Dynamic flux balance analysis of high cell density fed-batch culture of Escherichia coli BL21 (DE3) with mass spectrometry-based spent media analysis.

Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism-specific genome-scale metabolic models (GSMM) and by assuming instantaneous pseudo-steady states for processes that are inherently dynamic. This technique is well-suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed-batch, high-cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri-carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process.

Global Transcriptome-Guided Identification of Neutral Sites for Engineering Synechococcus elongatus PCC 11801.

Engineered cyanobacteria are attractive hosts for the phototrophic conversion of CO2 to chemicals. Synechococcus elongatus PCC11801, a novel, fast-growing, and stress-tolerant cyanobacterium, has the potential to be a platform cell factory, and hence, it necessitates the development of a synthetic biology toolbox. Considering the widely followed cyanobacterial engineering strategy of chromosomal integration of heterologous DNA, it is of interest to discover and validate new chromosomal neutral sites (NSs) in this strain. To that end, global transcriptome analysis was performed using RNA Seq under the conditions of high temperature (HT), carbon (HC), and salt (HS) and ambient growth conditions. We found upregulation of 445, 138, and 87 genes and downregulation of 333, 125, and 132 genes, under HC, HT, and HS, respectively. Following nonhierarchical clustering, gene enrichment, and bioinformatics analysis, 27 putative NSs were predicted. Six of them were experimentally tested, and five showed confirmed neutrality, based on unaltered cell growth. Thus, global transcriptomic analysis was effectively exploited for NS annotation and would be advantageous for multiplexed genome editing.

Cyanobacteria as cell factories: the roles of host and pathway engineering and translational research.

Cyanobacteria, a group of photoautotrophic prokaryotes, are attractive hosts for the sustainable production of chemicals from carbon dioxide and sunlight. However, the rates, yields, and titers have remained well below those needed for commercial deployment. We argue that the following areas will be central to the development of cyanobacterial cell factories: engineered and well-characterized host strains, model-guided pathway design, and advanced synthetic biology tools. Although several foundational studies report improved strain properties, translational research will be needed to develop engineered hosts and deploy them for metabolic engineering. Further, the recent developments in metabolic modeling and synthetic biology of cyanobacteria will enable nimble strategies for strain improvement with the complete cycle of design, build, test, and learn.

Transporter engineering for the development of cyanobacteria as cell factories: A text analytics guided survey.

Cyanobacteria are attractive candidates for photoautotrophic production of platform chemicals due to their inherent ability to utilize carbon dioxide as the sole carbon source. Metabolic pathways can be engineered more readily in cyanobacteria compared to higher photosynthetic organisms. Although significant progress has been made in pathway engineering, intracellular accumulation of the product is a potential bottleneck in large-scale production. Likewise, substrate uptake is known to limit growth and product formation. These limitations can potentially be addressed by targeted and controlled expression of transporter proteins in the metabolically engineered strains. This review focuses on the transporters that have been explored in cyanobacteria. To highlight the progress on characterization and application of cyanobacterial transporters, we applied text analytics to extract relevant information from over 1000 publications. We have categorized the transporters based on their source, their function and the solute they transport. Further, the review provides insights into the potential of transporters in the metabolic engineering of cyanobacteria for improved product titer.