Christoph  Benning  (Michigan)
Regulation  of  Triacylglycerol  Synthesis  and  Turnover  in  Microalgae
Microalgae  are  prolific  producers  of  triacylglycerols,  in  particular  when  they  encounter  nutrient  deprivation.
These  triacylglycerols  accumulate  in  lipid  droplets  and  can  serve  as  feedstock  for  the  production  of  biofuels.
To  harness  the  full  potential  of  microalgae  as  sustainable  biomass  for  biofuel  production,  fundamental   knowledge  of  the  cell  biology  and  biochemistry  of  lipid  droplet  formation  and  triacylglycerol  biosynthesis   and  turnover  is  required.  Our  current  focus  is  on  the  model  green  algae   Chlamydomonas  reinhardtii.
Following  N  deprivation  Chlamydomonas  cells  restructure,  photosynthetic  membranes  degrade,  and  large   vacuoles  and  lipid  droplets  form.  We  isolated  these  lipid  droplets  and  identified  associated  proteins  by  mass   spectrometry.  In  parallel,  we  compared  the  transcriptome  of  N-­‐replete  and  N-­‐deprived  cells  using  state-­‐of-­‐ the-­‐art  deep  sequencing  technology.  To  complement  our  global  studies,  we  generated  32,000  gene   disruption  lines  of  Chlamydomonas  and  conducted  a  forward  genetic  screen  to  identify  mutants  deficient  in
TAG  accumulation.
Creating  synergy  by  pursuing  these  approaches  in  parallel  has  yielded  striking  results.  For  example,  we   identified  a  major  lipid  droplet  associated  protein  (MLDP)  by  lipid  droplet  proteomics,  which  is  encoded  by  a   gene  that  is  strongly  induced  following  N  deprivation.  The  protein  is  specific  to  the  green  algal  phylum  and   inactivation  of  the   MLDP   gene  results  in  larger  lipid  droplets.  Lipase-­‐encoding  genes  have  emerged  as  some   of  the  most  regulated  following  N  deprivation.  In  addition,  one  of  the  first  tagged  genes  that  we  identified  in   our  mutant  collection  encodes  what  appears  to  be  a  polar  lipid  lipase.  Disruption  of  this  gene  reduces  TAG   accumulation  and  alters  lipid  composition  in  specific  ways  suggesting  a  role  in  membrane  lipid  turnover.  The   expression  of  the  gene  is  increased  following  N  deprivation.  Another  putative  lipase  associated  with  lipid   droplets  in  Chlamydomonas  can  partially  complement  a  growth  phenotype  in  a  TAG  lipase-­‐deficient  yeast   mutant.  Thus,  it  likely  encodes  a  triacylglycerol  lipase.  The  expression  of  its  gene  is  decreased  following  N   deprivation.  Although  we  are  only  at  the  beginning  our  analysis  in  Chlamydomonas,  a  rich  repertoire  of
interesting  factors  and  proteins  involved  in  lipid  droplet  formation  and  turnover  is  emerging.
Sam  Bryan  (QM/Imperial)
Spatial  localisation  of  the  hydrogenase  in  Synechocystis  sp.  PCC  6803
The  capacity  of  microorganisms  to  produce  hydrogen  is  mediated  by  metalloenzymes  called  hydrogenases.
The  soluble  or  loosely  membrane-­‐associated  bidirectional  hydrogenase  is  present  in  both  nitrogen  and  non-­‐ nitrogen  fixing  cyanobacteria.  It  is  a  heteropentameric  enzyme  encoded  by  the  hoxEFUYH  genes,  and  it  has  a
Nickel-­‐Iron  [NiFe]  centre  in  its  catalytic  site.  There  is  still  extensive  debate  on  the  location  of  the   hydrogenase,  whether  it  is  in  the  thylakoid  lumen  or  cytoplasmic  membrane.  Maturation  of  the  complex   may  also  occur  in  specific  compartments.  We  have  generated  a  HoxF-­‐GFP  chimera  for  spatial  resolution  (by   confocal  microscopy).  We  are  now  utilising  real  time  dynamic  monitoring  to  observe  the  localisation  of  the   hydrogenase  under  different  conditions  including  dark-­‐light  transitions.
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Donald  A.  Bryant  (Penn  State)
Synechococcus  sp.   PCC  7002:  a  robust,  versatile,  and  cosmopolitan  cyanobacterial  platform  for  biofuels   production
Synechococcus  sp.  PCC  7002  is  an  ideal  cyanobacterium  for  functional  genomics  and  biotechnological   applications  through  metabolic  engineering.  This  cyanobacterium  is  euryhaline,  unicellular  and  capable  of
(photo)heterotrophic  growth  on  glycerol.  The   Synechococcus  7002  genome  (~3.4  Mb)  has  been  sequenced   and  contains  about  3450  genes.  It  is  comprised  of  a  3.0-­‐Mb  chromosome  and  6  plasmids  that  range  in  size   from  4.8  to  186  kb.   Synechococcus  7002  is  naturally  transformable  and  is  competent  for  DNA  uptake   throughout  a  batch  growth  cycle.  Neutral-­‐site  integration  platforms  have  been  used  to  introduce  foreign   genes  into  all  of  these  plasmids  by  transformation,  and  using  this  strategy,  a  simple,  effective  and  flexible   gene  (over)expression  system  has  been  developed.   Synechococcus  7002  has  two  remarkable  properties  that   distinguish  it  from  other  cyanobacteria.  Firstly,  it  tolerates  extremely  high  light  intensities  so  long  as  the  cells   are  not  CO
2
-­‐limited,  and  it  is  also  very  tolerant  of  attendant,  elevated  levels  of  reactive  oxygen  and  nitrogen   species  (ROS/RNS).  Secondly,   Synechococcus  7002  grows  faster  than  other  well-­‐characterized  cyanobacteria
(~2.6-­‐h  doubling  time  with  urea  or  ammonium  and  4-­‐h  doubling  time  with  nitrate).  Understanding  the   genetic  and  physiological  bases  for  its  rapid  and  robust  growth  could  reveal  new  strategies  for  metabolic   engineering  and  biofuels  production  in  many  organisms.
We  are  employing  systems  biology  approaches  to  develop  a  deeper  understanding  of  the  physiology  of
Synechococcus  7002.  Based  on  cDNA  sequencing  using  SOLiD  methodologies,  we  have  performed  high-­‐ throughput  transcription  profiling.  More  than  45  conditions  and  strains  have  been  profiled.  The  resulting   data  are  being  compared  to  proteomic  data  and  are  being  used  to  improve  the  annotation  and  a  metabolic   model  incorporating  ~600  reactions  (collaboration  with  Pacific  Northwest  National  Laboratory  and  Dr.
Jennifer  Reed).  The  transcription  data  suggest  that   Synechococcus  7002  may  be  tolerant  of  ROS/RNS  and   high  light  because  it  constitutively  expresses  enzymes  for  protection  against  oxidative  stress  at  a  relatively   high  level.  It  also  produces  multiple  types  of  xanthophyll  carotenoids  (zeaxanthin,  myxoxanthophyll,  and   synechoxanthin)  that  all  contribute  to  light  and  ROS/RNS  tolerance.  The  profiling  data  have  provided  direct   information  about  useful  promoters  for  gene  expression  and  have  also  suggested  approaches  ( e.  g.
