Research attention is shifting towards renewable products made from microbial organisms altered using recombinant DNA technology. In metabolic engineering, these processes are optimized through iterative synthesis and analysis of strains. Photoautotrophic cyanobacteria have been investigated as host organisms. However, their growth rates and productivities are often lower than current industrial microorganisms, and a limited range of tools is available for addressing these metabolic inefficiencies.
This work focused on analysis methods. First, a metabolic flux analysis (MFA) simulation was analyzed to determine criteria for yielding reliable flux estimates. Isotopically nonstationary MFA estimates fluxes based on transient incorporation of carbon 13 into intracellular metabolites following introduction of labeled substrate. This query showed that enzyme substrate and product measurements directly improved reliability. Interestingly, latter time points approaching steady-state labeling more influenced reliability than the early, transient 13C incorporation.
Second, an isobutyraldehyde-producing strain of Synechococcus elongatus sp. PCC 7942 was analyzed. Genes from a pathway bypassing a bottleneck in upstream pyruvate production were singly overexpressed within this strain. Growth rates, aldehyde productivity, and metabolites pool sizes were compared to determine the effect of overexpressing phosphoenolpyruvate carboxylase, malate dehydrogenase (MDHox), and malic enzyme (MEox). Compared to the base strain, growth did not change significantly, but MEox increased aldehyde production 1.2-fold. The metabolite pool size analysis highlighted a limitation in malate conversion to pyruvate, leading to an increased malate pool in MDHox (10-fold compared to the wild-type strain) and decreased in MEox (6-fold). This identified as pivotal for increasing flux to pyruvate and its derivatives.