7 international symposium on industrial microbiology & biotechnolgy

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7
th
INTERNATIONAL
SYMPOSIUM ON INDUSTRIAL
MICROBIOLOGY &
BIOTECHNOLGY
IN HONOUR OF
PROFESSOR ARNOLD L.DEMAIN
Melbourne , Australia
The Bio21 Institute is pleased to be hosting the 7
th
International
symposium on Industrial Microbiology and Biotechnology.
Bringing together a number of internationals across industry
and research from Japan, Taiwan, China, Mexico, Germany and
Australia,many of whom are former research students,
postdocs, colleagues and friends of Professor Arnold Demain a
retired MIT Professor with a career spanning almost six decades
across industry and research sectors.
Arny’s Army & Friends Organising Committee
July 1 – 3 , 2010
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 2
Contents
Committee.................................................................................................................................3
Locations....................................................................................................................................4
Sponsors ……………………………………………………………………………………………………………………………………………6
A Tribute to Professor Arnold L.Demain - a lifetime in industrial microbiology……………………………… 7
Programme Outline................................................................................................................11
Detailed Programme July 2......................................................................................................12
Detailed Programme July 3......................................................................................................14
Contact Details.........................................................................................................................17
Abstracts - General Program Presentations...........................................................................21
Abstracts - Poster Presentations............................................................................................39
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 3
Committee
The 7
th
International Symposium on Industrial Microbiology & Biotechnology was organised by
Professor Margaret Britz with assistance from Ms Alex Waters (UTAS) and Dr Susan Pepper.
Arny’s Army & Friends would like to thank everyone for their participation and help.
Professor Margaret Britz also wishes to acknowledge Microbiology Australia as the original source of
the article on Professor Demain and that this article is being reproduced with the kind permission of
The Australian Society for Microbiology.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 4
Locations
Melbourne Convention and Exhibition Centre
The Melbourne Convention and Exhibition Centre is located at 1 Convention Centre Place.
Welcome Mixer
Feddish Cafe Bar & Restaurant
River Terrace Yarra Building
Federation Square
Feddish Restaurant
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 5
Bio21 Institute
30 Flemington Rd (near corner of Park Drive) Parkville,Victoria 3010, Australia.
Transport:From the CBD, take the 59 tram (along Elizabeth St) or take the 55 tram (along WilliamSt)
to Park Drive. Tram Stop 15 is closest to Bio21.
Parking:No public parking is available on site at Bio21. Limited street and meter parking is available
in the surrounding streets.
Airport:25 minutes fromMelbourne International Airport via freeway.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 6
Sponsors
ARP, Boston, USA (Peter Souw)

BioIndustry Partners, Australia
(Director: Margaret Britz)


Arny’s Army & Friends (at MIT 2007)
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 7
Article originally published in Microbiology Australia. Reproduced with the kind permission of The Australian Society for Microbiology.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 8
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 9
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 10
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 11
Programme Outline
July 1, 2010
6.00pm -8.00pm
Conference Mixer
All registrants can also attend the closing Plenary Session of th
e GIM
Symposium presented by Professor Demain at 4.00pm in the Melbourne
Conference and Convention Centre, and then join the Mixer for the 7
th
International Symposium on Industrial Microbiology and Biotechnology.
Registrants attending the Plenary Session at GIMS need to pick up a
visitor’s tag from the GIM registration desk
Mixer
Location
‘Feddish’
, River terrace Yarra Building

Federation Square
Corner Flinders Street and Swanston Street Tel: 03 9654 5855
(Federation Square is opposite Flinders Street Train Station)
July 2 , 2010
8.30am-5.00pm
Symposium Presentations
Symposium Location
Bio21
Molecular Science & Biotechnology Institute
The University of Melbourne
30 Flemington Road
Parkville VIC 3010
Australia
http://www.bio21.unimelb.edu.
au/
Transport:
From the CBD, take the 59 tram (along Elizabeth St) or take the
55 tram (along William St) to Park Drive. Tram Stop 15 is closest to Bio21.
July 3, 2010
9.00am-5.00pm
7.00pm
Symposium Presentations
Symposium Dinner
Symposium Lo
cation
Bio21 Molecular Science & Biotechnology Institute
30 Flemington Road, Parkville
Dinner Location

Feddish
’, River Terrace Yarra Building

Federation Square
Corner Flinders Street and Swanston Street Tel: 03 9654 5855
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 12
Detailed Programme July 2
July 2 , 2010
8:30am – 9:00am
Registration
9:00-10:30 Welcome and Opening Plenary Presentations
9.00am - 9.10am
Introduction
Professor Margaret Britz, Dean of Science, Engineering and Technology,
University of Tasmania
9.10am - 9.20am
Welcome and Opening Remarks
Professor Tony Bacic, Director, Bio21 Institute
9.20am

9.55am
Opening Plenary Presentations
Professor Sergio Sanchez
Departamento de Biologia Molecular y Biotecnologia Instituto de
Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico
‘Insights into the carbon source regulation of biochemical and
morphological differentiation in Streptomyces coelicolor’
Sergio Sánchez, Adan Chávez, Angela Forero, Yolanda García, Mauricio
Sánchez, Brenda Sánchez and Beatriz Ruiz
9.55
am

10.30am
Zhang Lixin
Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing,
People’s Republic of China
‘Biodiscovery in China’
10.30am
Morning Tea Break
11:00 – 12:30 General Program - Session One - Pharmaceuticals - toxins,
antibodies, antibiotics and natural products
11.00am

11:30am
Eric A. Johnson
‘Botulinum neurotoxin: from poison to medicine’
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th
International Symposium of Industrial Microbiology and Biotechnology Page 13
July 2 , 2010
Continued
11:30am

12:00pm
Yuanrong Cheng
‘Microbial conversion of sirolimus’
12:00pm

12:30pm
Bita Zaferanloo
‘Endophytes from an Australian native plant as a promising source of
industrially useful enzymes’
12:30pm
Lunch Break – Ground Floor
12:30pm – 1:30pm General Program - Session Two

- Prebiotics and
Probiotics
1.30pm

2:00pm
Nagendra Shah
‘Use of probiotic cultures in Cheddar cheese’
2.00pm

2:30pm
Aaron Gosling
‘Sugars to increase health: chemical engineering of the production of
galacto-oligosaccharides’
2.30pm

3:00pm
Margaret Britz
‘Stress responses in bacteria – when cell surfaces count’
3:00 pm
Afternoon Tea Break and Poster Session – Ground Floor
3:30pm – 5:00pm General Program Session Three – Mycotoxins
This session also includes a guided tour of Bio21
3.30pm

4:00pm
Tour of Bio 21 Institute
4.30pm

5:00pm
Avishay Stark
‘Molecular mechanisms of detection of aflatoxins and other mycotoxins’
End of Day One
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International Symposium of Industrial Microbiology and Biotechnology Page 14
Detailed Programme July 3
July 3, 2010
9:00-10:30 General Program Session Four -"White biotechnology" - from
cellulose bioconversion to next generation sequencing
9.00am - 9.30am
Masako Shinjoh
‘Next-generation Sequencing in White Biotechnology: An Integrative
System for Genomic Design in Fermentation-based Industrial Applications’
9.30am - 10.00am
J. H. David Wu
‘The Clostridium thermocellumCellulosome: A Molecular Machine for
Biomass Degradation’
10:00am

