Production of Aromatic Compounds in E. coli Strains Lacking Interconversion of PEP and Pyr When Glucose and Acetate Are Coutilized

Phosphoenolpyruvate
(PEP) is a precursor involved in the biosynthesis of aromatics and other
valuable compounds in <i>E. coli </i>. However, the PEP:carbohydrate
phosphotransferase system (PTS), is the largest PEP consumer. In this regard,
our group has generated <i>E. coli </i> JM101 mutants devoid of PTS
by deleting the <i>ptsHIcrr</i>operon (PB11 and PB12
strains), a strategy that, in theory, may double PEP availability. In these <i>ptsHIcrr^- </i> strains, the glycolytic and gluconeogenic pathways
function simultaneously, allowing the coutilization of secondary carbon sources
in the presence of glucose due to the absence of the EIIA ^Glc component. Taking into account this capacity, the
physiological and transcriptional response of blocking carbon skeletons
interchange between PEP and pyruvate (PYR) in these <i>ptsHIcrr^- </i> strains was investigated by deleting the <i>pykA</i>, <i>pykF</i> and <i>ppsA</i> genes, during simultaneous utilization of
glucose and acetate. It was shown that under this condition, in the PB11 <i>pykAF^- ppsA^-</i> strain
glycolysis and the TCA cycle appear to coexist independently. The expression profile of this derivative showed that all metabolic central
pathways are downregulated in the mixture. Apparently, the increase in PEP
availability could inhibit some glycolytic genes. In contrast, a partial separation of glycolysis and
the TCA cycle was achieved in the PB12 <i>pykAF^- ppsA^-</i> strain,
which upregulates the <i>aceBAK</i> operon and the
<i>sfcA</i> gene in
order to reroute the local flux towards the synthesis of PYR. In order to determine the effects of the
modifications at the PEP-PYR node on PEP availability, <i>ptsHIcrr^- pykAF^- ppsA^-</i> engineered derivatives were generated and tested for total aromatic
compounds (TAC) production. The engineered PB12 <i>pykAF^- ppsA^- tyrR^- pheA^ev2+</i>/pJLB<i>aroG^fbrtktA</i> derivative
achieved a 4-fold higher TAC yield on glucose and acetate (Y_TAC/Glc+Ace) compared with its control strain, representing 65%
of the theoretical maximum. In contrast, in the PB11 <i>pykAF^- ppsA^- tyrR^- pheA^ev2+</i>/pJLB<i>aroG^fbrtktA</i> derivative there was no benefit on
aromatics production since this strain reduced its q_Glc by 47%,
and it could cause lower intracellular PEP concentrations. However, when we
overexpressed the <i>glk</i> and
<i>galP</i> genes in the
engineered PB11 derivative in order to increase glucose consumption, the PB11 <i>pykAF^- ppsA^- tyrR^- pheA^ev2+</i>/pJLB<i>aroG^fbrtktA</i>/pv5GalP5Glk
strain increased 3-fold its Y_TAC/Glc+Ace compared with its control strain, representing 48% of
the theoretical maximum. Furthermore, the higher q_Glc of the former allowed an increase of
6-fold in the Y_TAC/Glc+Ace with respect to the PB11 <i>pykAF^- ppsA^- tyrR^- pheA^ev2+</i>/pJLB<i>aroG^fbrtktA</i>, the same derivative without the <i>glk</i> and <i>galP</i> genes.