Microbial cell factories have been used for the production of valuable chemical compounds using a classical metabolic engineering approach, but this requires much time and cost, and laborintensive processes to make cell factories industrially competitive. Systems metabolic engineering is an upgraded version, which understands the cell as a complex system in which networks of genes, transcripts, proteins, and metabolites are connected, facilitating the analysis of potential cell factories. However, efficient cell factory design, which aims for industrial-scale production, requires a comprehensive system, which goes beyond metabolism and considers industrial production challenges. A review is provided here of the developments and challenges in the application of systems biology for metabolic engineering and in recovery and purification processes for scaling up bio-based chemical production. Then, a new design, build, test, and learn prediction cycle for metabolic engineering is proposed, for the design of efficient cell factories. This considers system-wide characteristics and relies upon the integration of upstream (strain development), midstream (fermentation), and downstream (recovery and purification) analysis for strain design. In addition to this cycle, three issues should be taken into consideration: (i) The use of simple, available, and inexpensive materials; (ii) the identification and elimination of bottlenecks using non-complex recovery and purification processes; (iii) the assessment of commercial and chemical industry requirements from the perspective of system efficiency. In this context, highly efficient microbial cell factories should be developed to produce compounds with improved production performance to meet industrial application requirements.

BACKGROUND: Succinic acid production has been studied from a metabolic engineering or a downstream processing perspective, separately. The concentration of succinic acid and other by-products obtained after the strain design influences the production cost during the recovery and purification stage. A metabolic engineering-downstream coupling evaluation is important when selecting the metabolic targets for the strain design. In this in silico study, the metabolic engineering of an Escherichia coli strain to produce succinic acid using glycerol as a carbon source in the downstream process was evaluated in terms of operational cost and energy consumption. (0.3068, 0.0576, 0.1089 h -1 ) and succinate productivity (2.7534, 6.0772, 5.5661 mmol g -1 DW h -1 ), respectively. The results showed that the succinic acid productivity constituted a central parameter when selecting the appropriate gene targets for deletion, despite the presence of organic acids in the downstream process and the biomass growth rate. RESULTS: Three strain scenarios were selected using a bi-level linear optimization problem solved by Mixed Integer Linear Programing, and simulated in a transient fashion with dynamic flux balance analysis considering both biomass growth rate CONCLUSION: A metabolism-downstream coupled model shows that the bioproduct productivity and fermentation timeare key points when considering the operational cost and energy consumption involved in the engineering of strains for industrial-scale production.Metabolic engineering targets were obtained using OptKnock, which is a bi-level linear optimization problem solved by MILP. 33

Clostridium ( Ruminiclostridium ) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335′s transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log 2 fold change (FC) >|1| and 106 highly DEGs (log 2 FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD , were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes ( tktAB ) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation–reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.