10.30am
Yuval Shoham
‘Engineering glycoside hydrolases for novel applications and properties’
10.30am
Morning Tea Break
11:00 – 12:30 General Program - Session Five - Industrial Biotechnology
11.00a
m

11:30am
Munish Puri
‘Molecular characterisation and enzymatic hydrolysis of flavanoid
extracted from Citrus waste’
11:30am

12:00pm
Duen Gang Mou
‘Ambient Gas Exchange Enabled Stirred Culture Bottle -- A S.T.R.
Alternative To Shake Flask For High Gas-Liquid Mass Transfer’
12:00pm

12:30pm
Song Hae Bok
‘Slim and Sling PHW derived from vegetable and fruits can reduce body
fat and lipids in mammals’
12:30pm
Lunch Break
12:30pm – 1:30pm General Program - Session Six - Bioprocessing
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International Symposium of Industrial Microbiology and Biotechnology Page 15
July 3, 2010
Continued
1.30pm

1:45pm
Zheng-Xiang Wang
‘Microbial Resources as Starters for the Fermentation Industry -
Industrial microbial resource: collection, sharing and strain development’
1:45pm

2:00pm
Lixin Zhang
‘Discovery and developing synergistic medicines from marine microbial
natural products‘
2
.00
p
m

2:30pm
Arnold L. Demain

The trials and tribulations of an MIT professor’
2
.
3
0
p
m

3:00pm
Closing Remarks
3:00 pm
Afternoon Tea Break and Poster Session
4.00pm
End of Day Two
7.00pm
‘Fed
dish’
, River terrace Yarra Building

