ARTICLE Efficient Production of Free Fatty Acids from Soybean Meal Carbohydrates Dan Wang,1,2 Chandresh Thakker,3 Ping Liu,1 George N. Bennett,3 Ka-Yiu San1,4 1
Department of Bioengineering, Rice University, 6100 Main Street, MS-362, Houston, Texas 2 College of Chemistry and Chemical Engineering, Chongqing University, Chongqing, P. R. China 3 Department of BioSciences, Rice University, Houston, Texas 4 Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005-1892; telephone: þ1-713-348-5361; fax: þ1-713-348-5877; e-mail: [email protected]
Introduction ABSTRACT: Conversion of biomass feedstock to chemicals and fuels has attracted increasing attention recently. Soybean meal, containing significant quantities of carbohydrates, is an inexpensive renewable feedstock. Glucose, galactose, and fructose can be obtained by enzymatic hydrolysis of soluble carbohydrates of soybean meal. Free fatty acids (FFAs) are valuable molecules that can be used as precursors for the production of fuels and other value-added chemicals. In this study, free fatty acids were produced by mutant Escherichia coli strains with plasmid pXZ18Z (carrying acyl-ACP thioesterase (TE) and (3R)-hydroxyacyl-ACP dehydratase) using individual sugars, sugar mixtures, and enzymatic hydrolyzed soybean meal extract. For individual sugar fermentations, strain ML211 (MG1655 fadD- fabR-)/pXZ18Z showed the best performance, which produced 4.22, 3.79, 3.49 g/L free fatty acids on glucose, fructose, and galactose, respectively. While the strain ML211/pXZ18Z performed the best with individual sugars, however, for sugar mixture fermentation, the triple mutant strain XZK211 (MG1655 fadD- fabR- ptsG-)/pXZ18Z with an additional deletion of ptsG encoding the glucose-specific transporter, functioned the best due to relieved catabolite repression. This strain produced approximately 3.18 g/L of fatty acids with a yield of 0.22 g fatty acids/g total sugar. Maximum free fatty acids production of 2.78 g/L with a high yield of 0.21 g/g was achieved using soybean meal extract hydrolysate. The results suggested that soybean meal carbohydrates after enzymatic treatment could serve as an inexpensive feedstock for the efficient production of free fatty acids. Biotechnol. Bioeng. 2015;112: 2324–2333. ß 2015 Wiley Periodicals, Inc. KEYWORDS: free fatty acids; Escherichia coli; soybean meal hydrolysate; mixed sugars; catabolite repression
Correspondence to: Ka-Yiu San Contract grant sponsor: United Soybean Board (USB) Contract grant sponsor: China Scholarship Council Received 16 January 2015; Revision received 5 April 2015; Accepted 29 April 2015 Accepted manuscript online 5 May 2015; Article first published online 31 July 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25633/abstract). DOI 10.1002/bit.25633
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Free fatty acids (FFAs) are valuable molecules which could be used as precursors for the production of fuels, oleochemicals, and special health care products (Lennen et al., 2010; Liu et al., 2010; Nickolau et al., 2008). The traditional production of free fatty acids relies on the chemical or biological catalytic processes, such as the catalytic hydrolysis of triglycerides, but sources are mostly limited to animal fats and plant oils (Ooi and Pee, 1985). Alternatively, the fatty acid formation could occur in vivo in bacteria. Recently, many strategies are being made to increase the yield to approach theoretical levels and decrease the cost of bioconversion processes to make the biosynthetic process competitive with the traditional catalytic process (Janßen and Steinb€uchel, 2014; Lennen et al., 2010; PeraltaYahya et al., 2012). Wild type Escherichia coli strains do not normally accumulate free fatty acids while synthesizing membrane lipids (Voelker and Davies, 1994). However, it has been shown that the production of free fatty acids in E. coli could be induced by overexpression of an acyl-ACP thioesterase (TE) gene (Cho et al., 1995; Jiang and Cronan, 1994; Lu et al., 2008). Much work has been done to make fatty acids in E. coli of various chain lengths (Dellomonaco et al., 2011; Wu et al., 2014a), saturation status (Cao et al., 2010), and containing additional functional groups (Kim and Oh, 2013). Except for the availability of abundant metabolic tools to increase the fatty acids yield in E. coli, there is another advantage that makes E. coli an ideal host for fatty acids production. E. coli has been found to have the ability to metabolize many kinds of individual sugars and biomass hydrolysates to produce high-value added chemicals, such as ethanol, succinic acid, and lactic acid (Kim et al., 2011; Mazumdar et al., 2013; Thakker et al., 2013; Wang et al., 2011). By using an economical and abundant carbon source in the fermentation, the total process cost can be significantly reduced. Soybean derived sugars represent such an inexpensive source of carbon. Since the 1950s, global soybean production has increased 15 times over. The United States, Brazil, and Argentina together produce about 80% of the world’s soy. At commercial soybean ß 2015 Wiley Periodicals, Inc.
processing plants, soybean meal as a byproduct is produced in large quantities. Soybeans contain approximately 37% crude protein and 20% oil. Defatted soybean meal has been widely used as a protein source in poultry, swine, and other feed (Giannoccaro et al., 2006). Cellulose, hemicellulose, pectin, and trace amounts of starch represent insoluble carbohydrates of soybeans (Liu, 1997). Soluble carbohydrates consist of stachyose, raffinose, sucrose, glucose, fructose, and trace amounts of arabinose, rhamnose, fucose, ribose, xylose, and mannose (Eldridge et al., 1979; Karr-Lilienthal et al., 2005). Consumption of the indigestible oligosaccharides (raffinose and stachyose) results in flatulence and abdominal discomfort. These oligosaccharides are often removed during production of soy protein products (Thakker et al., 2013). Hence, soymeal carbohydrates will potentially serve as an inexpensive carbon source. In this study, soluble carbohydrates were extracted from soybean meal and fully hydrolyzed to hexose sugars by the addition of the enzyme, a-galactosidase, for the first time. Firstly, the individual sugar fermentations were executed in order to select the most promising strains for free fatty acids production. Then experiments using monosaccharide mixtures and the soybean meal extract hydrolysate were conducted to explore the feasibility of the strains for producing high yields of free fatty acids using soy-based renewable carbohydrate.
Materials and Methods Strains and Plasmids A list of strains and plasmids used in this study is shown in Table I. Strain ML103 was constructed by the deletion of gene fadD, which encoded the fatty acyl-CoA synthetase (Li et al., 2012). Strain ML190 was constructed with a ptsG deletion in ML103 (San and Li, 2013), while XZK211 was constructed by introducing the ptsG deletion in ML211 using the one-step inactivation method (Datsenko and Wanner, 2000). PtsG encodes the glucose-specific transporter, EIICBGlc, an important phosphorylation donor and acceptor in the glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) (Kim et al., 2010). ML211 has an additional fabR gene deletion based on ML190. FabR encodes the fatty acid biosynthesis regulator (Zhang et al., 2002).
Soybean Meal Extract and Soybean Meal Extract Enzymatic Hydrolysate Soybean meal was obtained from Hieden Feed and Supply, Inc. (Houston, TX). Twenty grams of soybean meal were added to 100 mL water and stirred at room temperature for 15 min. The content was centrifuged at 10,000 RPM for 10 min to remove wet soybean meal and the supernatant, herein referred to as Soybean meal extract, containing soluble carbohydrates was recovered. To prepare soybean meal extract hydrolysate, a-galactosidase enzyme powder (Deerland Enzymes, GA. 15,000 GAL U/g, Fridjonssson et al., 1999) was added to the soybean meal extract at a final concentration of 2 GAL U/mL and mixed by brief stirring at room temperature for 16–20 h. Then the content was centrifuged at 12,000 RPM for 15 min to remove debris. The supernatant, referred to as Soybean meal extract enzymatic hydrolysate, was recovered, neutralized to pH 7.5 with 8 M NaOH and autoclaved at 121 C for 20 min before used directly for fermentation.
Shake Flask Cultures Cells from glycerol stock were streaked onto LB agar plates supplemented with 100 mg/L ampicillin (Sigma, St. Louis, MO) and grown overnight in an incubator at 37 C. Three colonies selected from an agar plate were grown overnight in 5 mL of LB medium supplemented with 100 mg/L ampicillin (Sigma) in an orbital shaker operated at 250 RPM and 37 C. The culture was adopted as the seed inoculum to initiate fermentation. For experiments involving pure sugars, glucose, galactose, and fructose were added individually or together into LB medium at approximately 15 g/L (83.3 mM), and supplemented with 100 mg/L ampicillin. Soybean meal extract hydrolysate was used directly as the carbon source, and LB components were supplemented as additional nitrogen source. The addition of isopropyl-b-Dthiogalactopyranoside (IPTG) to a final concentration of 1 mM induced the expression of the acyl-ACP carrier protein thioesterase gene (TE) and (3R)-hydroxymyristoyl acyl carrier protein dehydratase gene (fabZ). The seed culture was added at 1% (v/ v) to a 250 mL flask containing 40 mL of culture medium except the inoculum was added at 10% (v/v) for strain XZK211 harboring pXZ18 or pXZ18Z. The flasks were placed in an orbital shaker operated at 250 rpm and 30 C. Experiments were performed in
Table I. List of strains and plasmids used in this study. Description or genotypea
Strains/plasmids Strains ML103 ML190 ML211 XZK211 Plasmids pXZ18 pXZ18Z
MG1655 MG1655 MG1655 MG1655
Source or reference
fadDfadD- ptsG- (ML103 with ptsG-) fadD- fabR- (ML103 with fabR-) fadD- fabR- ptsG- (ML211 with ptsG-)
Li et al. (2012) San and Li (2013) This study This study
pTrc99a carries an acyl-ACP thioesterase from Ricinus communis pTrc99a carries an acyl-ACP thioesterase from R. communis and the (3R)-hydroxyacyl-ACP dehydrase (fabZ) from E. coli
Zhang et al. (2011) San and Li (2013)
a fadD encodes the fatty acyl-CoA synthetase; ptsG encodes the glucose-specific phosphotransferase enzyme IICB component; fabR encodes the fatty acid biosynthesis regulator.
