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Increased Tolerance to Furfural by Introduction of Polyhydroxybutyrate Synthetic Genes to Escherichia coli
1Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea, 2Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul, Republic of Korea, 3New Drug Development Center, Osong Medical Innovative Foundation, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2019; 29(5): 776-784
Published May 28, 2019 https://doi.org/10.4014/jmb.1901.01070
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Lignocellulosic biomass is an abundant renewable resource for the production of biofuels, chemicals, and polymers [1-3]. Lignocellulose is mainly composed of three polymers, cellulose, hemicellulose, and lignin, together with other components, such as acetate, minerals, and phenolic substituents [4]. However, the main impediments in utilizing lignocellulose materials lie in the crystalline structure of cellulose sheathed by hemicellulose, degree of polymerization, biomass particle size, and recalcitrance of their bonding due to the protective covering of lignin [5]. To better utilize this biomass and efficiently extract carbohydrates, pretreatment processes are necessary [6-8]. Various pretreatment processes have been developed to purify sugar compounds, one of which is acid hydrolysis [9, 10]. However, in pretreatment processes, several unwanted inhibitors (
The polyhydroxyalkanoate (PHA) family of bio-based, biodegradable polymers is a promising next-generation product that can potentially substitute for petroleum-based plastics and is synthesized by many microorganisms as part of their natural metabolism [21, 22]. Previous studies on bacterial PHA accumulation have shown that it is largely affected by nutrient conditions, such as nitrogen and phosphate concentrations, and oxygen limitation [23-25]. Compared to petroleum-based plastics, PHA has many advantages including biodegradability and biocompatibility, while its thermal and mechanical properties are similar to those of petroleum-based plastics [26]. The most extensively studied member of the PHA family is poly(3-hydroxybutyrate)(PHB) [27]. The biosynthesis of PHB requires three reactions mediated by β-ketothiolase (BktB), acetoacetyl-CoA reductase (PhaB), and PHB polymerase (PhaC).
Bio-based resources are renewable and are expected to play a key role in the production of novel bio-based materials, contributing to a reduction in the negative environmental impact of petroleum-based products and thus addressing the bio-economy of the future [4]. The increasing worldwide need for bio-based plastic production will, therefore, be an important driver towards the use of renewable non-edible sources, such as lignocellulosic biomass [28]. To date, research has focused on the development of new bacterial strains and the discovery of cost-effective starting materials for PHB production [27, 29]. A number of bacteria that produce PHB from lignocellulose-derived monosaccharides have been identified [30-33]. However, their application is limited to using hydrolysates containing only small amounts of inhibitors or after eliminating toxic compounds [29, 31, 34]. Therefore, discovering an effective detoxification strategy remains important for efficient utilization of biomass hydrolysate for microbial growth and fermentation.
In this study, we investigated the feasibility of PHB production in
Materials and Methods
Bacterial Strains, Media, Reagents, and Culture Conditions
Strains and plasmids used in this study are listed in Table 1.
-
Table 1 . Bacterial strains, plasmids, and primers used in this study.
Strain or plasmid Description Reference E. coli strainsDH5α General cloning strain Invitrogen KSYH(DE3) BW25113 derivative containing DE3, Δ araBAD, ΔrhaBAD [35] KSYH(DE3)/pCDF KSYH(DE3) containing pCDFDuet-1 This study KSYH(DE3):: bktB KSYH(DE3) containing pCDF:: bktB This study KSYH(DE3):: phaB KSYH(DE3) containing pCDF:: phaB This study KSYH(DE3):: phaC KSYH(DE3) containing pCDF:: phaC This study YH090 KSYH(DE3) containing pLW487 [35] Plasmids pCDFDuet-1 A compatible spectinomycin-selectable plasmid carrying T7/ lac promoterNovagen pCDF:: bktB pCDFDuet-1 carrying bktB gene fromRalstonia eutropha H16This study pCDF:: phaB pCDFDuet-1 carrying phaB gene fromRalstonia eutropha H16This study pCDF:: phaC pCDFDuet-1 carrying phaC gene fromRalstonia eutropha H16This study pLW487 Spectinomycin-selectable pEP2-based plasmid carrying bktB ,phaB andphaC genes fromRalstonia eutropha H16 under the control oftrc promoter.[48]
-
Table 2 . Composition of lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree used in this study.
