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References

  1. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, et al. 2006. The path forward for biofuels and biomaterials. Science 311: 484-489.
    Pubmed CrossRef
  2. Lynd LR, Laser MS, Bransby D, Dale BE, Davison B, Hamilton R, et al. 2008. How biotech can transform biofuels. Nat. Biotechnol. 26: 169-172.
    Pubmed CrossRef
  3. Sims RE, Mabee W, Saddler JN, Taylor M. 2010. An overview of second generation biofuel technologies. Bioresour. Technol. 101: 1570-1580.
    Pubmed CrossRef
  4. Brodin M, Vallejos M, Opedal MT, Area MC, Chinga-Carrasco G. 2017. Lignocellulosics as sustainable resources for production of bioplastics-a review. J. Clean Prod. 162: 646-664.
    CrossRef
  5. Sharma HK, Xu C, Qin W. 2017. Biological pretreatment of lignocellulosic biomass for biofuels and bioproducts: an overview. Waste and Biomass Valorization.: 1-17.
    CrossRef
  6. Carroll A, Somerville C. 2009. Cellulosic biofuels. Annu. Rev. Plant Biol. 60: 165-182.
    Pubmed CrossRef
  7. Weijde Tvd, Alvim Kamei CL, Torres AF, Vermerris W, Dolstra O, Visser RGF, et al. 2013. The potential of C4 grasses for cellulosic biofuel production. Front. Plant Sci. 4: 107.
    Pubmed PMC CrossRef
  8. Bhatia SK, Gurav R, Choi T-R, Jung H-R, Yang S-Y, Moon Y-M, et al. 2019. Bioconversion of plant biomass hydrolysate into bioplastic (polyhydroxyalkanoates) using Ralstonia eutropha 5119. Bioresour. Technol. 271: 306-315.
    Pubmed CrossRef
  9. Kumar P, Barrett DM, Delwiche MJ, Stroeve P. 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. 48: 3713-3729.
    CrossRef
  10. Sun Y, Cheng J. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83: 1-11.
    Pubmed CrossRef
  11. Mills TY, Sandoval NR, Gill RT. 2009. Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnol. Biofuels 2: 26.
    Pubmed PMC CrossRef
  12. Saha BC. 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30: 279-291.
    Pubmed CrossRef
  13. Gorsich S, Dien B, Nichols N, Slininger P, Liu Z, Skory C. 2006. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 71: 339-349.
    Pubmed CrossRef
  14. Barciszewski J, Siboska GE, Pedersen BO, Clark BF, Rattan SI. 1997. A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA. FEBS Lett. 414: 457-460.
    Pubmed CrossRef
  15. Palmqvist E, Hahn-Hägerdal B. 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74: 25-33.
    CrossRef
  16. Zaldivar J, Martinez A, Ingram LO. 2000. Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol. Bioeng. 68: 524-530.
    Pubmed CrossRef
  17. Song H-S, Jeon J-M, Kim H-J, Bhatia SK, Sathiyanarayanan G, Kim J, et al. 2017. Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli. Bioresour. Technol. 245: 1430-1435.
    Pubmed CrossRef
  18. Seo H-M, Jeon J-M, Lee JH, Song H-S, Joo H-B, Park S-H, et al. 2016. Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural. J. Ind. Microbiol. Biotechnol. 43: 37-44.
    Pubmed CrossRef
  19. Wang X, Miller E, Yomano L, Zhang X, Shanmugam K, Ingram L. 2011. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Appl. Environ. Microbiol. 77: 5132-5140.
    Pubmed PMC CrossRef
  20. Wang X, Yomano LP, Lee JY, York SW, Zheng H, Mullinnix MT, et al. 2013. Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proc. Natl. Acad. Sci. USA 110: 4021-4026.
    Pubmed PMC CrossRef
  21. Madkour MH, Heinrich D, Alghamdi MA, Shabbaj II, Steinbuchel A. 2013. PHA recovery from biomass. Biomacromolecules 14: 2963-2972.
    Pubmed CrossRef
  22. Steinbuchel A, Fuchtenbusch B. 1998. Bacterial and other biological systems for polyester production. Trends. Biotechnol. 16: 419-427.
    Pubmed CrossRef
  23. Wen Q, Chen Z, Tian T, Chen W. 2010. Effects of phosphorus and nitrogen limitation on PHA production in activated sludge. J. Environ. Sci. 22: 1602-1607.
    Pubmed CrossRef
  24. Johnson K, Kleerebezem R, van Loosdrecht MC. 2010. Influence of the C/N ratio on the performance of polyhydroxybutyrate (PHB) producing sequencing batch reactors at short SRTs. Water Res. 44: 2141-2152.
    Pubmed CrossRef
  25. Ahn J, Jho EH, Nam K. 2015. Effect of C/N ratio on polyhydroxyalkanoates (PHA) accumulation by Cupriavidus necator and its implication on the use of rice straw hydrolysates. Environ. Eng. Res. 20: 246-253.
