S72 Special Abstracts / Journal of Biotechnology 150S (2010) S1S576
[I.10]
Figure 1. Inuence of carbohydrazide on effective 3HPA-production in repeated-
batch-fermentations each with 500 mM glycerol in 200-mL scale.
doi:10.1016/j.jbiotec.2010.08.186
[I.9]
Biocatalysis provides polymers with a broad range of solid-state
properties
M. Scandola 1, , M.L. Focarete 1 , R.A. Gross 2
1 Bologna University, Italy
2 Brooklyn Polytechnic University, United States
Keywords: Polymers; Biocatalysis; Solid-state properties; Bioma-
terials
High molecular weight polymers that cannot be obtained by
chemical routes are easily synthesized by lipase-catalyzed poly-
merization. Some lipases such as Candida antarctica Lipase B, when
used in ring opening polymerization, allow incorporation of differ-
ent monomers along the chain leading to copolymers with dened
composition and microstructure. Control over microstructure is
critical to the ultimate goal of tailoring the physical, mechanical,
and biological properties of copolymers. Hydrophilic/hydrophobic
balance can be adjusted by a suitable choice of the two monomers
and of composition, leading to materials with tunable hydrolytic
degradation rate for environmental and biomedical applications.
Copolymers of -pentadecalactone with -caprolactone, valerolac-
tone, dioxanone and trimethylenecarbonate are highly crystalline
over the whole composition range, an unusual behavior due
to co-crystallization of the co-monomer units. Copolyestercar-
bonates, copolyesteramides and polyol-containing copolyesters
synthesized by lipase-catalysed polycondensation show strongly
composition dependent physical properties, that can be easily tai-
lored by composition control and cover the whole range from hard
solid materials down to gluelike substances.
Financial support of Italian Ministry of Foreign Affairs (Direc-
torate General for Cultural Promotion and Cooperation
Signicant Bilateral Project Italia-USA) is gratefully acknowledged
doi:10.1016/j.jbiotec.2010.08.187
A strategy for coproduction of succinate and polyhydroxyalka-
noates
Zhen Kang, Cuijuan Gao, Huimin Liu, Qian Wang, Qingsheng Qi
Life Science School, Shandong University, China
Many chemicals and materials could be generated biologi-
cally from renewable substrates. Among them, succinic acid and
polyhydroxyalkanoates (PHA) are two important products with
application potential. In this study, a co-production system of succi-
nate and polyhydroxybutyrate (PHB) was constructed in Escherichia
coli. To drive carbon ux toward succinate formation and realize
succinate accumulation under aerobic condition, sdhA, encoding
a succinate dehydrogenase A subunit, poxB, encoding a pyruvate
oxidase, pta gene, which encodes a phosphate acetyltransferase
and iclR gene, encoding a repressor, were all inactivated in E.coli
MG1655. Meanwhile, in order to providing more oxalacetate for
succinate accumulation and for future mix-sugar fermentation,
ptsG gene, which encodes glucose-specic transporter of the phos-
photransferase system, was also knocked out. Then, the PHB
biosynthesis pathway was constructed in this engineered E. coli
by transforming the plasmid containing the phbCAB operon from
Ralstonia eutropha. Batch cultivation showed that the engineered
strain was able to accumulate 41.3 wt% PHB. PHB accumulation not
only minimized pyruvate and acetate secretion, but also improved
succinate production to 24.6 g l1 . The co-production of succinate
and PHB not only produce two products in one fermentation step
but also made the use of the cell debris, which was usually treated
as waste. To our knowledge, this is the rst report of co-production
organic acid and polymers with engineered E. coli. This biorenery
strategy may also applicable to other bioprocesses. Further study
towards coproduction of succinate and polyhydroxyalkanoates are
under way.
doi:10.1016/j.jbiotec.2010.08.188
[I.11]
Metabolic engineering of sucrose utilizing Escherichia coli for
polyhydroxybutyrate production
Y. Arin , H. Sugiarto, S. Sabri, J.O. Kromer, C.E. Vickers, L.K.
Nielsen
Australian Institute for Bioengineering and Nanotechnology, Univer-
sity of Queensland, Australia
Keywords: Metabolic engineering; Escherichia coli; Sucrose; Poly-
hydroxybutyrate
Polyhydroxyalkanoates (PHAs) are biopolymers with many
applications, including biodegradable plastics, textile bers, med-
ical implants, and biofuel additives. Polyhydroxybutyrate (PHB)
is a model PHA compound. Most industrial production of PHB
occurs via fermentation of glucose using recombinant Escherichia
coli. In terms of the carbon source used for fermentation,
sucrose from sugarcane has several advantages over glucose from
corn starch, both technically and economically. However, most
PHB-producing industrial E. coli strains are unable to utilize
sucrose.
In this study, we have used an E. coli strain that is able to utilize
sucrose for PHB production. This strain possesses a native sucrose
operon that consists of four csc genes encoding a sucrose perme-
ase, an invertase, a fructokinase, and a repressor protein. Ralstonia
eutrophus genes for PHB production were expressed from a plas-
mid vector. A fed-batch fermentation was performed in R/2 mineral
 Special Abstracts / Journal of Biotechnology 150S (2010) S1S576 S73