Biomass Components Concentration (g/l) Miscanthus Monosugars Glucose 102.19 ± 0.54 Xylose 20.16 ± 0.25 Galactose 1.42 ± 0.21 Arabinose 2.97 ± 0.17 Mannose 1.55 ± 0.21 Byproducts Formic acid - Acetic acid 0.24 ± 0.01 Levulinic acid - 5-Hydroxymethylfurfural 0.19 ± 0.01 Furfural 0.48 ± 0.01 Barley straw Monosugars Glucose 135.80 ± 0.04 Xylose 13.164 ± 0.67 Galactose - Arabinose 0.34 ± 0.01 Mannose 0.70 ± 0.04 Byproducts Formic acid 0.17 ± 0.03 Acetic acid 0.14 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.18 ± 0.01 Furfural 0.09 ± 0.01 Pine tree Monosugars Glucose 127.44 ± 0.22 Xylose 14.02 ± 0.33 Galactose - Arabinose - Mannose 2.95 ± 0.23 Byproducts Formic acid - Acetic acid 0.88 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.15 ± 0.01 Furfural 0.17 ± 0.01
DNA Manipulation
Gene cloning was conducted according to standard protocols [38]. PHB synthetic genes (
Analytical Methods
PHB production was determined by gas chromatography and a slightly modified version of a previously described method [39, 40]. For analysis, culture samples were centrifuged at 10,000 ×
Results
Effects of PHB on Furfural Resistance
It is well known that pretreatment of lignocellulose generates several potentially toxic compounds, such as organic acids and aldehydes (
-
Fig. 1.
Enhancement of polyhydroxybutyrate (PHB) production by furfural. Cell growth and PHB production inE. coli YH090 (pLW487) were investigated in the presence of furfural. Furfural concentration ranged from 0 to 20 mM. (A ) DCW (dry cell weight, g/l), (B ) PHB content (w/w %), (C ) Residual biomass (g/l), and (D ) PHB concentration (g/l). Cells were grown for 72 h. Error bars represent the standard deviation of two replicates.
To confirm that PHB production is increased in the presence of furfural, we monitored PHB production with or without 15 mM furfural in
-
Fig. 2.
Time-course profiles of polyhydroxybutyrate (PHB) production in (E. coli YH090 in the presence or absence of furfural.A ) DCW (dry cell weight, g/l) and (B ) PHB concentration (g/l). Error bars represent the standard deviation of two replicates.
Investigation of Furfural Resistance Induced by Individual Genes and the Protective Effect against Lignocellulose-Derived Inhibitors
It is widely known that many reductases are effective for inducing furfural resistance [19, 20, 44]. Similarly, we expected that PhaB, an NADPH-dependent acetoacetyl-CoA reductase, would be effective in restoring cell growth and improving PHB production in the presence of furfural. Thus, to determine which gene was responsible for increased cell growth and PHB production in the presence of furfural, individual gene (
-
Fig. 3.
Effects of overexpression of PHB synthetic genes on cell growth and furfural consumption. (A ) Optical density (OD) at 600 nm and (B ) Furfural concentration (mM). Cells were grown for 24 h. Error bars represent the standard deviation of two replicates.
To test whether introducing PHB synthetic genes might affect resistance to other lignocellulose-derived inhibitors, such as vanillin and 4-hydroxybenzaldehyde, cell growth of
-
Fig. 4.
Increased resistance to lignocellulose-derived inhibitors. Inhibitory effects of lignocellulose-derived inhibitors and protective effects of PHB synthesis were investigated. 4-Hydroxybenzaldehyde (4-Hb, 5 mM) and vanillin (10 mM) were used. DCW, dry cell weight, g/l. Cells were grown for 48 h.
Effects of Lignocellulose Hydrolysates on PHB Production
To evaluate the effects of lignocellulose hydrolysates on PHB production,
Discussion
For efficient utilization of lignocellulose as a sugar source, a pretreatment process, such as acid hydrolysis, is required [6, 7]. However, in the pretreatment process, several fermentation inhibitors are formed in addition to monosaccharides [11, 12]. In particular, furfural is considered one of the major inhibitors of
Here, we report the protective effect of PHB on growth inhibition by furfural and the stimulating effect of furfural on PHB production. Although the mechanisms underlying these effects still need to be investigated, we demonstrated the synergetic effects of furfural and PHB production and the feasibility of PHB production in the presence of lignocellulose hydrolysates. We showed that these effects were not related to a supplemental carbon source (Fig. 1C) or to only one of the enzymes in the PHB synthesis pathway (Fig. 3). In other words, the cell growth inhibition effect of furfural was restored by overexpression of three PHB synthetic genes in
Multiple reports have described that PHA formation and mobilization enhance stress tolerance [45-47]. A previous study indicated that a complete PHB mobilization system (expression of PHB synthetic genes and PHB depolymerase) serves as an intracellular energy and carbon storage system in
Acknowledgments
This study was supported by the Research Program for Solving Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234), National Research Foundation of Korea (NRF) (NRF-2015M1A5A1037196, NRF2016R1D1A1B03932301). Consulting service from the Microbial Carbohydrate Resource Bank (MCRB, Seoul, Korea) was kindly appreciated. This work was also supported by the Polar Academic Program (PAP,PE18900).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2019; 29(5): 776-784
Published online May 28, 2019 https://doi.org/10.4014/jmb.1901.01070
Copyright © The Korean Society for Microbiology and Biotechnology.