    CrossRef
  26. Sudesh K, Abe H, Doi Y. 2000. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25: 1503-1555.
    CrossRef
  27. Du C, Sabirova J, Soetaert W, Lin C. 2012. Polyhydroxy-alkanoates production from low-cost sustainable raw materials. Curr. Chem. Biol. 6: 14-25.
    CrossRef
  28. Broeren M. Production of Bio-ethylene−Technology Brief. IEA-ETSAP & IRENA, International Renewable Energy Agency.
  29. Cesário MT, Raposo RS, de Almeida MCM, van Keulen F, Ferreira BS, da Fonseca MMR. 2014. Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates. N. Biotechnol. 31: 104-113.
    Pubmed CrossRef
  30. Yu J, Stahl H. 2008. Microbial utilization and biopolyester synthesis of bagasse hydrolysates. Bioresour. Technol. 99: 8042-8048.
    Pubmed CrossRef
  31. Silva L, Taciro M, Ramos MM, Carter J, Pradella J, Gomez J. 2004. Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose and sugarcane bagasse hydrolysate. J Ind. Microbiol. Biotechnol. 31: 245-254.
    Pubmed CrossRef
  32. Dietrich D, Illman B, Crooks C. 2013. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava. BMC Res. Notes 6: 219.
    Pubmed PMC CrossRef
  33. Wang W, Yang S, Hunsinger GB, Pienkos PT, Johnson DK. 2014. Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator. Front. Microbiol. 5: 247.
    Pubmed PMC CrossRef
  34. Dietrich K, Dumont M-J, Schwinghamer T, Orsat V, Del Rio LF. 2017. Model study to assess softwood hemicellulose hydrolysates as the carbon source for PHB production in Paraburkholderia sacchari IPT 101. Biomacromolecules 19: 188-200.
    Pubmed CrossRef
  35. Yang YH, Brigham C, Song E, Jeon JM, Rha C, Sinskey A. 2012. Biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase. J. Appl. Microbiol. 113: 815-823.
    Pubmed CrossRef
  36. Jeon JM, Kim HJ, Bhatia SK, Sung C, Seo HM, Kim JH, et al. 2017. Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly (3-hydroxybutyrate-co-3-hydroxyvalerate). Bioproc. Biosyst. Eng. 40: 781-789.
    Pubmed CrossRef
  37. Mezzina MP, Wetzler DE, Almeida A, Dinjaski N, Prieto MA, Pettinari MJ. 2015. A phasin with extra talents: a poly-hydroxyalkanoate granule-associated protein has chaperone activity. Environ. Microbiol. 17: 1765-1776.
    Pubmed CrossRef
  38. Phylactides M. 1997. Molecular biology series 3. Tools of molecular biology: gene cloning. Br. J. Hosp. Med. 57: 49-50.
    Pubmed
  39. Braunegg G, Sonnleitner B, Lafferty R. 1978. A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Appl. Microbiol. Biotechnol. 6: 29-37.
    CrossRef
  40. Bhatia SK, Kim J, Song H-S, Kim HJ, Jeon J-M, Sathiyanarayanan G, et al. 2017. Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01. Bioresour. Technol. 233: 99-109.
    Pubmed CrossRef
  41. Zaldivar J, Ingram LO. 1999. Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioeng. 66: 203-210.
    Pubmed CrossRef
  42. Song HS, Jeon JM, Choi YK, Kim JY, Kim W, Yoon JJ, et al. 2017. L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli. J. Microbiol.Biotechnol. 27: 2165-2172.
    Pubmed CrossRef
  43. Nieves LM, Panyon LA, Wang X. 2015. Engineering sugar utilization and microbial tolerance toward lignocellulose conversion. Front. Bioeng. Biotechnol. 3: 17.
    Pubmed PMC CrossRef
  44. Moon J, Liu ZL. 2015. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae. Yeast 32: 399-407.
    Pubmed CrossRef
  45. Han M-J, Yoon SS, Lee SY. 2001. Proteome analysis of metabolically engineeredescherichia coli producing poly (3-hydroxybutyrate). J Bacteriol. 183: 301-308.
    Pubmed PMC CrossRef
  46. Wang Q, Yu H, Xia Y, Kang Z, Qi Q. 2009. Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application. Microb. Cell Fact. 8: 47.
    Pubmed PMC CrossRef
  47. James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R. 1999. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ Molecular biology series 3. Tools of molecular biology: gene cloning. Microbiol. 65: 822-827.
    Pubmed PMC CrossRef
  48. Yang YH, Brigham C, Willis L, Rha C, Sinskey A. 2011. Improved detergent-based recovery of polyhydroxyalkanoates (PHAs). Biotechnology Lett. 33: 937-942.
    Pubmed CrossRef

