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Noda Institute for Scientiﬁc Research GRANT
2011 Young Investigator Research Grant

Genetic engineering for copolymerized bio-polyester production from methanol
Izumi ORITA
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology

Research aims

Results

Polyhydroxyalkanoate (PHA) is an intracellular biopolyester, which is synthesized by various microorganisms as
the energy storage compound. This biopolyester can be produced from sugar and plant oil derived by biomass; furthermore, it has biodegradability and biocompatibility. Therefore, PHA is attracting interest as a substitute for petroleum-derived plastics. The most representative PHA ispoly(3-hydroxybutyrate)[P(3HB)], which is the homopolymer composed of only C4 monomer unit. However, P(3HB)
is not suitable for practical use because its physical property is stiff and brittle. On the other hand, copolyesters copolymerized with medium-chain-length monomer (C6–C10)
have ﬂexible properties. In this study, genetic engineeringof microorganisms was performed for efﬁcient productionof PHA copolymer composed of C4 and C6 monomer units
from methanol, which is inexpensive and sustainable feedstock. For this purpose, three kinds of methylotrophic bacteria (methylotrophs), capable of growth on methanol as asole carbon source, and a non-methylotrophic representative PHA producer were used as the host strains.

1. Genetic engineering of M. extorquens and P. denitriﬁcans.
First of all, we have reanalyzed PHAs produced by M.
extorquens AM1 wild-type strain and a previously constructed mutant strain, AM1CAc, in which its PHA synthasegene was replaced by a gene of the Aeromonas caviae PHAsynthase (phaCAc) with a broad speciﬁcity. The PHA produced by M. extorquens AM1 from methanol was previously thought to be P(3HB). Surprisingly, our results revealed that M. extorquens AM1 synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer copolymerized with C5 monomer unit. On the otherhand, the mutant strain AM1CAc accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) [P(3HB-co-3HV-co-3HHx)] terpolymer composedof C4, C5 and C6 monomer units (Table 1). Next, the genes
encoding enzymes for supplying the precursor of second
monomer focusing on CoA intermediate on the ethylmalonyl-CoA pathway, which is for regeneration of glyoxylate in this bacterium2), were introduced in M. extorquens
AM1CAc. As a result, when the gene encoding crotonylCoA carboxylase/reductase was expressed, C6 monomer
composition was increased 2 times compared with the parent strain, though C6 monomer composition was still low.
We also succeeded in increasing PHA production and C5
monomer composition by expression of the phaA and phaB
genes encoding monomer supplying enzymes (Table 1).
It was reported that P. denitriﬁcans accumulates P(3HB)
from methanol3). Its PHA polymerase gene was replacedwith the phaCAc gene or its mutant phaCNSDG gene by homologous recombination. However, unfortunately, thesetransformants accumulated only P(3HB) from methanol
like the parent strain.

Methods
General genetic manipulation was performed using
Escherichia coli DH5a and S17-1. Three methylotrophs,
Methylobacterium extorquens AM1, Paracoccus denitriﬁcans, and Methylobacillus ﬂagellatus and hydrogen oxidizing bacterium Ralstonia eutropha (non-methylotroph)were used as the host strains. The target genes wereampliﬁed by PCR and then ligated with modiﬁed pCM801)
or pK18mobsacB for gene expression or homologous
recombination, respectively. The accumulated PHAs wereanalyzed as follows. The cells were grown in nitrogenlimited medium containing 0.5% or 1% (v/v) methanol.
After the cultivation, the cells were harvested, followed bylyophilized. Cellular PHA content and composition weredetermined by GC and GC-MS after methanolysis of thedried cells in the presence of 15% sulfuric acid. Furthermore, the extracted PHAs were analyzed by GPC andNMR.

2.

Conferring PHA synthesis ability on M. ﬂagellatus
M. ﬂagellatus is an obligate methylotroph, which cangrow on methanol very efﬁciently, but is unable to synthesize PHA. In this methylotroph, we expressed the phaA andphaB genes from R. eutropha and the phaCNSDG genes
under the control of its own methanol dehydrogenase promoter. As the result of cultivation, the transformant accumulated 7 wt% P(3HB), though its growth rate was lower
than that of the parent strain.

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Table 1.

Biosynthesis of PHA from methanol by M. extorquens AM1 and recombinant strains.

StrainAM1AM1CAc
AM1CAc/pCM80-ccr
AM1CAc/pCM80Km-phaAB

Cultivation
timea(h)

DCWb
(g/l)

RCWc
(g/l)

PHAcontent(wt%)

Composition (mol%)
3HB (C4)3HV (C5)

3HHx (C6)

72
72
9672

0.699
0.874
0.720
0.416

0.642
0.580
0.539
0.241

8.38
32.6
25.1
43.6

95.7
99.2
99.5
96.6

3.92
0.710
0.528
3.23

00.2800.528
0.201

aCells were cultured on the medium containing 0.5% methanol.
Dry cell weight.
cResidual cell weight.
b3.

Conferring methanol utilization ability on R. eutropha
R. eutropha accumulates more than 80 wt% PHA whengrown on sugar and plant oil, but is unable to grow onmethanol. Analyses of the genome sequence revealed that
this bacterium has homologs of enzymes involved inmethanol oxidation. Therefore, we overexpressed them inR. eutropha. Characterization of the transformants is inprogress.

only short-chain-length monomers but also C6 monomerunit. We also succeeded in conferring PHA production ability on a strain of methylotroph, which is originally unable
to synthesize PHA. However, both composition of C6
monomer and PHA productivity are still low. Further strain
improvement through genetic engineering is required.
References1)

Conclusion
In this study, genetic engineering techniques were applied to strains of methylotroph and non-methylotroph for
efﬁcient PHA copolymer production from methanol. We
succeeded in constructing a strain of methylotroph, which
can efﬁciently synthesize PHA copolymer composed of notMarx, C. J., and M. E. Lidstrom. (2001) Development of improvedversatile broad-host-range vectors for use in methylotrophs and otherGram-negative bacteria. Microbiology 147: 2065–2075.
2) Peyraud, R., P. Kiefer, P. Christen, S. Massou, J. C. Portais, and J. A.
Vorholt. (2009) Demonstration of the ethylmalonyl-CoA pathway byusing 13C metabolomics. Proc Natl Acad Sci U S A 106: 4846–4851.
3) Yamane, T., X. F. Chen, and S. Ueda. (1996) Polyhydroxyalkanoate
synthesis from alcohols during the growth of Paracoccus denitriﬁcans. FEMS Microbiology Letters 135: 207–211.