A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.


Supplemental tables Table S1. (Separate file)
The annotated metabolites of fatty alcohol biosynthesis

Table S2. (Separate file)
The annotated metabolites of bioluminescence light Table S3.(Separate file) The annotated metabolites of alkane biosynthesis Table S4.E. coli strains used in this study.

Supplemental figures and legendsFigure S1 .
Figure S1.Sugar reduction by XR using D-glucose and D-galactose as substrates.A-B, The extracted ion chromatograms (EIC) and exact mass spectra at m/z 181.0 ([M-H] -) identified sorbitol as a product from reduction of D-glucose by XR.The reactions were carried out under ambient temperature for 10 min in 100 mM potassium phosphate buffer, pH 7.5 containing 5 µM XR, 0.25 mM NADPH, and 1 mM glucose.The control reaction was performed without addition of XR and NADPH.The sorbitol product was analyzed by an LC-MS triple quadrupole mass spectrometer in negative mode.C-D, The extracted ion chromatogram (EIC) and exact mass spectra at m/z 181.0 ([M-H] -) identified galactitol as a product from the reduction of D-galactose by XR.The reactions were carried out under ambient temperature for 10 min in 100 mM potassium phosphate buffer, pH 7.5 containing 5 µM XR, 0.25 mM NADPH, and 1 mM galactose.The control reaction was performed without addition of XR or NADPH.The galactitol product was analyzed by LC-MS with a triple quadrupole mass spectrometer in negative mode.E, The extracted ion chromatogram (EIC) and exact mass spectra at m/z 181.0 ([M-H] -) of a standard sorbitol analyzed by an LC-MS triple quadrupole mass spectrometer in negative mode.The standard compounds and samples were diluted in acetonitrile (ratio 1:1) before being analyzed on an Agilent 1200 series LC 6470 triple quadrupole mass spectrometer.The Agilent Poroshell 120 HILIC-Z 150´ 2.1 mm, 2.7 µm (particle size) column was used to achieve optimal separation.The mobile phase was 10 mM ammonium acetate in water pH 9.0 containing 2.5 µM deactivator (mobile phase A) and 10 mM ammonium acetate in 85% ACN pH 9.0 containing 2.5 µM deactivator (mobile phase B) with a flow rate of 0.3 ml/min.The gradient was changed from 4% mobile phase A / 96% mobile phase B to 35% mobile phase A / 65% mobile phase B in 24 min, and the column was maintained at 35˚C.The mass spectrometer was operated in scan mode to detect sugar alcohol products based on their retention times and m/z values compared to standard compounds.MS parameters were as follows: gas temperature, 250˚C; gas flow, 12 L/min; nebulizer, 45 psi; sheath gas flow, 12 L/min; capillary voltage, 3000 V; and VCharging, 1000.

Figure S2 .
Figure S2.Amount of fatty alcohol (mM) produced by E. coli-far and E. coli-far-xr at various time points of bioconversion using various types of sugars.E. coli-far and E. coli-far-xr were induced with 1 mM lactose at 220 rpm, 25˚C for 6 h and then harvested and employed as biocatalysts for fatty alcohol production.The bioconversion reactions were carried out in 0.1 M potassium phosphate buffer at pH 7.5 containing cells with OD600 of 30/ml and 10 mM lactose, 20 mM D-glucose, 10 mM each of D-glucose/Dgalactose, 10 mM each of D-glucose/D-fructose, and 10 mM each of D-glucose/L-arabinose.The reactions were incubated at 220 rpm, 25˚C for 2, 4, and 6 h with 10 mM each of D-glucose/D-galactose, 20 mM Dglucose, and, and for 2 to 4 h for 10 mM each of D-glucose/D-fructose and 10 mM each of D-glucose/Larabinose. Fatty alcohol products were extracted with 2 ml of ethyl acetate containing internal standard (200 µM of tetradecane).Data are shown as mean ± s.d., n = 2 replicate cultures.The addition of XR clearly improved the yield of fatty alcohol production by the whole-cell biocatalysts with all tested sugars, particularly with mixed D-glucose/D-galactose and lactose utilization.This might be due to XR conversion of glucose into its corresponding alcohol (sorbitol), alleviating the glucose repression effects and allowing the cell to co-utilize other carbon sources for creating the necessary precursors simultaneously for fatty alcohol synthesis.It should be noted that when D-glucose was used as a sole substrate, the enhancement effect in production of fatty alcohol by E. coli-far-xr compared to E. coli-far was less than that seen for the conditions using lactose, D-glucose with D-galactose, D-glucose with D-fructose, or D-glucose with Larabinose.The data imply that XR can enhance fatty alcohol production greater in the presence of mixed carbohydrate substrates.

Figure S3 .
Figure S3.Oxidative and osmotic stress test on E. coli-far and E. coli-far-xr.E coli strain FAR and FAR-XR protein overexpression was induced with 1 mM lactose for 6 h and used for an oxidative and osmotic stress assays by adding H2O2 and NaCl, respectively.A, A 0.5-ml reaction contained the biocatalyst with final OD600 of 0.4/ml and 5 mM H2O2 in 0.1 M Kpi pH 7.5.After incubation for 90 min at room temperature, the viable cells were measured by colony forming unit (CFU) counting on an LB agar plate.The results suggested that the cells containing XR had higher CFUs than the cells without XR.Data are shown as mean ± s.d., n = 3 replicate cultures.Asterisk indicates significant differences by t test (p < 0.05).B, A 0.5-ml reaction contained biocatalyst with a final OD600 of 0.4/ml, NaCl, trehalose (0.5 g/L), or glutathione (30 µM) in 0.1 M Kpi pH 7.5.After incubation for 3 h at room temperature, the number of viable cells were measured by colony forming unit (CFU) counting on an LB agar plate.Most of the results for the two cell types were similar except that E. coli-far showed a higher percentage of cell viability than E. coli-far-xr in the presence of 5% (w/v) NaCl (without addition of trehalose or glutathione) (p < 0.05, t test, Bonferroni-Dunn method).The results suggest that the up-regulation of trehalose and glutathione found in E. coli-far-xr does not enhance resistance to osmotic stress.Data are shown as mean ± s.d., n = 5 replicate cultures.