Increased Tolerance to Furfural by Introduction of Polyhydroxybutyrate Synthetic Genes to Escherichia coli
Hye-Rim Jung 1, Ju-Hee Lee 1, Yu-Mi Moon 1, Tae-Rim Choi 1, Su-Yeon Yang 1, Hun-Suk Song 1, Jun-Young Park 1, Ye-Lim Park 1, Shashi Kant Bhatia 1, 2, Ranjit Gurav 1, Byoung Joon Ko 3 and Yung-Hun Yang 1, 2*
1Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea, 2Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul, Republic of Korea, 3New Drug Development Center, Osong Medical Innovative Foundation, Republic of Korea
Correspondence to:Yung-Hun Yang
seokor@konkuk.ac.kr
Abstract
Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.
Keywords: Polyhydroxybutyrate, furfural, resistance, Escherichia coli, lignocellulose hydrolysate
Introduction
Lignocellulosic biomass is an abundant renewable resource for the production of biofuels, chemicals, and polymers [1-3]. Lignocellulose is mainly composed of three polymers, cellulose, hemicellulose, and lignin, together with other components, such as acetate, minerals, and phenolic substituents [4]. However, the main impediments in utilizing lignocellulose materials lie in the crystalline structure of cellulose sheathed by hemicellulose, degree of polymerization, biomass particle size, and recalcitrance of their bonding due to the protective covering of lignin [5]. To better utilize this biomass and efficiently extract carbohydrates, pretreatment processes are necessary [6-8]. Various pretreatment processes have been developed to purify sugar compounds, one of which is acid hydrolysis [9, 10]. However, in pretreatment processes, several unwanted inhibitors (
The polyhydroxyalkanoate (PHA) family of bio-based, biodegradable polymers is a promising next-generation product that can potentially substitute for petroleum-based plastics and is synthesized by many microorganisms as part of their natural metabolism [21, 22]. Previous studies on bacterial PHA accumulation have shown that it is largely affected by nutrient conditions, such as nitrogen and phosphate concentrations, and oxygen limitation [23-25]. Compared to petroleum-based plastics, PHA has many advantages including biodegradability and biocompatibility, while its thermal and mechanical properties are similar to those of petroleum-based plastics [26]. The most extensively studied member of the PHA family is poly(3-hydroxybutyrate)(PHB) [27]. The biosynthesis of PHB requires three reactions mediated by β-ketothiolase (BktB), acetoacetyl-CoA reductase (PhaB), and PHB polymerase (PhaC).
Bio-based resources are renewable and are expected to play a key role in the production of novel bio-based materials, contributing to a reduction in the negative environmental impact of petroleum-based products and thus addressing the bio-economy of the future [4]. The increasing worldwide need for bio-based plastic production will, therefore, be an important driver towards the use of renewable non-edible sources, such as lignocellulosic biomass [28]. To date, research has focused on the development of new bacterial strains and the discovery of cost-effective starting materials for PHB production [27, 29]. A number of bacteria that produce PHB from lignocellulose-derived monosaccharides have been identified [30-33]. However, their application is limited to using hydrolysates containing only small amounts of inhibitors or after eliminating toxic compounds [29, 31, 34]. Therefore, discovering an effective detoxification strategy remains important for efficient utilization of biomass hydrolysate for microbial growth and fermentation.
In this study, we investigated the feasibility of PHB production in
Materials and Methods
Bacterial Strains, Media, Reagents, and Culture Conditions
Strains and plasmids used in this study are listed in Table 1.
-
Table 1 . Bacterial strains, plasmids, and primers used in this study..