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

Received: January 31, 2019; Accepted: April 24, 2019

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 (e.g., furfural, 4-hydroxybenzaldehyde, vanillin, and acetate) are also produced [11, 12]. The quantity of these inhibitors depends on the type of biomass and the hydrolysis condition [13]. Among them, furfural is a major toxic byproduct from pretreated lignocellulose. Furfural is derived from pentose, and it damages DNA and inhibits glycolysis and sugar metabolism [13-15]. Furfural is converted to the less toxic furfuryl alcohol through reduction by aldehyde reductases [16]. To overcome the inhibitory effects of furfural, genes encoding NADH-dependent furfural reductase (FucO), NADPH-dependent oxidoreductase (YqhD), membrane-bound transhydrogenase interconverting NADH and NADPH (PntAB), and NAD salvage pathway (PncB, NadE) have been used to provide furfural resistance [17-20].

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 Escherichia coli in the presence of inhibitors such as furfural. Interestingly, we found an association between furfural and PHB production in E. coli, in which PHB synthetic genes provided high resistance to furfural and the compound itself stimulated PHB production.

Materials and Methods

Bacterial Strains, Media, Reagents, and Culture Conditions

Strains and plasmids used in this study are listed in Table 1. E. coli DH5α and KSYH(DE3) were used as host strains for gene cloning and PHB production, respectively [35-37]. For cell preparation and selection of transformants, the strains were cultured in lysogeny broth (LB) agar and/or liquid broth. LB agar was prepared by dissolving 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of distilled water. For evaluation of furfural resistance and PHB production, trans-formants were cultured in M9 minimal medium containing 2% glucose and 0.1% yeast extract, which had an initial pH of 6.8. Appropriate antibiotics (100 μg/ml spectinomycin and 25 μg/ml chloramphenicol for E. coli transformants) were added when required, and 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added at the beginning of culture. For preculture, a single colony from an LB agar plate was inoculated into 3 ml of LB broth. The culture was incubated overnight in a shaking incubator at 37°C and 200 rpm. For flask culture, grown cells were inoculated into 100 ml of production medium in 250-ml screw-cap flasks at a dilution of 1:100 (v/v). The culture was continuously shaken in an incubator at 200 rpm and 30°C. Restriction enzymes and DNA polymerase were purchased from Enzynomics (Korea), plasmid extraction and gel purification kits from GeneAll (Korea), medium components from Bacto or Difco (USA), and furfural, vanillin, and 4-hydroxybenzaldehyde from Sigma-Aldrich (USA). Lignocellulose hydrolysates (Miscanthus, barley straw, and pine tree) were purchased from Sugaren (Korea) and their composition is described in Table 2 [8].

Table 1 . Bacterial strains, plasmids, and primers used in this study..

Strain or plasmidDescriptionReference
E. coli strains
DH5αGeneral cloning strainInvitrogen
KSYH(DE3)BW25113 derivative containing DE3, ΔaraBAD, ΔrhaBAD[35]
KSYH(DE3)/pCDFKSYH(DE3) containing pCDFDuet-1This study
KSYH(DE3)::bktBKSYH(DE3) containing pCDF::bktBThis study
KSYH(DE3)::phaBKSYH(DE3) containing pCDF::phaBThis study
KSYH(DE3)::phaCKSYH(DE3) containing pCDF::phaCThis study
YH090KSYH(DE3) containing pLW487[35]
Plasmids
pCDFDuet-1A compatible spectinomycin-selectable plasmid carrying T7/lac promoterNovagen
pCDF::bktBpCDFDuet-1 carrying bktB gene from Ralstonia eutropha H16This study
pCDF::phaBpCDFDuet-1 carrying phaB gene from Ralstonia eutropha H16This study
pCDF::phaCpCDFDuet-1 carrying phaC gene from Ralstonia eutropha H16This study
pLW487Spectinomycin-selectable pEP2-based plasmid carrying bktB, phaB and phaC genes from Ralstonia eutropha H16 under the control of trc promoter.[48]


Table 2 . Composition of lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree used in this study..