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Figure 1. FFAs production and yield at 48 h using glucose, fructose, and galactose as a sole carbon source by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) glucose; (B) fructose; (C) galactose. Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. *P < 0.05; ns, not significant. Genes
ML103/pXZ18
ML103/pXZ18Z
ML190/pXZ18
ML190/pXZ18Z
ML211/pXZ18
ML211//pXZ18Z
XZK211/pXZ18
XZK211/pXZ18Z
fadD fabR ptsG TE fabZ
D
D
D
D
þ þ
D þ þ
D D
þ
D þ
D D þ
þ þ
D D D þ
D D D þ þ
þ, overexpression; D, knockout; blank, without manipulation.
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triplicate and samples were taken at 0, 24, 48, and 72 h for analysis of optical density, sugar concentrations, metabolites, and fatty acids. Analysis Method Culture was diluted to the linear range with 0.15 M NaCl, and optical density was measured at 600 nm with a spectrophotometer (DU800 Beckman Coulter). Procedure used to analyze extracellular metabolites was adapted from previous studies (Balzer et al., 2013; Lin et al., 2005; Sanchez et al., 2005). 1 mL culture was centrifuged at 13,000 RPM for 10 min, and the supernatant was filtered through a 0.2-mm syringe filter for HPLC
analysis. For glucose and acetic acid analysis, the HPLC system (Shimadzu-10A Systems, Shimadzu, Columbia, MD) was equipped with a cation-exchange column (Aminex HPX-87H, Bio Rad Labs, CA), a UV detector (ShimadzuSPD-10A) and a differential refractive index (RI) detector (Waters 2410, Waters, Milford, MA). A mobile phase of 2.5 mM H2SO4 solution at a 0.5 mL/min rate was used and the column operated at 55 C. For galactose and fructose analysis, the HPLC system was equipped with a cross linked polystyrene/divinylbenzene resin ion exchange column (Supercogel Ca, Sigma–Aldrich, LA), a UV detector and a differential refractive index (RI) detector. A mobile phase of HPLC-grade water at a 0.5 mL/min rate was used and the column was operated at 80 C.
Figure 2. FFAs compositions at 48 h using glucose, fructose, and galactose as a sole carbon source by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) glucose; (B) fructose; (C) galactose. Data are means SD (n ¼ 3).
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Total Fatty Acids Analysis
The fatty acids yield was calculated as g/g total sugar. The fatty acids composition was calculated as mass percentage of total at 48 h. Estimation of g fatty acid/g sugar yield for sugar mixture was based on the method reported previously by Wu et al. (2014a).
Fatty acids were extracted from culture using a procedure adapted from O’Fallon et al. (2007). Fermentation broth (1 mL) was mixed with 0.5 mL C15:0 as internal standards (1 g/L in methanol), 2 mL methanol, 0.7 mL of 10 N KOH in a Kimax tube, which was shaken vigorously for 3–5 min. Then the tube was incubated at 55–60 C for 1.5 h with intermittent shaking. Then 0.58 mL of 24 N H2SO4 was added to esterify the fatty acids, and the tubes were placed in a 55–60 C incubator for another 1.5 h with intermittent vortexing. After that, 2.0 mL of water and 2.0 mL of hexane was added and mixed. The tubes were centrifuged at 10 C, 4,000 RPM for 5 min to separate the liquid into two layers. The top hexane layer containing FAME was passed through a Pasteur pipette packed with sodium sulphite, and the sample was collected for GC analysis. The samples were analyzed and quantified by GC-FID system (GC QP 2010 from Shimadzu Scientific, Columbia, MD) using a FID detector. A DB-5MS column (30 cm 0.25 mm 0.25 mm, Agilent Co., Santa Clara, CA) was directly connected to the flame ionization detector (FID). Helium was used as the carrier gas with a flow rate of 1 mL/min. The oven temperature was initially held at 55 C for 2 min. Thereafter, it was raised with a gradient of 4 C/min to 220 C, where the temperature was held for 10 min. The system was also used at a 280 C interface temperature and a 250 C ion source temperature. The program GC-MS Postrun Analysis (Shimadzu, Columbia, MD) processed mass spectrometry data to obtain a spectrum (Li et al., 2012; Wu et al., 2014a).
Results and Discussion The Enzymatic Hydrolysate of Soybean Meal Extract In the enzymatic hydrolysate of soybean meal extract, glucose, galactose, and fructose are the three major monosaccharides. HPLC analysis showed the hydrolysate contains 72 mM glucose, 57 mM galactose, and 66 mM fructose, which corresponds to a total hexose concentration of 195 mM (35.1 g/L). A total hexose concentration of nearly 190 mM was obtained when 0.3% (v/v) concentrated H2SO4 was used to hydrolyze the soybean meal extract (Thakker et al., 2013). However, the hydrolysate was comprised of 1 mM stachyose, 13 mM raffinose, 25 mM sucrose, 55 mM galactose and/or fructose, and 42 mM glucose. Stachyose, raffinose, and sucrose were hard to be digested by E. coli, and additional genes were needed to make E. coli to consume them, (Schmid et al., 1976, 1988; Thakker et al., 2013), which was attributed to an increase in the cell’s metabolic burden. The enzymatic hydrolysis strategy developed in this study for soybean meal treatment could efficiently transform the soluble oligosaccharides to monosaccharides, which could be readily utilized by E. coli strains to produce high-value added chemicals.
Table II. Free fatty acids production using individual sugars as carbon source. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains ML103/pXZ18
ML103/pXZ18Z
ML190/pXZ18
ML190/pXZ18Z
ML211/pXZ18
ML211/pXZ18Z
XZK211/pXZ18
XZK211/pXZ18Z
Carbon Source
Cell density (OD600)
Sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose
9.40 0.57 9.00 0.75 8.55 0.32 9.63 0.33 7.58 0.31 10.75 0.17 8.14 0.12 5.11 0.51 7.05 0.25 6.64 0.20 6.30 0.19 7.95 0.33 7.16 0.29 7.76 0.15 10.25 0.70 6.47 0.49 6.14 0.26 7.26 0.33 5.05 0.46 5.82 0.09 7.05 0.36 5.75 0.17 5.78 0.16 6.97 0.08
87.95 0.27 82.49 0.16 76.50 0.02 83.05 0.52 85.65 0.02 84.48 0.21 87.95 0.27 82.49 0.16 76.50 0.02 83.18 0.57 85.50 0.25 84.48 0.40 83.85 0.25 82.27 0.49 76.13 0.22 83.64 0.44 82.49 0.16 74.42 0.47 83.39 0.65 82.13 0.22 70.86 0.35 86.90 0.52 82.28 0.61 71.00 0.44
37.88 1.55 36.06 0.02 37.02 0.03 48.58 2.12 31.65 0.87 40.48 0.60 12.48 0.48 33.42 0.91 35.35 1.75 45.83 0.65 38.92 0.86 48.64 2.78 18.03 1.13 17.15 0.26 16.21 0.24 36.76 2.17 35.89 1.14 29.55 1.87 34.29 3.12 35.01 1.11 26.80 1.67 41.81 1.01 33.02 0.61 34.05 1.06
36.24 0.92 31.25 0.05 30.57 0.06 11.05 0.95 29.77 1.45 32.29 0.58 40.84 1.93 30.52 0.68 36.06 0.91 10.46 1.41 18.01 0.86 10.24 0.86 50.96 2.45 43.54 0.86 37.89 0.56 30.97 0.82 17.10 0.65 15.85 1.18 35.04 0.70 30.95 1.33 26.80 1.67 17.24 0.07 28.99 0.35 28.85 0.80
19.26 0.96 24.32 0.36 23.49 0.43 38.85 0.69 29.37 1.36 19.68 0.80 22.98 0.48 26.67 0.33 19.94 1.05 39.74 1.15 38.64 0.81 34.33 2.04 17.64 1.05 22.05 0.63 25.91 1.25 24.74 0.58 41.72 1.46 47.33 0.79 22.89 2.17 25.49 1.51 27.53 0.81 36.63 0.33 28.59 0.90 26.27 0.44
6.62 0.24 8.37 0.10 8.90 0.12 1.52 0.04 9.20 0.38 7.55 0.24 23.70 0.73 9.39 0.12 8.65 0.71 3.96 0.03 4.43 0.08 6.79 0.25 13.38 1.04 17.25 0.98 19.98 0.90 7.53 0.09 5.29 0.18 7.27 0.74 7.77 0.34 8.55 1.86 11.88 0.15 4.32 0.06 9.40 0.21 10.83 0.55
42.86 0.63 39.62 0.07 40.48 0.09 12.57 0.49 38.97 0.98 39.83 0.84 64.54 0.43 39.92 0.20 44.71 0.55 14.43 1.15 22.45 0.89 17.03 0.73 64.33 0.70 60.79 0.54 57.87 0.38 38.50 0.50 22.39 0.50 23.12 0.23 42.81 2.23 39.49 2.29 45.67 1.57 21.56 0.59 38.39 0.63 39.48 1.12
Data are means standard deviation.