,   elimination  or  overproduction  of  sigma  factors)  that  could  help  to  alter  cellular  metabolism  in  a  global   manner.  A  collection  of  sigma  factor  and  transcription  factor  null  mutants  is  being  created,  and  the   construction  of  strains  overproducing  sigma  factors  is  planned.  Transcription  profiling  of  the  mutant  strains   has  begun  and  should  produce  additional  insights  for  manipulating  metabolism  globally.  A  dicistronic  operon   has  been  identified  that  may  encode  two  enzymes  that  can  replace  the  missing  2-­‐oxo-­‐glutarate   dehydrogenase  activity  of  the  TCA  cycle.  These  proteins  have  been  overproduced  in   E.  coli  and  are  being   assayed  for  the  ability  to  convert  2-­‐oxo-­‐glutarate  into  succinate.
Synechococcus  7002  was  isolated  on  Magueyes  Island,  Puerto  Rico,  from  mud  flats  associated  with  a  tilapia   farm.  We  have  recently  sequenced  the  genomes  of  four  closely  related   Synechococcus  sp.:  strains  PCC  7003,
7117,  73109,  and  8807.  These  strains  were  isolated  from  coastal  environments  in  Greenwich,  CT;  Port
Hedland,  Western  Australia;  City  Island,  NY;  Port  Gentil,  Gabon,  Africa,  respectively.  In  spite  of  their   geographic  diversity,  the  four  strains  are  surprising  similar  physiologically  to   Synechococcus  7002.  All  have   high  PS  II:  PS  I  ratios,  exhibit  very  rapid  growth,  and  have  similar  gene  contents  and  arrangements.  One   strain  grows  about  10%  faster  than   Synechococcus  7002,  possibly  because  it  has  slightly  higher  chlorophyll   a   content  per  cell.  Three  of  the  strains  can  grow  in  the  absence  of  vitamin  B the  presence  of  a  gene  for  vitamin  B
12 under  the  control  of  a  B
12
12
cosmopolitan  in  their  distribution  and  strikingly  similar  in  their  properties.
,  a  trait  that  is  correlated  with
-­‐independent  methionine  synthase  ( metE ),  which  is  encoded  on  pAQ7
-­‐riboswitch.  The  data  suggest  that  these   Synechococcus  sp.  strains  are
2

Nigel  Burroughs  (Warwick)
Systems  Biology  modelling:  from  omics  to  regulons
With  the  development  of  microarrays,  the  elucidation  global  regulatory  networks  became  possible  although   it  still  remains  to  a  large  degree  a  specialist  area.  This  is  in  part  because  of  the  difficulty  of  extracting  reliable   information  from  sparse  noisy  data.    In  this  talk  I  will  review  methods  of  regulatory  network  inference  from   gene  expression  data,  integrating  time  series  data,  bioinformatic  predictions  and  condition/strain  data.  In   this  analysis  we  find  that  the  most  accurate  method  depends  on  the  complexity  of  the  processes  and  the   underlying  transcription/translation  rates.
Arvind  Chali  (Virginia)
Functional  integration  of  transcriptome  data  reveals  broad  evolutionary  conservation  in  metabolic
subsystems  of  Chlamydomonas  reinhardtii
Michelle  Chang  (Berkeley)
Building  new  chemical  function  in  E.  coli
Living  systems  have  evolved  the  capacity  to  carry  out  many  chemical  transformations  of  interest  to  synthetic   chemistry  if  they  could  be  redesigned  for  targeted  purposes.  However,  our  ability  to  mix  and  match  enzymes   to  construct   de  novo  pathways  for  the  cellular  production  of  small  molecule  targets  is  limited  by  insufficient   understanding  how  chemistry  works  inside  a  living  cell.  Our  group  is  interested  in  using  synthetic  biology  as   a  platform  to  understand  the  molecular  principles  needed  to  design  high-­‐flux  synthetic  pathways.  Towards   these  goals,  we  have  built  a  robust  pathway  for  the  production  of   n -­‐butanol  from  individual  enzyme   components  and  explore  how  enzyme  mechanism  can  be  used  as  a  kinetic  control  element  to  push  a
reversible  pathway  to  high  yielding  production  of  second-­‐generation  biofuels.
Neil  D.  Clarke  (Singapore)
Transcriptome  and  lipidome  analysis  in  model  and  non-­‐model  algae
In  order  to  develop  a  ‘systems-­‐level’  description  of  lipid  metabolism  in  algae,  we  are  performing  combined   transcriptome  and  lipidome  analysis  of  in  the  model  alga  Chlamdomonas,  as  well  as  in  a  diverse  set  of  other   algal  species  that  are  less  amenable  to  genetic  perturbation.  The  strategy  in  Chlamydomonas  is  to  obtain   lipid  and  gene  expression  profiles  for  diverse  genotypes,  using  artificial  microRNAs  to  knockgene  expression   of  genes  involved  in  lipid  biosynthesis,  lipid  transport,  autophagy,  nitrogen-­‐  starvation  induced  signal   transduction,  and  other  relevant  processes.  These  data  are  being  used  to  develop  a  functional  metabolic   network  that  correlates  gene  expression  differences  with  features  in  the  lipid  profiles.
We  are  also  extending  this  work  to  other  species  through  our  participation  in  the  One  Thousand  Plant  (1KP)   transcriptome  project,  which  is  producing  transcriptome  sequences  for  a  large  number  of  diverse  algal   species.  For  many  of  these  species,  we  are  also  performing  detailed  lipidomic  analyses.  Similarities  and   differences  in  lipid  metabolism,  as  inferred  from  the  lipid  profile  and  the  transcriptome  sequences,  will  be
described.
Paul  A  Dalby  (UCL)
De  novo   pathway  design  and  evolution
Concepts  for  the  design  of   de  novo  pathways  for  the  synthesis  of  new  chemicals  will  be  introduced.    The   aminodiol  functionality  is  present  in  many  natural  and  synthetic  biologically  active  molecules  including   antibiotics,  alkaloids  and  amino  sugars.    An  elegant  de  novo  biocatalytic  pathway  has  enabled  the  synthesis   of  chiral  aminodiols  which  is  now  being  enhanced  to  an  industrially  useful  scale.  Transketolase  (TK)  mutants   have  been  obtained  by  directed  evolution  to  accept  new  substrates  and  to  both  improve  and  reverse
3

enantioselectivity  for  the  product.    Meanwhile  new  transaminase  (TAm)  variants  have  also  been  isolated   that  are  capable  of  processing  a  multitude  of  aldehyde  substrates  into  chiral  aminodiols.  These  enzyme   variants  have  been  re-­‐combined  into  the  pathway  to  diversify  the  range  of  products  that  can  be  synthesised.
General  strategies  for  the  directed  evolution  of  component  enzymes,  and  also  the  impact  of  re-­‐introducing
evolved  enzymes  into  pathways  will  be  discussed.