BiomassComponentsConcentration (g/l)
MiscanthusMonosugarsGlucose102.19 ± 0.54
Xylose20.16 ± 0.25
Galactose1.42 ± 0.21
Arabinose2.97 ± 0.17
Mannose1.55 ± 0.21
ByproductsFormic acid-
Acetic acid0.24 ± 0.01
Levulinic acid-
5-Hydroxymethylfurfural0.19 ± 0.01
Furfural0.48 ± 0.01
Barley strawMonosugarsGlucose135.80 ± 0.04
Xylose13.164 ± 0.67
Galactose-
Arabinose0.34 ± 0.01
Mannose0.70 ± 0.04
ByproductsFormic acid0.17 ± 0.03
Acetic acid0.14 ± 0.02
Levulinic acid-
5-Hydroxymethylfurfural0.18 ± 0.01
Furfural0.09 ± 0.01
Pine treeMonosugarsGlucose127.44 ± 0.22
Xylose14.02 ± 0.33
Galactose-
Arabinose-
Mannose2.95 ± 0.23
ByproductsFormic acid-
Acetic acid0.88 ± 0.02
Levulinic acid-
5-Hydroxymethylfurfural0.15 ± 0.01
Furfural0.17 ± 0.01


DNA Manipulation

Gene cloning was conducted according to standard protocols [38]. PHB synthetic genes (bktB, phaB, and phaC) were from Ralstonia eutropha H16. In brief, each target gene was amplified by PCR, and the amplification product was purified and digested with restriction enzymes. The digested fragment was ligated into a plasmid, which was digested with the same restriction enzymes. Ligated plasmids were transformed into E. coli DH5α using the heat-shock method. Constructed plasmids were used for further study only after they were confirmed by sequencing. Detailed information is shown in Table 1.

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 ×g for 10 min, washed with deionized water twice, and suspended in 1 ml of water. The suspended samples were lyophilized, weighed, and placed in Teflon-stoppered glass vials. For methanolysis of PHB samples, 1 ml of chloroform and 1 ml of a methanol/sulfuric acid (85:15, v/v) mixture were added to the vials, which were then incubated at 100°C for 2 h. Samples were cooled to room temperature and incubated on ice for approximately 10 min. After adding 1 ml of ice-cold water, samples were thoroughly vortex-mixed for 1 min and centrifuged at 2,000 ×g. The (lower) organic phase was collected using a pipette and transferred to a clean borosilicate glass tube containing anhydrous sodium sulfate (Na2SO4). Then, the samples were injected into a gas chromatograph (Young Lin Tech, Korea) equipped with a DB-Wax column (30 m × 0.32 mm × 0.5 μm) (Agilent Technologies, USA). The split ratio was 1:10. Helium was used as a carrier gas at a flow rate of 3.0 ml/min. Two microliters of the organic phase was injected using an autosampler. The inlet was maintained at 210°C. The column oven was held at 80°C for 5 min, heated to 220°C at 20°C/min, and then held at 220°C for 5 min. Peak detection was conducted using a flame ionization detector, which was maintained at 230°C. Furfural concentrations were determined by gas chromatography as mentioned above, except that the column oven was held at 50°C for 5 min, heated to 230°C at 20°C/min, and then held at 230°C for 5 min.

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 (e.g., acetic acid, levulinic acid, 4-hydroxybenzaldehyde, vanillin, and furfural) [33]. Furfural is considered the most potent inhibitor of E. coli growth [41] and has various cell-inhibitory effects [17, 18, 42]. It damages DNA, affects the hydrophobicity of intracellular membranes and depletes NAD(P)H in the cell [18]. In addition to general toxicity, because both PHB synthesis and furfural detoxification require NADH and NADPH as cofactors, cofactor depletion affects cell growth and productivity in E. coli and R. eutropha H16 [17, 18, 30, 33, 42]. Therefore, when cell growth and PHB content were monitored in E. coli YH090 containing PHB synthetic genes in the presence of different concentrations of furfural, we expected to observe a decrease in cell growth. However, interestingly, in the presence of 15 mM furfural, E. coli YH090 cell growth and PHB content were respectively 1.2-fold and 1.3-fold higher than in the absence of furfural, and the highest PHB concentration of 0.85 g/l was noted in this condition (Figs. 1A, 1B, and 1D). When more than 20 mM furfural was used, introducing PHB synthetic genes had no effect on furfural resistance. The residual biomass, defined as dry cell weight excluding that of PHB, was constant at different concentrations of furfural (Fig. 1C). These results are consistent with previous reports that E. coli does not contain furfural oxidative degradation pathways and is unable to catabolize furfural as a carbon source [43]. Although it is well known that furfural inhibits sugar metabolism, such as glycolysis, carbon assimilation is accelerated along with PHB accumulation in the presence of furfural (Figs. 1B and 1D) [13].

