
 e.  coli 10beta ( new england biolabs inc ) was used for cloning and plasmid propagation . to select e.  coli and p.  putida transformants ,
l 
  ( for e.  coli ) or 50  mg  l 
  ( for p.  putida ) . for induction of the rhapbad promoter , 0.2  % ( w / v ) filteredsterilized lrhamnose was added to the culture media .
alkbgt expression was induced by the addition of 0.025  % ( v / v ) dicyclopropyl ketone ( dcpk ) .
recombinant dna work was carried out according to standard protocols.15 a shuttle vector for replication in e.  coli and p.  putida was constructed from the cloning vector pacyc184 .
this new vector contains the pvs1origin for replication in p.  putida , with the chloramphenicol resistance gene replaced by a kanamycin resistance gene . to produce rhamnolipids , a synthetic operon consisting of the rhlabc genes from p. 
this operon was ligated into the new shuttle vector under the control of the rhapbad promoter .
the pbt10 plasmid contained the alkane monooxygenase genes ( alkbfg and alkst operons ) in the broadhost range vector pcom10 ( km).16 the plasmids were transferred into e.  coli and p.  putida kt2440 ( atcc 47054 ) through electroporation , finally yielding strain boapp002 . to determine the product concentration in the different examples ,
the fermentation broth was diluted immediately after sampling with acetone at a volume ratio of 1:1 and centrifuged at 21  000  g and 4  c for 2  min .
the sample supernatant was then subjected to hplc analysis [ agilent technologies 1200 series ; evaporative light scattering detector sedex ltelsd model 85lt ; zorbax sbc8 rapid resolution column ( 4.6150  mm , 3.5  m ) ] .
mobile phase  a : aqueous 0.1  % tfa ; mobile phase  b : methanol .
gradient : start with 70  % b in a to 100  % b within 15  min at a flow rate of 1  ml  min followed by 5  min of reequilibration with 70  % b in a. reference materials were used , and the identity and purity were checked by using hplc 
agar plate containing 50  mg  l 
  each of tetracycline and kanamycin , an inoculation loop full of glycerol cryoculture of strain p.  putida boapp002 was streaked .
nine 100  ml shake flasks were filled with 25  ml of lb medium containing 50  mg  l 
  each of tetracycline and kanamycin , and each was inoculated with a single colony of the overgrown agar plate and incubated in a shaking incubator for 24  h at 30  c and 200  rpm .
nine 1  l flasks were each used to mix 75  ml of m9 medium ( composition : 15  g  l 
  glucose , 6.8  g  l 
  na2po4 , 3  g  l 
  kh2po4 , 0.5  g  l 
  nacl , 2  g  l 
  nh4cl , 15  g  l 
  yeast extract , 0.49  g  l 
  mgso47  h2o , 50  mg  l 
  kanamycin sulfate , 15  ml  ll traceelement solution us3 consisting of 36.5  g  l 
  of 37  % strength hydrochloric acid , 11.91  g  l 
  mncl24  h2o , 1.87  g  l 
  znso47  h2o , 0.84  g  l 
  naedta2  h2o , 0.3  g  l 
  h3bo3 , 0.25  g  l 
  na2moo42  h2o , 4.7  g  l 
  cacl22  h2o , 17.3  g  l 
  feso47  h2o , 0.15  g  l 
  cucl22  h2o ) and the preculture from the 100  ml flasks .
the cultures were incubated at 30  c and 200  rpm . after 3  h of incubation , expression of the alkbgt genes
the cultures were incubated for a further 16  h at 30  c and 200  rpm .
the cultures of the nine 1  l flasks were combined and centrifuged at 5500  g at room temperature for 10  min .
the supernatant was discarded and the pellet was resuspended in 100  ml of modified m9 medium ( composition as provided above , without glucose and yeast extract ) .
this washing step was repeated for the complete removal of glucose and other possible carbon sources . in three 300  ml fermenters , 180  ml of modified m9 medium ( composition of which
is provided above , without glucose and yeast extract ) as well as 50  mg  l 
  each of tetracycline and kanamycin were added .
the fermenters were inoculated with a large volume of preculture suspension from the earlier step to reach a start od600 of 4 for the experiments with butyrate ( a ) and 1butanol ( b ) , or a start od600 of 8 for the experiment with nbutane ( c ) .
