Modular optimization of multi-gene pathways for fatty acids production in E. coli

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…Our engineered cell factory can be further optimized to produce a higher proportion of LCDCAs with specific chain lengths or degrees of desaturation. This can be achieved in two ways: by introducing heterologous thioesterase-coding genes that have long chain fatty acid specificity (e.g., Cocos nucifera fatty acyl-ACP thioesterase CnFatB2 and Iris germanica acyl-ACP thioesterase AAG43857 [124]) or by manipulating the native desaturase and elongase systems. The LEU2 gene was disrupted for enabling a successful site-specific multiple gene integration and testing various expression systems.…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…Optimization of ribosome binding site for NudB [38] E. coli Geraniol 2.0 g L À1 Fed-batch, 68 h Two-phase fermentation platform [43] S. cerevisiae Geraniol 293 mg L À1 Fed-batch, 48 h Overexpression of idi1 and tHMG1 [44] E. coli Limonene 650 mg L À1 Batch, 72 h Principal Component Analysis of proteomics data to optimize MVA pathway protein expression levels [46] E. coli Myrcene 58 mg L À1 Batch, 72 h Heterologous expression of myrcene synthase from Quecrus ilex [96] E. coli Cineol 653 mg L À1 Batch, 48 h Chromosomal mutation of ispA; heterologous expression of cineol synthase from Streptomyces clavuligerus [49] E. coli Linalool 505 mg L À1 Batch, 48 h Chromosomal mutation of ispA; heterologous expression of cineol synthase from Streptomyces clavuligerus [49] E. coli Pinene 140 mg L À1 Batch, 24 h Evolved pinene synthase from Pinus taeda to decrease substrate inhibition [48] E. coli Sabinene 2.7 g L À1 Fed-batch, 24 h Heterologous expression of gpps2 from Abies grandis and sabinene synthase from Salvia pomifera [52] S. cerevisiae Sabinene 18 mg L À1 Batch a) Altering a squalene synthase (erg20p) for GPP specificity [53] E. coli Farnesene 1.1 g L À1 Batch, 96 h In vitro measurement of MVA enzyme activity; balanced expression based on in vitro activity of heterologous pathway [55] S. cerevisiae Farnesene 130 g L À1 Fed-batch, 5-6 d a) Rewiring central carbon metabolism to enhance cytosolic CoA availability [56] E. coli Bisabolene 1.2 g L À1 Batch, 72 h Principal Component Analysis of proteomics data to optimize MVA pathway protein expression levels [46] E. coli b-Caryophyllene 1.5 g L À1 Fed-batch, 72 h Balanced overexpression of MVA and DXP pathway enzymes [58] Fatty acids E. coli Fatty acids 5.2 g L À1 Batch, 72 h Dynamic regulation and control; tuning expression of FadR [62,66] E. coli Fatty acids 8.6 g L À1 Fed-batch, 70 h Optimization of transcription levels in three arbitrary modules within fatty-acid biosynthesis [67] E. coli Fatty acids 3.9 g L À1 Fed-batch, 44 h Dynamic control using transcriptional regulator FapR [61] E. coli Fatty acids 7 g L À1 Batch, 24 h Reversed b-oxidation cycle; overexpression of FadBA and select thioesterases in strain RB03 (RB02 DyqhD DfucO DfadD) [64] E. coli Branched fatty acids 276 mg L À1 Batch, 48 h Incomplete lipoylation of 2-oxoacid dehydrogenases [76] E. coli Fatty acids 694 mg L À1 Batch, 48 h Heterologous expression of Val, Leu, Ile biosynthetic pathways; overexpression of bFabH2 and 'TesA [74] E. coli Fatty acids 21.5 g L À1 Fed-batch, 43 h Ensemble-based selection of bacterial strains u...…”

“…[61] A high titer (8.6 g L À1 ) fatty acid production was reported in E. coli, where combinatorial optimization of pathway modules tuned the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. [67] Most recently, 21.5 g L À1 fatty acids production was achieved in E. coli by linking upregulated FadR to growth performance, which subsequently selects highperforming non-genetic variants among the heterogeneous ensemble of cells. [68] In addition to the efforts to dynamically regulate carbon flux toward fatty acids, advances in pathway modification also improved efficiency of the fatty acid-based biofuel production.…”

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…24 Especially today, the rapid development of synthetic biology 25 and metabolic engineering 26 require efficient, flexible, and faithful DNA assembly approaches because of the low capacity of traditional restriction, digestion, and ligation methods. In fact, modular and combinatorial assemblies of various genetic segments, particularly the assembly of large DNA fragments without scars by restriction, digestion, and ligation methods are extremely difficult.…”