Figure 1. Enhancement of polyhydroxybutyrate (PHB) production by furfural. Cell growth and PHB production in E. 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 E. coli YH090 for 72 h (Fig. 2). During the initial 24 h of cultivation, cell growth and PHB production were lower in the presence of 15 mM furfural than in the absence of furfural. However, after 24 h of cultivation in the presence of 15 mM furfural, cell growth and PHB content sharply increased and, at 72 h, cell growth was 1.55-fold higher and the PHB concentration was 4-fold higher than in the absence of furfural (Fig. 2). Therefore, addition of 15 mM furfural strongly stimulated cell growth and PHB production. The mechanisms underlying these effects, however, remain to be elucidated.

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 (phaB) of the PHB synthesis pathway was overexpressed separately in E. coli KSYH(DE3), and cell growth and the furfural conversion rate were evaluated for 24 h (Fig. 3). Contrary to our expectations, KSYH(DE3) strains containing individual gene (phaB) separately showed no clear difference in cell growth and furfural conversion rate (Fig. 3). In contrast, E. coli YH090 strain containing the three PHB synthetic genes showed notably improved cell growth and the highest furfural conversion rate during 24 h cultivation. During the initial 6 h of cultivation, this YH090 strain showed significantly lower cell growth and furfural conversion rate than the other strains; however, after 12 h cultivation, cell growth was completely restored, and the furfural conversion rate was the highest observed. These results were consistent with the findings that E. coli YH090 showed a longer lag phase in cell growth and PHB production in the presence of furfural as discussed above (Fig. 2). Based on these findings, we conclude that furfural resistance depends on the overexpression of the three PHB synthetic genes, not any individual gene of the PHB synthesis pathway.

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 E. coli KSYH(DE3) and YH090 was evaluated in medium containing 4-hydroxybenzaldehyde (5 mM) or vanillin (10 mM) [42]. Similar to our findings for furfural, when the three PHB synthetic genes were introduced, cell growth was increased by 1.58-fold and 2-fold in the presence of 4-hydroxybenzaldehyde and vanillin, respectively (Fig. 4). We conclude that the introduction of PHB synthetic genes confers resistance not only to furfural, but also to vanillin and 4-hydroxybenzaldehyde.

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, E. coli YH090 was grown in M9 minimal medium containing lignocellulose hydrolysates. The composition of hydrolysates from Miscanthus, barley straw, and pine tree, which contain several inhibitors, are shown in Table 2. As the initial glucose concentration was adjusted to 2%, each lignocellulose hydrolysate was diluted appropriately. Because only small amounts of pentose sugars (xylose and arabinose) were not detected by HPLC, their concentrations were ignored. To evaluate the effects of lignocellulose hydrolysates, cell growth and PHB production in synthetic medium containing M9 minimal medium and 2% glucose as a sole carbon source, without inhibitors, were also investigated. PHB production was significantly increased with increasing fermentation time, especially in the presence of hydrolysates of Miscanthus, barley straw, or pine tree. Especially, in the presence of Miscanthus hydrolysate, the PHB concentration reached 0.67 g/l at 24 h, 1.06 g/l at 48 h, and 1.29 g/l at 72 h, corresponding to an approximately 2-fold increase compared to synthetic medium without any inhibitor. When ligno-cellulose hydrolysates containing well-known fermentation inhibitors (formic acid, 5-hydroxymethylfurfural, and furfural) were used, PHB production was increased compared to that in synthetic medium. Although it cannot be definitively concluded that the inhibitors increase PHB production, the results indicate that PHB production is possible in lignocellulose hydrolysates in the presence of inhibitors such as furfural, vanillin, and 4-hydroxy-benzaldehyde.

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 E. coli growth [41].

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 E. coli KSYH(DE3). In particular, PHB production was increased in the presence of furfural compared to in its absence.

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 E. coli, and it increases tolerance to stress conditions, such as carbon starvation, heat shock, and osmotic pressure [46]. In addition, PHA mobilization influences chaperone protein levels [37, 45]. It is well known that PHB accumulation in recombinant E. coli can cause stress in cells and induces the expression of various protective proteins related to stress resistance (e.g., heat shock proteins, such as GroEL, GroES, and DnaK) [45]. Therefore, stress responses might be responsible for the increased cell growth and PHB production in the presence of furfural. In conclusion, PHB synthetic genes can improve tolerance to toxic inhibitors, such as furfural, vanillin, and 4-hydroxybenzaldehyde. In addition, although these are well-known fermentation inhibitors, they could be used to stimulate PHB production. PHB production by E. coli YH090 in the presence of lignocellulose hydrolysates was approximately 2-fold higher than that in the synthetic medium. Consequently, PHB production in recombinant E. coli using lignocellulose hydrolysates could be useful for cost-effective bio-based plastic production.

