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Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass
Bioresource Technology Reports 9 (2020) 100395
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Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass Tingting Liu, Bingyin Peng, Shuangcheng Huang, Anli Geng
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School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Saccharomyces cerevisiae Xylose-fermentation Ethanol Shikimate pathway Cis,cis-muconic acid Oil palm empty fruit bunch (OPEFB)
New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.
1. Introduction Muconic acid, also known as 2,4-hexadienedioic acid, is an unsaturated dicarboxylic acid. It can be converted to terepthalic acid, which is a precursor for the production of polyethylene terephthalate (PET) (Burk et al., 2011; Curran et al., 2013; Xie et al., 2014; Averesch and Krömer, 2018; Henard et al., 2019). In addition, muconic acid can also be converted to adipic acid, a chemical precursor used for the production of nylon, lubricants, coatings, plastics, and plasticizers (Niu et al., 2002; Weber et al., 2012; Curran et al., 2013; Xie et al., 2014; Bart and Cavallaro, 2015a, 2015b; Deng et al., 2016; Averesch and Krömer, 2018; Henard et al., 2019). Muconic acid is produced primarily from petroleum feedstock through chemical synthesis and it is environmentally unfriendly and non-renewable (Curran et al., 2013). As an alternative, metabolic engineering is able to solve this demand through the development of microorganisms producing a diverse variety of chemicals from renewable plant-based carbon sources, such as crops and lignocellulosic biomass (Jang et al., 2012; Curran et al., 2013; Lane et al., 2018; Pyne et al., 2018; Kwak et al., 2019). Aromatic compounds, such as benzoate, toluene, and benzene, are oxidized by some bacteria and produce catechol as the central aromatic intermediate (Harwood and Parales, 1996). The ortho-cleavage of catechol performed by catechol 1,2-dioxygenase (EC 3.1.11.1, CatA) yields cis,cis-muconic acid (CCM) (Vaillancourt et al., 2006; Wells Jr
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and Ragauskas, 2012). Benzoate is stable, water soluble and non-volatile. It is therefore one of the most common raw materials for CCM production. Several bacteria, such as Arthrobacter and Pseudomonas species, are known to endogenously degrade aromatic compounds into CCM (Mizuno et al., 1988; Xie et al., 2014). However, in terms of using carbohydrates as the substrate, CCM is not endogenously produced by any known microorganisms. A common synthetic route using renewable resources such as glucose was established in Escherichia coli (Niu et al., 2002). This heterologous CCM biosynthetic pathway was based on the expression of three heterologous genes that encode 3-dehydroshikimate (3-DHS) dehydratase (AroZ) and protocatechuic acid (PCA) decarboxylase (AroY) from Klebsiella pneumoniae, and catechol 1,2-dioxygenase (CatA) from Acinetobacter calcoaceticus. Another novel artificial pathway in E. coli for CCM biosynthesis included the conversion of anthranilate, the first intermediate in the tryptophan biosynthetic branch, to catechol and subsequently to muconic acid by anthranilate 1,2-dioxygenase (AntABC) and catechol 1,2-dioxygenase (CatA), respectively (Sun et al., 2013). Even though these E. coli strains are able to generate considerable amounts of CCM under neutral-pH conditions, they do not allow a costcompetitive industrial production process because purification of CCM from the fermentation broth in its undissociated form is conducted at low pH and substantial acidification of the fermentation broth with subsequent recycling is necessary (Hsieh, 1984). On the other hand,
Corresponding author at: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, 599489, Singapore. E-mail address: [email protected] (A. Geng).
https://doi.org/10.1016/j.biteb.2020.100395 Received 1 January 2020; Received in revised form 28 January 2020; Accepted 28 January 2020 Available online 29 January 2020 2589-014X/ © 2020 Elsevier Ltd. All rights reserved.
