Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Abstract

Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7-and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA-derived compounds.biofuels | dynamic metabolic control | transcriptional regulation A grand challenge in synthetic biology is to move the design of biomolecular circuits from purely genetic constructs toward systems that integrate different levels of cellular complexity, including regulatory networks and metabolic pathways (1). Despite the fact that a large volume of regulatory architectures and motifs has been discovered (2, 3), little has been accomplished in pathway engineering to improve cellular productivity and yield by exploiting dynamic pathway regulation and metabolic control (4). One essential part in implementing synthetic metabolic control in pathway engineering is to engineer novel metabolite sensors with desired input-output relationships. For example, have designed and applied a regulatory circuit that can sense the glycolytic pathway hallmark metabolite acetylphosphate to control the lycopene biosynthetic pathway (5) and generate oscillatory gene expression (6) as well as achieve artificial cell-cell communication (7). Dahl et al. (8) have used stressresponse promoters to improve farnesyl pyrophosphate production, and Tsao et al. (9) have rewired the Escherichia coli native quorumsensing regulon for autonomous induction of recombinant proteins.Traditional metabolic engineering is largely focused on the overexpression of rate-limiting steps (10

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...…”

“…Various metabolic engineering approaches were used to increase titers and yields of free fatty acid production, including dynamic pathway regulation, [61,62] organismal growth control, [63] and pathway modification. [64,65] High yields of fatty acid production were reported in two studies.…”

“…[66] A slightly higher yield was achieved by implementing a malonyl-CoA sensor, FapR. [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.…”

Glycolysis and Its Metabolic Engineering Applications

“…Incorporation of a malonylCoA response regulator in E. coli is able to dynamically control the expression of critical enzymes involved in the supply and consumption of malonylCoA and efficiently redirect carbon flux toward fatty acid biosynthesis. 131 Recently, biosensors for various metabolites or natural products have been revealed, which are potential candidates that could be engineered into systems like monitoring of biosynthesis processes, real time detection of products, and dynamical control of metabolic pathways [ Figure 1.3(d)]. …”

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

“…In the fi rst method, to analyze total fatty acids including free fatty acid and O -acyl lipids in the cells, acidcatalyzed esterifi cation and transesterifi cation were used as previously described using 1% H 2 SO 4 /methanol ( 11 ). The samples were heated at 80°C for 2 h. In the second method, esterifi ed microorganisms based on fatty acid biosynthesis and assembly frameworks provides a promising solution to bioproducts from microorganisms to replace petrochemicals, as fatty acids and derivatives share many similar chemical properties with petroleum fuels and petrochemicals such as reduced state of hydrocarbons and high density of energy (12)(13)(14)(15)(16). Therefore, metabolic engineering of microbial systems has potential in providing an alternative to plant sources for hydroxy fatty acid for industrial uses ( 17,18 ).…”