medium using sucrose as a sole carbon source under pH-stat [I.13]

feeding control for high level PHB production. In semi-optimized
conditions, the wild-type strain was capable of producing 30 g L1 A novel bacterial tyrosinase for biomaterials and biocatalysis
PHB at a productivity of 1.1 0.1 g L1 h1 . In order to improve M. Fairhead , L. Thoeny-Meyer
sucrose utilization, the sucrose operon repressor gene was knocked
out. The engineered strain produced 46 g L1 PHB at a produc- Swiss Federal Laboratories for Materials Testing and Research, Switzer-
tivity of 1.8 0.1 g L1 h1 , a 50% increase in yield over the wild land
type. Keywords: Tyrosinase; Melanin; Immobilized enzymes; Protein
To further improve PHB production, we have engineered cen- engineering
tral carbon metabolism to increase carbon ux to PHB precursors.
Tyrosinase (EC 1.14.18) is a type 3 copper oxidase enzyme that
Specically, we have knocked out pgi and overexpressed eda, fbaA,
converts tyrosine and DOPA into dopaquinone, which is a precursor
and tpiA. Preliminary data shows that tpiA overexpression on its
in melanin biosynthesis (Claus and Decker, 2006). Tyrosinases have
own can increase PHB production by 50%, while the other genes
many potential biotechnological applications, one of them being
have insignicant effect on PHB production on their own. We are
the ability to catalyze proteinprotein or proteinpolysaccharide
currently evaluating overexpression of the gene targets in the pgi
cross-linking (Halaouli et al., 2006).
knock-out strain.
We report on the successful expression of a tyrosinase-like
gene from the aquatic bacterium Verrucomicrobium spinosum in
doi:10.1016/j.jbiotec.2010.08.189 Escherichia coli (RCTEBT, 2010). The gene codes for a 57 kDa pro-
tein (full length unprocessed form) which bioinformatic analysis
[I.12] suggests has a TAT signal peptide, the two copper binding motifs
typical of the tyrosinase protein family and a C-terminal extension
Potential applications of microbial biomass and PHA elastomer similar to that found in the pro-form of plant and fungal polyphenol
from glycerol to obtain biodegradable and compostable lms oxidases. The protein was puried in a cytoplasmic form to homo-
Rosa Palmeri 1, , Manuela Fragal 1 , Stefano La Porta 2 , Antonino geneity and shown to contain 1.72 copper atoms per polypeptide,
Felice Catara 1 in agreement with a binuclear copper site. Both mono-and diphe-
nol oxidase activites were present as shown by spectrophotometric
1Parco Scientico e Tecnologico della Sicilia, Italy assays for tyrosine hydroxylation.
2Universit di Messina, Italy We expressed various mutants of the recombinant enzyme in E.
Keywords: Biopolymer; Glycerol; Paper; Organic waste coli and found that removal of the C-terminal extension by genetic
engineering or limited trypsin digest of the pro-form results in a
Due to the great concern about the long lasting time for the
more active enzyme, i.e. 30100 fold increase in monophenolase
degradation of plastic materials different approaches are under
and diphenolase activities.
evaluation to nd friendly solutions to the problem and assure a
In addition, cross-linking experiments demonstrate that this
sustainable growth. The use of 100% biodegradable lmsappears as
enzyme may be used for the formation of cross-linked enzyme
a challenging alternative for specic applications, as organic agri-
aggregates. The protein can also be stamped onto surfaces allowing
culture and composting. Among them is the coating of different
the formation of thin melanin lms in-situ.
supports with different blends of oils, polymers and natural prod-
ucts to improve their performances. Based on successful conversion
of fatty acids in PHA by Pseudomonas corrugata CFBP5454, we have
improved the quality of the polymer by approaching the metabolic
pathway of fatty acids through the conversion of glycerol with P
mediterranea CFBP5447 strain. A high biomass fermentation pH-
stat fed-batch type was settled in order to obtain an unbalance of
carbon (glycerol)/ammonia source. At the end of fermentation, the
accumulation of intracellular PHA was detected with Nile-red and References
centrifuged to spin and wash the broth. The biomass was freeze-
dried and later acetone extracted to recover the elastomer. Claus, H., Decker, H., 2006. Bacterial tyrosinases. Syst Appl Microbiol. 29,
Different solutions of biomass or PHA were sprayed or cal- 314.
Halaouli, S., Asther, M., Sigoillot, J.C., Hamdi, M., Lomascolo, A., 2006. Fungal
endered on paper and ber sheets and tested for their potential tyrosinases: new prospects in molecular characteristics, bioengineering and
applications, as mulching lms or moist organic waste bags. Results biotechnological applications. J Appl Microbiol. 100, 219232.
show that both coatings reduces the water and oil absorption Role of the C-terminal extension in a bacterial tyrosinase. (2010) Michael Fairhead
and Linda Thny-Meyer. FEBS J. In press.
improving the resistance and maintaining a good air permeation as
compared with polyethylene lms and untreated paper. Biomass
coated paper sheets were unbroken up to 96 hours whereas the doi:10.1016/j.jbiotec.2010.08.191
control was broken within ve minutes. Biomass coated paper and
ber bags of different size appear very promising to manage moist
organic wastes.

doi:10.1016/j.jbiotec.2010.08.190