Federation Square
Corner Flinders Street and Swanston Street Tel: 03 9654 5855
(Federation Square is opposite Flinders Street Train Station)
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 16
Poster Presentations
Poster Presentations
M. C. Cárdenas
, C. Wacher,
B. Barkla, B. Ruíz, R. Rodríguez
-
Sanoja
‘Depletion of Zeins, a Strategy to Simplify a Fermented Corn Dough sample to Proteomic Study’
S.
-
J. Chang, D. Rouch, S. Brigg, S. Pepper,
M. L. Britz
‘Insertional Inactivation of a type I Fatty Acid Synthase in Corynebacterium glutamicumand
Identification of Two fas Genes in the Genome’
Kunyu Jiang, Jian Zhang, Changyan Yu, Fabao Liu, Nianfa Gao, Yan Zhu, Shiru Jia, Tongcun Zhang,
Qiang Gao
‘Construction of Metabolically-engineered Zymomonas mobilis CP4 Strain for L-lactic acid
Production’
Ho Wei Loon, Chan Kai Wai, Bernard Tzing Ziang Vui, Lim Siok Har,
Ngao Wee Chen, Tong Mei Ling,
Teh Soo Chin, Vun Su Chiun, Mak Ken Hing, Jenifer Rolland L., Celistha Fay J. Ferdinand Deosing,
Jessica Peter,Hew Chaw Sen, Lai Ngit Shin, Lee Ping-Chin and Ho Coy Choke
‘Screening for Potential Microbial Inhibitors against Prokaryotic and Eukaryotic Signal Transduction
and Isocitrate Lyase in Mycobacterium from Danum Valley, Sabah’
J
.
W
.
Park
,
S
.
R
.
Park
and
Y.J. Yoon
‘Complete Reconstruction and Engineering of the 4,6-disubstitued 2-deoxystreptamine Containing
Aminoglycoside Antibiotics’
S. Pepper and
M. L. Britz
Identification of a Cell-Surface Located Protein with Growth at Low pH in Bifidobacterium animalis
(syn. lactis) Strain Bb12
Iman Rusmana, Alina Akhdiya, and
Nisa Rachmania Mubarik
‘Characterization of Antimicrobial Substances Inhibiting Vibrio harveyi and Escherichia coli Growth
Produced by Bacillus spp. Isolated from Shrimp Pond and Their Molecular Identification’
Sánc
hez Sergio, Chávez Adán, Ruiz Beatriz,
García
-
Huante Yolanda
‘Possible Relation of SCO2127 with Morphological Differentiation in Streptomyces coelicolor’
S. Sánchez, R. Rodríguez
-
Sanoja and
B. Ruiz
‘Physiology of Formation of Glucose Kinases in Streptomyces peucetius var. caesius’
Contact Details
Surname
First Name
Institution
email address
Agayn Venetka INNOSO, the Netherlands info@innoso.org
Bacic Tony Bio21 Institute, the University of Melbourne, Australia Tel 83442225 - Helen Varnavas
Bayly Ron Monash University, Melbourne, Australia Ron.Bayly@monash.edu
Best Geoffrey RMIT University, Melbourne, Australia geoffrbest@gmail.com
Britz Margaret University of Tasmania, Hobart, TAS, Australia
Margaret.Britz@utas.edu.au and
margaret@margaretbritz.com
Cardenas Maria Catalina
Instituto de Investigaciones Biomedicas Universidad Nacional
Autonoma de Mexico
catycardenas@hotmail.com
Cheng Yuanrong Fujian Institute of Microbiology, PR China fim@pu3.fz.fj.cn
Cheshire Bill Virbac (Australia) Pty Ltd, Sydney, Australia bill.cheshire@virbac.com.au
Demain Arnold Drew University, NJ, USA ademain@drew.edu
Qiang Gao
Key Laboratory of Industrial Microorganisms, Ministry of Education
& Tianjin City College of Biotechnology, Tianjin University of Science
and Technology Mail Box 8 No 29, 13th Avenue TEDA, Tianjin 300457
PR China
gaoqiang@tust.edu.cn
qgaoamtf@gmail.com
Gosling Aaron
Chemical and Biomolecular Engineering, Bio21,
The University
of Melbourne, Parkville Victoria 3010, Australia
agosling@unimelb.edu.au
Ho Coy Choke Biotechnology Program, University of Malaysia, Sabah hocoychoke@yahoo.com
Huang Jie Fujian Institute of Microbiology, PR China
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th
International Symposium of Industrial Microbiology and Biotechnology Page 18
Surname
First Name
Institution
email address
Huang Baowen Fujian Provincial Intellectual Property Office, PR China
Garcia-Huante Yolander
Instituto de Investgaciones Biomedicas, Distrito Federal Mexico
04510
yori_gh@yahoo.com
Jiang Hong Fujian Institute of Microbiology, PR China
Johnson Eric and Mary
Department of Bacteriology, University of Wisconsin Madison
Madison WI, USA
eajohnso@wisc.edu
Kay Nicole Pfizer Pty Ltd, Melbourne, Australia nicole.kay@pfizer.com
MacKinnon Cindy Van Eyk Pty Ltd, Melbourne, Australia cindy@vaneyk.net
Mou Duen Gang Moubio Inc, 10F-1, 90 Jokon Street, Taipei Taiwan 11649 dgmou@ms33.hinet.net
Mubarik Nisa Rachmania
Department of Biology, Faculty of Mathematics and Natural
Sciences, Bogor Agricultural University, Jalan Agatis, IPB, Darmaga,
Bogor, West Java, Indonesia
nrachmania@ipd.ac.id
mubariknisa@yahoo.com
Palombo Enzo Swinburne University Victoria 3122 Australia epalombo@swin.edu.au
Puri Munish Centre for Biotechnology ITRI, Deakin Univeristy Geelong Vic 3217 muish.puri@deakin.edu.au
7
th
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Surname
First Name
Institution
email address
Ruiz-Villafan Beatriz
Instituto de Investgaciones Biomedicas, Universdad Nacional
Autonoma de Mexico
brvillafan@ccorreo.unam.mx
Sanchez Sergio
Instituto de Invesigaciones Biomedicas,UNAM 3er Circuito Exterior
s/n Mexico Distrito Federal 04510 Mexico
sersan@unam.mx
Sanchez Brenda
Instituto de Investigaciones Biomedicas Cuidad Universitaria S/N
Mexico DF Mexico DF 04510 Mexico
bss_qa@yahoo.com
Shah Nagendra
School of Biomedical and Health Sciences, Faculty of Heath
Engineering and Science,Victoria University, Melbourne, Australia
nagendra.shah@vu.edu.au
Shinjoh Masako
MS BioConsulting Graduate School of Biological Sciences, Nara
Institute of Science and Technology, Japan
shinjohm@qf7.so-net.ne.jp or
masako.shinjoh@genedata.com
Shoham Yuval Biotechnology and Food Engineering Technion, Haifa Israel 32000 yshoham@tx.technion.ac.il
Stark Avishay Department of Biochemistry,Tel Aviv University,Israel avis@tauex.tau.ac.il
Summerville Zoe Virbac (Australia) Pty Ltd, Sydney, Australia
7
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International Symposium of Industrial Microbiology and Biotechnology Page 20
Surname
First Name
Institution
email address
Wang Zheng Xiang Jiangnan University Wuxi, PR China zxwang@jiangnan.edu.cn
Wu J.H.David University of Rochester,NJ, USA davidwu@che.rochester.edu
Yoon Yeo Joon
Ewha Womans Univeristy, Department of Chemistry and Nano
Science, Seoul, South Korea
joonyoon@ewha.ac.kr
Zaferanloo Bita Swinburne University Victoria 3122 Australia bzaferanloo@swin.edu.au
Zhao Xinqing
School of Life Science and Biotechnology Dalian University of
Technology Dalian 116024 PR China
xqzhao@dlut.edu.cn
Zhang Lixin
Institue of Microbiology Chinese Academy of Sciences, Beijing, PR
China
Lzhang03@gmail.com
Zhu Jian
Hangzhou Huadong Medicine Group Biotechnology Institute Co,Ltd,
PR China
Jianghong709@yahoo.com.cn
Abstracts - General Program Presentations
Botulinum Neurotoxin: From Poison to Medicine
Eric A. Johnson
a
a
Department of Bacteriology, University of Wisconsin, Madison, WI, USA
E-mail:eajohnso@wisc.edu
“In poison there is physic”
-Shakespeare
Northumberland, Act I, Scene I
Toxins are increasingly being used as valuable tools for analysis of cellular physiology
and some are used medicinally in humans for treatment of disease. In particular, botulinum
toxin, the most poisonous substance known, is used for treatment of a myriad of human
neuronal disorders characterized by including involuntary muscle spasms, pain, and
inflammation. Since approval of type A botulinum toxin by the FDA in December 1989 for
three disorders (strabismus, blepharospasm and hemifacial spasm), the number of
indications being treated has increased greatly to include numerous focal dystonias,
spasticity, tremors, cosmetic applications, migraine and tension headaches, and other
maladies. Many of these diseases were previously refractory to pharmacological and
surgical approaches of treatment. The remarkable therapeutic utility of botulinum toxin lies
in its ability to specifically and potently inhibit involuntary muscle activity for an extended
duration. The clostridia produce more protein toxins than any other bacterial genus and are
a rich reservoir of toxins for research and medicinal uses. Research is underway to utilize
clostridial toxins for drug delivery, prevention of food poisoning, and for the treatment of
cancer and other diseases. The remarkable success of botulinum toxin as a therapeutic has
created a new field of investigation in microbiology.
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Microbial conversion of sirolimus
Jie Huang, Hong Jiang, Kualiang Li, Guoxin Yang, Yuanrong Cheng
a
*
a
Fujian Institute of Microbiology, Fuzhou 350007, China
*Corresponding author E.mail: fim@pub3.fz.fj.cn
, xlms2003@yahoo.com.cn
Sirolimus, also known as Rapamycin (Fig.1), produced by Streptomyces hygroscopicus AY
B-994 and our strain FC904 is a 36-membered macrolide immunosuppressant with
antifungal, antiproliferative and antitumor activity. Interestingly, Harrison et al (2009)
reported rapamycin extended longevity in old age mice. The great achievement has
tremendously increased scientists interest in study on extending the human life span with
sirolomus and/or its derivatives.As sirolimus has multifunctional effect and practical use, its
structural modifications have come to professional great attention. Several novel mTOR
inhibitors by chemical semi-synthesis such as RAD001, CCI-779 and AP23573 (Fig.1) have
been used as drug-eluting stents to prevent restenosis of coronary arteries and to develop a
new anticancer targeting drug. Besides the chemical synthesis, microbial conversion of
sirolimus acts as another approach to the search for novel analogs of sirolimus.
In this paper, it showed that more than one hundred different types of microorganisms
were screened for the microbial conversion of sirolimus and we found that 15 strains of
Micromonospora sp., 3 strains of Streptomyces, 4 strains of Actinoplanes and 3 strains of
Bacteria as well as 4 strains of Fungi were capable of transforming sirolimus respectively.
Micromonospora sp. FIM03-712 was selected as a candidate and classified as
Micromonospora chersina. The conversion conditions of sirolimus by Micromonospora sp.
FIM03-712 were studied. One loop of the culture was inoculated into a seed medium,
incubated at 32¥on a rotary shaker (240rpm) after incubation for 72hr and the vegetative
mycelia were transferred into a fermentation medium and incubated at the same conditions.
After 36hr, a sirolimus methanol solution was added and made the final concentration at
300μg/ml. The growth further continued for 72hr. The harvested broth was extracted and
analysed by HPLC. One major conversion product, FJ900, was isolated, purified and by
means of physico-chemical properties and spectral analyses.Compound FJ900 was
identified with 14-deoxorapamycin (Fig.2).
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International Symposium of Industrial Microbiology and Biotechnology Page 23
This compound was obtained from a new rapamycin producer Actinoplanes sp.N902-
109 by adding Cytochrome P450 inhibitor to the fermentation broth. In comparison with
sirolimus,compound FJ900 had less antifungal, anticancer and immunosuppressive activities.
Another bacterium strain No.287 was very capable as a sirolimus converting microorganism,
and classified as Bacillus megaterium 287. The method for converting sirolimus was similar
to those in Micromonospora chersina FIM03-712 except for the incubation conditions,
sirolimus adding time and concentrations. Bacillus megaterium No.287 transformed
sirolimus to three analogs, 287-p1, 287-p2 and 287-p3. Among them, compound 287-p1
was further purified and identified as 29, 42 -bis-O-demethylrapamycin (Fig.3) by means of
physical-chemical properties and spectral analyses. Regarding to the biological activity and
the enzymology work are in progress.
Key words: Sirolimus; microbial conversion; 14-deoxorapamycin; 29, 42 -bis-O-
demethylrapamycin