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Individual Sugar Fermentation for Free Fatty Acids In this study, strains ML103, ML190, ML211, XZK211 harboring plasmids pXZ18, or pXZ18Z were first examined for their metabolism of the individual sugars. As it can be seen from Figure 1A, all four strains with plasmids pXZ18 or pXZ18Z can consume glucose and produce free fatty acids efficiently. Comparing the fatty acid production of strains ML103/pXZ18 and ML103/ pXZ18Z, the latter could produce 3.81 g/L with a yield of 0.26 g/g, which was more than 70% of the maximum theoretical value. It was increased by 31.4% and 44.4% compared to the production of
ML103/pXZ18, which produced 2.90 g/L with a yield of 0.18 g/g. The highest concentration of FFAs was achieved by strain ML211/ pXZ18Z, which was 4.22 g/L with a yield of 0.28 g/g and was more than 75% of the theoretical value. The data demonstrated that the co-expression of TE and fabZ functioned well compared with the individual TE expression batch. These results were in agreement with previous investigations (San and Li, 2013; Wu et al., 2014a). Strain ML211/pXZ18Z also performed best in the fructose and galactose fermentation, which produced 3.80 g/L free fatty acids with a yield of 0.26 g/g on fructose, and 3.78 g/L with a yield of 0.28 g/g on galactose (Figs. 1B and C). Compared to the strain
Figure 3. FFAs production and yield using mixed sugars by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) final titer at 48 h (g/L); (B) yield (g/g total sugar); (C) compositions (g/L). Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. The data are statistically significant with P < 0.05 unless stated as ns (not significant).
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ML103/pXZ18Z, which produced 3.81, 3.17, 3.10 g/L fatty acids on glucose, fructose, and galactose, strain ML211/pXZ18Z increased the fatty acids production nearly 10.5%, 19.9%, 21.9%, respectively. This experiment indicated that the additional fabR gene deletion contributed to the increase in FFAs production. FabR is reported to regulate the synthesis of unsaturated fatty acids (Feng and Cronan, 2011; Zhang et al., 2002). Similar results were also reported by Wu et al. (2014b) when glycerol was used as the carbon source. Deletion of ptsG increased the FFAs production about 11.4% and 10.3% on glucose and fructose in TE overexpressed strain ML190/ pXZ18 compared to strain ML103/pXZ18 (Figs. 1A and B). However, when TE and fabZ were co-expressed in strain ML103/ pXZ18Z and ML190/pXZ18Z, the advantage of the ptsG mutation was not as obvious. There was no significant difference in FFAs production between these two strains (Fig. 1C). For strain ML211/ pXZ18Z and XZK211/pXZ18Z (strains with fabR deletion), the ptsG deletion even caused a decrease in free fatty acids production of about 16.4% on glucose (Fig. 1A), 11.1% on fructose (Fig. 1B) and 13.9% on galactose (Fig. 1C). The total fatty acids content and compositions for engineered strains at 48 h on individual sugars are shown in Figure 2 and Table II. Typical fatty acid production patterns at 24 h by the engineered strains on individual sugars were shown in Figure S1. Figure 2 showed the concentration in g/L for C14, C16, C16:1, and C18:1 straight chain fatty acids as major products. All eight strains produced very low levels of C18:1 FFAs while strain ML190/pXZ18 and ML211/pXZ18 could produce 0.23–0.76 g/L C18:1 FFAs. Comparing the data in Figure 2 and Table II, all strains harboring plasmids pXZ18 produced a nearly equivalent quantity of saturated and unsaturated fatty acids on the different sugars, while strains harboring plasmids pXZ18Z produced saturated tetradecanoic acid (C14) and hexadecanoic acid (C16) as the major components of FFAs. For strains ML103/pXZ18Z, ML190/pXZ18Z, ML211/pXZ18Z, and XZK211/pXZ18Z, the total C16 fatty acids production reached to 0.87–1.57 g/L on glucose, 0.64–1.57 g/L on fructose, and 0.93–1.53 g/L on galactose. The total C14 fatty acids production was even higher, about 1.30– 1.85 g/L on glucose, and 1.00–1.32 g/L on fructose, 0.98–1.56 g/L on galactose. The proportion of total unsaturated fatty acids of ML103/pXZ18Z, ML211/pXZ18Z, and strain XZK211/pXZ18Z dropped to less than 40%, while it dropped to less than 23% for strain ML190/pXZ18Z.
Fermentation of Free Fatty Acids with Sugar Mixture Substrates In sugar mixture cultivation, cellular metabolism is more complicated due to catabolite repression (Shimizu, 2013; Stephanopoulos, 2007). Before the hydrolysate fermentation experiment was executed, mixed sugar fermentation was performed to explore the promising strains for FFAs production using multiple sugars simultaneously. The mixed sugar fermentation medium was composed of about 31.9 mM (5.75 g/L) glucose, 24.8 mM (4.50 g/L) galactose, and 26.8 mM (4.75 g/L) fructose, and the total concentration of sugar was about 83.4 mM (15.0 g/L). The ratio of individual sugar was prepared according to the observed ratio in the soybean meal extract enzymatic hydrolysate. Strains ML103, ML190, ML211, XZK211 harboring plasmids, pXZ18 or pXZ18Z as indicated, were investigated for use of mixed sugars for FFAs production. While the strain ML211/pXZ18Z consistently gave better fatty acid production performance in individual sugar fermentation (Fig. 1), it was not the most effective strain for mixed sugars fermentation (Fig. 3A and B). The strain ML211/pXZ18Z produced only 2.83 g/L FFAs with a yield of 0.19 g/g, which is 1.37-fold of the yield of strain ML211/pXZ18. Two ptsG mutant strains, ML190/ pXZ18Z and XZK211/pXZ18Z, performed even better; these two strains reached higher FFAs concentrations of 3.16 and 3.18 g/L, respectively, with a yield of 0.21 g/g and 0.22 g/g at the end of the fermentation. These results suggested ptsG mutation contributed to improved performance in mixed sugars fermentation. For production of other chemicals, such as succinate and lactate, a ptsG mutation strain also showed relieve of catabolite repression on sugar mixtures and performed better to form a high concentration of the desired chemicals (Andersson et al., 2007; Dien et al., 2002; Wang et al., 2011). Figure 3C and Table III showed the distribution of different chain length FFAs of these eight engineered strains, which behaved differently. A lower percentage of unsaturated FFAs produced by strains harboring pXZ18Z, 12.2% for strain ML103/pXZ18Z, 37.8% for strain ML190/pXZ18Z, 45.2% for strain ML211/pXZ18Z, and 39.1% for strain XZK211/pXZ18Z were observed. This proportion decreased about 6.85% to 69.9% compared with the corresponding strains harboring pXZ18. It could be found that the unsaturated fatty acids proportion of strains ML211/pXZ18 and
Table III. Free fatty acids production using mixed sugars as carbon source. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains
Cell density (OD600)
Total sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
ML103/pXZ18 ML103/pXZ18Z ML190/pXZ18 ML190/pXZ18Z ML211/pXZ18 ML211/pXZ18Z XZK211/pXZ18 XZK211/pXZ18Z
7.36 0.37 7.58 0.51 7.17 0.27 8.35 0.51 4.41 0.45 6.82 0.44 7.37 0.04 7.04 0.17
73.24 0.66 84.66 0.14 79.05 0.77 82.83 0.17 83.49 0.53 82.45 0.63 73.14 0.72 75.26 0.24
38.41 1.46 56.79 0.37 33.06 0.71 35.40 1.34 15.74 0.48 12.97 0.68 35.16 2.26 36.88 0.75
33.43 1.74 10.24 0.58 30.09 1.33 28.88 1.19 40.08 1.05 31.03 2.33 34.65 0.98 31.02 0.62
21.02 1.51 31.00 2.07 26.38 2.79 26.82 1.88 24.14 0.67 41.81 1.23 21.84 0.91 24.02 1.61
7.14 1.01 2.00 0.08 10.47 1.41 8.90 0.54 20.04 0.01 14.19 0.32 8.35 1.21 8.08 0.08
40.57 1.33 12.22 0.90 40.56 1.06 37.78 0.32 60.12 0.26 45.23 1.09 43.00 1.08 39.11 2.09
Data are means standard deviation.
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ML211/pXZ18Z all increased compared with the strains ML190/ pXZ18 and ML190/pXZ18Z. This result might be caused by the additional fabR deletion in ML211, which removed the encoded fatty acid biosynthesis regulator gene. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis (McCue et al., 2001). So transcription of both fabA and fabB in a fabR deletion strain would increase relative to the parental strain, and the unsaturated fatty acid production will be enhanced (Feng and Cronan, 2011; Zhang et al., 2002). The effect of carbon source on fatty acid distribution by the engineered strains is shown in Table S1. The effect of carbon source is strain dependent. Soybean Meal Extract Enzymatic Hydrolysate Fermentation Soybean meal extract enzymatic hydrolysate produced in our lab was investigated for FFAs production by the strains ML103/pXZ18, ML190/pXZ18Z, ML211/pXZ18Z, and XZK211/pXZ18Z. The enzymatic hydrolyzed biomass was diluted about 2.4-fold to give an initial total sugar of 15 g/L (83.3 mM) in the fermentation medium. The FFAs concentrations, yields and distribution were shown in Figure 4 and Table IV. Similar to the fermentation results with sugar mixture as the substrates, strains ML190/pXZ18Z and XZK211/pXZ18Z were able to produce higher FFAs among the four strains, which were 2.73 and 2.78 g/L, with high yields of 0.20 g/g and 0.21 g/g, respectively (Figs. 4A and B). However, the final concentrations of FFAs they reached were 13.6% and 12.6% less than those obtained on pure sugar mixtures. The possible reason might be that some salts, proteins, and complex compounds released in the hydrolysate functioned as inhibitors for FFAs fermentation, and finally decreased the utilization efficiency of the sugars (J€onsson et al., 2013; Zha et al., 2012). The FFAs distribution was shown in Figure 4C. The strain ML190/pXZ18Z produced unsaturated fatty acids with a proportion of 34.8% of the total FFAs, while strain ML211/pXZ18Z produced a proportion of 62.1%. It might be deduced that the additional fabR deletion in ML211 could contribute to the increased proportion of the unsaturated FFAs in the total FFAs for soybean meal extract hydrolysate fermentation. The results were consistent with the fermentation data on pure mixed sugar substrate. The idea of renewable resources has inspired research over a long period of time, among which fatty acid biosynthesis with the aim to produce free fatty acids or derivatives for substitution of diesel is a major subject (Janßen and Steinb€uchel, 2014). Although this method offers a great potential for industrialization due to its short production time and very low land-use, the use of cellulose, lignin, hemicellulose, and other non-food carbon source still needs further investigation to reduce the production cost. Here, the combination of enzymatic hydrolysis and metabolic engineering to make soybean meal, a non-food carbon source, to be a good substrate for high-level free fatty acids production is desirable and indicates its general possibility. An approximate cost comparison of soybean meal carbohydrates with other feedstock is shown in Table S2.
