Systems Biotechnology: Combined in Silico and Omics Analyses for the Improvement of Microorganisms for Industrial Applications - Systems Biology: Networks, Models, and Applications - page 218

Table 7.2 Results of flask cultures of recombinant E. coli strains producing
PHB a

Dry cell PHB conc. PHB content

Strain (Plasmid b ) Medium c weight (g/l) (g/l) (wt%)

KS272 (pJC4 + pACYC184) LB 4.13 3.12 75.9

KEDA (pJC4 + pACYC184) LB 4.40 2.70 61.9

KEDA (pJC4 + pAC104Eda) LB 6.10 4.40 72.5

KS272 (pJC4 + pACYC184) MR 4.30 2.50 60.0

KEDA (pJC4 + pACYC184) MR 4.50 2.00 44.0

KEDA (pJC4 + pAC104Eda) MR 6.20 4.00 64.5

a These data were taken from [95].
b pJC4 is a high copy number plasmid harboring the A. latus PHB biosynthesis operon.
pAC104Eda is a pACYC184 derivative harboring the eda gene. Plasmids pJC4 and pACYC184
are compatible in E. coli .
c Glucose was added to 20 g/l in both media. Abbreviations are: LB medium, Luria-Bertani
medium [101]; MR medium, a chemically defined medium [102].

These results verify the essential role of the ED pathway during
PHB biosynthesis in recombinant E. coli as predicted by in silico exper-
iment. The increase in the ED pathway flux during PHB production
is also experimentally supported by the proteome analysis of a different
E. coli strain, XL1-Blue, which showed an increase in Eda expression
level under PHB-producing condition [31]. It should be noticed that
there exists the possibility that metabolic pathways might be differ-
ently controlled in different E. coli strains, which can also be studied
in a similar way. This case study demonstrates the effectiveness of the
systems biotechnological approach to elucidate previously unknown
metabolic characteristics.

CASE STUDY 4: INCREASING THE METABOLIC FLUX TO SUCCINIC ACID IN
E. coli BY SYSTEMS BIOTECHNOLOGICAL RESEARCH CYCLE

There have been many successful cases of the development of meta-
bolically engineered E. coli strains for the production of succinic acid
(C4), which is used as a food additive, an ion chelator, and a precursor
of polymers, and has been chemically produced from maleic anhydride
[98]. Recently, metabolic engineering of E. coli by heterologous gene
expression (the pyc gene encoding pyruvate carboxylase), amplification
of an inherent gene (the ppc gene encoding phosphoenolpyruvate
carboxylase), and deletion of several genes ( ptsG , ldhA , pfl ) has shown
the possibility for the biotechnological production of succinic acid. In
this case study, the aim is to develop an E. coli strain that is able to over-
produce succinic acid by means of comparative genomics and
metabolic flux prediction, followed by subsequent gene knockout and
cultivation experiments. This approach follows the systems biotechno-
logical research cycle toward stain improvement as shown in figure 7.12.