Harry J. Gilbert University of Southampton B.Sc in Physiology and Biochemistry (1972- 1975) Why do a PhD? – Absolutely loved biochemistry and physiology.

Harry J. Gilbert

• University of Southampton B.Sc in Physiology and Biochemistry (1972-1975)

• Why do a PhD?– Absolutely loved biochemistry and physiology– Wanted to know more. – Also girlfriend (now wife) had another year of her

French degree to do at Southampton

Rosie

Me

PhD in enzymology and molecular genetics University of Southampton (1975-1978)

• Protein biochemistry• Enzymology• Molecular genetics

– No gene cloning, gene sequencing, reverse transcriptase cloning or PCR.

– No Structural biology (crystallography or NMR)

How was my PhD structured

• How to do science– Dr Terry Drabble was my supervisor– Intelligent, clear thinker and most unambitious scientist I’ve

met– No postdocs his only PhD student– Like learning to ride a bike– 1st year outlined the project and the first tranche of

experiments and taught me the techniques needed– 2nd year Discussed experiments but left me to get on with it– 3rd year completely independent let me design and carry

out experiments no input – Helped me to write scientific English which I found

incredibly hard

What did I learn from my PhD?• How to develop a logical series of experiments to answer

the question.• Not so much let’s see what happens more how can we

answer the question• Not naturally gifted technically so needed to develop my

expertise• Think of all the appropriate controls• Work extremely hard as science is tough

– 12 hour days routinely during week and at least Saturday in the lab. Maybe 3 weeks holiday a year.

• Learn to read the literature to develop ideas and techniques– No computers so difficult to access material

• Journal clubs showed me how to present material and whether I really understood papers. Great lessons learnt

Biggest lesson I learnt

• When things went wrong I learnt far more about science than when things worked– 1st nine six months went very well

• Star of the year. Learnt techniques but little else

– Then absolutely nothing worked for 18 months• Hero to zero

– During those 18 months I learnt• Technical rigour• Logical experimental design• Controls• Perseverance• Problem solving• Eventually found that department changed its source of casamino acids. • It contained peptides that induced E. coli proteinases• Cleaved my enzyme in half and changed quaternary structure

Developing skills and product

• Motivation was to learn and enjoy science• Developed skills that may make me

employable; laughable now– Molecular genetics– Detailed protein chemistry, affinity purification– Enzymology, assay development, active site

inhibitors• Published 3 papers in decent journals

What other things did I do and not do?

• Things I did– Went to one conference

• SGM; Hull in January• SGM meeting in Southampton

• Things I didn’t do– No training in transferable skills– No PhD society etc

• Advice– Don’t get distracted. Focus on learning about and doing science– Remember people like me will just be interested in the papers you have

published, what skills you have and if you have a passion for science.– They won’t be interested in your other skills you might have picked up

• We had a bit of an argument about this last Friday about this!

What to do when PhD finished in 1978-9

Refused to do a post doc as no jobs in academiaand a post doc would make me unemployable.

Gave myself a year to get a permanent job in science

~20 permenant PhD researchjobs at PHLS Porton Down

Appointed maybe because Isaid I played cricket and theyneeded someone youngish inthe team

• Thatcher came to power and dismantled Universities; no lectureships now or in the future

• No biotech industry although one hoped it was coming

Basic/Senior Microbiologist at PHLS Porton Down1979-1984

Developing protein products for the NHSPhenylalanine ammonia lyase (phenylketonuria)

Erwinia asparaginase (treatment of acute lymphoid leukemia)

Growth hormone (dwarfism and burns)

Learnt molecular biology: construction and screening of genomic and cDNA libraries; DNA

sequencing, use of expression vectors to produce proteins in E. coli

Given the opportunity of learning molecular biology techniques. Just thought it would be interesting and fun.

Moved to Newcastle University in 1985 Why?

PHLS Porton down was becoming more development less researchI missed not being in a University environmentWanted to work with UG and PhD students

New Blood Lectureships: introduce newexpertise into University departments

New Blood Lectureship in Department of Agricultural Biochemistry and Nutrition

Was this a good move?• Introducing molecular biology in livestock

agriculture was novel in 1985My Prof. in Southampton said: “Harry if you went to do molecular biology in a mainstream biochemistry department you would be one of many. To do it in an a livestock agricultural department will give you more opportunity to stand out.”