Oliver  Ebenhoeh  (Aberdeen)
Mathematical  models  of  metabolism  and  photosynthetic  acclimation  of  Chlamydomonas
The  talk  will  be  devided  in  two  parts,  the  first  part  focussing  on  the  reconstruction  and  improvement  of  the   metabolic  network  of  Chlamydomonas,  the  second  part  covering  ongoing  research  activities  regarding   models  of  photosynthetic  acclimation.  The  method  of  network  expansion,  introduced  previously  by  our   group,  is  a  structural  analysis  method  to  assess  biochemical  production  capabilities  of  metabolic  networks.  I   will  give  a  short  summary  about  how  this  method  was  used  to  assess  and  classify  networks  of  different   organisms  and  how  it  may  be  applied  to  infer  minimal  nutrient  requirements,  before  covering  in  more  detail   how  it  was  employed  to  improve  the  genome  annotation  of  Chlamydomonas  by  identifying  inconsistencies   in  its  draft  metabolic  network.  Our  approach  integrates  genomics,  proteomics  and  metabolomics  data  with   bioinformatics  and  modelling  techniques  to  identify  missing  parts  of  the  network  and  to  compute  possible   solutions  how  these  gaps  may  be  filled.
Photosynthetic  organisms  have  developed  acclimation  mechanisms  which  allow  them  to  cope  with  rapidly   changing  light  conditions.  The  most  prominent  mechanisms  on  the  time-­‐scale  of  seconds  to  minutes  are   non-­‐photochemical  quenching  (NPQ)  and  state  transitions.  Despite  active  research,  the  precise  molecular   mechanisms  and  implications  of  these  processes  are  not  yet  fully  understood.  To  support  the  investigation   of  these  important  regulatory  processes  from  a  theoretical  side,  we  have  developed  mathematical  models   describing  these  central  acclimation  mechanisms.  I  will  present  preliminary  results  which  allow  hypotheses   on  some  molecular  mechanisms  and  shed  new  light  on  the  different  adaptive  strategies  of  plants  and  green
algae.
John  H  Golbeck  (Penn)
A  Hybrid  Biological/Organic  Photochemical  Half-­‐Cell  for  Generating  Dihydrogen
In  this  talk,  I  will  describe  our  work  on  the  development  of  a  hybrid  biological/organic  photo-­‐electrochemical
.  The  device  will  couple  Photosystem  I  (PSI),   half-­‐cell  that  carries  out  the  reaction:  2H
(H
+  +  2 e  –  +  2hν
→
H
2 which  captures  and  stores  energy  derived  from  sunlight,  with  a  catalyst  such  as  a  [FeFe]-­‐hydrogenase
2 ase)  or  a  [NiFe]-­‐H
2 ase,  which  generate  H
2
with  an  input  of  two  electrons  and  two  protons.  The  technical   challenge  is  to  deliver  the  electrons  from  the  acceptor  side  of  PSI  to  the  catalytic  module  rapidly,  using  a   method  that  does  not  depend  on  diffusion  chemistry.  To  accomplish  this  goal,  we  have  devised  a  technology   based  on  a  molecular  wire,  which  serves  to  tether  the  photochemical  module  to  the  catalytic  module  at  a   fixed  distance  so  that  an  electron  can  quantum  mechanically  tunnel  between  the  F distal  [4Fe-­‐4S]  cluster  of  a  H
P
700
+  and  F
B
–
2
B
cluster  of  PSI  and  the   ase  enzyme  at  a  rate  faster  than  the  competing  charge  recombination  between
.  To  link  the  photochemical  and  catalytic  modules  of  our  half-­‐cell,  a  short  aliphatic  or  aromatic   dithiol  molecule  forms  a  coordination  bond  with  an  exposed  Fe  of  the  F exposed  Fe  of  the  distal  [4Fe-­‐4S]  cluster  of  a  H
2
B
cluster  of  a  PSI  variant  and  with  an   ase  variant.  This  is  practically  achieved  by  changing  a  ligating
Cys  residue  of  the  surface-­‐located  [4Fe-­‐4S]  cluster  of  each  protein  to  a  Gly,  thereby  exposing  the  Fe  atom  for   chemical  rescue  by  the  added  dithiolate-­‐containing  molecular  wire.  Our  latest  results  show  that  when  Cyt   is  cross-­‐linked  to  PSI  and  ascorbate  is  the  sacrificial  donor,  the  PSI—wire—[FeFe]-­‐H
2
,  which  is  equivalent  to  a  throughput  of  142   e –  PS  I -­‐1  s -­‐1 c ase  construct  evolves  H
6
2
.    Putting  this  into   at  a  rate  of  2885  µmoles  mg  Chl -­‐1  h -­‐1 perspective,  cyanobacteria  evolve  O throughput  of  46   e –  PS  I -­‐1  s -­‐1
2
at  a  rate  of  ~400  µmoles  mg  Chl -­‐1  h -­‐1 ,  which  is  equivalent  to  a
in   Synechococcus  sp  PCC  7002  (assuming  a  ratio  of  PS  I:PS  II  of  1.8).  The   significantly  greater  electron  throughput  by  our  hybrid  biological/organic  nanoconstruct  over   in  vivo
4

oxygenic  photosynthesis  validates  the  concept  of  tethering  proteins  through  their  physiologically  relevant   redox  cofactors  to  overcome  diffusion-­‐based  rate  limitations  on  electron  transfer.  We  are  currently   extending  this  work  by  (i)  docking  the  PSI—molecular  wire—[FeFe]-­‐H
2 ase  construct  on  a  Au  electrode  so   that  it  functions  as  the  cathode  of  a  photo-­‐electrochemical  half-­‐cell;  (ii)  engineering  a  His → Gly  mutation  in   an  oxygen-­‐tolerant  [NiFe]-­‐H
PSI—wire—[NiFe]-­‐H
2
2 ase  from   Ralstonia  eutropha  and  tether  this  [NiFe]-­‐H
2 ase  to  the  PSI—molecular   wire  module;  and  (iii)  assembling  a  PSI—molecular  wire  module   in  vivo  in  preparation  for  a  living  cell-­‐based
ase  construct.   This  research  was  funded  by  the  U.S.  Department  of  Energy,  Basic  Energy
Sciences,  Division  of  Materials  Sciences  and  Engineering,  under  Contract  DE-­‐  FG-­‐05-­‐05-­‐ER46222.
Martin  Hagemann  (Rostock)
Metabolic  and  transcriptomic  phenotyping  of  inorganic  carbon  acclimation  among  cyanobacteria
The  amount  of  available  inorganic  carbon  is  one  of  the  main  limiting  environmental  factors  for   photosynthetic  organisms  such  as  cyanobacteria.  Using  the  cyanobacterial  model  strains   Synechocystis  sp.
PCC  6803  and   Synechococcus   elongatus  PCC  7942,  we  characterized  metabolic  and  transcriptomic  changes  in   cells  that  had  been  shifted  from  high  to  low  CO
2
levels.  Metabolic  phenotyping  indicated  an  activation  of   glycolysis,  the  oxidative  pentose  phosphate  cycle  and  glycolate  metabolism  at  lowered  CO
2
levels.  The   metabolic  changes  coincided  with  a  general  reprogramming  of  gene  expression,  which  included  not  only   increased  transcription  of  inorganic  carbon  transporter  genes  but  also  genes  for  enzymes  involved  in   glycolytic  and  photorespiratory  metabolism.  In  contrast,  the  mRNA  content  for  genes  from  N-­‐assimilatory   pathways  decreased.  These  observations  indicated  that  cyanobacteria   control  homeostasis  of  the  C/N  ratio.