Figure 4. FFAs production and yield using soybean meal enzyme hydrolysate by by ML103, ML190, ML211, XZK211 harboring pXZ18Z. (A) final titer at 48 h (g/L); (B) yield (g/g total sugar); (C) compositions (g/L). Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. The data are statistically significant with P < 0.05 unless stated as ns (not significant).
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Table IV. Free fatty acids production using soybean meal enzyme hydrolysate. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains
Cell density (OD600)
Sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
ML103/pXZ18Z ML190/pXZ18Z ML211/pXZ18Z XZK211/pXZ18Z
7.36 0.37 7.84 0.32 7.35 0.10 7.44 0.44
76.41 0.42 74.57 0.18 74.04 0.20 74.65 0.24
36.80 0.30 38.59 1.25 17.07 0.71 38.86 0.09
25.74 0.14 28.04 0.54 43.87 1.03 28.54 0.93
29.11 0.34 26.64 0.72 20.88 1.02 25.72 1.22
8.35 0.13 6.72 0.25 18.19 0.62 6.88 0.42
34.09 0.07 34.76 0.38 62.05 0.41 35.42 0.43
Data are means standard deviation.
Conclusions In this study, free fatty acids were produced from individual sugars and enzymatic hydrolysate of soybean meal extract by engineered E. coli strains ML103, ML190, ML211, XZK211 plus plasmid pXZ18 or pXZ18Z. It was shown that overexpression of fabZ could further enhance the free fatty acids production besides TE overexpression. The unsaturated fatty acids proportion in the total fatty acids could be decreased with fabZ overexpression or increased with fabR deletion. The fadD, ptsG, fabR triple mutant strain XZK211/pXZ18Z, carrying fabZ behind the TE from R. communis, can produce FFAs efficiently from the soybean meal carbohydrates with a high concentration of 2.78 g/L and a high yield of 0.21 g/g. This work was supported in part by the United Soybean Board (USB), and the China Scholarship Council.
References Andersson C, Hodge D, Berglund KA, Rova U. 2007. Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli. Biotechnol Progr 23:381–388. Balzer G, Thakker C, Bennett GN, San KY. 2013. Metabolic engineering Escherichia coli to minimize byproduct formate and improving succinate productivity through NADH availability by heterologous expression of NAD(þ)-dependent formate dehydrogenase. Metab Eng 20:1–8. Cao Y, Yang J, Xian M, Xu X, Liu W. 2010. Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Appl Microbiol Biotechnol 87:271–280. Cho H, Cronan J, Jr. 1995. Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 270:4216–4219. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Nat Acad Sci 97:6640–6645. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R. 2011. Engineering reversal of the b-oxidation cycle for the synthesis of fuels and chemicals. Nature 476:355–359. Dien BS, Nichols NN, Bothast RJ. 2002. Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of Llactate acid. J Ind Microbiol Biotechnol 29:221–227. Eldridge AC, Black LT, Wolf JW. 1979. Carbohydrate composition of soybean flours, protein concentrates, and isolates. J Agric Food Chem 27:799–802. Feng Y, Cronan JE. 2011. Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters. Mol Microbiol 80:195–218. Fridjonssson O, Watzlawick H, Gehweiler A, Rohrhirsch T, Mattes R. 1999. Cloning of the gene encoding a novel thermostable galactosidase from Thermus brockianus I TI360. Appl Environ Microbiol 65:3955–3963. Giannoccaro E, Wang YJ, Chen P. 2006. Effects of solvent, temperature, time, solventto-sample ratio, sample size, and defatting on the extraction of soluble sugars in soybean. J Food Sci 71:C59–C64.
2332
Biotechnology and Bioengineering, Vol. 112, No. 11, November, 2015
Janßen HJ, Steinb€uchel A. 2014. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels 7:7–33. Jiang P, Cronan J, Jr. 1994. Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action. J Bacteriol 176:2814–2821. J€onsson L, Alriksson B, Nilvebrant N. 2013. Bioconversion of lignocelluloses: Inhibitors and detoxification. Biotechnol Biofuels 6:16–25. Karr-Lilienthal LK, Grieshop CM, Spears JK, Fahey GC. 2005. Amino acid, carbohydrate, and fat composition of soybean meals prepared at 55 commercial U.S. soybean processing plants. J Agric Food Chem 53:2146– 2150. Kim JH, Block DE, Mills DA. 2010. Simultaneous consumption of pentose and hexose sugars: An optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl Microbiol Biotechnol 88:1077–1085. Kim KR, Oh DK. 2013. Production of hydroxyl fatty acids by microbial fatty acidhydroxylation enzymes. Biotechnol Adv 31:1473–1485. Kim NJ, Li H, Jung K, Chang HN, Lee PC. 2011. Ethanol production from algal hydrolysates using Escherichia coli KO11. Bioresour Technol 102:7466– 7469. Lennen R, Braden G, West R, Dumesic J, Pfleger B. 2010. A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 106:193–202. Li M, Zhang X, Agrawal A, San K. 2012. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acyl-ACP thioesterase from Ricinus communis. Metab Eng 14:380–387. Lin H, Bennett GN, San K. 2005. Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab Eng 7:116–127. Liu K. 1997. Soybeans: Chemistry, technology, and utilization. New York: Chapman and Hall. Liu T, Vora H, Khosla C. 2010. Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 12:378–386. Lu X, Vora H, Khosla C. 2008. Overexpression of free fatty acids in E. coli: Implications for biodiesel production. Metab Eng 10:333–339. Mazumdar S, Bang J, Oh MK. 2013. L-Lactate production from seaweed hydrolysate of Laminaria japonica using metabolically engineered Escherichia coli. Appl Biochem Biotechnol 172:1938–1952. McCue L, Thompson W, Carmack C, Ryan MP, Liu JS, Derbyshire V, Lawrence CE. 2001. Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes. Nucleic Acids Res 29:774–782. Nickolau B, Perera M, Brachova L, Shanks B. 2008. Platform chemicals for a biorenewable chemical industry. Plant J 54:536–545. O’Fallon JV, Busboom JR, Nelson ML, Gaskins CT. 2007. A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils, and feedstuffs. J Anim Sci 85:1511–1521. Ooi S, Pee H. 1985. Processing for industrial fatty acids-II. J Am Oil Chem Soc 62:348–351. Peralta-Yahya PB, Zhang F, del Cardayre SB, Keasling JD. 2012. Microbial engineering for the production of advanced biofuels. Nature 488:320–328. San KY, Li M. 2013. Bacteria and method for synthesizing fatty acids. Vol. WO/2013/ 059218, USA.
Sanchez AM, Bennett GN, San K. 2005. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng 7:229–239. Schmid K, Schupfner M, Schmitt R. 1982. Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12. J Bacteriol 151:68–76. Schmid K, Schmitt R. 1976. Raffinose metabolism in Escherichia coli K12. Purification and properties of a new a-galactosidase specified by a transmissible plasmid. Eur. J Biochem 67:95–104. Shimizu K. 2013. Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism. ISRN Biochem 645983:1–47. Stephanopoulos G. 2007. Challenges in engineering microbes for biofuels production. Science 315:801–804. Thakker C, San K, Bennett GN. 2013. Production of succinic acid by engineered E. coli strains using soybean carbohydrates as feedstock under aerobic fermentation conditions. Bioresour Technol 130:398–405. Voelker T, Davies H. 1994. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of plant medium-chain acyl-acyl carrier protein thioesterase. J Bacteriol 176:7320–7327. Wang D, Li Q, Yang M, Zhang Y, Su Z, Xing J. 2011. Efficient production of succinic acid from corn stalk hydrolysates by a recombinant Escherichia coli with ptsG mutation. Process Biochem 46:365–371. Wu H, San KY. 2014. Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli. Biotechnol Bioeng 9999:1–11.