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Table 1 Plasmids used in this work. Plasmid name
Characteristics
References
pUG6 pSH47 pJPPP-XK pJFE12 pGEMK14
E. coli plasmid containing KanMX gene with loxP sites Plasmid containing Cre recombinase under control of GAL1 promoter for removal of KanMX from integration cassettes pUC19, genomic integrative plasmid used to overexpress Saccharomyces cerevisiae endogenous genes RPE1, RKI1, TAL1, TKL1 and XKS1 pGBT9-based yeast shuttle plasmid, ADH1p-Gal4- ADH1t were replaced with TEF1p and CYC1t pUC19-based yeast integration plasmid, loxP–KanMX4–loxP, codon-optimized Aro4 from S. cerevisiae, Aro1 with AroE harbouring a stop codon from S. cerevisiae pUC19-based yeast integration plasmid with ura3-TDH3p-BvuXylA-PGK1t expression cassette pJFE12-based yeast shuttle plasmid containing AroZ-Neu, AroY-Com and CatA-Cup genes
Güldener et al., 1996 Güldener et al., 1996 Peng et al., 2015 This study This study
pXbUN pJAA9N
This study This study
fermenting S. cerevisiae for CCM biosynthesis.
Saccharomyces cerevisiae might be an ideal host microorganism for CCM production because fermentation can be performed at low pH, like lactic acid production (Lee et al., 2015; Nugroho et al., 2016). Moreover, it offers advantages of withstanding low temperature, tolerance to low pH and high osmatic pressure, and suitability in large-scale fermentation and industrial application (Liu et al., 2019). Significant advancement was achieved in engineering S. cerevisiae to produce ethanol from lignocellulosic sugars such as glucose and xylose (Lane et al., 2018; Kwak et al., 2019). Continuous efforts were made in engineering S. cerevisiae to produce muconic acid using glucose as the substrate and good progress was achieved (Weber et al., 2012; Curran et al., 2013; Horwitz et al., 2015; Leavitt et al., 2017; Pyne et al., 2018; Kildegaard et al., 2019). The first CCM-producing recombinant yeast was constructed by Weber and his colleagues by screening AroZ, AroY and CatA genes from a few microorganisms and 1.56 mg/L CCM was obtained (Weber et al., 2012). Up to 141 mg/L CCM were obtained in shake-flask fermentation using an engineered S. cerevisiae strain constructed by importing AroZ from Podospora anserina, AroY from Enterobacter cloacae, and CatA from Candida albicans, and overexpression of a feedback-resistant mutant of Aro4 (Curran et al., 2013). About 1 g/ L CCM was obtained with catechol feeding using a recombinant CCMproducing recombinant yeast strain constructed via CRISPR-Cas9 tools (Horwitz et al., 2015). By a combined biosensor-enabled strain directed evolution and overexpression of a truncated Aro1 gene, 0.5 g/L CCM was obtained by shake-flask batch fermentation and 2.1 g/L CCM was obtained by fed-batch fermentation in a bioreactor (Leavitt et al., 2017). Through the engineering of the Aro1 protein in the engineered yeast and overexpression of Pad1, which is responsible for activation of AroY, 1.2 g/L CCM was obtained under prototrophic conditions and 5.1 g/L CCM was obtained when amino acids were supplemented in a fed-batch bioreactor (Pyne et al., 2018). Recently, a few xylose-fermenting yeast strains were constructed in our research group and they were successfully applied in converting lignocellulosic biomass, such as oil palm empty fruit bunch (OPEFB), to ethanol (Peng et al., 2015; Liu et al., 2018; Huang et al., 2019). To further advance our research in converting lignocellulosic biomass to chemicals and fuels, here we established both heterologous CCM biosynthesis and xylose fermentation pathways in S. cerevisiae INVSc1. A three-step CCM biosynthetic pathway comprised of genes encoding AroZ from Neurospora crassa (AroZ-Neu, GenBank Accession Number KHE84566.1), AroY from Commensalibacter intestini A911 (AroY-Com, GenBank Accession Number EHD13070.1), and CatA from Cupriavidus necator (CatA-Cup, GenBank