Sirolimus
R=H RAD001 R=
CCI-779 R= AP23573 R=
Fig.1.
Sirolimus and other three
mTOR inhibitors
O O
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Fig.2. 14-deoxorapamycin Fig.3. 29, 42 -bis-O-demethylrapamycin
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Endophytes from an Australian native plant as a promising source of
industrially useful enzymes
B. Zaferanloo
, N.K. Mahato, P.J. Mahon and E.A. Palombo
a
a
Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne
University of Technology, Hawthorn VIC 3122, Australia
E-mail:bzaferanloo@swin.edu.au
Endophytes are microorganisms that live as symbionts in the intercellular spaces of plant
tissues. They are becoming increasingly recognised as sources of novel secondary
metabolites with potential application in medicine, agriculture and industry. There is
limited information about endophytes from Australian native plants or the bioactive
metabolites produced by these microorganisms. We have therefore identified endophytic
fungi isolated from the Australian native plant Eremophila longifolia and screened them for
enzyme activities of industrial interest.Seventeen fungal strains were isolated from the
leaves of E.longifolia and identified by the amplification and sequencing of the internal
transcribed spacer regions of the ribosomal DNA. The fungi were screened for selected
enzyme activities (amylase, protease, cellulase and xylanase) using minimal medium agar
plates containing a single substrate for each target class of enzyme. Enzyme activities were
investigated at three different incubation temperatures (9°C, 25°C and 37°C) at pH 5.5. Over
two-thirds of the isolates showed amylase activity at 25°C and around one-third exhibited
protease activity. None of the isolates was able to degrade crystalline cellulose or birch
xylan. The active fungi showed the highest relative enzyme activities at 9°C and 37°C, which
might be correlated to the fact that the host plant grows in arid conditions. Australian
native plants, many of which have been used for as traditional medicines by aboriginal
people, are thus potential novel sources of endophytic fungi with promising enzyme
activities capable of breaking down recalcitrant substrates, and may be useful for industrial
applications.To our knowledge, this is the first time endophytes from Australian native
plants have been screened for their enzyme activities.
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International Symposium of Industrial Microbiology and Biotechnology Page 26
Use of Probiotic Cultures in Cheddar Cheese
Nagendra Shah
School of Biomedical and Health Sciences, Faculty of Health Engineering and Science
Victoria University
Email nagendra.shah@vu.edu.au
Abstract not received
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Sugars to increase health: chemical engineering of the production of
galacto-oligosaccharides
Aaron Gosling
ab*
, Geoff Stevens
a
, Andrew Barber
c
, Sandra Kentish
a
, Sally Gras
ab
a
Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010.
b
Bio21,The University of Melbourne, Victoria 3010.
c
Innovative Food and Plants Division, SARDI, Days Road, Regency Park, SA 5010.
*Presenting author, Email: agosling@unimelb.edu.au
Functional foods give a health benefit above and beyond their nutritional value.
Prebiotics are examples of this group of foods. These carbohydrates pass undigested to the
large intestine, where they selectively increase the activity of beneficial microflora. This
imparts many health benefits including increased immune function, increased mineral
absorption and perhaps even decreased risk of cancer, providing a health benefit above and
beyond the nutrition expected from carbohydrates.
Functional foods that modulate the activity of beneficial gut microflora are
commercially important, with an estimated global retail market of US$15.4 billion in 2008;
an increase of 12.5% from 2007. A major class of commercial prebiotics are galacto-
oligosaccharides (GOS), which are produced on a thousand tonne scale from the dairy sugar
lactose using the enzyme β-galactosidase. Despite this process having commercial success,
the science underlying GOS formation, particularly in complex media such as milk is not fully
understood.
Our interest lies in understanding the effect of industrially relevant processing
parameters on the reaction performed in complex dairy media. We have recently used an
understanding of the role of temperature to develop a pre-treatment step of a commercial
enzyme preparation known as Biolacta FN5, giving a facile way to increase GOS yield. We
are continuing to develop understanding of the roles of substrate identity and concentration
to facilitate engineering the product yield and prebiotic efficacy in complex media.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 28
Stress responses in bacteria: when cell surfaces count
M. Hosseini Nezhad
a,b
, S.J. Pepper
a
, M.A. Hussain
a
and M.L. Britz
a,c
a
The University of Melbourne, Parkville, Victoria, Australia;
b
Khorasan Research Institute for
Food Science and Technology, Mashad, Iran;
c
Faculty of Science, Engineering and Technology,
University of Tasmania, Hobart, Tasmania, Australia
E-mail: Margaret.Britz@utas.edu.au
Responses to environmental stress in bacteria can include generalized stress
responses (e.g.induction of chaperone proteins) and induction of condition-specific
pathways. These responses can have several impacts, besides regeneration of correctly-
folded functional proteins that aid cell recovery and survival plus removal of denatured
proteins, including: changes in membrane lipid composition, altered septation patterns
(resulting in filament formation) and heightened resistance to stressors. In industrial
processing, bacteria are exposed to several stressors that may result in the physiological and
biochemical changes that impact on performance. This paper addresses some of the
changes seen in Lactobacillus casei strains exposed to nutritional and acid stress. Members
of the Lb. casei group are important in dairy processing either as adjuncts that are added to
fermented milk products or as adventitious microflora. They contribute to the flavour and
functionality traits of final products, such as aged cheeses and yogurt, including exhibiting
probiotic activity.
Nutritional stress was examined by proteomic analysis of cytoslic fractions of cells
cultured in the absence of lactose, the normal carbon source used to culture Lb. casei.
Glycolic and pentose phosphate pathway enzymes were induced, indicating that
metabolism was switched to scavenging other carbon sources in the medium, which
included tween 80 and amino acids. This was consistent with the metabolites formed,
which included acetic, caproic and octanoic acids plus aldehydes derived from metabolism
of aromatic amino acids.
Growth at low pH (4) caused induction of several cell-surface proteins, where the
pattern of proteins induced varied between strains. In some, a surface antigen (MW 42 kDa)
was strongly expressed throughout growth, and this was likely to be a membrane protein
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 29
(from hydrophobicity analysis and removal on centrifugation with cell debris after lysis). N-
terminal sequence analysis and DNA sequencing of the cloned gene, showed that one of the
proteins was a surface protein of unknown function. However, a conserved domain
belonging to the NPLC_P60 superfamily, which occurs in bacterial lipoproteins, was detected:
this domain occurs in amidases associated with cell wall and peptidoglycan metabolism.
Other proteins were extracted into 5M LiCl, demonstrating an extracellular location in the S-
layer. 2DE of LiCl extracts showed strong up-regulation of enolase- and GAPDH-like proteins,
both known to bind fibronectin and contribute to immune-modulation in probiotic LAB.
Heat shock failed to modulate expression of these proteins, demonstrating a specific cellular
response to growth at low pH that favoured synthesis of surface proteins. Other proteins
involved in pyruvate metabolism (Ldh) and GroEL also increased at the cell surface.
These results show that growth conditions can significantly modify the relative
expression of cell surface proteins by Lb casei, and this has implications for improving
probiotic activity in different strains and in dairy products.
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th
International Symposium of Industrial Microbiology and Biotechnology Page 30
Molecular mechanisms of detection of aflatoxins and other mycotoxins
A.-A. Stark
Department of Biochemistry, Tel Aviv University, Ramat Aviv 60078, Israel
E-mail: avis@tauex.tau.ac.il
Today, we are able to detect and quantify minute amounts of mycotoxins using
sophisticated liquid chromatography-mass spectrometry systems. Likewise, polymerase
chain reaction-based systems are available for the identification of toxigenic fungi. These
systems are rare in the less affluent, developing countries. The introduction of new portable
rapid one step assay [ROSA] kits that are based on immunological and nanotechnological