Wu H, Lee J, Karanjikar M, San KY. 2014a. Efficient free fatty acid production from woody biomass hydrolysate using metabolically engineered Escherichia coli. Bioresour Technol 169:119–125. Wu H, Karanjikar M, San KY. 2014b. Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol. Metab Eng 25:82–91. Zha Y, Muilwijk B, Coulier L, Punt PJ. 2012. Inhibitory compounds in lignocellulosic biomass hydrolysates during hydrolysate fermentation process. J Bioprocess Biotechniq 2:1–11. Zhang XJ, Li M, Agrawal A, San KY. 2011. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterase. Metab Eng 13: 713–722. Zhang YM, Marrakchi H, Rock CO. 2002. The FabR (YijC) transcription factor regulates unsaturated fatty acid biosynthesis in Escherichia coli. J Biol Chem 277:15558–15565. Zhang YM, Rock CO. 2009. Transcriptional regulation in bacterial membrane lipid synthesis. J Lipid Res 50:S115–S119.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
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Department of Bioengineering, Rice University, 6100 Main Street, MS-362, Houston, Texas 2 College of Chemistry and Chemical Engineering, Chongqing University, Chongqing, P. R. China 3 Department of BioSciences, Rice University, Houston, Texas 4 Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005-1892; telephone: þ1-713-348-5361; fax: þ1-713-348-5877; e-mail: [email protected]
Introduction ABSTRACT: Conversion of biomass feedstock to chemicals and fuels has attracted increasing attention recently. Soybean meal, containing significant quantities of carbohydrates, is an inexpensive renewable feedstock. Glucose, galactose, and fructose can be obtained by enzymatic hydrolysis of soluble carbohydrates of soybean meal. Free fatty acids (FFAs) are valuable molecules that can be used as precursors for the production of fuels and other value-added chemicals. In this study, free fatty acids were produced by mutant Escherichia coli strains with plasmid pXZ18Z (carrying acyl-ACP thioesterase (TE) and (3R)-hydroxyacyl-ACP dehydratase) using individual sugars, sugar mixtures, and enzymatic hydrolyzed soybean meal extract. For individual sugar fermentations, strain ML211 (MG1655 fadD- fabR-)/pXZ18Z showed the best performance, which produced 4.22, 3.79, 3.49 g/L free fatty acids on glucose, fructose, and galactose, respectively. While the strain ML211/pXZ18Z performed the best with individual sugars, however, for sugar mixture fermentation, the triple mutant strain XZK211 (MG1655 fadD- fabR- ptsG-)/pXZ18Z with an additional deletion of ptsG encoding the glucose-specific transporter, functioned the best due to relieved catabolite repression. This strain produced approximately 3.18 g/L of fatty acids with a yield of 0.22 g fatty acids/g total sugar. Maximum free fatty acids production of 2.78 g/L with a high yield of 0.21 g/g was achieved using soybean meal extract hydrolysate. The results suggested that soybean meal carbohydrates after enzymatic treatment could serve as an inexpensive feedstock for the efficient production of free fatty acids. Biotechnol. Bioeng. 2015;112: 2324–2333. ß 2015 Wiley Periodicals, Inc. KEYWORDS: free fatty acids; Escherichia coli; soybean meal hydrolysate; mixed sugars; catabolite repression
Correspondence to: Ka-Yiu San Contract grant sponsor: United Soybean Board (USB) Contract grant sponsor: China Scholarship Council Received 16 January 2015; Revision received 5 April 2015; Accepted 29 April 2015 Accepted manuscript online 5 May 2015; Article first published online 31 July 2015 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25633/abstract). DOI 10.1002/bit.25633
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Free fatty acids (FFAs) are valuable molecules which could be used as precursors for the production of fuels, oleochemicals, and special health care products (Lennen et al., 2010; Liu et al., 2010; Nickolau et al., 2008). The traditional production of free fatty acids relies on the chemical or biological catalytic processes, such as the catalytic hydrolysis of triglycerides, but sources are mostly limited to animal fats and plant oils (Ooi and Pee, 1985). Alternatively, the fatty acid formation could occur in vivo in bacteria. Recently, many strategies are being made to increase the yield to approach theoretical levels and decrease the cost of bioconversion processes to make the biosynthetic process competitive with the traditional catalytic process (Janßen and Steinb€uchel, 2014; Lennen et al., 2010; PeraltaYahya et al., 2012). Wild type Escherichia coli strains do not normally accumulate free fatty acids while synthesizing membrane lipids (Voelker and Davies, 1994). However, it has been shown that the production of free fatty acids in E. coli could be induced by overexpression of an acyl-ACP thioesterase (TE) gene (Cho et al., 1995; Jiang and Cronan, 1994; Lu et al., 2008). Much work has been done to make fatty acids in E. coli of various chain lengths (Dellomonaco et al., 2011; Wu et al., 2014a), saturation status (Cao et al., 2010), and containing additional functional groups (Kim and Oh, 2013). Except for the availability of abundant metabolic tools to increase the fatty acids yield in E. coli, there is another advantage that makes E. coli an ideal host for fatty acids production. E. coli has been found to have the ability to metabolize many kinds of individual sugars and biomass hydrolysates to produce high-value added chemicals, such as ethanol, succinic acid, and lactic acid (Kim et al., 2011; Mazumdar et al., 2013; Thakker et al., 2013; Wang et al., 2011). By using an economical and abundant carbon source in the fermentation, the total process cost can be significantly reduced. Soybean derived sugars represent such an inexpensive source of carbon. Since the 1950s, global soybean production has increased 15 times over. The United States, Brazil, and Argentina together produce about 80% of the world’s soy. At commercial soybean ß 2015 Wiley Periodicals, Inc.
processing plants, soybean meal as a byproduct is produced in large quantities. Soybeans contain approximately 37% crude protein and 20% oil. Defatted soybean meal has been widely used as a protein source in poultry, swine, and other feed (Giannoccaro et al., 2006). Cellulose, hemicellulose, pectin, and trace amounts of starch represent insoluble carbohydrates of soybeans (Liu, 1997). Soluble carbohydrates consist of stachyose, raffinose, sucrose, glucose, fructose, and trace amounts of arabinose, rhamnose, fucose, ribose, xylose, and mannose (Eldridge et al., 1979; Karr-Lilienthal et al., 2005). Consumption of the indigestible oligosaccharides (raffinose and stachyose) results in flatulence and abdominal discomfort. These oligosaccharides are often removed during production of soy protein products (Thakker et al., 2013). Hence, soymeal carbohydrates will potentially serve as an inexpensive carbon source. In this study, soluble carbohydrates were extracted from soybean meal and fully hydrolyzed to hexose sugars by the addition of the enzyme, a-galactosidase, for the first time. Firstly, the individual sugar fermentations were executed in order to select the most promising strains for free fatty acids production. Then experiments using monosaccharide mixtures and the soybean meal extract hydrolysate were conducted to explore the feasibility of the strains for producing high yields of free fatty acids using soy-based renewable carbohydrate.
Materials and Methods Strains and Plasmids A list of strains and plasmids used in this study is shown in Table I. Strain ML103 was constructed by the deletion of gene fadD, which encoded the fatty acyl-CoA synthetase (Li et al., 2012). Strain ML190 was constructed with a ptsG deletion in ML103 (San and Li, 2013), while XZK211 was constructed by introducing the ptsG deletion in ML211 using the one-step inactivation method (Datsenko and Wanner, 2000). PtsG encodes the glucose-specific transporter, EIICBGlc, an important phosphorylation donor and acceptor in the glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) (Kim et al., 2010). ML211 has an additional fabR gene deletion based on ML190. FabR encodes the fatty acid biosynthesis regulator (Zhang et al., 2002).
Soybean Meal Extract and Soybean Meal Extract Enzymatic Hydrolysate Soybean meal was obtained from Hieden Feed and Supply, Inc. (Houston, TX). Twenty grams of soybean meal were added to 100 mL water and stirred at room temperature for 15 min. The content was centrifuged at 10,000 RPM for 10 min to remove wet soybean meal and the supernatant, herein referred to as Soybean meal extract, containing soluble carbohydrates was recovered. To prepare soybean meal extract hydrolysate, a-galactosidase enzyme powder (Deerland Enzymes, GA. 15,000 GAL U/g, Fridjonssson et al., 1999) was added to the soybean meal extract at a final concentration of 2 GAL U/mL and mixed by brief stirring at room temperature for 16–20 h. Then the content was centrifuged at 12,000 RPM for 15 min to remove debris. The supernatant, referred to as Soybean meal extract enzymatic hydrolysate, was recovered, neutralized to pH 7.5 with 8 M NaOH and autoclaved at 121 C for 20 min before used directly for fermentation.
Shake Flask Cultures Cells from glycerol stock were streaked onto LB agar plates supplemented with 100 mg/L ampicillin (Sigma, St. Louis, MO) and grown overnight in an incubator at 37 C. Three colonies selected from an agar plate were grown overnight in 5 mL of LB medium supplemented with 100 mg/L ampicillin (Sigma) in an orbital shaker operated at 250 RPM and 37 C. The culture was adopted as the seed inoculum to initiate fermentation. For experiments involving pure sugars, glucose, galactose, and fructose were added individually or together into LB medium at approximately 15 g/L (83.3 mM), and supplemented with 100 mg/L ampicillin. Soybean meal extract hydrolysate was used directly as the carbon source, and LB components were supplemented as additional nitrogen source. The addition of isopropyl-b-Dthiogalactopyranoside (IPTG) to a final concentration of 1 mM induced the expression of the acyl-ACP carrier protein thioesterase gene (TE) and (3R)-hydroxymyristoyl acyl carrier protein dehydratase gene (fabZ). The seed culture was added at 1% (v/ v) to a 250 mL flask containing 40 mL of culture medium except the inoculum was added at 10% (v/v) for strain XZK211 harboring pXZ18 or pXZ18Z. The flasks were placed in an orbital shaker operated at 250 rpm and 30 C. Experiments were performed in
Table I. List of strains and plasmids used in this study. Description or genotypea
Strains/plasmids Strains ML103 ML190 ML211 XZK211 Plasmids pXZ18 pXZ18Z
MG1655 MG1655 MG1655 MG1655
Source or reference
fadDfadD- ptsG- (ML103 with ptsG-) fadD- fabR- (ML103 with fabR-) fadD- fabR- ptsG- (ML211 with ptsG-)
Li et al. (2012) San and Li (2013) This study This study
pTrc99a carries an acyl-ACP thioesterase from Ricinus communis pTrc99a carries an acyl-ACP thioesterase from R. communis and the (3R)-hydroxyacyl-ACP dehydrase (fabZ) from E. coli
Zhang et al. (2011) San and Li (2013)
a fadD encodes the fatty acyl-CoA synthetase; ptsG encodes the glucose-specific phosphotransferase enzyme IICB component; fabR encodes the fatty acid biosynthesis regulator.