Accession Number WP_153957085.1), and xylose-fermenting pathway (Peng et al., 2015) were engineered in the yeast for CCM biosynthesis and ethanol production from both glucose and xylose (Fig. S1). To further optimize CCM production, the strain was modified by the overexpression of a truncated Aro1 gene, Aro1AroEΔ (Aro1 gene with AroE gene modified with a stop codon) (Leavitt et al., 2017). In addition, a feedback-resistant mutant of Aro4 gene from S. cerevisiae, Aro4opt, was overexpressed in the yeast, which caused a single lysine-to-leucine substitution at position 229 in the Aro4 enzyme (Hartmann et al., 2003; Luttik et al., 2008; Curran et al., 2013). To our knowledge, this is the first report on the construction of a xylose-
2. Materials and methods 2.1. Strains and media The commercial diploid S. cerevisiae strain INVSc1 (MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52) (Invitrogen, Carlsbad, CA, USA) was used as the host strain in this study. The yeast strain was routinely propagated at 30 °C in yeast extract peptone dextrose (YPD) medium or yeast synthetic minimal (SM) medium. YPD medium contained 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and/or was supplemented with 200 mg/L G418. SMG medium contained 6.7 g/L Difco yeast nitrogen base, 20 g/L glucose, and 76 mg/L of each individual amino acid, such as histidine (His), uracil (Ura), tryptophan (Trp) and leucine (Leu), to form SMG + His +Ura + Trp + Leu, SMG + His+Trp + Leu or SMG + His+Leu media, depending on the requirement for auxotrophy selection. SMX medium contained 6.7 g/L Difco yeast nitrogen base, defined concentration of xylose, 76 mg/L His and 76 mg/L Leu each. The pH of all yeast SM media was adjusted to 6.0. Strain E. coli DH5α was obtained from Life Technologies (Rockville, MD, USA) and used for all gene cloning and plasmid propagation. Luria–Bertani (LB) broth and agar (Thermo Fisher Scientific, Singapore) supplemented with 100 μg/mL ampicillin was used to select and culture positive E. coli transformants. All chemicals were of analytical grade and were obtained from SigmaAldrich (Singapore) unless otherwise stated. 2.2. Plasmid construction Plasmids and primers used in this work are listed in Tables 1 and S1. Plasmid maps are provided in Fig. S2. The polymerase chain reactions (PCRs) were conducted using Phusion High-Fidelity DNA Polymerase from New England Biolabs (Ipswich, MA, USA) following manufacturer's instructions. Primers were synthesized from Integrated DNA Technologies (Singapore). The DNA fragments, ADH1p, PGK1t, FBA1p, TPI1t, CYC1t, AroEΔ, Trp1u (-571,-226) and Trp1d (1365) were amplified from the genomic DNA of S. cerevisiae strain INVSc1. The loxPKanMX4-loxP cassette was amplified from the plasmid pUG6 (Güldener et al., 1996). Gene Aro4 encoding 3-deoxy-D-arabino-heptulosonate-7phosphate synthase was amplified from S. cerevisiae INVSc1. It was fused with a DNA fragment encoding a single lysine-to-leucine substitution at position 229 of Aro4 enzyme through overlapping extension PCR (OE-PCR) to obtain Aro4opt. Through OE-PCR of the above DNA fragments, gene cassettes of Trp1u-loxP-KanMX4-loxP, ADH1p-Aro4opt-PGK1t and FBA1p-Aro1AroEΔ-TPI1t-Trp1d were sequentially cloned into plasmid pUC19, resulting in plasmid pGEKA14. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The pJFE12 vector was constructed by replacing the ADH1p-Gal4ADH1t cassette with TEF1p-CYC1t into SphI-digested plasmid pGBT9 (Fields and Song, 1989). Genes AroZ-Neu, AroY-Com, and CatA-Cup were codon-optimized for their expression in S. cerevisiae and 2
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conducted in 100-mL shaking flasks containing 50 mL SMX medium (50 g/L xylose) with an initial OD600 of 2.0 (0.48 g/L cell dry weight). The flasks were capped with rubber stoppers to maintain oxygen-limited conditions and incubated at 200 rpm and 30 °C.