techniques makes it possible to identify and quantify major mycotoxins such as aflatoxins,
ochratoxin, T2-toxin, zearalenone, deoxynivelenol and fumoisin in the field. This important
development caters to the needs of those who suffer the most from mycotoxin intoxications
of humans and farm animals.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 31
Next-generation Sequencing in White Biotechnology:An Integrative System
for Genomic Design in Fermentation-based Industrial Applications
H.-P.Fischer
b
,
T.Hartsch
b
, S. Ribrioux
2
, L. Macko
b
,F.Staubli
a
and M.Shinjoh
a
a
Genedata KK, 2-7-11-603 Nishi-Gotanda, Shinagawa-Ku, Tokyo 141-0031, Japan
b
Genedata AG, Magarethenstrasse 38, 4053-Basel, Switzerland
E-mail:masako.shinjoh@genedata.com
Development of fermentation-based methodologies is being assisted by high-
throughput technologies, which are generating vast amounts of data, for example from
production strain re-sequencing projects. However, the sheer volume of data generated by
these technologies can pose problems for scientists struggling to reach meaningful
conclusions. A key to meeting this challenge is the establishment of a generic and
integrative approach to data handling, visualization, and analysis.
In close collaboration with partners, we have developed a flexible, scalable, and
integrative solution to support and streamline research processes. The Genedata system
seamlessly integrates public and proprietary data in one database, and includes interactive
tools to analyze, store, and share data and results across an organization. This supports
projects using a system biology and genomic design approach to understand and improve
productivity by rational means.
We illustrate here,how standard genomics technologies together with next-
generation sequencing data of e.g. microorganism and fungi help elucidate metabolic
capacities, expression behaviour, and strain stability on a genome-wide level. The system
supports a cross-omics, cross-technology, knowledge-based metabolic engineering
approach to e.g.improve microbial or fungal strains for the production of antibiotics,
biofuels, and feed and food ingredients.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 32
The Clostridium thermocellumCellulosome: A Molecular Machine for
Biomass Degradation
J.H. David Wu
University of Rochester, 206 Gavett Hall, Rochester, NY 14627, USA
Email: davidwu@che.rochester.edu
Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic, and ethanogenic
bacterium that is capable of directly converting cellulosic materials into ethanol. The large
amounts of attention and resources devoted over the past 30 years to the Trichoderma-
Saccharomyces concept have perhaps interfered in the industrial development of the
potential of the cellulolytic, thermophilic anaerobic bacteria such as C. thermocellum and
their cellulases and hemicellulases.
C. thermocellumis capable of converting biomass into ethanol, acetic acid, lactic acid,
and hydrogen. The interest in this organism is due to several factors. First, C. thermocellum
can directly convert lignocellulosic biomass to fuels and chemicals in a single step. Second,
its anaerobic nature negates expensive oxygen transfer process for cellulase production.
Third, its growth temperature at 60°C facilitates ethanol recovery and reduces the cooling
requirement and potential of contamination in fermentation.
The C. thermocellum cellulase system exists as a multi-protein complex called the
cellulosome. More than 70 enzymatic subunits have been found to be associated with the
cellulosome, which include endoglucanases, xylanases, other hemicellulases, pectinases,
esterases, proteases, and exoglucanases. The core of the cellulosome is a 250-kDa non-
catalytic polypeptide, CipA, which binds to cellulose and serves as a scaffold for the catalytic
subunits. In addition, the bacterium produces free cellulases that are not associated with
the cellulosome. The bacterium is thus capable of producing a large number of glycosyl
hydrolases. However, very little is known about the regulation of these enzymes in C.
thermocellum. The presentation will review the current knowledge of the C. thermocellum
cellulase system and the regulation of its biosynthesis.
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th
International Symposium of Industrial Microbiology and Biotechnology Page 33
Engineering Glycoside Hydrolases for Novel Applications and Properties
Yuval Shoham
Dept. of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel
E-mail: yshoham@tx.technion.ac.il
Glycoside hydrolases cleave the glycosidic bond between two or more carbohydrates
via general acid catalysis and can accelerate the reaction rate by over 10
17
. Hemicellulases
are glycosidases that hydrolyze hemicellulose, the second most abundant natural polymer
on earth. Together with cellulases, these enzymes can serve as key components for utilizing
lignocellulolytic matter as a renewable source of energy with control of total CO
2
emission.
In this talk I will focus on selected enzymes from Geobacillus stearothermophilus, a
thermophilic bacterium that utilizes a complete and well-characterized hemicellulolytic
system. I will demonstrate how combining high-resolution crystal structures of enzyme-
substrate complexes together with biochemical studies can allow the elucidation of the
catalytic mechanism, and the structural features that control substrate specificity.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 34
Molecular characterisation and enzymatic hydrolysis of flavanoid
extracted from Citrus waste
Munish Puri
Centre for Biotechnology and Interdisciplinary Sciences, Institute of Technology Research and
Innovation (ITRI), Deakin University, Victoria 3217, Australia
E-mail: munish.puri@deakin.edu.au
The citrus fruit processing industry generates substantial quantities of waste rich in
phenolic substances, which is a valuable natural source of polyphenols (flavonoids) such as
naringin and its disposal is becoming a major problem. In the US alone, the juice processing
of oranges and grapefruit generates over 5 Mt of citrus waste every year.In the case of India,
about 2.15 Mt of citrus peel out of 6.28 Mt of citrus fruits are produced yearly from citrus
juice processing. In case of Australia, about 15-40% of citrus peel waste is generated by
processing of citrus fruit (0.85 Mt). Thus Isolation of functional compounds (mostly
flavanoids) and their further processing can be of interest to the food and pharmaceutical
industry.This peel is rich in naringin and may be used for rhamnose production by utilizing
α-
L
-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal
rhamnosyl groups from naringin to yield prunin and rhamnose. We recently purified
recombinant α-
L
-rhamnosidase from E. coli cells using immobilized metal-chelate affinity
chromatography (IMAC) and used it for naringin hydrolysis. The purified enzyme established
hydrolysis of naringin extracted from citrus peel and thus endorses its industrial applicability
for producing rhamnose. Infrared (IR) spectroscopy confirmed molecular characteristics of
naringin extracted from citrus peel waste.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 35
Ambient Gas Exchange Enabled Stirred Culture Bottle
-- A S.T.R. Alternative To Shake Flask For High Gas-Liquid Mass Transfer
D.G. Mou
Founder, Moubio Inc., 10F-1, 90 Jokon St., Taipei, Taiwan 11649
E-mail: dgmou@alum.mit.edu
Through a gas vent riser tube or a gas intake downer tube, spent gas exhaust and
ambient substrate gas intake in a stirred bottle culture are an order of magnitude enhanced
without line gas sparging when compared to similarly sized shake flask cultures. In addition
to realistic impeller mixing, the novel in-situ ambient gas pumping design, tested in a 500 mL
wide-mouth bottle, produced an E. coli growth at 90% working volume equal or better than
that of a reciprocating 250ml Erlenmeyer flask at 20% working volume through enhanced
gas ventilation and gas hold-up. A peer-reviewed short communication has been published
in April 2010 issue of Industrial Biotechnology.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 36
Slim and Sling PHW derived from vegetable and fruits can reduce body fat
and lipids in mammals
Song Hae Bok
Abstract not received
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 37
Discovery and developing synergistic medicines from marine microbial
natural products
Lixin Zhang
Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, People’s Republic of
China
E-mail: Lzhang03@gmail.com
There has been an explosion of information about novel bioactive compounds
isolated from marine microbes in an effort to further explore the relatively untapped marine
microbes and their secondary metabolites for drug discovery. A diversified marine
microbial natural product library was built for screening bioactive compounds in a high
throughput manner. The production of drugable secondary metabolites was significantly
improved by precision engineering. For example, we precisely identified that σhrdB was
responsible for regulating the production of avermectins by transcriptional profiling.