the following parameters were set during fermentation : temperature 30  c ( all ) , gassing with air 3  nl  h ( a and b ) or with nbutane / air mixture ( 25  % /75  % ) 2  nl  h ( c ) , stirrer speed set at 700  rpm ( a and b ) or 900  rpm ( c ) , ph value of 7.1 regulated with 1  m phosphoric acid ( a ) or with 5  % ammonia solution ( b ) or an initial ph value of 7.4 , not regulated throughout the experiment ( c ) . a continuous feed with sodium butyrate solution ( a : 0.25  g  l 
 h ) or 1butanol solution ( b : 0.17  g  l 
 h ) was applied .
after 15  h ( a and b ) or 18  h ( c ) , the expression of rhamnolipid production genes was induced by adding 2  g  l 
  of rhamnose .
all experiments were run in duplicate and the average values for rhamnolipid titers are given .
for aeration , stainlesssteel filter / sparger tips with an outer diameter of 6.35  mm , a length of 25.4  mm , and a pore size of 10  m were used .
the used nbutane / air mixture ( 25  % /75  % ) ensures that the reaction always takes place under nonexplosive conditions .

 e.  coli 10beta ( new england biolabs inc ) was used for cloning and plasmid propagation . to select e.  coli and p.  putida transformants ,
l 
  ( for e.  coli ) or 50  mg  l 
  ( for p.  putida ) . for induction of the rhapbad promoter , 0.2  % ( w / v ) filteredsterilized lrhamnose was added to the culture media .
alkbgt expression was induced by the addition of 0.025  % ( v / v ) dicyclopropyl ketone ( dcpk ) .
recombinant dna work was carried out according to standard protocols.15 a shuttle vector for replication in e.  coli and p.  putida was constructed from the cloning vector pacyc184 .
this new vector contains the pvs1origin for replication in p.  putida , with the chloramphenicol resistance gene replaced by a kanamycin resistance gene . to produce rhamnolipids , a synthetic operon consisting of the rhlabc genes from p. 
this operon was ligated into the new shuttle vector under the control of the rhapbad promoter .
the pbt10 plasmid contained the alkane monooxygenase genes ( alkbfg and alkst operons ) in the broadhost range vector pcom10 ( km).16 the plasmids were transferred into e.  coli and p.  putida kt2440 ( atcc 47054 ) through electroporation , finally yielding strain boapp002 .

 e.  coli 10beta ( new england biolabs inc ) was used for cloning and plasmid propagation . to select e.  coli and p.  putida transformants ,
l 
  ( for e.  coli ) or 50  mg  l 
  ( for p.  putida ) . for induction of the rhapbad promoter , 0.2  % ( w / v ) filteredsterilized lrhamnose was added to the culture media .
alkbgt expression was induced by the addition of 0.025  % ( v / v ) dicyclopropyl ketone ( dcpk ) .
recombinant dna work was carried out according to standard protocols.15 a shuttle vector for replication in e.  coli and p.  putida was constructed from the cloning vector pacyc184 .
this new vector contains the pvs1origin for replication in p.  putida , with the chloramphenicol resistance gene replaced by a kanamycin resistance gene . to produce rhamnolipids , a synthetic operon consisting of the rhlabc genes from p. 
this operon was ligated into the new shuttle vector under the control of the rhapbad promoter .
the pbt10 plasmid contained the alkane monooxygenase genes ( alkbfg and alkst operons ) in the broadhost range vector pcom10 ( km).16 the plasmids were transferred into e.  coli and p.  putida kt2440 ( atcc 47054 ) through electroporation , finally yielding strain boapp002 .
to determine the product concentration in the different examples , the fermentation broth was diluted immediately after sampling with acetone at a volume ratio of 1:1 and centrifuged at 21  000  g and 4  c for 2  min .
the sample supernatant was then subjected to hplc analysis [ agilent technologies 1200 series ; evaporative light scattering detector sedex ltelsd model 85lt ; zorbax sbc8 rapid resolution column ( 4.6150  mm , 3.5  m ) ] .
mobile phase  a : aqueous 0.1  % tfa ; mobile phase  b : methanol .
