# Analysis of Poly-3-Hydroxybutyrate Production with Different Microorganisms Using the Dynamic Simulations for Evaluation of Economic Potential Approach

## TL;DR

This paper introduces a new method called DySEEP to evaluate the economic potential of producing biodegradable plastics like PHB using different microorganisms.

## Contribution

The novel contribution is the DySEEP approach, which combines dynamic simulations with economic metrics to optimize bioplastic production.

## Key findings

- For recombinant E. coli, a PHB yield of 0.37 g/g leads to positive cash flow, and 0.50 g/g maximizes theoretical profit.
- Genetic modifications such as knocking out specific metabolic pathways could enhance PHB production in E. coli.
- Optimal bioreactor strategies suggest using 20% of glucose in the growth phase and 80% in the production phase for maximum profit.

## Abstract

Concerns with sustainability have led to increasing interest
in
bioprocesses in the last decades. In particular, environmental problems
with plastic disposal have been a major issue. Bioplastics such as
poly-3-hydroxybutyrate (PHB) are potential substitutes since they
are biodegradable and less toxic. However, their production costs
are high and optimization is required. Many works have therefore aimed
to build strains capable of higher product yields. But in the case
of products that share a precursor with biomass and in the case of
intracellular metabolites, a trade-off may occur. Not only yield but
also final biomass, titer, and productivity have to be considered.
Therefore, this work presents an approach named Dynamic Simulations
for Evaluation of Economic Potential (DySEEP), which uses dynamic
flux balance analysis (DFBA) and an economic metric as a function
of the bioprocess parameters for evaluation of the production of bioproducts
of industrial interest. As a case study, the PHB production potential
of recombinant Cupriavidus necator, Escherichia coli, and Saccharomyces
cerevisiae was analyzed. While some key polymer properties
cannot be predicted due to the nature of DFBA simulations, the PHB
production is a good case study to highlight the trade-off between
biomass and product formation. It was identified that for growth-associated
production with recombinant E. coli and the NADPH-dependent PHB synthesis pathway, the scenario that
starts positive cash flows is when a yield of 0.37 g/g and its respective
final biomass, titer, and productivity is achieved, and the maximum
theoretical profit would be achieved when a yield of 0.50 g/g and
its respective parameters is reached. Furthermore, comparison between
the internal flux distribution of the best scenario identified and
the flux distributions of a simulation constrained with experimental
data from the literature allowed the suggestion of genetic modifications
that could enhance PHB production, such as knockouts for interruption
of the oxidative phase of the pentose phosphate pathway, of the acetate
production reaction, and of the reaction catalyzed by the 2-oxoglutarate
dehydrogenase enzyme or downregulation of the TCA cycle, setting therefore
potential targets for metabolic engineering strategies. For nongrowth-associated
production with both recombinant E. coli and C. necator, the scenario at which
cash flow starts to become positive is when 40% (mol) of the available
glucose is used in the growth phase and the remaining 60% is used
in the production phase, and the scenario that leads to the maximum
theoretical profit, within a realistic maximum PHB content, is when
20% (mol) is used in the growth phase and the remaining 80% in the
production phase, information that sets targets for bioreactor operation
strategies such as defining the moments for nutrient limitation and
for synthetic biology by showing when to activate genetic toggle-switches.