Through manipulation of the hrdB gene by error-prone polymerase chain reaction, two
superior recombinants were identified with 46% and 52% more avermectin production,
respectively. Re-introduction of the recovered plasmids reconfirmed the overproduction of
avermectin B1a. Sequencing analysis showed that the mutant sites were conserved.
Engineering of the hrdB gene not only provides a route to recognize the complex regulatory
system of secondary metabolites production, but also presents a promising strategy for
improving the yield of active compounds in other Streptomyces species.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 38
Microbial Resources as Starter for Fermentation Industry
----Industrial microbial resource: collection, sharing and strain development
Zheng-Xiang Wang
Center for Bioresource & Bioenergy, School of Biotechnology, Jiangnan University, Wuxi, China
Culture & Information Center of Industrial Microorganisms of China Universities, Wuxi, China
The Key Laboratory of Industrial Biotechnology, the Ministry of Education, Wuxi, China
It is well-known that microbial strains are the fundamentals of fermentation industry
establishment and development. Microbial strains for fermentation industry can be
developed by either native strains through strain screening or genetically improved strains
modified via modern genetic procedures. Although the genetically improved strains are
more and more frequently being applied, a good parent (wild type) strain is essential and
sometimes critical for a successful strain development. Based on this consideration, a
microbial collection of biodiversity has been established in which more than 13,000 isolates
have been identified, classified and long-term storied, and more than 400 molecular cloning
materials and gene cloning hosts have been collected, and part of them has been opened to
public through internet.
Based on the collection, several interesting findings and achievements have been
obtained in the development of the specific industrial strains,for instance, for production of
D-lactic acid, thermostable -amylase, bacterial -amylase or ethanol. 1) a more suitable
host for overproduction of recombinant -amylase was obtained by using a rational strain
selection strategy, which exists in the environmental biotope via spontaneous mutation
accumulation and/or natural genome shuffling; 2) better parent strains for strain
development of D-lactic acid or ethanol production were selected out based on
“biodiversity of metabolism” in different isolated strain belonging to the same species; 3) a
high-efficient molecular procedure for multi-locus disruption and multiple genes expression
was developed and successfully applied in the strain development for D-lactic acid or
ethanol production.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 39
Abstracts - Poster Presentations
Depletion of Zeins, a Strategy to Simplify a Fermented Corn Dough
sample to Proteomic Study
M. C. Cárdenas
a
, C. Wacher
b
, B. Barkla
c
, B. Ruíz
a
, R. Rodríguez- Sanoja
a
a
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones
Biomédicas UNAM. Ciudad Universitaria, C.P 04510, Apto. Postal 70228, México, D.F.
b
Departamento de Alimentos y Biotecnología, Facultad de Química UNAM. Ciudad
Universitaria, C.P 04510, México, D.F.
c
Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Colonia Miraval, 62250 Cuernavaca, Morelos, México.
E-mail: romina@correo.biomedicas.unam.mx, catycardenas@hotmail.com
The preparation of high-quality protein samples for proteomic studies are of high
importance. We are interested in the study of the amylases present during the process of
pozol fermentation. However, the amount of protein supplied by the substratum is bigger
than the protein supplied by the microbiota. Moreover approximately 50% of substratum
proteins have biopolymer properties. All these conditions generate many manipulation
problems and interferences with the identification and characterization of important low-
abundant proteins. As well the high-abundant proteins showed a partial mobility due to its
properties. These proteins, for these molecular weight and abundance correspond to zeins,
a major storage protein in kernel of corn.
The most abundant proteins in pozol are the zeins, storage protein in kernels. To
eliminate them we use a method previously reported for selective extraction of zeins, with
the use of ethanol-solution 70 % + 0.5% sodium acetate. However, this method extracts less
than a half of total zeins. To efficient the extraction we use sequential extractions for
gradual elimination of zeins.
The result showed that the depletion with EtOH 70% + 0.5% sodium acetate is a
good method to extract only zeins. We removed approximately 50% of total protein by
depleting the 4 most abundant proteins (zeines). This procedure results in the simplification
of pozol system and enabling the detection of low-level proteins.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 40
Insertional Inactivation of a type I Fatty Acid Synthase in Corynebacterium
glutamicumand Identification of Two fas genes in the Genome
S.-J. Chang
ab
, D. Rouch
b
, S. Brigg
b
, S. Pepper
b
, M. L. Britz
b
a
Celltrion, South Korea;
b
The University of Melbourne
E-mail: margaret@margaretbritz.com
Corynebacterium glutamicum has been used in the industrial production of amino
acids for over 50 years. A distinguishing feature of this species and other species of the
nocardioform group of bacteria is the production of mycolic acids which occur in an outer
sheaf on cells and confer resistance to physical and chemical assault. The biosynthesis of
corynomycolates involves the condensation of C
16
to C
18
fatty acids to form α-branched, β-
hydroxy lipids. This paper reports an investigation on the chirality of condensation of fatty
acids involved in formation of the major corynomycolates using mutants inactivated in one
of the two fas genes present in the genome.
The mutations obtained in C.glutamicum were complemented by oleic acid.
Sequence analysis of the PCR product used in inactivation and restriction enzyme analysis of
the region surrounding this showed that the insertion occurred in a fas gene (locus tag
cg0957) annotated as fas 1B. A second fas gene (cg 2743, annotated fas 1A) patterns
showed 48% sequence identity and a similar domain structure. Analysis of fragmentation
patterns of corynomycolates by GC-MS following growth on
13
C-oleic acid showed that this
label was not incorporated into C
32:0
(showing that oleic acid was not broken down into
palmitic acid but was incorporated in C
34:1
). The fragmentation products for C
34:1
were a
mixture of
13
C and
12
C.
These results show that of the two fas genes in the C. glutamicum genome cg0957 is
associated with oleic acid biosynthesis. The patterns of
13
C from oleic acid and
12
C from
glucose into fragmentation products indicated that the second fas gene was functional and
associated with palmitic acid and steric acid synthesis.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 41
Construction of metabolically-engineered Zymomonas mobilis CP4 strain for
L-lactic acid production
Kunyu Jiang
a,b
, Jian Zhang
a
, Changyan Yu
b
, Fabao Liu
b
, Nianfa Gao
a
, Yan Zhu
a
, Shiru Jia
b
,
Tongcun Zhang
a
, Qiang Gao
a,b
*
a
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology,
Tianjin University of Science and Technology, Tianjin 300457, China;
b
Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China
*Corresponding author: Tel: +86-22-60601599; Fax: +86-22-60602298
Email: gaoqiang@tust.edu.cn
Lactic acid is a versatile organic acid with a wide range of applications in food,
pharmaceutical, cosmetic,leather, textile and many other industries. Also, it is the precursor
of the biodegradable plastic polylactic acid. For bacterial lactic acid fermentation, L-lactate
dehydrogenase (LDH, EC1.1.1.27) plays a key role in the last step of anaerobic glycolysis by
converting pyruvate and NADH to L-lactate and NAD
+
.
The Gram-negative bacterium Zymomonas mobilis CP4 is able to efficiently and rapidly
produce ethanol from simple sugars through its unique Entner-Doudoroff pathway under
anaerobic condition. However, using engineered Z. mobilis to produce L-lactic acid has not
ever been reported during previous studies.
Z. mobilis has 13 different constitutive expression genes linked to Entner-Doudoroff
pathway.Among these highly expressing promoters, we chose adhB promoter (PadhB) from
Z. mobilis CP4 with the LDH gene (ldhL) from Lactobacillus casei ATCC 334 to construct a
metabolically-engineered Z. mobilis strain for L-lactic acid production. In brief,PCR-
amplified PadhB and ldhL gene were adjacenty inserted in the MCS region of the broad-
host-range plasmid pBBR1MCS3, where the tandem PadhB-ldhL fragment formed as a joint
gene, to generate the plasmid pBBR1MCS3-PadhB-ldhL, which was subsequently
transformed into Z. mobilis CP4 by electroporation. Bio-Rad Gene Pulser™ apparatus was
used to generate exponential decay pulses with the parameters of 1.5kV/cm field strength,
25μF capacity, 400Ω resistor and 5-10ms time constant in a 2-mm gap electroporation
