y.: Biosynthesis of polyhydroxyalkanoate (PHA)
copolymer from fructose using wild-type and laboratory-evolved
PHA synthases; Macromol. Biosci., 5, 112–117 (2005)