The combined strategy using push/pull and block increased FFA production, but the strain showed approximately 0.048 g FFA/g glucose, corresponding to 14% of the theoretical yield, indicating that further manipulations are needed to improve the Increase FFA production. The use of the mutant thioesterase overcame the limited FFA production caused by protein-protein interaction in the fatty acid synthesis pathway, resulting in twofold higher FFA production than the use of wild-type thioesterase. Finally, identifying and engineering membrane transport systems, which are indirectly involved in increased FFA production, improved both extracellular and total FFA production.
Further engineering including overexpression of the transcriptional regulator FadR and phosphoenolpyruvate carboxylase (PPC) increased FFA production. The combinatorial manipulation of the strategies increased the yield of FFA production (0.24 g FFA/g glucose, corresponding to 69% of the theoretical yield) which is 5 times higher than that of the standard strain. While success was achieved in an increase in FFA production titer and yield, the optimized FFA-producing strain could be constructed through further engineering such as increasing the NADPH pool, developing the active stationary phase expression system, and reprogramming the metabolic pathway. .
Introduction-overview of fatty acid production
- Motivation
- Fatty acid biosynthesis pathway in Escherichia coli
- Successful engineering to increase fatty acid production
- Objectives
A valuable molecule for fatty acid synthesis is acetyl-CoA, which could act as an initiation and elongation precursor in initiation and elongation stages. Fatty acid synthesis could be initiated by the conversion of acetyl-CoA to malonyl-CoA by the action of acetyl-CoA carboxylase (ACC) [7]. Malonyl-CoA must then be transferred to an acyl carrier protein (ACP) to participate in fatty acid initiation and elongation.
The next step for fatty acid synthesis is the condensation reaction of acetyl-CoA and malonyl-ACP to synthesize acetoacetyl-ACP. Thioesterase catalyzes the hydrolysis of the thioester bond in acyl-ACP or acyl-CoA to produce free fatty acid and ACP or CoA. Thus, the use of thioesterase not only accelerates FFA production, but also changes the type of fatty acid produced.
Introduction of an acetyl-CoA carboxylation bypass
- Abstract
- Introduction
- Materials and methods
- Bacterial strains and plasmids
- Media and cultivation conditions
- Metabolite quantification
- Results and Discussion
- MMC-overexpressing E. coli showed improved FFA production
- Redirecting TCA cycle intermediates into fatty acid synthesis increased FFA production production
- Conclusions
The MMC bypass succeeded in enhancing FFA production (Fig. 2.3) and reducing acetate formation (Fig. 2.2d, Table 2.3), presumably indicating that the MMC expression redirects metabolic flux from PEP to malonyl-CoA with reduced flux to acetyl -CoA. The MMC bypass improved the FFA production titer and yield compared to ACC-mediated acetyl-CoA carboxylation (Table 2.3). It is hypothesized that improving the MMC bypass by supplying OAA will further improve FFA production.
Thus, precursor alteration and diverted carbon flux caused by MMC bypass serve to increase FFA production titer and yield (Table S2). In contrast, no increase in free fatty acid production was observed in cells overexpressing ACC, as expected (Figure 2.6). The combination of MMC expression with the addition of aspartate, which is converted to OAA, resulted in an approximately 2.5-fold increase in FFA production.
- Abstract
- Introduction
- Methods
- Bacterial strains and plasmids
- Media and cultivation conditions
- Mutant library construction
- Enrichment and isolation of ‘TesA mutants with increased activity
- Analysis of FFA and glucose
- Enzyme expression and kinetic analysis
- Results and discussion
- Construction and characterization of a high-throughput screening system
- Genotypic and phenotypic analysis of isolated ‘TesA mutants
- The effect of mutation at Arg 64 of ‘TesA on enzymatic activity
- Increased FFA production driven by high specific activity of ‘TesA R64C
- Conclusions
- Abbreviations used
An optimal molar ratio of fatty acid synthase (FAS) components is key to maximizing FAS activity. We hypothesized that engineering TesA with a high specific activity is necessary for further improvement of FFA production (Fig. 3.1b). The response of the fatty acid sensing system to fatty acids was measured as described previously [83].
Overnight-grown cells in the minimal medium were harvested and resuspended in 70 ml of the fresh M9 medium. Briefly, two overlapping PCR products were generated from tesA using the primer sets (TesA FP, TesA SD RP, TesA SD FP and TesA RP). The fastest growth and highest RFP intensity of the SBF05 should be the result of its high FFA production.
This increased population of the FFA-overproducing strain (SBF05) clearly demonstrated that the constructed sensing system detected the level of FFAs and selectively supported the growth of strains synthesizing a large amount of FFAs. Thus, the change of Asp74 may increase the FFA production in a manner similar to that of the SBF07. Kinetic parameters of the 'TesA and 'TesAR64C were analyzed in tests with palmitoyl-CoA to confirm the correlation between the increased FFA production level and increased enzymatic activity.
Therefore, these kinetic parameters can be considered as supporting evidence of the maximum amount of FFA synthesis in SBF08. The motion of the switch loop (residues 75–80) affects substrate specificity by stabilizing the Michaelis complex (MC) when 'TesA interacts with its substrate [ 22 ]. It has been reported that the relative abundance ratios of the proteins in the FAS components are critical to generate the greatest synergy for fatty acid synthesis [73].