cuvette.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 42
Although the evaluation of L-lactic acid production by Z. mobilis CP4 (pBBR1MCS3-
Padh-ldhL) recombinant is still undergoing, the introduction of this plasmid with the joint
gene PadhB-ldhL into Z. mobilis CP4 host lays the foundation to the research of high lactic
acid producing Z. mobilis CP4 recombinants. Considering the obviously influence of
expressive foreign genes to the L-lactate expression, the LDH from Bacillus coagulans can be
used to replace the LDH from L. casei ATCC 334 because of its high lactic acid yield in the
future.
Key Words:L-lactic acid,Zymomonas mobilis CP4, adhB promoter, Lactobacillus casei ATCC
334, L-lactate dehydrogenase, joint gene PadhB-ldhL, plasmid pBBR1MCS3, electroporation
Acknowledgement
This study is financially supported by National 973 Basic Research Program of China
(2007CB714305), National 863 High-Tech R & D Program of China (2008AA10Z336) and Key
Project of Tianjin Natural Science Foundation of China (08JCZDJC15100).
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 43
Screening for Potential Microbial Inhibitors against
Prokaryotic and Eukaryotic Signal Transduction and
Isocitrate Lyase in Mycobacterium from Danum Valley, Sabah
Ho Wei Loon, Chan Kai Wai, Bernard Tzing Ziang Vui, Lim Siok Har, Ngao Wee Chen, Tong Mei Ling,
Teh Soo Chin, Vun Su Chiun, Mak Ken Hing, Jenifer Rolland L., Celistha Fay J. Ferdinand Deosing,
Jessica Peter,Hew Chaw Sen, Lai Ngit Shin, Lee Ping-Chin and Ho Coy Choke
a*
a
Biotechnology Programme School of Science and Technology Universiti Malaysia Sabah
Locked Bag 2073
88999 Kota Kinabalu
Sabah, Malaysia
* e-mail: hocoychoke@yahoo.com
A diversity of actinomycetes and microfungi was isolated from various sites
throughout the Danum Valley during expedition I (25/9–27/9/2004) and II (28/10–
29/10/2004).A total of 217 soil samples were collected under trees that had been identified
to species or genus level in Danum Valley Conservation Area; 535 actinomycetes and 93
microfungi were isolated using selective isolation media to obtain pure cultures. These pure
isolates were grown aerobically to produce microbial secondary metabolites.
Acetone crude extracts were screened for potential inhibitors against prokaryotic
serine/threonine protein kinase (AfsK/AfsR) of Streptomyces griseus, the two component
signal transduction systems (KdpE/KdpD and PhoP/PhoR) and isocitrate lyase (ICL) in
glyoxylate shunt of Mycobacterium. This research also screened for potential inhibitors
against proteins involved in eukaryotic (yeast and human) signal transduction systems.
These include glycogen synthase kinase-3β (GSK-3β) cascade, protein phosphatase 1 (PP1),
MAP kinase cascade (MKK1 and MSG5) and Ras/Raf-1 protein-protein interaction. Four
fungal strains (H9386, H9387, H9407 and H9409) inhibited AfsK/AfsR of Streptomyces
griseus. One actinomycete strain (H11588) was found to be toxic to KdpE/KdpD in M.
smegmatis. Actinomycete strain H11855 showed positive inhibitory towards PhoP/PhoR and
15 strains were toxic to M. smegmatis. Meanwhile, 1 fungal strain and 26 actinomycete
strains showed toxicity to ICL in glyoxylate pathway in M. smegmatis. In PP1 screening, 37
extracts were toxic to yeast and 3 actinomycete extracts (H11533, H11534 and H11735)
were found to be potential inhibitors.
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th
International Symposium of Industrial Microbiology and Biotechnology Page 44
In GSK-3β screening, 32 strains were toxic in yeast and 9 actinomycete strains
(H11462, H11490, H11526, H11668, H11686, H11695,H11720, H11785 and H11809) were
found to be positive in the test. Five strains and seven strains of actinomycete showed
toxicity in MKK1 and MSG5 respectively,and 37 actinomycete strains were found to be toxic
in Ras/Raf-1 protein-protein interaction. No strains were found to inhibit KdpE/KdpD and ICL
in M. smegmatis,MKK1, MSG5 and Ras/Raf-1 protein-protein interaction in yeast.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 45
Complete reconstruction and engineering of the 4,6-disubstitued 2-
deoxystreptamine containing aminoglycoside antibiotics
J.W. Park, S.R. Park and Y.J. Yoon
a
a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750,
Republic of Korea
E-mail: joonyoon@ewha.ac.kr
Although 2-deoxystreptamine-containing aminoglycoside such as gentamicin and
kanamycin is an important class of clinically valuable antibiotics, its biosynthetic pathway
still remains unclear. Therefore, we reconstructed this pathway through the heterologous
expression of combinations of putative biosynthetic genes in non-aminoglycoside-producing
Streptomyces venezuelae to reveal the complete biosynthetic route, which is a totally
unpredicted symmetric pathway. We were also able to modulate the aminoglycoside
biosynthetic flux for the increased production of a valuable minor component by swapping
glycosyltransferases. Co-expression of various heterologous aminoglycoside genes with
selected sets of kanamycin biosynthetic genes led to the in vivo biosynthesis of 3’-
deoxykanamycins including tobramycin, and the S-4-amino-2-hydroxybutyric acid (AHBA)-
conjugated kanamycins, amikacin and the novel derivative, which exhibits greater activity
than amikacin against a panel of Gram-negative bacteria. This work demonstrates the
potential for direct in vivo production of clinically useful antibiotics and combinatorial
biosynthesis of more robust aminoglycosides
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 46
Identification of a Cell-Surface Located Protein with Growth at Low pH in
Bifidobacterium animalis (syn. lactis) Strain Bb12
S. Pepper and M. L. Britz
a
a
The University of Melbourne
E-mail:
margaret@margaretbritz.com
Bifidobacterium animalis (syn. lactis) has been used as a starter culture in several
manufactured product lines including yoghurts and anti-diarrhea tablets. As a starter
culture it has been selected through a series of mutations and propogations to make it
suitable for use in dairy products. This strain was studied to determine the affect of a
sudden decrease in pH or continuous exposure to low pH.
Protein profiles of membrane and cell-surface associated fractions, for growth at
both neutral pH (7.0) and mildly acidic conditions were essentially identical. At pH 5.0 an
increase was noted in the density of a band around 33kDa. The same band was markedly
increased during growth at pH 4.0. A 14 fold increase was observed in the relative amount
of the protein present in cultures grown at pH 4.0.
Marked up-regulation of a protein of a protein of approximately 33kDa was a feature
of SDS-PAGE gels of cells of Bifidobacterium animalis (syn. lactis) strain VUP Bb 12 grown
under strongly acidic conditions.
7
th
International Symposium of Industrial Microbiology and Biotechnology Page 47
Characterization of Antimicrobial Substances Inhibiting Vibrio harveyi and
Escherichia coli Growth Produced by Bacillus spp. Isolated from Shrimp Pond
and Their Molecular Identification
Iman Rusmana
a
, Alina Akhdiya
b
, and Nisa Rachmania Mubarik
a
a
Department of Biology, Bogor Agricultural University , Darmaga, Bogor 16680, Indonesia
b
Indonesian Research Center for Agriculture Biotechnology and Genetic Resources, Cimanggu Bogor
16680, Indonesia
email: nrachmania@ipb.ac.id
Application of bacterial probiotic is an alternative solution to control the growth of
bacterial pathogens in shrimp culture. Bacillus sp. is one group of bacteria that has been
used widely as a probiotic in aquaculture. This bacteria can produce antimicrobial
polipeptides such as bacteriocins that can inhibit growth of other bacteria.
Isolation from several samples of pond water, pond sediment and shrimp intestines
resulted in a collection of 175 isolates of Bacillus spp. Verification assay showed that
Bacillus sp. LTS 40 had the biggest antimicrobial activity. Competition assay showed that
Bacillus sp LTS 40 isolate could inhibit the growth of V. harveyi and E. coli up to 81,8% and
85,5% respectively.
The antimicrobial substances produced by Bacillus sp. LTS 40 isolate was a bacteriocin
produced during its growth phase. The bacteriocin produced by Bacillus sp. LTS 40 was
stable in the pH range of 3 - 11 and this antimicrobial substance remained stable after
heating at 100
0
C for 20 minutes. Based on purification result using chromatografic filtration
technique and SDS PAGE analysis showed that protein fraction with molecular weight of
47,3 kDa was effective to inhibit V. harveyi growth and fraction with molecular weight of
34,83 kDa was effective to inhibit E. coli growth .
Molecular identification based on 16S rRNA gene sequences showed that Bacillus sp
LTS40 isolates had similarity to Bacillus subtilis up to 98 %.
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International Symposium of Industrial Microbiology and Biotechnology Page 48
Possible role of SCO2127 in the physiology of Streptomyces coelicolor
Sánchez Sergio, Chávez Adán, Ruiz Beatriz, García-Huante Yolanda
a
a
Instituto de Investigaciones Biomédicas, UNAM. Mexico D.F. 04510.
E-mail: yori_gh@yahoo.com