Therefore,  results  obtained  from  the  wild-­‐type  strains  were  compared  to  the   Synechocystis  6803  mutants
D glcD1  and  D ccmM  with  defective  photorespiratory  glycolate  dehydrogenase  and  carbon  concentrating   mechanisms,  respectively,  and  the  MP2  mutant  of   Synechococcus  7942,  which  is  defective  for  the  central
C/N-­‐regulating  PII-­‐protein.  These  mutants  showed  distinct  changes  in  levels  of  metabolites  and  or  of   transcripts  compared  to  wild-­‐type  cells,  which  may  shed  light  on  possible  regulatory  mechanisms  of  central
C-­‐  and  N-­‐metabolism.  On  example,  the  PII-­‐signaling  appears  to  down-­‐regulate  the  N-­‐metabolism  at  lowered
CO
2
,  whereas  the  specific  shortage  of  inorganic  carbon  is  recognized  by  different  mechanisms  using
metabolite  signals  such  as  glycolate.
Klaus  Hellgardt  (Imperial)
Multiple  energy  vectors  from  algae  processing
Chris  Herring  (Mascoma)
The  role  of  OMICs  in  the  development  of  Thermoanaerobacterium  saccharolyticum  for  production  of   ethanol  from  pretreated  hardwood
Thermoanaerobacterium  saccharolyticum  is  a  thermophilic  anaerobic  bacterium  that  was  engineered  to   produce  ethanol  at  near-­‐theoretical  yield  from  a  wide  array  of  biomass-­‐derived  sugars.  I  will  present  findings   of  OMICs  studies  conducted  as  part  of  a  project  to  develop  this  organism  for  the  production  of  ethanol  from   pretreated  hardwood.  To  begin,  a  genome  sequence  was  generated  and  resequencing  was  performed  on  6   mutagenized  and  selected  strains.  An  in  silico  reconstruction  of  metabolism  was  created  based  on  the   existing  reconstruction  of  Clostridium  thermocellum.  Microarrays  were  designed  and  used  to  study  gene   expression  profiles  in  >20  different  steady  state  and  “shock”  conditions  relevant  to  biomass  fermentation.
Methods  for  sampling  cultures  for  intracellular  metabolite  profiling  by  GC/MS  were  validated  and  then  used   to  analyze  the  effects  of  the  inhibitors  HMF  and  furfural.  Examples  will  be  shown  of  combined  expression   and  metabolite  analysis  of  fermentations  of  cellobiose  conducted  to  determine  why  ethanol  production   ceased.  Perturbations  caused  by  extracts  containing  inhibitors  and  hemicellulose  were  also  analyzed.
Clusters  of  upregulated  genes  mostly  comprised  combinations  of  transporters,  regulators,  glycosyl
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hydrolases  and  carbohydrate-­‐specific  catabolic  genes,  but  also  included  oxidoreductases  that  may  be  related   to  inhibitor  detoxification  or  intracellular  redox  balancing.  The  importance  of  intra-­‐  and  extracellular  redox   in  biofuel  production  will  be  further  discussed,  including  its  importance  in  directing  carbon  flux  and  its  effect   on  SSF  performance.
Patrik  Jones  (Turku)
Engineering  model  systems  for  biofuel  production  and  related  fundamental  studies  in  prokaryotes
The  talk  will  describe  efforts  to  engineer  prokaryotes,  E.  coli  and  various  cyanobacteria,  for  biofuel   production.  Our  group  is  operating  somewhere  in  between  fundamental  and  applied  sciences  and  utilizes   both  wet-­‐lab  experimental  and  computational  approaches  (although  the  wet-­‐lab  work  is  dominant).  Topics   that  will  be  covered  include  the  development  of  basic  tools  for  cyanobacteria  engineering,  the  metabolism
of  (1)  Fe-­‐S  clusters,  (2)  NADPH,  (3)  H2  and  (4)  hydrocarbon  fuels,  and  utilization  of  computational  tools  to  aid   engineering.
Olaf  Kruse  (Bielefeld)
Biofuels  from  algae-­‐  challenges  for  industrial  levels  of  productivity
Solar  to  fuels  with  microalgae  are  nowadays  considered  as  a  promising  alternative  to  the  traditional  use  of   crop  plants  for  bioenergy  production.  However,  to  achieve  the  goals  of  profitable  production  lines  and   positive  energy  balances  with  algae,  new  bio-­‐refinery  concepts  are  needed  to  combine  the  production  of   bio-­‐fuels  such  as  bio-­‐methane  and  bio-­‐hydrogen  with  the  extraction  of  oils  for  biodiesel  and/or  the   separation  of  valuable  co-­‐products  (Stephens  et  al.,  2010;  Kruse  &  Hankamer,  2010).  Such  bio-­‐refinery   concepts  can  be  designed  with  the  aim  of  achieving  CO2  neutral  systems  in  which  CO2  and  essential   nutrients  such  as  P  and  N  are  recycled.  Since  H2  and  CH4  are  volatile  products  that  can  be  easily  collected,   these  gaseous  fuels  can  be  considered  excellent  components  of  such  new  bio-­‐refineries.  The  improvement   of  fuel  production  from  microalgae  includes  the  systematic  analysis  of  metabolic  pathways  as  well  as   targeted  metabolic  engineering  with  transformable  microalgae  to  overcome  potential  bottlenecks.
Stéphane  Lemaire  (CNRS  Paris)
Redox  based  post-­‐translational  modifications:  a  central  role  in  the  regulation  of  cell  metabolism
Cells  must  constantly  adapt  to  their  environment  and  have  developed  many  systems  to  perceive   extracellular  factors  or  changes  in  environmental  conditions.  Redox  signaling  mechanisms  play  a  central  role   in  these  processes  and  act  mainly  through  reversible  posttranslational  modification  of  protein  thiols  under   the  control  of  small  disulfide  oxidoreductases  named  thioredoxins  (TRXs)  and  glutaredoxins  (GRXs).  Protein   glutathionylation  is  a  reversible  post-­‐translational  modification  controlled  by  GRXs  and  consisting  in  the   formation  of  a  mixed  disulfide  between  glutathione  and  a  protein  cysteine  residue.  Emerging  evidence   suggests  that  it  could  constitute  an  important  mechanism  of  regulation  and  signaling  in  photosynthetic   organisms.
We  developed  several  proteomic  approaches  based  on  radiolabeling,  biotinylation  or  affinity  purification  to   identify  proteins  regulated  by  TRXs  or  undergoing  glutathionylation  in  the  unicellular  green  alga
Chlamydomonas  reinhardtii .  These  methods  allowed  identification,   in  vivo   or  in  vitro ,  of  55  putative  targets   of  TRXs  and  more  than  200  glutathionylated  proteins  involved  in  numerous  cell  processes  and  metabolic   pathways  including  photosynthesis.  We  further  analyzed  the  redox  regulation  of  several  putative  targets   related  to  carbon  metabolism,  including  A
4
-­‐glyceraldehyde-­‐3-­‐phosphate  dehydrogenase,  TRX  f  and  isocitrate   lyase,  by  kinetic  analyses,  mass  spectrometry,  site-­‐directed  mutagenesis  and  western  blotting.  The  activity   of  all  the  proteins  analyzed  was  found  to  be  regulated  by  one  or  several  redox  post-­‐translational   modifications.  TRXs  and  GRXs  were  also  found  to  exhibit  distinct  biochemical  properties  and  reactivities.  All   these  data  indicate  that  redox  post-­‐translational  modifications  likely  constitute  an  important  mechanism  of
6

regulation  and  signaling  in  photosynthetic  organisms.  TRXs  and  GRXs  appear  as  central  regulators  allowing,   depending  on  the  intracellular  redox  state,  a  fine  tuning  of  cell  metabolism  under  stress  conditions.
