The  impact  of  orthogonal  gene  expression  on  heterologous  pathway  productivity  

 
The   goals   of   metabolic   engineering   are   often   at   odds   with   the   hostâ??s   own   cellular  
objectives.   For   example,   the   heterologous   overproduction   of   a   secondary   metabolite  
such  as  lycopene  in  Escherichia  coli  directly  competes  with  the  growth  objective  of  E.  coli  
by   diverting   carbon   flux   away   from   the   native   metabolic   network   (i.e.,   biomass  
formation)  and  toward  lycopene  accumulation  through  the  DXP  pathway.  A  significant  
amount   of   work   in   the   metabolic   engineering   community   has   been   carried   out   to  
address   this   problem   largely   through   investigating   carbon   and   redox   balancing  
between   heterologous   pathways   and   the   hostâ??s   metabolism.   While   this   is   essential   for  
successful   pathway   engineering,   very   little   attention   has   been   given   to   balancing   the  
energy  and  material  flux  through  the  process  of  heterologous  gene  expression,  which  is  
typically   shared   with   the   host.   That   is,   heterologous   expression   traditionally   relies   on  
native  RNA  polymerases  and  ribosomes.  If  the  energetic  cost  or  burden  of  expression  is  
at  least  partially  due  to  the  process  of  gene  expression  itself,  and  not  solely  the  product  
(e.g.,   pathway   enzymes),   then   expressing   a   heterologous   pathway   using   orthogonal  
cellular   machinery   (i.e.,   RNAPs   and   ribosomes   that   act   only   on   the   heterologous  
pathway   genes)   should   be   less   costly   to   the   host   than   expressing   the   same   pathway  
enzymes   via   native   transcription   and   translation.   In   particular,   orthogonal   gene  
expression  systems  that  provide  a  stable,  dedicated  supply  of  cellular  machinery  should  
alleviate   burden   and   increase   growth   rate   (and   therefore   productivity)   if   the  
concentrations  of  available  native  RNA  polymerases  and  ribosomes  in  the  cell  are  rate  
limiting.   Building   from   recent   work   in   synthetic   biology   that   has   demonstrated  
functional   orthogonal   transcription-­â?â??translation   processes,   our   work   experimentally  
investigates   the   impact   of   orthogonally   expressing   three   heterologous   enzymes   for  
lycopene  biosynthesis  in  E.  coli  on  fermentation  productivity,  yield  and  titer.