Several industrially important secondary metabolites, such as antibiotics, antitumor
drugs, immunomodulators, etc, are produced by Streptomyces and their synthesis is
suppressed by carbon catabolite repression (CCR). Streptomyces coelicolor is the genetically
best characterized strain in this genus. In this model, it has been established that glucose
kinase (Glk) plays a key role in the mechanism of CCR. Conversely, Streptomyces peucetius
var. caesius mutants resistant to 2-deoxyglucose exhibit glucose transport deficiency, low
Glk activity, and insensitivity to CCR. This phenotype can be pleiotropically complemented
by a 576 bp gene encoding SCO2127 from S. coelicolor, suggesting important participation
of this protein in the CCR process. Considering our lack of knowledge about the synthesis
and regulation of SCO2127, the aim of this work was to determine its expression along the
fermentation process in a chemically defined medium supplemented with different carbon
sources. Cell-free extracts from cultures grown in the presence of glucose, arabinose,
mannitol or glutamate were analyzed by western blot using anti-SCO2127 polyclonal
antibodies. In agreement with its possible involvement in the CCR process, SCO2127 was
detected during the logarithmic growth phase of S. coelicolor cultures grown in glucose and
found to be linked to the sugar concentration present in the culture medium. In addition,
the presence of SCO2127 was detected in all extracts except those obtained from cultures
grown in glutamate. Considering that sco2127, does not seems encode for a Glk nor for a
glucose permease, our results suggest that SCO2127 likely produces biochemical signals
involved in sugar, but not amino acid, consumption as a necessary step to elicit CCR in the
genus Streptomyces. However, using the enhanced green fluorescence protein (EGFP) as
reporter gene regulated by the promoter region of sco2127, we observed a signal at 24 and
32 h using glutamate as carbon source, indicating that its expression could take place in a
constitutive manner, but was not high enough to be detected using polyclonal antibodies
anti SCO2127. Since we have recently found that SCO2127 can bind proteins involved in cell
differentiation (Chavez et al., in preparation), SCO127 could be a sensor protein for
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International Symposium of Industrial Microbiology and Biotechnology Page 49
nutrients. In other words, when nutrients are available in the culture media, these are
incorporated and metabolized by the cell producing key catabolites or intermediates, which
may be the signal for SCO2127 to commence a series of reactions that could elicit CCR. Once
the concentration of these metabolites decreases, the conditions turn out to be unfavorable,
generating a signal that causes the interaction of SCO2127 with proteins involved with
morphological differentiation and spore formation. All this could explain the pleiotropic
effect attributed to this protein.
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International Symposium of Industrial Microbiology and Biotechnology Page 50
Physiology of formation of glucose kinases in Streptomyces peucetius var.
caesius
S. Sánchez, R. Rodríguez-Sanoja and B. Ruiz
a
a
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones
Biomédicas, UNAM. México, D.F. A.P. 70228, C.P. 04510 México.
E-mail: brvillafan@correo.unam.mx
Glucose kinase (Glk) catalyzes glucose phosphorylation to produce glucose 6-
phosphate in the glycolytic pathway. In addition to its catalytic activity, in Streptomyces
coelicolor, Glk has been implicated in carbon catabolite repression (CCR) of many genes for
utilization of other carbon sources such as agarose or xylose (1, 2). Despite its involvement
in CCR, little is known about how Glk exerts this function. Using mass spectrometry, two Glk
proteins were found in Streptomyces peucetius var. caesius NRRL B-5337, one ATP-
dependent Glk and the other containing a polyphosphate-dependent activity. These
enzymes are encoded by separate genes in the Streptomyces genome. The temporal activity
of these enzymes was confirmed through a time course fermentation of S. peucetius var.
caesius. The ATP-dependent Glk activity occurred during the logarithmic growth phase,
while the polyphosphate-dependent Glk showed maximum expression during the mid-
stationary growth phase. Both Glk enzymes where present in glucose supplemented
cultures,but not in glutamate. The presence of two Glk activities in this Streptomyces strain,
suggests that both enzymes may be involved in CCR mechanisms in this microorganism.
References
1. Angell, S., Lewis, C.G.; Buttner, M. J. and Bibb, M. J.1994. Glucose repression in
Streptomyces coelicolor A3 (2): a likely regulatory role for glucose kinase. Mol. Gen. Genet.
244:135-43.
2. Kwakman, J. H. J. M. and P. W. Postma.1994.Glucose kinase has a regulatory role in
carbon catabolite repression in Streptomyces coelicolor. J. Bacteriol. 176(9):2694-8.