Radhakrishnan  Mahadevan  (Toronto)
Model-­‐based  Analysis  and  Design  of  Metabolism  for  Biofuels  and  Biochemicals
Bioprocess  development  for  biofuels  and  biochemicals  typically  requires  several  rounds  of  metabolic   engineering  to  meet  process  targets  including  product  yield,  titer  and  productivity,  all  of  which  impact  the   process  economics.  Recent  advances  in  experimental  and  computational  technologies  have  enabled  the   detailed  characterization  of  biological  systems.  In  particular,  the  molecular  components  of  these  systems   including  the  list  of  genes,  proteins  they  encode,  and  compounds  that  interact  with  these  proteins  can  be   determined.  Similar  advances  in  computational  modeling  techniques  have  allowed  the  development  of   genome-­‐scale  models  of  metabolism  in  several  organisms.    In  this  talk,  the  use  of  such  models  for  metabolic   engineering  will  be  presented.  Model  refinement  through  the  incorporation  of  a  fundamental  physical   constraint  that  accounts  for  membrane  area  will  be  described.  In  the  first  part,  a  rational  approach  based  on   bi-­‐level  optimization  to  enhance  bioprocess  productivity  by  forcing  co-­‐utilization  of  substrates  will  be  shown.
Experimental  results  from  the  application  of  this  approach  to  enforce  substrate  co-­‐utilization  in   Escherichia   coli  will  be  discussed.  The  second  part  of  the  talk  will  focus  on  the  use  of  synthetic  biology  tools  such  as  the   toggle  switch  to  manipulate  bacterial  metabolism  and  apply  optimization  &  control  principles  at  the  genetic   level  by  “re-­‐wiring”  the  bacterial  machinery  to  improve  process  productivity  in   E.  coli  and  respiration  rates  in
Geobacter .    In  the  last  part  of  the  talk,  a  novel  nested  nonlinear  optimization  method  for  metabolic   engineering  resulting  in  over  hundred  different  strain  design  strategies  for  succinate  production  will  be
presented.
Conrad  Mullineaux  (QM,  London)
Colocalisation  of  electron  transport  complexes  in  bioenergetic  membranes  -­‐  does  distribution  at  the  100     nm  length  scale  control  the    partitioning  of  reducing  power?
We  have  used  fluorescent  protein  tagging  and  fluorescence  microscopy  to  probe  the  distribution  of  electron   transport  complexes  in  the  cytoplasmic  membrane  of  Escherichia  coli  and  the  thylakoid  membranes  of   cyanobacteria.    Complexes  studied  include  all  the  major  OXPHOS  complexes  of  E.  coli,  and  the  bidirectional   hydrogenase  and  a  number  of  respiratory  complexes  in  cyanobacteria.    Although  the  fluorescence   microscopic  techniques  used  have  relatively  low  resolution,  they  have  the  considerable  advantages  that  they   allow  the  complete  distribution  and  dynamics  of  specific  proteins  to  be  observed  in  living  cells.    Quantitative   electron  transport  measurements  show  that  in  most  cases  electron  transport  function  is  not  perturbed  by   the  tag.  A  distribution  pattern  frequently  observed  is  the  clustering  of  electron  transport  complexes  into   specific  zones  in  the  membrane,  typically  of  the  order  of  100  nm  in  diameter  and  containing  tens  to   hundreds  of  copies  of  the  tagged  complex.  In  cyanobacteria,  we  have  observed  instances  where  the   distribution  of  the  complex  is  under  physiological  control,  and  changes  in  the  distribution  of  the  complex   seem  to  correlate  with  changes  in  electron  transport  activity.  The  case  of  cyanobacteria  is  particularly   intriguing,  since  respiratory  and  photosynthetic  complexes  share  the  same  membrane,  giving  multiple   possible  pathways  of  electron  transport.  We  propose  that  electron  transport  is  kinetically  confined  to  length
scales  of  around  100  nm  or  less,  and  that  co-­‐localisation  on  these  scales  is  a  major  determinant  of  electron   transport  pathways.  Efficient  re-­‐routing  of  electron  transport  for  biofuel  production  will  require   understanding  and  manipulation  of  the  factors  that  control  the  distribution  of  protein  complexes  in  the   membrane.
7

Norio  Murata  (Okazaki)
Stress  sensitivity  of  photosynthesis  and  gene-­‐engineered  improvement  of  stress  tolerance  in  cyanobacteria
The  productivity  of  microalgae  primarily  depends  on  the  efficieincy  of  photosynthesis  and  the  stability  of   photosynthetic  machineries.  However,  they  are  limited  by  various  kinds  of  environemtal  stress,  such  as   stong  light,  high  and  low  temperatures,  and  high  salt.  Previous  studies  have  demonstrated  that  photosystem
II  (PSII)  is  a  main  target  of  such  environmental  stresses  and  is  damaged  by  strong  visible  light  and  UV  light.
However,  algal  cells  cope  with  such  damaging  effects  of  light  by  a  strong  ability  of  repair,  which  includes   removal  of  the  damaged  PSII  and  de  novo  synthesis  of  necessary  proteins  for  reassembly  of  PSII.  We  have   demonstrated  that  various  kinds  of  stress,  such  as  extreme  temperatures  and  salt  stress,  inhibit  the  repair   mainly  by  inactivating  the  translation  machinery,  in  particular,  at  the  elongation  factor  G  (EF-­‐G).  Our  recent   studies,  in  use  of  cyanobacteria,  have  demonstrated  that  several  gene-­‐engineering  methods  strengthen  the   algal  photosysnthesis  against  environmental  stresses;  namely,  (1)  Desaturation  of  fatty  acids  in  membrane   lipids  by  genetic  engineering  of  fatty  acid  desaturases;  (2)  Introduction  of  the   codA  gene  for  synthesis  of   glycinebetaine;  (3)  Overexpression  of  peroxide-­‐scavenging  enxymes;  (4)  Overexpression  of  EF-­‐G.  In  this   workshop,  we  will  discuss  these  methods  on  the  basis  of  the  newly  developed  scheme  for  the  mechanism  of   photoinhibition.  We  will  discuss  also  other  potential  methods  for  the  improvement  of  stress  tolerance  of  the
photosynthetic  machinery  in  cyanobacteria.
