Mid-Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects.

Mid-to-long-chain dicarboxylic acids (DCAi, i ≥ 6) are organic compounds in which two carboxylic acid functional groups are present at the terminal position of the carbon chain. These acids find important applications as structural components and intermediates across various industrial sectors, including organic compound synthesis, food production, pharmaceutical development, and agricultural manufacturing. However, conventional petroleum-based DCA production methods cause environmental pollution, making sustainable development challenging. Hence, the demand for eco-friendly processes and renewable raw materials for DCA production is rising. Owing to advances in systems metabolic engineering, new tools from systems biology, synthetic biology, and evolutionary engineering can now be used for the sustainable production of energy-dense biofuels. Here, we explore systems metabolic engineering strategies for DCA synthesis in various chassis via the conversion of different raw materials into mid-to-long-chain DCAs. Subsequently, we discuss the future challenges in this field and propose synthetic biology approaches for the efficient production and successful commercialization of these acids.

Enhancing surfactin production in Bacillus subtilis: Insights from proteomic analysis of nitrate-induced overproduction and strategies for combinatorial metabolic engineering.

Surfactin biosynthesis in Bacillus subtilis is intricately regulated by environmental conditions. In the present study, addition of nitrate, a nitrogen source, increased the production of surfactin in B. subtilis ATCC 21332, whereas its absence resulted in minimal or no surfactin production. Proteomics revealed the mechanism underlying nitrate-induced surfactin overproduction, identifying three key differential proteins (preprotein translocase subunit SecA, signal recognition particle receptor FtsY, and cell division adenosine triphosphate-binding protein FtsE) relevant to surfactin transport and regulation. Combinatorial metabolic engineering strategies (enhanced nitrate reduction, fatty acid hydroxylation, rational transporter engineering, and feeding) led to a 41.4-fold increase in surfactin production compared with the initial production in the wild-type strain. This study provides insights into the molecular mechanism of nitrate-induced surfactin overproduction and strategies to enhance the performance of surfactin-producing strains.

Metabolic engineering of an auto-regulated Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid.

One challenge in metabolic engineering for industrial applications is the construction of highly efficient microbial cell factories. For this purpose, dynamic regulation of metabolic flux may be indispensable. In this study, an auto-regulated Corynebacterium glutamicum chassis for 5-aminolevulinic acid (5-ALA) biosynthesis was constructed. First, the expression of critical genes involved in 5-ALA synthesis and cofactor regeneration was precisely modulated. Furthermore, odhA expression was controlled using the strategies of static metabolic engineering (SME, with a weak promoter), dynamic metabolic engineering (DME, with a temperature-sensitive plasmid), and auto-inducible metabolic engineering (AME, with a growth-related promoter). The AME strategy showed the best effect and dynamically balanced the tradeoff between cell growth and 5-ALA synthesis. Additionally, the expression of exporter-encoding rhtA was regulated using AME strategy by the two-component system HrrSA in response to extracellular heme. The final strain A30 achieved the highest 5-ALA production (3.16 g/L) ever reported in C. glutamicum through C5 pathway.

High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering.

4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5.12 g/L but a considerable amount of L-isoleucine still accumulated in the culture. We then dynamically modulated the activity of the α-ketoglutarate dehydrogenase complex (ODHC) by employing L-isoleucine-responsive transcription or attenuation strategies. The best-engineered strain, HIL18, produced 34.21 g/L 4-HIL with a negligible accumulation of byproducts, including approximately 0.6 g/L L-isoleucine. This study achieved the highest production and yield of 4-HIL, and optimizing the TCA cycle by dynamically modulating the activity of ODHA can be a powerful strategy to balance the carbon flux and achieve efficient production of α-ketoglutarate and derivatives.