Figure S4 .
Figure S4.Comparison of the fatty alcohol production in the bioconversion by different cofactor regenerating systems in the engineered cell.The bioconversion was described in Experimental procedures.Fatty alcohol was detected during the bioconversion in E. coli-far (red), E. coli-far-xr (blue), E. coli-far-gdh when 10 mM lactose was used as a substrate (green) and when 20 mM D-glucose was used as a substrate (orange) in E. coli-far-gdh.Analytical methods were described in Experimental procedures.Data are shown as mean ± s.d., n = 3 replicate cultures.

Figure S5 .
Figure S5.Time-course analysis of ATP in E. coli-luxCDEAB and E. coli-luxCDEAB-xr after adding 10 mM lactose for 4, 8, 12, and 24 h.The samples were diluted in acetonitrile (ratio 1:1) before being analyzed by the LC 6470 triple quadrupole mass spectrometer (Agilent 1200 series).An Agilent Poroshell 120 HILIC-Z 150 ´ 2.1 mm, 2.7 µm (particle size) column was used to achieve optimal separation.The flow was 0.3 ml/min with a mobile phase of 10 mM ammonium acetate in water pH 9.0 containing 2.5 µM deactivator (mobile phase A) and 10 mM ammonium acetate in 85% ACN pH 9.0 containing 2.5 µM deactivator (mobile phase B).The gradient was changed from 4% of mobile phase A / 96% of mobile phase B to 35% of mobile phase A / 65% of mobile phase B over 24 min., and the column was maintained at 35˚C.The mass spectrometer was operated in a SIM mode to detect ATP based on the parameters of retention time and m/z by comparison to values of the standard compound.MS parameters were as follows: gas temperature, 250°C; gas flow, 12 L/min; nebulizer, 45 psi; sheath gas flow, 12 L/min; capillary voltage, 3000 V; and VCharging, 1000.Data are shown as mean ± s.d., n = 5 replicate cultures; error bars show s.d; asterisk denotes significant differences by multiple t test (p < 0.05).

Figure S6 .
Figure S6.Tridecane production (mM) from tetradecanoic acid by E. coli harboring FAP with/without XR overexpressed in the plasmid system.Plasmids of pCDFDuet-fap and pRSFDuet-gstxr (pRSFDuet in case of biocatalyst without XR) were co-transformed into E. coli BL21 (DE3) to generate E. coli-pfap and E. coli-pfap-pxr.A single colony of the resulting transformants was inoculated in LB broth in the presence of streptomycin 25 µg/ml and kanamycin 34 µg/ml at 220 rpm, 37˚C for 17 h.The starter cultures were then sub-cultured with 1% inoculant into TB media containing a half-dose of the antibiotics mentioned above and grown at 37˚C until the OD600 reached 0.6-0.7.The cultures were then induced with 10 mM lactose and incubated further with shaking at 220 rpm, 25˚C for 12 h, then harvested and employed as biocatalysts for alkane production.The bioconversion reactions were carried out in potassium phosphate buffer (0.1 M, pH 7.0) containing a cell biocatalyst at an OD600 of 60/ml and 10 mM tetradecanoic acid.The reaction was set in a capped 20 ml clear glass vial and incubated under blue light (PPFD-B 20 µmolphotons/m 2 /s) with shaking at 100 rpm, 25˚C.Fatty acid and alkane were extracted using 4 ml of ethyl acetate containing the internal standard (200 µM of tetradecane) and analyzed by GC-FID equipped with HP-5 column.Data are shown as mean ± s.d., n = 3 replicate cultures.Three hours after the reaction was initiated, the productivity of E. coli-pfap-pxr (blue) was 1.3 mmol/L/h, which is higher than 1 mmol/L/h of E. coli-pfap (red) by 1.3-fold.To improve the use of the XR/lactose system as an enhancer system for producing the active FAP (FAP:FAD) without adding extra antibiotic and creating a more stable biocatalyst, we further created a strain of E. coli BL21 (DE3) with an XR integrated genome (see construction details in Experimental procedures).The use of the XR genome integrated cell version for production of tridecane was discussed in the Main text.

Figure S7 .
Figure S7.Level of intermediates in flavin biosynthesis during protein overexpression of E. coli-fap and E. coli-fap-xr.After 75 mM of lactose was added into the TB culture of E. coli-fap and E. coli-fap-xr, the cells (at equivalent cell amounts based on OD600 in 1 ml) were taken from the culture at 9, 13, and 20 hours.The metabolites were extracted from the samples and analyzed by LC-IM-QTOF as described in the Experimental procedures.The levels of metabolites were obtained from the peak area by Find by the Formular (FBF) function with mass tolerance ±30 ppm in the MassHunter Qualitative software (Agilent), with the exception of DMLZ, for which levels were obtained from the abundance value as identified by Mass Profiler.Data are shown as mean ± s.d., n = 6 technical replicates (2 biological replicates), and the asterisk denotes a significant difference by multiple t test (p < 0.05, Bonferroni-Dunn method).