Construction of high FFA-producing strain with combined manipulation
5.4. Results and discussion
5.4.1. Combined manipulation of the strategies to increase FFA production
When the MMC complex was overexpressed in SBF25 (ΔenvR, ΔgusC, and ΔmdlA triple deletion strain), the strain (SBF40) produced 2.1 g/L, which is 2.9-fold increase in FFA production over that of SBF06 (0.7 g/L) (Fig. 5.1a). The SBF40 exhibited similar growth (Fig. 5.1b), but low glucose consumption (Fig. 5.1c), compared with those of SBF06. Increased FFA production titer and yield in SBF40 might be due to function of MMC bypass that redirects metabolic flux from PEP to malonyl-CoA via OAA. When the ‘TesA was replaced to ‘TesAR64C in SBF40, the strain (SBF41) produced 3.3 g/L, which is 1.6-fold higher than that of SBF40 (Fig. 5.1a). Employing the ‘TesAR64C increased FFA production as observed in the Chapter 3 that the change of amino acid residue in non- conserved region increases catalytic activity up to two-fold and then overcomes limited FFA production driven by protein-protein interaction in the fatty acid synthetic pathway. Additional expression of PPC in SBF41 (designated SBF42) to increase carbon flux toward oxaloacetate from phosphoenolpyruvate further improved FFA production titer and yield by 1.17- and 1.18-fold, compared with SBF41 (Fig. 5.1a and 5.1d). However, the titer in SBF42 was 23% lower than that of SBF44, highest FFA-yielding strain in previous literatures. The strain harbors overexpression of ‘TesA and FadR (transcriptional factor of fatty acid biosynthesis), and inactive β-oxidation pathway. Up- regulated expression of FadR led to increased expression level of genes involved in fatty acid synthetic pathway, resulting in 7-fold higher FFA production than that of control strain without FadR overexpression [72]. Thus, FadR was overexpressed to enhance activity of fatty acid biosynthesis in SBF42. When FadR was overexpressed in SBF42, the strain (SBF43) produced 6.9 g/L, which is 9.5- and 1.4-fold higher than those of SBF06 and SBF44, respectively. Interestingly, both strains , SBF43 and SBF44 overexpressing FadR, exhibited higher cell density with faster glucose consumption, indicating that overexpression of FadR also increases activity of pathways associated with biomass formation. Finally, the strain (FadR overexpressing SBF43) consumed almost glucose in 72 h and produced higher FFA production titer, leading to 1.2-fold higher production yield than SBF44.
Furthermore, the strain SBF43 exhibited around 5-fold higher production yield than the strain (ACC↑,
‘TesA↑, and ∆fadD) in benchmark study [35]. To the best of our knowledge, SBF43 is an engineered strain that exhibited the highest FFA production titer and yield at flask-level batch cultivation.
Figure 5.1. Batch culture of engineered strains at 72 h post-induction. (a) FFA production titer, (b) cell growth, (c) residual glucose, and yield (at the end of cultivation) of engineered strains [SBF06 (strain with overexpression of ‘TesA, ●), SBF25 (strain with overexpression of ‘TesA and deletion of envR, gusC, and mdlA, ■), SBF40 (strain with overexpression of
‘TesA and MMC and deletion of envR, gusC, and mdlA, ♦), SBF41 (strain with overexpression of ‘TesAR64C and MMC and deletion of envR, gusC, and mdlA, ▲), SBF42 (strain with overexpression of ‘TesAR64C, MMC, and PPC and deletion of envR, gusC, and mdlA, ○), SBF43 (strain with overexpression of ‘TesAR64C, MMC, PPC, FadR and deletion of envR, gusC, and mdlA, □), SBF43 (with two-phase reaction, △), and SBF44 (strain with overexpression of ‘TesA and FadR and deletion of fadE, ◇)] were measured. Expression of ‘TesA, ‘TesAR64C, and FadR was induced by addition of 0.2 mM IPTG.
Expression of MMC and PPC was induced by the addition of 25 nM tetracycline at an OD600 of 1.5. Dodecane layer was added after 24 h post-induction.
Figure 5.2. Fed-batch fermentation of SBF43 strain. Time courses of cell growth (OD), glucose consumption, and free fatty acid production were measured in 0.5 L fermentor. Each symbol indicates [OD600 (●), FFA (■), and glucose (▲)]. After fermentation, all particles on wall of the fermentor were resuspended. The red square indicates FFA production at the end of fermentation.