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Figure 1. FFAs production and yield at 48 h using glucose, fructose, and galactose as a sole carbon source by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) glucose; (B) fructose; (C) galactose. Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. *P < 0.05; ns, not significant. Genes
ML103/pXZ18
ML103/pXZ18Z
ML190/pXZ18
ML190/pXZ18Z
ML211/pXZ18
ML211//pXZ18Z
XZK211/pXZ18
XZK211/pXZ18Z
fadD fabR ptsG TE fabZ
D
D
D
D
þ þ
D þ þ
D D
þ
D þ
D D þ
þ þ
D D D þ
D D D þ þ
þ, overexpression; D, knockout; blank, without manipulation.
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triplicate and samples were taken at 0, 24, 48, and 72 h for analysis of optical density, sugar concentrations, metabolites, and fatty acids. Analysis Method Culture was diluted to the linear range with 0.15 M NaCl, and optical density was measured at 600 nm with a spectrophotometer (DU800 Beckman Coulter). Procedure used to analyze extracellular metabolites was adapted from previous studies (Balzer et al., 2013; Lin et al., 2005; Sanchez et al., 2005). 1 mL culture was centrifuged at 13,000 RPM for 10 min, and the supernatant was filtered through a 0.2-mm syringe filter for HPLC
analysis. For glucose and acetic acid analysis, the HPLC system (Shimadzu-10A Systems, Shimadzu, Columbia, MD) was equipped with a cation-exchange column (Aminex HPX-87H, Bio Rad Labs, CA), a UV detector (ShimadzuSPD-10A) and a differential refractive index (RI) detector (Waters 2410, Waters, Milford, MA). A mobile phase of 2.5 mM H2SO4 solution at a 0.5 mL/min rate was used and the column operated at 55 C. For galactose and fructose analysis, the HPLC system was equipped with a cross linked polystyrene/divinylbenzene resin ion exchange column (Supercogel Ca, Sigma–Aldrich, LA), a UV detector and a differential refractive index (RI) detector. A mobile phase of HPLC-grade water at a 0.5 mL/min rate was used and the column was operated at 80 C.
Figure 2. FFAs compositions at 48 h using glucose, fructose, and galactose as a sole carbon source by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) glucose; (B) fructose; (C) galactose. Data are means SD (n ¼ 3).
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Total Fatty Acids Analysis
The fatty acids yield was calculated as g/g total sugar. The fatty acids composition was calculated as mass percentage of total at 48 h. Estimation of g fatty acid/g sugar yield for sugar mixture was based on the method reported previously by Wu et al. (2014a).
Fatty acids were extracted from culture using a procedure adapted from O’Fallon et al. (2007). Fermentation broth (1 mL) was mixed with 0.5 mL C15:0 as internal standards (1 g/L in methanol), 2 mL methanol, 0.7 mL of 10 N KOH in a Kimax tube, which was shaken vigorously for 3–5 min. Then the tube was incubated at 55–60 C for 1.5 h with intermittent shaking. Then 0.58 mL of 24 N H2SO4 was added to esterify the fatty acids, and the tubes were placed in a 55–60 C incubator for another 1.5 h with intermittent vortexing. After that, 2.0 mL of water and 2.0 mL of hexane was added and mixed. The tubes were centrifuged at 10 C, 4,000 RPM for 5 min to separate the liquid into two layers. The top hexane layer containing FAME was passed through a Pasteur pipette packed with sodium sulphite, and the sample was collected for GC analysis. The samples were analyzed and quantified by GC-FID system (GC QP 2010 from Shimadzu Scientific, Columbia, MD) using a FID detector. A DB-5MS column (30 cm 0.25 mm 0.25 mm, Agilent Co., Santa Clara, CA) was directly connected to the flame ionization detector (FID). Helium was used as the carrier gas with a flow rate of 1 mL/min. The oven temperature was initially held at 55 C for 2 min. Thereafter, it was raised with a gradient of 4 C/min to 220 C, where the temperature was held for 10 min. The system was also used at a 280 C interface temperature and a 250 C ion source temperature. The program GC-MS Postrun Analysis (Shimadzu, Columbia, MD) processed mass spectrometry data to obtain a spectrum (Li et al., 2012; Wu et al., 2014a).
Results and Discussion The Enzymatic Hydrolysate of Soybean Meal Extract In the enzymatic hydrolysate of soybean meal extract, glucose, galactose, and fructose are the three major monosaccharides. HPLC analysis showed the hydrolysate contains 72 mM glucose, 57 mM galactose, and 66 mM fructose, which corresponds to a total hexose concentration of 195 mM (35.1 g/L). A total hexose concentration of nearly 190 mM was obtained when 0.3% (v/v) concentrated H2SO4 was used to hydrolyze the soybean meal extract (Thakker et al., 2013). However, the hydrolysate was comprised of 1 mM stachyose, 13 mM raffinose, 25 mM sucrose, 55 mM galactose and/or fructose, and 42 mM glucose. Stachyose, raffinose, and sucrose were hard to be digested by E. coli, and additional genes were needed to make E. coli to consume them, (Schmid et al., 1976, 1988; Thakker et al., 2013), which was attributed to an increase in the cell’s metabolic burden. The enzymatic hydrolysis strategy developed in this study for soybean meal treatment could efficiently transform the soluble oligosaccharides to monosaccharides, which could be readily utilized by E. coli strains to produce high-value added chemicals.
Table II. Free fatty acids production using individual sugars as carbon source. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains ML103/pXZ18
ML103/pXZ18Z
ML190/pXZ18
ML190/pXZ18Z
ML211/pXZ18
ML211/pXZ18Z
XZK211/pXZ18
XZK211/pXZ18Z
Carbon Source
Cell density (OD600)
Sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose Glucose Fructose Galactose
9.40 0.57 9.00 0.75 8.55 0.32 9.63 0.33 7.58 0.31 10.75 0.17 8.14 0.12 5.11 0.51 7.05 0.25 6.64 0.20 6.30 0.19 7.95 0.33 7.16 0.29 7.76 0.15 10.25 0.70 6.47 0.49 6.14 0.26 7.26 0.33 5.05 0.46 5.82 0.09 7.05 0.36 5.75 0.17 5.78 0.16 6.97 0.08
87.95 0.27 82.49 0.16 76.50 0.02 83.05 0.52 85.65 0.02 84.48 0.21 87.95 0.27 82.49 0.16 76.50 0.02 83.18 0.57 85.50 0.25 84.48 0.40 83.85 0.25 82.27 0.49 76.13 0.22 83.64 0.44 82.49 0.16 74.42 0.47 83.39 0.65 82.13 0.22 70.86 0.35 86.90 0.52 82.28 0.61 71.00 0.44
37.88 1.55 36.06 0.02 37.02 0.03 48.58 2.12 31.65 0.87 40.48 0.60 12.48 0.48 33.42 0.91 35.35 1.75 45.83 0.65 38.92 0.86 48.64 2.78 18.03 1.13 17.15 0.26 16.21 0.24 36.76 2.17 35.89 1.14 29.55 1.87 34.29 3.12 35.01 1.11 26.80 1.67 41.81 1.01 33.02 0.61 34.05 1.06
36.24 0.92 31.25 0.05 30.57 0.06 11.05 0.95 29.77 1.45 32.29 0.58 40.84 1.93 30.52 0.68 36.06 0.91 10.46 1.41 18.01 0.86 10.24 0.86 50.96 2.45 43.54 0.86 37.89 0.56 30.97 0.82 17.10 0.65 15.85 1.18 35.04 0.70 30.95 1.33 26.80 1.67 17.24 0.07 28.99 0.35 28.85 0.80
19.26 0.96 24.32 0.36 23.49 0.43 38.85 0.69 29.37 1.36 19.68 0.80 22.98 0.48 26.67 0.33 19.94 1.05 39.74 1.15 38.64 0.81 34.33 2.04 17.64 1.05 22.05 0.63 25.91 1.25 24.74 0.58 41.72 1.46 47.33 0.79 22.89 2.17 25.49 1.51 27.53 0.81 36.63 0.33 28.59 0.90 26.27 0.44
6.62 0.24 8.37 0.10 8.90 0.12 1.52 0.04 9.20 0.38 7.55 0.24 23.70 0.73 9.39 0.12 8.65 0.71 3.96 0.03 4.43 0.08 6.79 0.25 13.38 1.04 17.25 0.98 19.98 0.90 7.53 0.09 5.29 0.18 7.27 0.74 7.77 0.34 8.55 1.86 11.88 0.15 4.32 0.06 9.40 0.21 10.83 0.55
42.86 0.63 39.62 0.07 40.48 0.09 12.57 0.49 38.97 0.98 39.83 0.84 64.54 0.43 39.92 0.20 44.71 0.55 14.43 1.15 22.45 0.89 17.03 0.73 64.33 0.70 60.79 0.54 57.87 0.38 38.50 0.50 22.39 0.50 23.12 0.23 42.81 2.23 39.49 2.29 45.67 1.57 21.56 0.59 38.39 0.63 39.48 1.12
Data are means standard deviation.