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.

Figure 1.Enhancement of polyhydroxybutyrate (PHB) production by furfural. Cell growth and PHB production in E. 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.
Journal of Microbiology and Biotechnology 2019; 29: 776-784https://doi.org/10.4014/jmb.1901.01070

Fig 2.

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.
Journal of Microbiology and Biotechnology 2019; 29: 776-784https://doi.org/10.4014/jmb.1901.01070

Fig 3.

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.
Journal of Microbiology and Biotechnology 2019; 29: 776-784https://doi.org/10.4014/jmb.1901.01070

Fig 4.

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.
Journal of Microbiology and Biotechnology 2019; 29: 776-784https://doi.org/10.4014/jmb.1901.01070

Fig 5.

Figure 5.Polyhydroxybutyrate (PHB) production using lignocellulose hydrolysates. Cell growth and PHB production in E. coli YH090 (pLW487) using lignocellulose hydrolysates were compared. Lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree were used. (A) DCW (dry cell weight, g/l) and (B) PHB concentration (g/l). Cells were grown for 72 h. Error bars represent the standard deviation of two replicates.
Journal of Microbiology and Biotechnology 2019; 29: 776-784https://doi.org/10.4014/jmb.1901.01070

Table 1 . Bacterial strains, plasmids, and primers used in this study..

Strain or plasmidDescriptionReference
E. coli strains
DH5αGeneral cloning strainInvitrogen
KSYH(DE3)BW25113 derivative containing DE3, ΔaraBAD, ΔrhaBAD[35]
KSYH(DE3)/pCDFKSYH(DE3) containing pCDFDuet-1This study
KSYH(DE3)::bktBKSYH(DE3) containing pCDF::bktBThis study
KSYH(DE3)::phaBKSYH(DE3) containing pCDF::phaBThis study
KSYH(DE3)::phaCKSYH(DE3) containing pCDF::phaCThis study
YH090KSYH(DE3) containing pLW487[35]
Plasmids
pCDFDuet-1A compatible spectinomycin-selectable plasmid carrying T7/lac promoterNovagen
pCDF::bktBpCDFDuet-1 carrying bktB gene from Ralstonia eutropha H16This study
pCDF::phaBpCDFDuet-1 carrying phaB gene from Ralstonia eutropha H16This study
pCDF::phaCpCDFDuet-1 carrying phaC gene from Ralstonia eutropha H16This study
pLW487Spectinomycin-selectable pEP2-based plasmid carrying bktB, phaB and phaC genes from Ralstonia eutropha H16 under the control of trc promoter.[48]

Table 2 . Composition of lignocellulose hydrolysates from Miscanthus, barley straw, and pine tree used in this study..

BiomassComponentsConcentration (g/l)
MiscanthusMonosugarsGlucose102.19 ± 0.54
Xylose20.16 ± 0.25
Galactose1.42 ± 0.21
Arabinose2.97 ± 0.17
Mannose1.55 ± 0.21
ByproductsFormic acid-
Acetic acid0.24 ± 0.01
Levulinic acid-
5-Hydroxymethylfurfural0.19 ± 0.01
Furfural0.48 ± 0.01
Barley strawMonosugarsGlucose135.80 ± 0.04
Xylose13.164 ± 0.67
Galactose-
Arabinose0.34 ± 0.01
Mannose0.70 ± 0.04
ByproductsFormic acid0.17 ± 0.03
Acetic acid0.14 ± 0.02
Levulinic acid-
5-Hydroxymethylfurfural0.18 ± 0.01
Furfural0.09 ± 0.01
Pine treeMonosugarsGlucose127.44 ± 0.22
Xylose14.02 ± 0.33
Galactose-
Arabinose-
Mannose2.95 ± 0.23
ByproductsFormic acid-
Acetic acid0.88 ± 0.02
Levulinic acid-
5-Hydroxymethylfurfural0.15 ± 0.01
Furfural0.17 ± 0.01