Strain or plasmid Description Reference E. coli strainsDH5α General cloning strain Invitrogen KSYH(DE3) BW25113 derivative containing DE3, Δ araBAD, ΔrhaBAD [35] KSYH(DE3)/pCDF KSYH(DE3) containing pCDFDuet-1 This study KSYH(DE3):: bktB KSYH(DE3) containing pCDF:: bktB This study KSYH(DE3):: phaB KSYH(DE3) containing pCDF:: phaB This study KSYH(DE3):: phaC KSYH(DE3) containing pCDF:: phaC This study YH090 KSYH(DE3) containing pLW487 [35] Plasmids pCDFDuet-1 A compatible spectinomycin-selectable plasmid carrying T7/ lac promoterNovagen pCDF:: bktB pCDFDuet-1 carrying bktB gene fromRalstonia eutropha H16This study pCDF:: phaB pCDFDuet-1 carrying phaB gene fromRalstonia eutropha H16This study pCDF:: phaC pCDFDuet-1 carrying phaC gene fromRalstonia eutropha H16This study pLW487 Spectinomycin-selectable pEP2-based plasmid carrying bktB ,phaB andphaC genes fromRalstonia eutropha H16 under the control oftrc promoter.[48]
-
Table 2 . Composition of lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree used in this study..
Biomass Components Concentration (g/l) Miscanthus Monosugars Glucose 102.19 ± 0.54 Xylose 20.16 ± 0.25 Galactose 1.42 ± 0.21 Arabinose 2.97 ± 0.17 Mannose 1.55 ± 0.21 Byproducts Formic acid - Acetic acid 0.24 ± 0.01 Levulinic acid - 5-Hydroxymethylfurfural 0.19 ± 0.01 Furfural 0.48 ± 0.01 Barley straw Monosugars Glucose 135.80 ± 0.04 Xylose 13.164 ± 0.67 Galactose - Arabinose 0.34 ± 0.01 Mannose 0.70 ± 0.04 Byproducts Formic acid 0.17 ± 0.03 Acetic acid 0.14 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.18 ± 0.01 Furfural 0.09 ± 0.01 Pine tree Monosugars Glucose 127.44 ± 0.22 Xylose 14.02 ± 0.33 Galactose - Arabinose - Mannose 2.95 ± 0.23 Byproducts Formic acid - Acetic acid 0.88 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.15 ± 0.01 Furfural 0.17 ± 0.01
DNA Manipulation
Gene cloning was conducted according to standard protocols [38]. PHB synthetic genes (
Analytical Methods
PHB production was determined by gas chromatography and a slightly modified version of a previously described method [39, 40]. For analysis, culture samples were centrifuged at 10,000 ×
Results
Effects of PHB on Furfural Resistance
It is well known that pretreatment of lignocellulose generates several potentially toxic compounds, such as organic acids and aldehydes (
-
Figure 1.
Enhancement of polyhydroxybutyrate (PHB) production by furfural. Cell growth and PHB production inE. coli YH090 (pLW487) were investigated in the presence of furfural. Furfural concentration ranged from 0 to 20 mM. (A ) DCW (dry cell weight, g/l), (B ) PHB content (w/w %), (C ) Residual biomass (g/l), and (D ) PHB concentration (g/l). Cells were grown for 72 h. Error bars represent the standard deviation of two replicates.
To confirm that PHB production is increased in the presence of furfural, we monitored PHB production with or without 15 mM furfural in
-
Figure 2.
Time-course profiles of polyhydroxybutyrate (PHB) production in (E. coli YH090 in the presence or absence of furfural.A ) DCW (dry cell weight, g/l) and (B ) PHB concentration (g/l). Error bars represent the standard deviation of two replicates.
Investigation of Furfural Resistance Induced by Individual Genes and the Protective Effect against Lignocellulose-Derived Inhibitors
It is widely known that many reductases are effective for inducing furfural resistance [19, 20, 44]. Similarly, we expected that PhaB, an NADPH-dependent acetoacetyl-CoA reductase, would be effective in restoring cell growth and improving PHB production in the presence of furfural. Thus, to determine which gene was responsible for increased cell growth and PHB production in the presence of furfural, individual gene (
-
Figure 3.
Effects of overexpression of PHB synthetic genes on cell growth and furfural consumption. (A ) Optical density (OD) at 600 nm and (B ) Furfural concentration (mM). Cells were grown for 24 h. Error bars represent the standard deviation of two replicates.
To test whether introducing PHB synthetic genes might affect resistance to other lignocellulose-derived inhibitors, such as vanillin and 4-hydroxybenzaldehyde, cell growth of
-
Figure 4.