Figure 5.3. Schematic diagram of developed strain in this study. The manipulations carried out are 1) introduction of the methylmalonyl-CoA carboxyltransferase to provide alternative pathway for malonyl-CoA synthesis by avoiding regulations, altering precursor, and redirecting carbon flux from TCA cycle to FFA synthesis, 2) improved specific activity of the ʹTesA (thioesterase that catalyzes hydrolysis of acyl-ACP into FFA) to overcome limits of stoichiometric interaction of enzymes involved in FFA synthesis, 3) increased expression level of the transcriptional activator FadR to enhance expression level of genes in fatty acid synthetic pathway and significantly reinforce carbon flux toward FFA synthesis, and 4) inactivated function of transcriptional repressor EnvR to increase efflux rate of FFA by inducing expression of the AcrAB, multidrug efflux pump.
The addition of dodecane layer in culture medium has previously increased production of FFA and its derivatives [38, 115]. The dodecane layer prevents FFA accumulation in the culture medium and increases FFA production by 1.5-fold. As expected, the addition of dodecane layer in culture medium of SBF43 at 24 h post-induction exhibited 15% higher FFA production compared with that without the dodecane layer (Fig. 5.1a).
Next, the performance of SBF43 was tested in a fed-batch fermentation process. The cells were cultivated in the M9 minimal medium for 72 h post-induction. The strain produced 18.4 g/L FFA with 84 g/L glucose consumption (Fig. 5.2), leading to 0.22 g of FFA/g of glucose, which is equivalent to 65% of theoretical yield. Furthermore, the FFA productivity of SBF43 was 0.26 g/L/h, which is 2.6-fold higher than that of strain in benchmark study [35], indicating that the developed
‘push/pull and block’ strategy is better than that of the benchmark study to improve FFA production.
Table 5.2. Literature summary of FFA production
Strain Genotype Thioesterase Media OD
Titer (g/L)
Productivity (g/L/h)
Yield (%)
Specific productivitya
(g FFA/g DCW/h) Reference
MG1655 ΔfadBA FadM
Minimal with 3% glucose (Batch culture)
7.5 b 7 0.12 28 0.04 [138]
DH1 FadR+ ‘TesA
Minimal with 2% glucose (Batch culture)
N.Ic 5.2 0.07 26 N.Ic [72]
BL21(DE3) Modular
design CnFatB2
MK with 2%
glucose1% YE (Fed-batch)
90.8 8.6 0.12 7.8 0.004 [74]
DH10B
ΔfadE, POPQC
system
‘TesA
M9 with 2%
glucose (Fed-batch)
130 b 21.5 0.5 15 0.01 [139]
MG1655
ΔenvR, ΔgusC, ΔmdlA MMC+
FadR+
PPC+
TesAR64C
M9 with 2%
glucose 0.1% YE (Fed-batch)
47 18.4 0.26 22 0.015 This study
a. Dry cell weight (DCW) was calculated by following empirical rule that OD600 1 is 0.36 g/L [140].
b. Author’s supposition based on the literature.
c. N.I, not indicated in the literature.
The SBF43 strain has been constructed based on ‘push/pull and block’ approach (Fig. 5.3).
First, introduction of the MMC bypass avoided regulation in native acetyl-CoA carboxylation in E.
coli. It also altered precursor specificity and redirected carbon flux from TCA cycle to the FFA synthesis, Second, mutant thioesterase (‘TesAR64C) accelerated the FFA production by overcoming limitation driven by protein-protein interaction in the fatty acid synthetic pathway. Third, overexpressing transcriptional factor FadR further increased FFA production by enhancing activity of fatty acid synthase as previously reported [72]. Finally, deletion of envR, gusC, and mdlA that are identified in this thesis increased both total FFA production and extracellular FFA production.
Combination of the strategies has resulted in highest productivity and yield in FFA production (Table 5.2).
Tremendous advances have been made in microbial FFA production by identifying key enzymes, understanding regulations involved, and manipulating metabolic pathway. As a specific productivity of biofuels needs to be over 0.1 g of FFA/g of dry cell weight (DCW) per an hour for commercial manufacturing process of biofuel production [141], current strain (SBF43 exhibiting 0.015 g FFA/g DCW per an hour) has to be further engineered to increase titer, productivity, and yield.
There are several potential strategies to improve performance of the strain, including cofactor regeneration and stable expression of fatty acid synthase in stationary phase. The detailed strategies are discussed in Chapter 6.