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Individual Sugar Fermentation for Free Fatty Acids In this study, strains ML103, ML190, ML211, XZK211 harboring plasmids pXZ18, or pXZ18Z were first examined for their metabolism of the individual sugars. As it can be seen from Figure 1A, all four strains with plasmids pXZ18 or pXZ18Z can consume glucose and produce free fatty acids efficiently. Comparing the fatty acid production of strains ML103/pXZ18 and ML103/ pXZ18Z, the latter could produce 3.81 g/L with a yield of 0.26 g/g, which was more than 70% of the maximum theoretical value. It was increased by 31.4% and 44.4% compared to the production of
ML103/pXZ18, which produced 2.90 g/L with a yield of 0.18 g/g. The highest concentration of FFAs was achieved by strain ML211/ pXZ18Z, which was 4.22 g/L with a yield of 0.28 g/g and was more than 75% of the theoretical value. The data demonstrated that the co-expression of TE and fabZ functioned well compared with the individual TE expression batch. These results were in agreement with previous investigations (San and Li, 2013; Wu et al., 2014a). Strain ML211/pXZ18Z also performed best in the fructose and galactose fermentation, which produced 3.80 g/L free fatty acids with a yield of 0.26 g/g on fructose, and 3.78 g/L with a yield of 0.28 g/g on galactose (Figs. 1B and C). Compared to the strain
Figure 3. FFAs production and yield using mixed sugars by ML103, ML190, ML211, XZK211 harboring pXZ18 or pXZ18Z. (A) final titer at 48 h (g/L); (B) yield (g/g total sugar); (C) compositions (g/L). Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. The data are statistically significant with P < 0.05 unless stated as ns (not significant).
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ML103/pXZ18Z, which produced 3.81, 3.17, 3.10 g/L fatty acids on glucose, fructose, and galactose, strain ML211/pXZ18Z increased the fatty acids production nearly 10.5%, 19.9%, 21.9%, respectively. This experiment indicated that the additional fabR gene deletion contributed to the increase in FFAs production. FabR is reported to regulate the synthesis of unsaturated fatty acids (Feng and Cronan, 2011; Zhang et al., 2002). Similar results were also reported by Wu et al. (2014b) when glycerol was used as the carbon source. Deletion of ptsG increased the FFAs production about 11.4% and 10.3% on glucose and fructose in TE overexpressed strain ML190/ pXZ18 compared to strain ML103/pXZ18 (Figs. 1A and B). However, when TE and fabZ were co-expressed in strain ML103/ pXZ18Z and ML190/pXZ18Z, the advantage of the ptsG mutation was not as obvious. There was no significant difference in FFAs production between these two strains (Fig. 1C). For strain ML211/ pXZ18Z and XZK211/pXZ18Z (strains with fabR deletion), the ptsG deletion even caused a decrease in free fatty acids production of about 16.4% on glucose (Fig. 1A), 11.1% on fructose (Fig. 1B) and 13.9% on galactose (Fig. 1C). The total fatty acids content and compositions for engineered strains at 48 h on individual sugars are shown in Figure 2 and Table II. Typical fatty acid production patterns at 24 h by the engineered strains on individual sugars were shown in Figure S1. Figure 2 showed the concentration in g/L for C14, C16, C16:1, and C18:1 straight chain fatty acids as major products. All eight strains produced very low levels of C18:1 FFAs while strain ML190/pXZ18 and ML211/pXZ18 could produce 0.23–0.76 g/L C18:1 FFAs. Comparing the data in Figure 2 and Table II, all strains harboring plasmids pXZ18 produced a nearly equivalent quantity of saturated and unsaturated fatty acids on the different sugars, while strains harboring plasmids pXZ18Z produced saturated tetradecanoic acid (C14) and hexadecanoic acid (C16) as the major components of FFAs. For strains ML103/pXZ18Z, ML190/pXZ18Z, ML211/pXZ18Z, and XZK211/pXZ18Z, the total C16 fatty acids production reached to 0.87–1.57 g/L on glucose, 0.64–1.57 g/L on fructose, and 0.93–1.53 g/L on galactose. The total C14 fatty acids production was even higher, about 1.30– 1.85 g/L on glucose, and 1.00–1.32 g/L on fructose, 0.98–1.56 g/L on galactose. The proportion of total unsaturated fatty acids of ML103/pXZ18Z, ML211/pXZ18Z, and strain XZK211/pXZ18Z dropped to less than 40%, while it dropped to less than 23% for strain ML190/pXZ18Z.
Fermentation of Free Fatty Acids with Sugar Mixture Substrates In sugar mixture cultivation, cellular metabolism is more complicated due to catabolite repression (Shimizu, 2013; Stephanopoulos, 2007). Before the hydrolysate fermentation experiment was executed, mixed sugar fermentation was performed to explore the promising strains for FFAs production using multiple sugars simultaneously. The mixed sugar fermentation medium was composed of about 31.9 mM (5.75 g/L) glucose, 24.8 mM (4.50 g/L) galactose, and 26.8 mM (4.75 g/L) fructose, and the total concentration of sugar was about 83.4 mM (15.0 g/L). The ratio of individual sugar was prepared according to the observed ratio in the soybean meal extract enzymatic hydrolysate. Strains ML103, ML190, ML211, XZK211 harboring plasmids, pXZ18 or pXZ18Z as indicated, were investigated for use of mixed sugars for FFAs production. While the strain ML211/pXZ18Z consistently gave better fatty acid production performance in individual sugar fermentation (Fig. 1), it was not the most effective strain for mixed sugars fermentation (Fig. 3A and B). The strain ML211/pXZ18Z produced only 2.83 g/L FFAs with a yield of 0.19 g/g, which is 1.37-fold of the yield of strain ML211/pXZ18. Two ptsG mutant strains, ML190/ pXZ18Z and XZK211/pXZ18Z, performed even better; these two strains reached higher FFAs concentrations of 3.16 and 3.18 g/L, respectively, with a yield of 0.21 g/g and 0.22 g/g at the end of the fermentation. These results suggested ptsG mutation contributed to improved performance in mixed sugars fermentation. For production of other chemicals, such as succinate and lactate, a ptsG mutation strain also showed relieve of catabolite repression on sugar mixtures and performed better to form a high concentration of the desired chemicals (Andersson et al., 2007; Dien et al., 2002; Wang et al., 2011). Figure 3C and Table III showed the distribution of different chain length FFAs of these eight engineered strains, which behaved differently. A lower percentage of unsaturated FFAs produced by strains harboring pXZ18Z, 12.2% for strain ML103/pXZ18Z, 37.8% for strain ML190/pXZ18Z, 45.2% for strain ML211/pXZ18Z, and 39.1% for strain XZK211/pXZ18Z were observed. This proportion decreased about 6.85% to 69.9% compared with the corresponding strains harboring pXZ18. It could be found that the unsaturated fatty acids proportion of strains ML211/pXZ18 and
Table III. Free fatty acids production using mixed sugars as carbon source. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains
Cell density (OD600)
Total sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
ML103/pXZ18 ML103/pXZ18Z ML190/pXZ18 ML190/pXZ18Z ML211/pXZ18 ML211/pXZ18Z XZK211/pXZ18 XZK211/pXZ18Z
7.36 0.37 7.58 0.51 7.17 0.27 8.35 0.51 4.41 0.45 6.82 0.44 7.37 0.04 7.04 0.17
73.24 0.66 84.66 0.14 79.05 0.77 82.83 0.17 83.49 0.53 82.45 0.63 73.14 0.72 75.26 0.24
38.41 1.46 56.79 0.37 33.06 0.71 35.40 1.34 15.74 0.48 12.97 0.68 35.16 2.26 36.88 0.75
33.43 1.74 10.24 0.58 30.09 1.33 28.88 1.19 40.08 1.05 31.03 2.33 34.65 0.98 31.02 0.62
21.02 1.51 31.00 2.07 26.38 2.79 26.82 1.88 24.14 0.67 41.81 1.23 21.84 0.91 24.02 1.61
7.14 1.01 2.00 0.08 10.47 1.41 8.90 0.54 20.04 0.01 14.19 0.32 8.35 1.21 8.08 0.08
40.57 1.33 12.22 0.90 40.56 1.06 37.78 0.32 60.12 0.26 45.23 1.09 43.00 1.08 39.11 2.09
Data are means standard deviation.
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ML211/pXZ18Z all increased compared with the strains ML190/ pXZ18 and ML190/pXZ18Z. This result might be caused by the additional fabR deletion in ML211, which removed the encoded fatty acid biosynthesis regulator gene. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis (McCue et al., 2001). So transcription of both fabA and fabB in a fabR deletion strain would increase relative to the parental strain, and the unsaturated fatty acid production will be enhanced (Feng and Cronan, 2011; Zhang et al., 2002). The effect of carbon source on fatty acid distribution by the engineered strains is shown in Table S1. The effect of carbon source is strain dependent. Soybean Meal Extract Enzymatic Hydrolysate Fermentation Soybean meal extract enzymatic hydrolysate produced in our lab was investigated for FFAs production by the strains ML103/pXZ18, ML190/pXZ18Z, ML211/pXZ18Z, and XZK211/pXZ18Z. The enzymatic hydrolyzed biomass was diluted about 2.4-fold to give an initial total sugar of 15 g/L (83.3 mM) in the fermentation medium. The FFAs concentrations, yields and distribution were shown in Figure 4 and Table IV. Similar to the fermentation results with sugar mixture as the substrates, strains ML190/pXZ18Z and XZK211/pXZ18Z were able to produce higher FFAs among the four strains, which were 2.73 and 2.78 g/L, with high yields of 0.20 g/g and 0.21 g/g, respectively (Figs. 4A and B). However, the final concentrations of FFAs they reached were 13.6% and 12.6% less than those obtained on pure sugar mixtures. The possible reason might be that some salts, proteins, and complex compounds released in the hydrolysate functioned as inhibitors for FFAs fermentation, and finally decreased the utilization efficiency of the sugars (J€onsson et al., 2013; Zha et al., 2012). The FFAs distribution was shown in Figure 4C. The strain ML190/pXZ18Z produced unsaturated fatty acids with a proportion of 34.8% of the total FFAs, while strain ML211/pXZ18Z produced a proportion of 62.1%. It might be deduced that the additional fabR deletion in ML211 could contribute to the increased proportion of the unsaturated FFAs in the total FFAs for soybean meal extract hydrolysate fermentation. The results were consistent with the fermentation data on pure mixed sugar substrate. The idea of renewable resources has inspired research over a long period of time, among which fatty acid biosynthesis with the aim to produce free fatty acids or derivatives for substitution of diesel is a major subject (Janßen and Steinb€uchel, 2014). Although this method offers a great potential for industrialization due to its short production time and very low land-use, the use of cellulose, lignin, hemicellulose, and other non-food carbon source still needs further investigation to reduce the production cost. Here, the combination of enzymatic hydrolysis and metabolic engineering to make soybean meal, a non-food carbon source, to be a good substrate for high-level free fatty acids production is desirable and indicates its general possibility. An approximate cost comparison of soybean meal carbohydrates with other feedstock is shown in Table S2.