References

  1. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, et al. 2006. The path forward for biofuels and biomaterials. Science 311: 484-489.
    Pubmed CrossRef
  2. Lynd LR, Laser MS, Bransby D, Dale BE, Davison B, Hamilton R, et al. 2008. How biotech can transform biofuels. Nat. Biotechnol. 26: 169-172.
    Pubmed CrossRef
  3. Sims RE, Mabee W, Saddler JN, Taylor M. 2010. An overview of second generation biofuel technologies. Bioresour. Technol. 101: 1570-1580.
    Pubmed CrossRef
  4. Brodin M, Vallejos M, Opedal MT, Area MC, Chinga-Carrasco G. 2017. Lignocellulosics as sustainable resources for production of bioplastics-a review. J. Clean Prod. 162: 646-664.
    CrossRef
  5. Sharma HK, Xu C, Qin W. 2017. Biological pretreatment of lignocellulosic biomass for biofuels and bioproducts: an overview. Waste and Biomass Valorization.: 1-17.
    CrossRef
  6. Carroll A, Somerville C. 2009. Cellulosic biofuels. Annu. Rev. Plant Biol. 60: 165-182.
    Pubmed CrossRef
  7. Weijde Tvd, Alvim Kamei CL, Torres AF, Vermerris W, Dolstra O, Visser RGF, et al. 2013. The potential of C4 grasses for cellulosic biofuel production. Front. Plant Sci. 4: 107.
    Pubmed KoreaMed CrossRef
  8. Bhatia SK, Gurav R, Choi T-R, Jung H-R, Yang S-Y, Moon Y-M, et al. 2019. Bioconversion of plant biomass hydrolysate into bioplastic (polyhydroxyalkanoates) using Ralstonia eutropha 5119. Bioresour. Technol. 271: 306-315.
    Pubmed CrossRef
  9. Kumar P, Barrett DM, Delwiche MJ, Stroeve P. 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. 48: 3713-3729.
    CrossRef
  10. Sun Y, Cheng J. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83: 1-11.
    Pubmed CrossRef
  11. Mills TY, Sandoval NR, Gill RT. 2009. Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnol. Biofuels 2: 26.
    Pubmed KoreaMed CrossRef
  12. Saha BC. 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechnol. 30: 279-291.
    Pubmed CrossRef
  13. Gorsich S, Dien B, Nichols N, Slininger P, Liu Z, Skory C. 2006. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 71: 339-349.
    Pubmed CrossRef
  14. Barciszewski J, Siboska GE, Pedersen BO, Clark BF, Rattan SI. 1997. A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA. FEBS Lett. 414: 457-460.
    Pubmed CrossRef
  15. Palmqvist E, Hahn-Hägerdal B. 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74: 25-33.
    CrossRef
  16. Zaldivar J, Martinez A, Ingram LO. 2000. Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol. Bioeng. 68: 524-530.
    Pubmed CrossRef
  17. Song H-S, Jeon J-M, Kim H-J, Bhatia SK, Sathiyanarayanan G, Kim J, et al. 2017. Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli. Bioresour. Technol. 245: 1430-1435.
    Pubmed CrossRef
  18. Seo H-M, Jeon J-M, Lee JH, Song H-S, Joo H-B, Park S-H, et al. 2016. Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural. J. Ind. Microbiol. Biotechnol. 43: 37-44.
    Pubmed CrossRef
  19. Wang X, Miller E, Yomano L, Zhang X, Shanmugam K, Ingram L. 2011. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Appl. Environ. Microbiol. 77: 5132-5140.
    Pubmed KoreaMed CrossRef
  20. Wang X, Yomano LP, Lee JY, York SW, Zheng H, Mullinnix MT, et al. 2013. Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proc. Natl. Acad. Sci. USA 110: 4021-4026.
    Pubmed KoreaMed CrossRef
  21. Madkour MH, Heinrich D, Alghamdi MA, Shabbaj II, Steinbuchel A. 2013. PHA recovery from biomass. Biomacromolecules 14: 2963-2972.
    Pubmed CrossRef
  22. Steinbuchel A, Fuchtenbusch B. 1998. Bacterial and other biological systems for polyester production. Trends. Biotechnol. 16: 419-427.
    Pubmed CrossRef
  23. Wen Q, Chen Z, Tian T, Chen W. 2010. Effects of phosphorus and nitrogen limitation on PHA production in activated sludge. J. Environ. Sci. 22: 1602-1607.
    Pubmed CrossRef
  24. Johnson K, Kleerebezem R, van Loosdrecht MC. 2010. Influence of the C/N ratio on the performance of polyhydroxybutyrate (PHB) producing sequencing batch reactors at short SRTs. Water Res. 44: 2141-2152.
    Pubmed CrossRef
  25. Ahn J, Jho EH, Nam K. 2015. Effect of C/N ratio on polyhydroxyalkanoates (PHA) accumulation by Cupriavidus necator and its implication on the use of rice straw hydrolysates. Environ. Eng. Res. 20: 246-253.