Based on this fact, we hypothesized that high fatty acid production could be achieved by optimal expression of 'TesA with high specific activity, but not by overexpression of wild-type 'TesA.
Disruption of membrane transport systems
- Abstract
- Introduction
- Materials and methods
- Bacterial strains and plasmids
- Mutant library construction
- Screening of the mutant library and identification of insertion sites
- Media and cultivation conditions
- Free fatty acid measurement and statistical analysis
- Results
- Screening and identification of high FFA-producing mutants
- Improvement of FFA production by envR deletion
- Enhanced FFA production by multiple gene disruption
- Discussion
- Conclusion
- Abbreviations used
Inactivation of FadD in the β-oxidation pathway has also been shown to enhance FFA production[ 38 ]. The dominant mutants, accounting for ten of the 16 mutants analyzed, exhibited a transposon insertion in gusC resulting in the largest increase in FFA production (1.7-fold) compared to the control strain SBF06. The mutants, accounting for four of the 16 mutants analyzed, exhibited a transposon insertion in envR resulting in a 1.4-fold increase in FFA production compared to the control strain.
Two mutant strains with a transposon insertion in mdlA showed a 1.2-fold increase in FFA production compared to the control strain. Consistent with this hypothesis, overexpression of AcrAB in SBF06 cells resulted in increased production of free fatty acids (both total and extracellular fatty acids) comparable to that of the envR deletion strain (Figure 4.1). We also investigated the time dependence of intracellular, extracellular and total production of free fatty acids in the envR deletion strain.
Total FFA production in the envR deletion strain was similar to that of the control strain (SBF06) up to 12 h post-induction (Fig. 4.4). However, total and extracellular FFA production in the envR deletion strain was significantly increased (approximately 1.4-fold and 2.0-fold, respectively) 48 h after induction (Fig. 4.4). A mutant with triple deletion of envR, gusC and mdlA showed total FFA production comparable to that of the gusC deletion strain (Fig. 4.5).
The triple deletion strain also showed increased total FFA production (160 mg/L/OD) compared to the envR deletion strain (133 mg/L/OD) after 48 h of culture. Inactivation of OmpF resulted in a small increase in FFA production (8%) with no change in the proportion of extracellular FFAs. Extracellular FFAs accounted for 50% of total FFA production in the quintuple deletion strain at 72 hours post-induction (Fig. 4.6).
Deletion of envR, gusC or mdlA resulted in increased total FFA production or 1.2-fold, respectively) without a deleterious effect on cell growth.
Construction of high FFA-producing strain with combined manipulation
- Abstract
- Introduction
- Methods
- Bacterial strains and plasmids
- Media, cultivation conditions, and metabolite quantification
- Fed-batch fermentation
- Results and discussion
- Combined manipulation of the strategies to increase FFA production
- Conclusions
Remarkable progress has been made through the 'push/pull and block' strategy to increase FFA production in E. coli. The combined manipulation using push/pull and blocking increased FFA production, but the strain exhibited about 0.048 g FFA/g glucose, which is equal to 14% of the theoretical yield [35], indicating that further manipulations are needed to increase Production of FFA. To increase FFA production in E. coli, the strategies used in this thesis were combined based on the 'push/pull and block' approach.
Combinatorial manipulation of the strategies increased the production yield of FFA (0.15 g FFA/g glucose, corresponding to 44% of the theoretical yield) with a 5-fold higher titer. Additional expression of FadR, a transcriptional activator of fatty acid synthesis, increased free fatty acid production 9.6-fold with 69% of theoretical yield compared to a control strain overexpressing wild-type thioesterase. Finally, the strain (FadR overexpressing SBF43) consumed almost all glucose within 72 h and produced a higher production titer of FFA, resulting in a 1.2-fold higher production yield than SBF44.
Culture of groups of engineered strains at 72 hours post-induction. a) FFA production titer, (b) cell growth, (c) residual glucose and yield (at the end of cultivation) of engineered strains [SBF06 (strains overexpressing 'TesA, ●), SBF25 (strain with overexpression of ' TesA and deletion of envR, gusC and mdlA, ■), SBF40 (strain with overexpression of. The dodecane layer prevents the accumulation of FFA in the culture medium and increases FFA production by 1.5-fold. As expected, the addition of the layer of dodecane in culture medium of SBF43 at 24 h after induction showed 15% higher FFA production compared to that without dodecane coating (Fig. 5.1a).
Third, overexpression of the FadR transcription factor further increased FFA production by increasing fatty acid synthase activity as previously reported [ 72 ]. Finally, deletion of envR, gusC and mdlA that have been identified in this thesis increased total FFA production and extracellular FFA production. The combination of strategies has resulted in the highest productivity and yield in FFA production (Table 5.2).
SBF43, the engineered strain in this study, showed a higher FFA production titer and productivity than the strain in the reference strain.
Summary and future perspectives
- Summary of the findings
- Recommendations for future work
- Increasing NADPH pool by controlling carbon flux between EMP and PPP Balance of cofactors and their regeneration are often required as production of desired
- Developing active expression system in stationary phase
- Reprogramming metabolic pathway to develop efficient microbial cell factories for efficient FFA production
- References
- Acknowledgements
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