Figure 4. FFAs production and yield using soybean meal enzyme hydrolysate by by ML103, ML190, ML211, XZK211 harboring pXZ18Z. (A) final titer at 48 h (g/L); (B) yield (g/g total sugar); (C) compositions (g/L). Data are means SD (n ¼ 3). Statistics were performed by the two-tailed student t-test. The data are statistically significant with P < 0.05 unless stated as ns (not significant).
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Table IV. Free fatty acids production using soybean meal enzyme hydrolysate. Compositions of free fatty acids (mass percentage of total at 48 h)
Strains
Cell density (OD600)
Sugars consumed (mM)
C14
C16:1
C16
C18:1
Total unsaturated fatty acid
ML103/pXZ18Z ML190/pXZ18Z ML211/pXZ18Z XZK211/pXZ18Z
7.36 0.37 7.84 0.32 7.35 0.10 7.44 0.44
76.41 0.42 74.57 0.18 74.04 0.20 74.65 0.24
36.80 0.30 38.59 1.25 17.07 0.71 38.86 0.09
25.74 0.14 28.04 0.54 43.87 1.03 28.54 0.93
29.11 0.34 26.64 0.72 20.88 1.02 25.72 1.22
8.35 0.13 6.72 0.25 18.19 0.62 6.88 0.42
34.09 0.07 34.76 0.38 62.05 0.41 35.42 0.43
Data are means standard deviation.
Conclusions In this study, free fatty acids were produced from individual sugars and enzymatic hydrolysate of soybean meal extract by engineered E. coli strains ML103, ML190, ML211, XZK211 plus plasmid pXZ18 or pXZ18Z. It was shown that overexpression of fabZ could further enhance the free fatty acids production besides TE overexpression. The unsaturated fatty acids proportion in the total fatty acids could be decreased with fabZ overexpression or increased with fabR deletion. The fadD, ptsG, fabR triple mutant strain XZK211/pXZ18Z, carrying fabZ behind the TE from R. communis, can produce FFAs efficiently from the soybean meal carbohydrates with a high concentration of 2.78 g/L and a high yield of 0.21 g/g. This work was supported in part by the United Soybean Board (USB), and the China Scholarship Council.
References Andersson C, Hodge D, Berglund KA, Rova U. 2007. Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli. Biotechnol Progr 23:381–388. Balzer G, Thakker C, Bennett GN, San KY. 2013. Metabolic engineering Escherichia coli to minimize byproduct formate and improving succinate productivity through NADH availability by heterologous expression of NAD(þ)-dependent formate dehydrogenase. Metab Eng 20:1–8. Cao Y, Yang J, Xian M, Xu X, Liu W. 2010. Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Appl Microbiol Biotechnol 87:271–280. Cho H, Cronan J, Jr. 1995. Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 270:4216–4219. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Nat Acad Sci 97:6640–6645. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R. 2011. Engineering reversal of the b-oxidation cycle for the synthesis of fuels and chemicals. Nature 476:355–359. Dien BS, Nichols NN, Bothast RJ. 2002. Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of Llactate acid. J Ind Microbiol Biotechnol 29:221–227. Eldridge AC, Black LT, Wolf JW. 1979. Carbohydrate composition of soybean flours, protein concentrates, and isolates. J Agric Food Chem 27:799–802. Feng Y, Cronan JE. 2011. Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters. Mol Microbiol 80:195–218. Fridjonssson O, Watzlawick H, Gehweiler A, Rohrhirsch T, Mattes R. 1999. Cloning of the gene encoding a novel thermostable galactosidase from Thermus brockianus I TI360. Appl Environ Microbiol 65:3955–3963. Giannoccaro E, Wang YJ, Chen P. 2006. Effects of solvent, temperature, time, solventto-sample ratio, sample size, and defatting on the extraction of soluble sugars in soybean. J Food Sci 71:C59–C64.
2332
Biotechnology and Bioengineering, Vol. 112, No. 11, November, 2015
Janßen HJ, Steinb€uchel A. 2014. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels 7:7–33. Jiang P, Cronan J, Jr. 1994. Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action. J Bacteriol 176:2814–2821. J€onsson L, Alriksson B, Nilvebrant N. 2013. Bioconversion of lignocelluloses: Inhibitors and detoxification. Biotechnol Biofuels 6:16–25. Karr-Lilienthal LK, Grieshop CM, Spears JK, Fahey GC. 2005. Amino acid, carbohydrate, and fat composition of soybean meals prepared at 55 commercial U.S. soybean processing plants. J Agric Food Chem 53:2146– 2150. Kim JH, Block DE, Mills DA. 2010. Simultaneous consumption of pentose and hexose sugars: An optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl Microbiol Biotechnol 88:1077–1085. Kim KR, Oh DK. 2013. Production of hydroxyl fatty acids by microbial fatty acidhydroxylation enzymes. Biotechnol Adv 31:1473–1485. Kim NJ, Li H, Jung K, Chang HN, Lee PC. 2011. Ethanol production from algal hydrolysates using Escherichia coli KO11. Bioresour Technol 102:7466– 7469. Lennen R, Braden G, West R, Dumesic J, Pfleger B. 2010. A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 106:193–202. Li M, Zhang X, Agrawal A, San K. 2012. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acyl-ACP thioesterase from Ricinus communis. Metab Eng 14:380–387. Lin H, Bennett GN, San K. 2005. Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab Eng 7:116–127. Liu K. 1997. Soybeans: Chemistry, technology, and utilization. New York: Chapman and Hall. Liu T, Vora H, Khosla C. 2010. Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 12:378–386. Lu X, Vora H, Khosla C. 2008. Overexpression of free fatty acids in E. coli: Implications for biodiesel production. Metab Eng 10:333–339. Mazumdar S, Bang J, Oh MK. 2013. L-Lactate production from seaweed hydrolysate of Laminaria japonica using metabolically engineered Escherichia coli. Appl Biochem Biotechnol 172:1938–1952. McCue L, Thompson W, Carmack C, Ryan MP, Liu JS, Derbyshire V, Lawrence CE. 2001. Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes. Nucleic Acids Res 29:774–782. Nickolau B, Perera M, Brachova L, Shanks B. 2008. Platform chemicals for a biorenewable chemical industry. Plant J 54:536–545. O’Fallon JV, Busboom JR, Nelson ML, Gaskins CT. 2007. A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils, and feedstuffs. J Anim Sci 85:1511–1521. Ooi S, Pee H. 1985. Processing for industrial fatty acids-II. J Am Oil Chem Soc 62:348–351. Peralta-Yahya PB, Zhang F, del Cardayre SB, Keasling JD. 2012. Microbial engineering for the production of advanced biofuels. Nature 488:320–328. San KY, Li M. 2013. Bacteria and method for synthesizing fatty acids. Vol. WO/2013/ 059218, USA.
Sanchez AM, Bennett GN, San K. 2005. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng 7:229–239. Schmid K, Schupfner M, Schmitt R. 1982. Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12. J Bacteriol 151:68–76. Schmid K, Schmitt R. 1976. Raffinose metabolism in Escherichia coli K12. Purification and properties of a new a-galactosidase specified by a transmissible plasmid. Eur. J Biochem 67:95–104. Shimizu K. 2013. Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism. ISRN Biochem 645983:1–47. Stephanopoulos G. 2007. Challenges in engineering microbes for biofuels production. Science 315:801–804. Thakker C, San K, Bennett GN. 2013. Production of succinic acid by engineered E. coli strains using soybean carbohydrates as feedstock under aerobic fermentation conditions. Bioresour Technol 130:398–405. Voelker T, Davies H. 1994. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of plant medium-chain acyl-acyl carrier protein thioesterase. J Bacteriol 176:7320–7327. Wang D, Li Q, Yang M, Zhang Y, Su Z, Xing J. 2011. Efficient production of succinic acid from corn stalk hydrolysates by a recombinant Escherichia coli with ptsG mutation. Process Biochem 46:365–371. Wu H, San KY. 2014. Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli. Biotechnol Bioeng 9999:1–11.
Wu H, Lee J, Karanjikar M, San KY. 2014a. Efficient free fatty acid production from woody biomass hydrolysate using metabolically engineered Escherichia coli. Bioresour Technol 169:119–125. Wu H, Karanjikar M, San KY. 2014b. Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol. Metab Eng 25:82–91. Zha Y, Muilwijk B, Coulier L, Punt PJ. 2012. Inhibitory compounds in lignocellulosic biomass hydrolysates during hydrolysate fermentation process. J Bioprocess Biotechniq 2:1–11. Zhang XJ, Li M, Agrawal A, San KY. 2011. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterase. Metab Eng 13: 713–722. Zhang YM, Marrakchi H, Rock CO. 2002. The FabR (YijC) transcription factor regulates unsaturated fatty acid biosynthesis in Escherichia coli. J Biol Chem 277:15558–15565. Zhang YM, Rock CO. 2009. Transcriptional regulation in bacterial membrane lipid synthesis. J Lipid Res 50:S115–S119.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
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