synthesized from GenScript (Singapore). TDH3p, PGK1p and CYC1t were amplified from the genomic DNA of S. cerevisiae. Gene fragments, AroZ-CYC1t, TDH3p-AroY-CYC1t and PGK1p-CatA, were fused through OE-PCR and sequentially cloned into plasmid pJFE12, resulting in plasmid pJAA9N. TDH3p-BvuXylA-PGK1t cassette encoding xylose isomerase (XylA) from B. vulgatus was amplified from pJFX11 (Peng et al., 2015) and DNA fragment ura3-pNTS was amplified from plasmid XXUN (Liu et al., 2018). DNA fragments of ura3-pNTS and TDH3p-BvuXylA-PGK1t were respectively cloned into BamHI/HindIII and SacI/BamHI sites of pUC19, resulting in plasmid pXbUN. URA3 was selected as an auxotrophic marker and pNTS was used as recombinant arm targeting to NTS2-2 following SwaI digestion in yeast.
2.5.2. Co-fermentation of glucose and xylose by strain Ic0.09-31 The preculture of the evolved recombinant S. cerevisiae strain was grown in SMX medium (50 g/L xylose). Cells in the exponential phase were centrifuged, washed twice, and subsequently used to inoculate 50 mL YP medium, which was supplemented with 88 g/L glucose and 50 g/L xylose. Fermentation was conducted in 100-mL shake flasks capped with rubber stoppers under oxygen-limited conditions at 200 rpm and 30 °C. To test effects of oxygen exposure on ethanol production and CCM biosynthesis, co-fermentation of glucose and xylose was performed in YP medium containing 88 g/L glucose and 50 g/L xylose as described above at an initial OD600 of 1.0 under both aerobic and oxygen-limited conditions. To examine the catechol feeding effects, fermentation was conducted in 50 mL SM medium containing 42.4 g/L glucose and 25.5 g/L xylose in 100-mL shake flasks capped with rubber stoppers under oxygen-limited conditions at 200 rpm and 30 °C. One gram per liter catechol was added to log-phase culture. A culture under the same conditions without catechol feeding was used as the negative control.
2.3. Strain construction Xylulokinase and non-oxidative pentose phosphate pathway were overexpressed by integrating SwaI-digested pJPPP-XK into S. cerevisiae INVSc1 (Peng et al., 2015). Sequentially, genes Aro1AroEΔ and Aro4opt were introduced into the yeast strain by transforming them both to SbfIdigested pGEKA14. The KanMX4 marker for the integrated pJPPP-XK and pGEKA14 was individually removed by transforming a Cre plasmid pSH47 containing Ura3. Later on, pSH47 was lost by streaking the yeast on YPD agar plates containing 1 g/L 5-Fluoroorotic acid (5-FOA) (Güldener et al., 1996). Genes BvuXylA, AroZ-Neu, AroY-Com, and CatA-Cup were introduced into the yeast strain by co-transforming SwaI-digested pXbUN and the episomal plasmid pJAA9N.
An Agilent 1200 series high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) was used to analyse the concentration of CCM, ethanol and pathway intermediates in S. cerevisiae cultures. The metabolites were separated by HPLC using an Aminex HPX-87H ion exchange column (Bio-Rad Laboratories, Woodinville, WA, USA) eluted at 65 °C using 5 mM of sulfuric acid at a flow rate of 0.6 mL/min. A 1200 series diode array detector was used for the detection of PCA (220 nm), catechol (220 nm), and CCM (250 nm). The detection of ethanol and other intermediates was performed using a 1200 series refractive index detector. Cell density was measured using a UV–visible spectrophotometer (Shimadzu, Tokyo, Japan) at the wavelength of 600 nm (OD600). One OD600 is equivalent to about 0.24 g/L dry cell weight (DCW) (Liu et al., 2018).