Ed  van  Niel  (Lund)
The  extreme  thermophilic  Caldicellulosiruptor  saccharolyticus:  a  promising  hydrogen  cell  factory
Dark  fermentation  of  hemicellulose-­‐containing  waste  to  hydrogen  is  considered  one  of  the  promising   avenues  of  biofuel  production.  The  extreme  thermophilic  bacterium   Caldicellulosiruptor  saccharolyticus   can   produce  hydrogen  from  carbohydrate-­‐rich  substrates  at  yields  close  to  the  theoretical  maximum  (i.e.,  4  mol
H
2
/mol  hexose).  This  strict-­‐anaerobic  Gram-­‐positive  bacterium  is  able  to  ferment  an  array  of  mono-­‐,  di-­‐  and   polysaccharides,  and  is  relatively  tolerant  to  high  partial  hydrogen  pressures,  making  it  a  potential  candidate   for  exploitation  in  a  biohydrogen  process.  Its  physiology  has  been  studied  in  various  environmental   conditions,  including  biomass  hydrolysates  as  feedstock.  However,  this  organism  bears  various  hallmarks  of   being  adapted  to  a  sugar-­‐lean  environment,  such  as  low  osmotolerance.  This  should  be  improved,  either  by   evolutionary  adaptation  or  metabolic  engineering,  before  this  organism  can  be  used  in  an  economical   feasible  process.
Together  with  other  research  groups  genome  annotation  and  transcriptomics  and  proteomics  protocols   have  been  accomplished  as  platforms  for  further  study  on  the  'enviromics'  of  the  organism.
We  followed  two  approaches  to  deepen  our  understanding  of  the  physiology  of  this  unique  organism.  One   was  recently  performed  through  reconstructing  and  analyzing  its  genome-­‐scale  metabolic  network,  which   consists  of  575  reactions,  involving  507  genes  and  596  metabolites.  The  reconstruction  process  identified   the  lack  of  auxotrophy  for  any  of  the  amino  acids  in   C.  saccharolyticus ,  which  was  confirmed  by  growing  the   organism  in  a  chemically  defined  medium  under  chemostat  conditions.  The  reconstruction  is  further   converted  to  a  computational  model  to  enable  constrained-­‐based  flux  analysis.  The  model  can  successfully   mimic  experimentally-­‐measured  fluxes  in  glucose-­‐limited  chemostat  cultures  and  is  able  to  capture  the   behaviour  of  the  cells  in  response  to  variation  in  sugar  type  in  the  medium.  Our  second  approach  is  through   kinetic  modelling  of  the  central  carbon  metabolism,  which  is  still  ongoing.  So  far,  a  picture  is  emerging  how   the  redox-­‐  and  energy  metabolism  are  highly  integrated  in   C.  saccharolyticus ,  which  comes  especially  to  the   fore  in  the  tight  regulation  of  hydrogen  production.  For  instance,  the  lactate  dehydrogenase  activity  is   controlled  on  enzyme  level  via  the  ratio  of  two  energy  carriers,  i.e.,  pyrophosphate  and  ATP.  The  latter   example  underlines  that  a  good  knowledge  of  'classical'  kinetics  of  enzyme  regulation,  in  addition  to  modern
genome-­‐scale  methods,  is  essential  to  make  sense  of  the  metabolic  system  of  cell.
8

Clemens  Posten  (Karlsruhe)
Process  development  for  hydrogen  production  with  Chlamydomonas  reinhardtii
Hydrogen  production  under  anaerobic  and  sulfur  deficient  conditions  is  intensively  investigated  in  many   working  groups.  For  a  commercial  process  specific  parameters  like  acetate  feeding  and  phase  shift  to   anaerobiosis  are  critical  factors.  In  this  contribution  a  two  phase  process  will  be  presented,  where  sulfur   limitation  is  induced  by  a  controlled  feed  either  in  batch  or  in  fed-­‐batch  process  policy.  Anaerobiosis  and  the   following  hydrogen  production  can  be  achieved  without  cell  separation  and  without  additional  acetate  feed.
The  discussion  will  include  the  stoichiometric  hydrogen  yield  and  the  necessity  for  a  controlled  water
splitting  and  respiration  activity  of  the  cells.
António  Roldão  (Chalmers)
Bringing  biofuels  closer  to  reality:  engineering  yeast  cell  factories  for  the  production  of  bio-­‐butanol  and   next  generation  of  bio-­‐diesel
To  surmount  the  global  dependency  on  oil-­‐derived  products,  renewable,  environmental  friendly  and   sustainable  energy  sources  are  required.  Potential  fuel  candidates  are  biomass-­‐derived  1-­‐butanol  and  next   generation  biodiesel.  They  present  several  advantages  over  traditional  and  worldwide  used  bioethanol.
These  include:
1)  biobutanol  –  can  be  blended  directly  into  gasoline  (Park  et  al.  1989),  is  highly  hydrophobic,  has  high   octane  rating  and  energy  content  (Ladisch  1991),  generates  few  volatile  organic  compound  emissions  and  is   not  highly  corrosive  making  its  distribution  through  pipelines  and  filling  stations  feasible;
2)  biodiesel  –  it  is  biodegradable,  can  be  used  in  any  diesel  vehicle,  accounts  for  significantly  less  emissions   than  standard  diesel  and  has  a  high  flash  point.
Traditionally,  biomass-­‐derived  butanol  is  produced  using  aerobic  fermentation  of  the  bacterium  Clostridium   acetobutylicum,  which  converts  carbohydrates  into  butanol  and  two  by-­‐products,  acetone  and  ethanol
(Jones  et  al.  1986;  Ezeji  et  al.  2007).  On  the  other  hand,  the  established  biodiesel  production  process  is   chemically  based  consisting  in  converting  vegetable  oil  (e.g.  soybean,  rapeseed  and  palm  oils)  or  animal  fat
(derived  from  poultry,  pork  or  beef)  into  fatty  esters  in  a  transesterification  step  with  methanol  while   generating  glycerol  as  by-­‐product  (Bajpai  D  et  al.  2006).  To  circumvent  process-­‐derived  limitations  (e.g.   formation  of  by-­‐products,  energy  and  chemicals  requirements,  geographical  and  seasonal  dependency  or   toxic  waste  water  generation)  and  enhance  yields  and  end-­‐product  titers,  different  production  workflows   based  on  yeast  as  cell  factory  (well  characterized  microorganism  and  availability  of  multiple  tools  for  its   genetic  manipulation)  are  being  pursued.  It  involves  feedstock  optimization  (e.g.  lignocellulosic  biomass),   superior  microbial  cultures/strain  generation  and  up-­‐  and  down-­‐stream  process  optimization.
In  the  global  project’s  objective  of  generating  an  yeast  cell  factory  for  butanol  production,  three  key  steps   are  currently  being  performed:
1)  reconstruct  a  functional  pathway  for  1-­‐butanol  in  Saccharomyces  cerevisiae  through  genetic  engineering,   testing  different  heterologous  enzymes  and  evaluating  pathway  performance  at  small-­‐scale  shake  flasks  or   fully  controlled  bioreactors;
2)  engineering  the  central  carbon  metabolism  of  S.  cerevisiae  for  maximizing  the  flux  towards  butanol;
3)  increase  the  tolerance  of  yeast  cells  to  1-­‐butanol  (toxic  above  a  specific  threshold)  by  evolutionary   engineering  (sequential  batch  cultures)  and  random  mutagenesis  (UV  light).
Regarding  biodiesel  production,  several  genetic  engineering  strategies  have  been  designed  to  reconstruct   the  heterologous  pathway  for  production  of  FAEEs  (biodiesel)  in  yeast.  In  addition,  the  lipid  and  central   carbon  metabolism  of  yeast  cells  is  being  engineered  to  direct  the  flux  towards  production  of  FAEEs,  thereby   maximizing  titers  and  yields.