gradient : start with 70  % b in a to 100  % b within 15  min at a flow rate of 1  ml  min followed by 5  min of reequilibration with 70  % b in a. reference materials were used , and the identity and purity were checked by using hplc 
on an lysogeny broth ( lb ) agar plate containing 50  mg  l 
  each of tetracycline and kanamycin , an inoculation loop full of glycerol cryoculture of strain p.  putida boapp002 was streaked .
nine 100  ml shake flasks were filled with 25  ml of lb medium containing 50  mg  l 
  each of tetracycline and kanamycin , and each was inoculated with a single colony of the overgrown agar plate and incubated in a shaking incubator for 24  h at 30  c and 200  rpm .
nine 1  l flasks were each used to mix 75  ml of m9 medium ( composition : 15  g  l 
  glucose , 6.8  g  l 
  na2po4 , 3  g  l 
  kh2po4 , 0.5  g  l 
  nacl , 2  g  l 
  nh4cl , 15  g  l 
  yeast extract , 0.49  g  l 
  mgso47  h2o , 50  mg  l 
  kanamycin sulfate , 15  ml  ll traceelement solution us3 consisting of 36.5  g  l 
  of 37  % strength hydrochloric acid , 11.91  g  l 
  mncl24  h2o , 1.87  g  l 
  znso47  h2o , 0.84  g  l 
  naedta2  h2o , 0.3  g  l 
  h3bo3 , 0.25  g  l 
  na2moo42  h2o , 4.7  g  l 
  cacl22  h2o , 17.3  g  l 
  feso47  h2o , 0.15  g  l 
  cucl22  h2o ) and the preculture from the 100  ml flasks .
the cultures were incubated at 30  c and 200  rpm . after 3  h of incubation , expression of the alkbgt genes
the cultures were incubated for a further 16  h at 30  c and 200  rpm .
the cultures of the nine 1  l flasks were combined and centrifuged at 5500  g at room temperature for 10  min . the supernatant was discarded and the pellet was resuspended in 100  ml of modified m9 medium ( composition as provided above , without glucose and yeast extract ) .
this washing step was repeated for the complete removal of glucose and other possible carbon sources . in three 300  ml fermenters ,
180  ml of modified m9 medium ( composition of which is provided above , without glucose and yeast extract ) as well as 50  mg 
the fermenters were inoculated with a large volume of preculture suspension from the earlier step to reach a start od600 of 4 for the experiments with butyrate ( a ) and 1butanol ( b ) , or a start od600 of 8 for the experiment with nbutane ( c ) .
the following parameters were set during fermentation : temperature 30  c ( all ) , gassing with air 3  nl  h ( a and b ) or with nbutane / air mixture ( 25  % /75  % ) 2  nl  h ( c ) , stirrer speed set at 700  rpm ( a and b ) or 900  rpm ( c ) , ph value of 7.1 regulated with 1  m phosphoric acid ( a ) or with 5  % ammonia solution ( b ) or an initial ph value of 7.4 , not regulated throughout the experiment ( c ) . a continuous feed with sodium butyrate solution ( a : 0.25  g  l 
 h ) or 1butanol solution ( b : 0.17  g  l 
 h ) was applied .
after 15  h ( a and b ) or 18  h ( c ) , the expression of rhamnolipid production genes was induced by adding 2  g  l 
  of rhamnose .
all experiments were run in duplicate and the average values for rhamnolipid titers are given .
for aeration , stainlesssteel filter / sparger tips with an outer diameter of 6.35  mm , a length of 25.4  mm , and a pore size of 10  m were used . the used nbutane / air mixture
( 25  % /75  % ) ensures that the reaction always takes place under nonexplosive conditions .
 abstractmicrobially derived surfactants , socalled biosurfactants , have drawn much attention in recent years and are expected to replace current petrochemical surfactants , owing to their environmental and toxicological benefits . 
 one strategy to support that goal is to reduce production costs by replacing relatively expensive sugars with cheaper raw materials , such as shortchain alkanes . 
 herein , we report the successful onepot total synthesis of rhamnolipids , a class of biosurfactants with 12  stereocenters , from butane as sole carbon and energy source through the design of a tailored wholecell biocatalyst . 
Experimental Section
Strain Construction
Bacterial Strains
DNA Methods
Analytics
Production of Rhamnolipids by using BOAPP002 and Butyric Acid, 1Butanol, or 