Increased resistance to lignocellulose-derived inhibitors. Inhibitory effects of lignocellulose-derived inhibitors and protective effects of PHB synthesis were investigated. 4-Hydroxybenzaldehyde (4-Hb, 5 mM) and vanillin (10 mM) were used. DCW, dry cell weight, g/l. Cells were grown for 48 h.
Effects of Lignocellulose Hydrolysates on PHB Production
To evaluate the effects of lignocellulose hydrolysates on PHB production,
Discussion
For efficient utilization of lignocellulose as a sugar source, a pretreatment process, such as acid hydrolysis, is required [6, 7]. However, in the pretreatment process, several fermentation inhibitors are formed in addition to monosaccharides [11, 12]. In particular, furfural is considered one of the major inhibitors of
Here, we report the protective effect of PHB on growth inhibition by furfural and the stimulating effect of furfural on PHB production. Although the mechanisms underlying these effects still need to be investigated, we demonstrated the synergetic effects of furfural and PHB production and the feasibility of PHB production in the presence of lignocellulose hydrolysates. We showed that these effects were not related to a supplemental carbon source (Fig. 1C) or to only one of the enzymes in the PHB synthesis pathway (Fig. 3). In other words, the cell growth inhibition effect of furfural was restored by overexpression of three PHB synthetic genes in
Multiple reports have described that PHA formation and mobilization enhance stress tolerance [45-47]. A previous study indicated that a complete PHB mobilization system (expression of PHB synthetic genes and PHB depolymerase) serves as an intracellular energy and carbon storage system in
Acknowledgments
This study was supported by the Research Program for Solving Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234), National Research Foundation of Korea (NRF) (NRF-2015M1A5A1037196, NRF2016R1D1A1B03932301). Consulting service from the Microbial Carbohydrate Resource Bank (MCRB, Seoul, Korea) was kindly appreciated. This work was also supported by the Polar Academic Program (PAP,PE18900).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

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Table 1 . Bacterial strains, plasmids, and primers used in this study..
Strain or plasmid Description Reference E. coli strainsDH5α General cloning strain Invitrogen KSYH(DE3) BW25113 derivative containing DE3, Δ araBAD, ΔrhaBAD [35] KSYH(DE3)/pCDF KSYH(DE3) containing pCDFDuet-1 This study KSYH(DE3):: bktB KSYH(DE3) containing pCDF:: bktB This study KSYH(DE3):: phaB KSYH(DE3) containing pCDF:: phaB This study KSYH(DE3):: phaC KSYH(DE3) containing pCDF:: phaC This study YH090 KSYH(DE3) containing pLW487 [35] Plasmids pCDFDuet-1 A compatible spectinomycin-selectable plasmid carrying T7/ lac promoterNovagen pCDF:: bktB pCDFDuet-1 carrying bktB gene fromRalstonia eutropha H16This study pCDF:: phaB pCDFDuet-1 carrying phaB gene fromRalstonia eutropha H16This study pCDF:: phaC pCDFDuet-1 carrying phaC gene fromRalstonia eutropha H16This study pLW487 Spectinomycin-selectable pEP2-based plasmid carrying bktB ,phaB andphaC genes fromRalstonia eutropha H16 under the control oftrc promoter.[48]
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Table 2 . Composition of lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree used in this study..
Biomass Components Concentration (g/l) Miscanthus Monosugars Glucose 102.19 ± 0.54 Xylose 20.16 ± 0.25 Galactose 1.42 ± 0.21 Arabinose 2.97 ± 0.17 Mannose 1.55 ± 0.21 Byproducts Formic acid - Acetic acid 0.24 ± 0.01 Levulinic acid - 5-Hydroxymethylfurfural 0.19 ± 0.01 Furfural 0.48 ± 0.01 Barley straw Monosugars Glucose 135.80 ± 0.04 Xylose 13.164 ± 0.67 Galactose - Arabinose 0.34 ± 0.01 Mannose 0.70 ± 0.04 Byproducts Formic acid 0.17 ± 0.03 Acetic acid 0.14 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.18 ± 0.01 Furfural 0.09 ± 0.01 Pine tree Monosugars Glucose 127.44 ± 0.22 Xylose 14.02 ± 0.33 Galactose - Arabinose - Mannose 2.95 ± 0.23 Byproducts Formic acid - Acetic acid 0.88 ± 0.02 Levulinic acid - 5-Hydroxymethylfurfural 0.15 ± 0.01 Furfural 0.17 ± 0.01
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