Strategies  for  developing  yeast  strains  capable  of  converting  pentose  and  hexose  sugars  derived  from  novel   substrates  (e.g.  lignocellulosic  biomass  such  as  such  xylose  or  wood  sugar)  into  target  biofuels  at  high  yields
are  also  being  designed/evaluated.
9

Alison  Smith  (Cambridge)
Sustainable  biodiesel  production  from  algae  -­‐  from  life-­‐cycle  assessment  to  life  cycle  biology
Algal  biofuel  production  has  a  number  of  attractions,  but  there  are  many  difficulties  still  to  be  overcome   before  they  can  be  a  commercial  reality.  One  aspect  is  environmental  sustainability  which  requires  an   efficient  production  system  whose  design  is  optimised  for  maximum  output  at  minimal  costs  to  the   environment.  A  pilot-­‐scale  Life  Cycle  Analysis  (LCA)  of  a  putative  algal  biodiesel  production  process  for  the
UK  was  conducted,  comparing  growth  in  open  raceway  ponds  versus  closed  tubular  photobioreactors
(PBRs).  We  found  cultivation  in  the  typical  outdoor  system  to  be  more  environmentally  sustainable  than  in   closed  air-­‐lift  tubular  bioreactors,  with  an  estimated  global  warming  potential  (GWP)  ~  80%  lower  than   fossil-­‐derived  diesel,  whereas  in  PBRs  the  GWP  was  much  greater  than  for  fossil  fuel.  However,  for  algal   growth  in  outdoor  facilities,  biomass  yields  are  often  reduced  by  contaminating  bacteria,  predating   zooplankton,  or  undesirable  algae  competing  for  the  resources.    An  understanding  of  algal  ecology,  which   can  be  employed  to  decrease  the  detrimental  effects  of  competition,  predation  and  contamination,  is   therefore  justified.  Not  all  bacteria  lead  to  a  decrease  in  algal  growth.    Algal-­‐bacterial  symbioses  have   previously  been  shown,  a  prominent  example  being  symbiosis  of  algal  vitamin  B12  auxotrophs  with  vitamin
B12  producing  bacteria.    Half  of  all  algal  species  have  an  obligate  requirement  for  vitamin  B12  for  growth,   and  none  can  synthesise  it  (Croft  et  al.  2005  Nature  438:90-­‐93).  We  have  shown  that  growth  of  vitamin  B12   dependent  algae  can  be  rescued  by  symbiotic  vitamin  B12  producing  bacteria.  We  investigated  the  dynamics   of  algae  (Lobomonas  rostrata)  and  bacteria  (Mesorhizobium  loti)  grown  under  symbiosis-­‐requiring   conditions.  The  two  organisms  were  observed  to  form  a  stable  equilibrium  in  terms  of  population  numbers   that  can  be  maintained  over  many  generations,  although  it  was  possible  to  perturb  it  by  addition  of  either   vitamin  B12  or  a  carbon  source  for  the  bacteria.  We  modelled  the  growth  of  algal  and  bacterial  populations   mathematically,  and  found  that  independent  growth  could  not  explain  growth  kinetics.  Parameters  for   nutrient  exchange  were  required  that  indicated  a  degree  of  species  interdependence,  lending  credence  to   the  model  of  symbiosis  that  we  developed.  The  stability  of  the  equilibrium  suggests  a  robustness  of  culture,   which  may  perhaps  be  more  stable  against  competitors.  We  have  begun  to  test  this  in  a  series  of
competition  experiments.
Alexander  Steinbüchel  (Münster)
Plasmid  addiction  systems  designed  to  allow  stable  production  of  products  during  microbial  fermentations
Biotechnological  production  processes,  which  rely  on  fermentations  of  microorganisms,  often  depend  on   foreign  genetic  information  located  on  plasmids.  Plasmids  are  separate  genetic  elements  and  autonomously   replicated  from  the  chromosomes.  Genetically  engineered  microorganisms  produce  important  chemicals,   biopolymers,  biofuels  and  high  value  proteins  like  insulin.  The  success  of  plasmid-­‐based  microbial  production   systems  significantly  depends  among  others  on  plasmid  stability.  Frequently  used  plasmids  harbour   antibiotic  resistance  genes  and  require  the  addition  of  antibiotics  to  the  cultivation  medium  for  plasmid   maintenance.  Whereas  this  procedure  is  feasible  at  the  laboratory  scale,  it  is  not  applicable  at  large  scale   cultivations  in  industry  due  to  the  high  costs  and  due  to  ecological  constraints.  Chromosomal  gene   integration  is  just  one  strategy  to  stabilize  foreign  genes.
Another  strategy  is  the  application  of  plasmid  addiction  systems  (PAS).  This  lecture  provides  an  overview  on
PAS,  which  could  be  used  in  microorganisms.    Examples  for  the  use  of  PAS  for  the  production  of  the   biopolymer  cyanophycin  will  be  provided.  However,  these  PAS  are  also  applicable  to  the  production  of  other   compounds.  Two  different  types  of  plasmid  addiction  systems  were  engineered:  (i)  In  catabolism-­‐based  PAS   a  gene  encoding  a  key  enzyme  essentially  required  for  the  utilization  of  a  carbon  source  is  deleted  in  the   chromosome  but  provided   in  trans  on  the  plasmid  that  encodes  also  the  cyanophycin  synthesis  gene.
Alternatively,  the  gene(s)  coding  for  an  alternative  pathway  to  utilize  this  carbon  source  are  provided  on  the   plasmid.  In   Ralstonia  eutropha  two  PAS  based  on  a  chromosomally  deleted  KDPG  aldolase  gene  ( eda )  with   the  same  gene  provided  on  the  plasmid  or  with  the  gene  for  a  bifunctional  xylulose-­‐5-­‐phosphate/fructose-­‐6-­‐ phosphate  phosphoketolase  gene  ( xfp )  from   Bifidobacterium  animalis ,  respectively,  were  engineered.    After
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having  in  the  past  established  the  Embden-­‐Meyerhof  pathway  for  carbohydrate  catabolism  for  a  second   time  a  completely  different  pathway  for  a  new  fructose  catabolism  route  was  thereby  functionally   established  in   R.  eutropha .  (ii)  In  anabolism-­‐based  PAS  a  gene  or  group  of  genes  encoding  genes  required  for   the  biosynthesis  of  an  essential  cell  component  were  deleted  in  the  chromosome,  but  a  gene  for  an   alternative  enzyme  or  a  group  of  genes  for  an  alternative  pathway  were  provided   in  trans  on  the  plasmid   encoding  the  cyanophycin  synthesis  gene.  In   Escherichia  coli  one  PAS  based  on  a  disrupted  succinylase   pathway  for  lysine  biosynthesis  with  deleted   dapE  gene,  which  was  complemented  by  the  plasmid  encoded   artificial  aminotransferase  pathway  mediated  by  the   dapL  gene,  was  engineered.  A  second  PAS  was  based   on  a  disrupted  4-­‐hydroxy-­‐3-­‐methylbut-­‐2-­‐enyl  diphosphate  reductase  gene  ( ispH )  yielding  an  impaired   deoxyxylulose  5-­‐phosphate  pathway  for  isopentenyl  pyrophosphate  (IPP)  biosynthesis.  In  this   E.  coli  mutant
a  completely  synthetic  and  episomal  mevalonate  pathway  was  established  for  provision  of  the  essential  IPP.