• Little pressure very relaxed environment– Very difficult to know what to do as a new area for me– Too much reading increased panic

Prof David Sherratt (now FRS) at meeting said don’t panic go into the lab and do some experiments and it will flow: Completely correct

Was this a good move?My Prof sent me on a tour of UK Agric institutes. Met Dr Geoff. Hazlewood at Babraham Cambridge. Anaerobic microbiologist interested in molecular biology. I was a molecular biologist/enzymologist who needed anaerobic microbiology. Natural rapport worked together for 10 years. Synergy made the team better than us individually

The problem

• Cellulose degradation critical• Need to increase cellulase

activity in rumen

Rate limiting step in cellulosic Biobiofuels

• Conventional = starch and sucrose• To increase cellulosic biofuel need more efficient enzymes• USA have invested $1 billion in cellulosic biofuels last year!

Rate limiting step is the enzymatic Hydrolysis of the wall

Lignocellulosic biofuels and Synthetic Biology

● Synthetic biology and enzyme technology● Improve enzymatic efficiency● Reduce pretreatment● Reduce economic/environmental costs

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● Synthetic biology and microbial strains

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Ethanol

Butanol

Advanced biofuels

● Develop Consolidated Bioprocessing systems

Science with an application

• Lots of ways to spin your science to have a long term application– Critical to get long term funding

• Other way is to be like Jeff Errington and do fundamental world class science– Sustained funding requires publishing regularly in

Nature/Science/Cell

The problem

Starch

Sucrose

The Plant Cell Wall

The Problem

• Yes chemical complexity• But also physical access

issues• Enzymes ~10000-fold

less active against substrate in the walls vs soluble substrate

Initial research (1986-1992)

• Clone cellulase genes– Rapidly diversify to other plant cell wall degrading

enzymes• Characterize the enzymes and appended

non-catalytic carbohydrate binding modules (CBMs)

• Sequence the genes to determine primary structures

First Cellulase gene sequenced revealed multiple serines

MINRSVLKIPALVKPLVQALVLVGCTLGVAQAEVGNPRVNQLGYIPNGDRIAVYKASNNSAQTWQLTHNG SLIASGQTIPKGSDASSGDNIHHIDLSSVTATGSGFTLTVGGDSSYPFSISSTTFNAAFYDALKYFYHNR SGIAIETPYTGGGRGSYASHSRWSRPAGHLNQGANKGDMNVPCWSGTCNYSLNVTKGWYDAGDHGKYVVNGISVWTLLNLYERAQHITGNLAAVADGSMNIPESGNGVADILDEARWQMEFMLAMQVPQGQAKAGMAHHKIHDVGWTGLPLAPHEDPQQRALVPPSTAATLNLAATAAQAARIWKDIDAGFAALCLTAAERAWNAAQANPNDIYSGNYDNGGGGYGDRFVADEFYWAAAELYITTGDSRYLPTINNYTLERTDFGWPDTELLGVMSLAVVPATHTNSLRIAARNHIQTIASTHLTTQSASGYPAPLSSLEYYWGSNSVIANKLVLMGLAYDFSGNQNFALGVSKGINYLFGSNVLSTSFITGLGTNTVAQPHHRFWAGALNSNYPWAPPGALSGGPNAGLEDSLSASSGCTSRPATCWLDSIDAWSTNEITINWNAPLAWVLGFYNDFAATQGGSSSSSSSSSSSVPVSSSSSSSIIPSSSSSSIQPSSSSSSMPSSSSSSSSVVASSSSSVSGGLRCNWYGTLYPLCVTTQSGWGWENSQSCISASTCSAQPAPYGIVGAASSSSQAANRSPTLQLSANATGFEGGSMVCCTLHINGAASDPDGDNLTYSWQVISGNTVVASGSSSSASIHVSNQRGYEVSMTVSDGRGGVATETTFVSVYFSDYFPGSSSSASNINSSSSSSSSSSSSAIVSSSSSVVSSSSSSAASGGNCQYVVTNQWNNGFTAVIRVRNNGSSAINGWSVNWSYSDGSRITNS WNANVTGNNPYAASALGWNANIQPGQTAEFGFQGTKGAGSAQVPAVTGSVCQ