    CrossRef
  26. Sudesh K, Abe H, Doi Y. 2000. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25: 1503-1555.
    CrossRef
  27. Du C, Sabirova J, Soetaert W, Lin C. 2012. Polyhydroxy-alkanoates production from low-cost sustainable raw materials. Curr. Chem. Biol. 6: 14-25.
    CrossRef
  28. Broeren M. Production of Bio-ethylene−Technology Brief. IEA-ETSAP & IRENA, International Renewable Energy Agency.
  29. Cesário MT, Raposo RS, de Almeida MCM, van Keulen F, Ferreira BS, da Fonseca MMR. 2014. Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates. N. Biotechnol. 31: 104-113.
    Pubmed CrossRef
  30. Yu J, Stahl H. 2008. Microbial utilization and biopolyester synthesis of bagasse hydrolysates. Bioresour. Technol. 99: 8042-8048.
    Pubmed CrossRef
  31. Silva L, Taciro M, Ramos MM, Carter J, Pradella J, Gomez J. 2004. Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose and sugarcane bagasse hydrolysate. J Ind. Microbiol. Biotechnol. 31: 245-254.
    Pubmed CrossRef
  32. Dietrich D, Illman B, Crooks C. 2013. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava. BMC Res. Notes 6: 219.
    Pubmed KoreaMed CrossRef
  33. Wang W, Yang S, Hunsinger GB, Pienkos PT, Johnson DK. 2014. Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator. Front. Microbiol. 5: 247.
    Pubmed KoreaMed CrossRef
  34. Dietrich K, Dumont M-J, Schwinghamer T, Orsat V, Del Rio LF. 2017. Model study to assess softwood hemicellulose hydrolysates as the carbon source for PHB production in Paraburkholderia sacchari IPT 101. Biomacromolecules 19: 188-200.
    Pubmed CrossRef
  35. Yang YH, Brigham C, Song E, Jeon JM, Rha C, Sinskey A. 2012. Biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase. J. Appl. Microbiol. 113: 815-823.
    Pubmed CrossRef
  36. Jeon JM, Kim HJ, Bhatia SK, Sung C, Seo HM, Kim JH, et al. 2017. Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly (3-hydroxybutyrate-co-3-hydroxyvalerate). Bioproc. Biosyst. Eng. 40: 781-789.
    Pubmed CrossRef
  37. Mezzina MP, Wetzler DE, Almeida A, Dinjaski N, Prieto MA, Pettinari MJ. 2015. A phasin with extra talents: a poly-hydroxyalkanoate granule-associated protein has chaperone activity. Environ. Microbiol. 17: 1765-1776.
    Pubmed CrossRef
  38. Phylactides M. 1997. Molecular biology series 3. Tools of molecular biology: gene cloning. Br. J. Hosp. Med. 57: 49-50.
    Pubmed
  39. Braunegg G, Sonnleitner B, Lafferty R. 1978. A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Appl. Microbiol. Biotechnol. 6: 29-37.
    CrossRef
  40. Bhatia SK, Kim J, Song H-S, Kim HJ, Jeon J-M, Sathiyanarayanan G, et al. 2017. Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01. Bioresour. Technol. 233: 99-109.
    Pubmed CrossRef
  41. Zaldivar J, Ingram LO. 1999. Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioeng. 66: 203-210.
    Pubmed CrossRef
  42. Song HS, Jeon JM, Choi YK, Kim JY, Kim W, Yoon JJ, et al. 2017. L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli. J. Microbiol.Biotechnol. 27: 2165-2172.
    Pubmed CrossRef
  43. Nieves LM, Panyon LA, Wang X. 2015. Engineering sugar utilization and microbial tolerance toward lignocellulose conversion. Front. Bioeng. Biotechnol. 3: 17.
    Pubmed KoreaMed CrossRef
  44. Moon J, Liu ZL. 2015. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae. Yeast 32: 399-407.
    Pubmed CrossRef
  45. Han M-J, Yoon SS, Lee SY. 2001. Proteome analysis of metabolically engineeredescherichia coli producing poly (3-hydroxybutyrate). J Bacteriol. 183: 301-308.
    Pubmed KoreaMed CrossRef
  46. Wang Q, Yu H, Xia Y, Kang Z, Qi Q. 2009. Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application. Microb. Cell Fact. 8: 47.
    Pubmed KoreaMed CrossRef
  47. James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R. 1999. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ Molecular biology series 3. Tools of molecular biology: gene cloning. Microbiol. 65: 822-827.
    Pubmed KoreaMed CrossRef
  48. Yang YH, Brigham C, Willis L, Rha C, Sinskey A. 2011. Improved detergent-based recovery of polyhydroxyalkanoates (PHAs). Biotechnology Lett. 33: 937-942.
    Pubmed CrossRef