Ralf  Steuer  (Manchester)
Systems  Biology  of  Cyanobacterial  Biofuel  Production
To  overcome  the  dependency  on  fossil  fuels,  alternative  sources  of  environmentally  safe,  renewable  and   affordable  energy  are  urgently  needed.  Biofuels  derived  from  plants  and  other  biological  materials  offer  a   potential  alternative  as  transportation  fuels,  but  are  currently  not  available  in  large  volumes  at  an  affordable   price.
Within  the  framework  of  FORSYS-­‐Partner,  we  have  established  a  systems  biology  research  initiative  to   explore  the  efficient  and  economic  production  of  biofuels  using  phototrophic  microorganisms.  Our  approach   is  to  combine  photosynthesis  with  the  synthesis  of  ethanol  in  one  cyanobacterial  cell.  An  integral  part  of  our   initiative  is  to  establish  a  systemic  understanding  of  selected  oxygenic  photosynthetic  prokaryotes,  based  on     experimental  characterization  of  photosynthetic  growth,  including  metabolomic  and  transcriptomic  analyses   combined  with  mathematical  modeling.
The  focus  of  our  project  is  Synechocystis  sp.  PCC  6803,  a  widely  used  model  organism  for  the  study  of   photosynthetic  and  metabolic  processes  and  their  regulation.  With  a  rich  compendium  of  genomic,   biochemical  and  physiological  data  available,  this  unicellular  cyanobacterium  is  an  ideal  candidate  for  a   systems-­‐level  description  of  primary  metabolism  and  its  regulation.  We  present  an  iterative  construction  of   a  computational  model  that  serves  as  a  first  step  towards  a  comprehensive  computational  description  of   cellular  metabolism  in  unicellular  autotrophs  and  will  describe  recent  advances  to  engineer  cyanobacteria  as   hosts  for  the  production  of  third  generation  biofuels.
Wim  Vermaas  (Arizona  State  University)
Solar-­‐Powered  Production  of  Biofuels  by  Cyanobacteria:  Stoichiometry  of  Reducing  Equivalents  and
Chemical  Energy,  and  Energy  Conversion  Efficiency
Cyanobacteria  are  a  promising  platform  for  solar-­‐powered,  CO
2
-­‐consuming  production  of  biofuels  and  other   useful  products  using  photosynthesis.    Efficient  production  of  such  compounds  requires  that  the   stoichiometry  of  reducing  equivalents  (NADPH)  and  chemical  energy  (ATP)  produced  as  a  result  of   photosynthetic  electron  transport  is  well-­‐matched  by  the  stoichiometry  of  reducing  equivalents  and   chemical  energy  required  for  production  of  the  desired  compounds.    Stoichiometry  requirements  are  met   when  linear  photosynthetic  electron  transport  is  used  to  produce  compounds  via  the  fatty  acid  or  isoprenoid   biosynthesis  pathways.    In  the  case  of  fatty  acid  production,  the  amount  of  energy  stored  in  the  fatty  acid   can  be  up  to  28%  of  the  energy  of  the  light  if  one  were  to  excite  with  680  nm  light  and  all  absorbed  light  was   used  for  fatty  acid  production.    Making  adjustments  for  solar  illumination  (only  ~50%  of  the  energy  can  be   used  for  photosynthesis),  blue-­‐photon  utilization,  and  losses  due  to  non-­‐photochemical  quenching  and  the   requirements  for  maintenance  energy,  the  solar  energy  conversion  efficiency  may  still  be  in  the  range  of
~7%,  which  is  superior  to  most  other  bio-­‐based  approaches.    However,  photohydrogen  production  that   directly  uses  reducing  equivalents  from  photosynthetic  electron  transfer  for  H
2
production  does  not  require
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ATP  and  thereby  is  not  properly  balanced  stoichiometrically.    An  additional  complexity  of  H
2
production  in   relatively  small  cyanobacterial  cells  at  somewhat  alkaline  pH  is  that  the  number  of  free  protons  in  a  cell  is   extremely  limited  (a  few  protons  per  cyanobacterial  cell  of  1  fL  at  pH  8.0).    However,  regardless  the  inherent   difficulties  of  light-­‐driven  H
2
production  in  cyanobacteria,  the  utilization  of  cyanobacteria  for  light-­‐driven
generation  of  carbon-­‐based  biofuels  and  related  products  can  be  efficient  and  is  very  promising.
Percival  Zhang  (Virginia)
Replacing  crude  oil  with  sugar  (before  we  run  out  of  oil)
Economically  viable  production  of  biofuels  from  renewable  energy  sources  are  typical  goal-­‐oriented  projects   with  numerous  (hidden)  constraints,  such  as  efficiency,  cost,  rate,  scale,  implementation  time,  competing   technology,  technology  maturation,  environmental,  safety,  and  so  on.    As  a  biochemical  engineer,  my   research  integrates  engineering  design  principles  with  protein  biochemistry,  microbiology,  and  modern   biotechnology  to  address  key  challenges  in  the  sustainability  revolution.  Since  cellulosic  ethanol  and  butanol   may  be  the  best  biofuel  candidate  in  short  terms.    To  overcome  biomass  recalcitrance,  we  are  developing   recombinant  cellulolytic   Bacillus  subtilis  that  can  produce  engineered  (non-­‐natural)  cellulase  complexes,   hydrolyze  cellulose,  and  produce  desired  biofuels  in  one  step.    We  have  created  the  first  real  recombinant   cellulolytic  microorganism  that  can  grow  on  solid  cellulose  by  using  its  recombinant  cellulase  without  help  of   any  other  organic  nutrient.
Since  hydrogen  is  an  ultimate  fuel  in  the  transport  sector,  we  have  designed  synthetic  enzymatic  pathways   that  can  produce  12  mol  hydrogen  per  glucose  unit  and  water  for  the  first  time.      In  addition  to  potentially   low-­‐cost  renewable  hydrogen  production,  high-­‐purity  hydrogen  produced  by  cascade  enzymes  may  also   solve  hydrogen  storage,  distribution,  and  safety  problems.    We  strongly  recommend  that  high-­‐product  yield   and  potentially  low-­‐cost  biofuels  will  be  produced  by  cell-­‐free  synthetic  enzymatic  pathway   biotransformation  (SyPaB)  that  can  implement  complicated  biochemical  reactions  that  living  entities  cannot   achieve  through   in  vitro  assembly  of  stable  enzymes  and  (biomimetic)  coenzymes.    Great  potential  markets   of  SyPaB,  include  chiral  compounds,  biodegradable  sugar  batteries,  sulfur-­‐free  jet  fuel,  hydrogen,  sugar   hydrogen  fuel  cell  vehicles,  high-­‐density  electricity  storage,  and  synthetic  starch,  are  motivating  to  solve
remaining  technical  obstacles.    The   in  vitro   assembly  of  numerous  enhanced  performance  and  stable   enzymes  in  one  bioreactor  that  can  last  a  very  long  reaction  time  (e.g.,  several  months)  would  be  an  out-­‐of-­‐ the-­‐box  solution  for  high-­‐yield  and  ultra-­‐low-­‐cost  biofuels  production.
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