Fell in love with cellulases no protein I knew contained such amino acid repeats

1992-2008• Structural biology• NMR and X-ray crystallography

Catalytic module Non-catalytic carbohydratebinding modules (CBMs)

Non-catalytic Carbohydrate Binding Modules (CBMs)

• CBMs 40-150 residues linked by flexible sequence to catalytic module

• Bind to components of the cell wall

• Can we use CBMs to target highly efficient glycoside hydrolases against the cell wall?

http://www.cazy.org/

Using synthetic biology to target enzymes to the plant cell wall

• Mannan stained with an antibody• Degradation of mannan reduces

amount of antibody bound

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CjMan26A (mannanase)CjMan26A-CBM27CjMan26A-CBM3a

Enzyme concentration (M)

Flu

ore

scen

se (

pix

els

x 10

5)

0 μM 0.07 μM 0.6 μM

0 μM 0.07 μM 0.6 μM

0 μM 0.07 μM 0.6 μM

CjMan26A (mannanse)

CjMan26A-CBM3a

CjMan26A-CBM27

Engineering novel specificity into the AXHd3 arabinofuranosidase

Modifying pocket topology• Active site binds xylose or

arabinose• Exo-activity conferred by active

site pocket topology• Tyr143 forms lip of the pocket• Mutate Tyr143 to Ala• Pocket much shallower• Can xylan chain enter the

active site?

Wild type

Y143A mutant

Was this a good move?• Unusual expertise enabled me to rapidly obtain

grant fundingFound the best postdoc in world. Technically brilliant, determined, hardworking, a strong and positive influence on the group. Logical and intelligent. JUDITH HALL

• Had some outstanding PhD students, many from overseas

• Senior lecturer after 6 years and Professor after 7 years

• University merged departments in ~2003 in resulted in me joining the Institute of Cell and Molecular Biosciences

In 55 needed a new challenge

• Asked to join the Complex Carbohydrate Research Centre at the University of Georgia in 2008– Fantastic experience:

learnt a lot on carbohydrate analysis

• Decided to come back to Newcastle in 2010

53,000 3rd largest soccer stadium in England 92,000 college football at UGA

November 10th 2008November 4th 2008

Microbiota glycanases

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OHOHO

Monosaccharides

Health

Complex glycans drive microbiota structure

Complex glycans

Disease

Crohn’s disease

Microbial fermentation

Model of yeast mannan depolymerization

• Presents a selfish mechanism of glycan utilization.• Activity of the GH76 mannanases is critical to this model• Mannose at base of branch removed ~1000-fold more efficiently by

BT3774 (periplasmic mannosidase ) than BT2799 (surface mannosidase)

GH76 mannanases optimized for selfish mannan utilization

• Surface GH76 endomannanases must cut infrequently– Do not lose surface mannan

• Periplasmic GH76s must cleave small oligosaccharides• Biochemistry supports this

Mannooligosaccharide D.P.

Lo

g1

0k c

at/K

M (

M-1

min

-1)

3 4 5 6 7 8100

101

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BT2623 (surface)BT2631 (periplasm)BT3782 (periplasm)BT3792 (surface)

BT

3782

-

Perip

lasm

ic

Stan

dar

d (S

) - BT 37

92 -

Surfa

ce

Man1

Man2

Man3

Man5

Limit mannan products

B. theta (Bt) does not share mannan with anyone

• B. cellulolyticus (Bc) grows efficiently on mannose

• B. xylanisolvens (Bx) grows on debranched mannan or mannose

• Neither organism can use S. cerevisiae mannan in the presence of B. theta

• Contrary to the general view of extensive synergy in the human microbiota

B. cellulolyticus

B. xylanisolvens

Dr Lis Lowe Max Temple Dr Fiona Cuskin

People who have worked for me30 PhD students and ~30 postdocs

15 permenant academic positions4 permenant positions in research institutes3 are CEOs of biotech companies6 research positions in industry3 scientific administrators1 scientific journalist12 current postdocs1 Science teacher

Advice• Evaluate career choices after PhD

– Don’t be timid as I was• If you Postdoc be very very choosy.

– Go to a world class lab and get 4-5 year position– Don’t go to a single posdoc single PhD student lab– Do science that is important

• Fundamental and/or applied

– See how you can be extremely productive– Have a game plan for fellowships after say 3 years

• Think very very carefully about embarking on a second postdoc