Page 521 - Refining Biomass Residues for Sustainable Energy and Bioproducts

Blue biotechnology: a vision for future marine biorefineries 475

in DNA leading to enhanced production of the desired metabolite. Rather than focus-
ing on a single gene, the pathway leading to the gene can also be modified rationally
with the help of metabolic engineering approaches. However, one of the prerequisites
for metabolic engineering is knowledge on the whole genome, transcriptomes, pro-
teins, and metabolites of the promising species. Although this was particularly a seri-
ous problem in blue biotechnology decades ago, the advancement in scientific tools
had provided fruitful results in recent times. Such modern toolkits include next-
generation sequencing, advanced mass spectrometric, and isotope labeling methods.
Thus the potentiality of metabolic engineering has started to unfold in recent times.
The following section explains the steps involved in a typical metabolic engineering
approach. The first step is the formation of metabolic pathway map representing all
intracellular and extracellular reactions in a species. If species are sequenced and genes
are annotated, this can be usually obtained from online repositories like Kyoto encyclo-
pedia of genes and genomes (KEGG) database (Ogata et al., 1999). However, for
unknown species, DNA has to be first sequenced and, subsequently, genes have to be
annotated. Each reaction in the metabolic pathway map should contain all the reactants
and products with proper stoichiometric coefficients. After which, mass balances are
applied around intracellular reactants and products. Finally, the measured values com-
prising the rates of substrate uptake and extracellular product formation are used for
flux calculation. The end result is the generation of the complete metabolic pathway of
given species with information on intracellular and extracellular fluxes.
After preparation of the metabolic pathway model, constraints including nutrient
uptake, intracellular flux level, growth media component’s concentration, thermo-
dynamic reaction limit, and experimentally measured flux levels can be added.
Finally, the flux model is provided with a goal of maximizing the objective func-
tion, typically this is biomass. Software are available for the development and anal-
ysis of flux models including Pathway Tools/MetaFlux, Simpheny, MetNetMAker,
COBRA toolbox, Surrey FBA, and Web-based FAME (the Flux Analysis and
Modeling Environment) (Copeland et al., 2012).
The generation of metabolic pathway map of given species has various applica-
tions. One of them is the identification of nodal rigidity. The knowledge on nodal
rigidity provides the users with information on the extent of flux ratio changes pos-
sible in a branch present in the metabolic pathway. In addition, applications includ-
ing alternate pathway identification, measurement of nonquantifiable extracellular
fluxes, and prediction of maximum yield of the desired product can also be per-
formed. Moreover, the effect of gene deletion on pathway fluxes and resulting
changes in the level of the desired metabolite can be simulated and validated using
metabolic engineering flux-model approaches and related analytical techniques.

21.7 Environmental considerations and social reflections

Species present in the ocean are highly diverse and abundant. They are a source of
important chemicals and valuable medicines. Successful establishment of the effi-
cacy of the product always leads to the major environmental problem of