2.5.3. Biomass hydrolysate fermentation Oil palm empty fruit bunch (OPEFB) was collected from Teck Guan Holdings Sdn Bhd (Tawau, Malaysia). OPEFB hydrolysate was prepared using crude cellulase from Trichoderma reesei Rut-C30 according to the protocols described in our earlier report (Wang et al., 2020). OPEFB hydrolysate was sterilized using 0.22 μm filter membrane (Merck Millipore, Singapore). It contained 22 g/L glucose, 15 g/L xylose and 5.6 g/ L acetic acid. It was supplemented with 7 g/L yeast extract 2 g/L peptone, 2 g/L (NH4)2SO4, 2.05 g/L KH2PO4, and 0.25 g/L Na2HPO4 to make the OPEFB hydrolysate fermentation medium as described in our earlier report (Liu et al., 2018). Fermentation experiments were conducted as described in the previous session under oxygen-limited conditions in 100-mL shake flasks containing 50 mL OPEFB hydrolysate medium. For all fermentation experiments, the initial cell density was 0.48 g/ L DCW (OD600 of 2.0) unless otherwise stated. Samples were taken at time intervals for OD600 measurement and metabolite content analysis. Experiments were conducted in triplicates and average results are reported.
2.5. CCM production using the recombinant yeast
2.6. Enzyme activity assays
2.5.1. Adaptive evolution of the recombinant yeast strain for enhanced xylose fermentation and CCM production Adaptive laboratory evolution was shown to be effective to reinforce the desired flux network in vivo from xylose to ethanol or chemicals in xylose selective medium (Dragosits and Mattanovich, 2013; Reyes et al., 2014; Peng et al., 2015). To enhance xylose fermentation and CCM biosynthesis, the recombinant S. cerevisiae strain was anaerobically evolved in a chemostat by continuous diluting cultivation according to the protocols described in our earlier report (Peng et al., 2015). Its dilution rate was increased from 0.01 to 0.1 1/h in SMX medium containing 20 g/L xylose at 30 °C and pH 6.0 (Fig. 1). Samples were taken periodically at varying dilution rates of 0.06–0.1 1/h for OD600 measurement and colony isolation. These colonies were then cultivated aerobically in 24-well plates containing 600 μL SMX medium (50 g/L xylose) in each well at 218 rpm and 30 °C. OD600 at 30 h was monitored using a microplate reader (TECAN Group, AG, Männedorf, Switzerland) (Fig. 2A) and seven colonies with higher absorbance at 15 h indicated in red dots were chosen for further investigation (Fig. 2B). Fermentation of these seven colonies was subsequently
Cells were grown to the exponential phase in the SMX medium containing 20 g/L xylose. Enzyme activities of XylA, and AroZ were assayed using total cell protein extract, which was obtained using the Pierce Y-PER Yeast Protein Extraction Reagent (Thermo Fisher Scientific, Singapore) and EDTA-free Halt Protease Inhibitor Cocktail Set V (Merck, Singapore). Protein concentration of the cell extracts was determined using a NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific, Singapore). Extraction of raw protein from the yeast strains was performed in duplicate. Spectrophotometric assays of enzyme activity were then performed at an ambient temperature below 30 °C (Curran et al., 2013). For the measurement of AroZ activity, 300 mg of the crude protein was added to a cuvette containing excessive Y-PER reagent and 0.1–0.75 mM 3-DHS in a total volume of 1 mL. The absorbance at 290 nm was recorded every 10 s for 3 min. For the measurement of XylA activity, the assay mixture (1 mL) contained extraction buffer, 0.15 mM nicotinamide adenine dinucleotide (NADH), 1 U sorbitol dehydrogenase (SDH), and 300 mg of the crude protein (Peng et al., 2015). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of the substrate per min.
2.4. Metabolite content analysis and cell density measurement
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Fig. 1. Anaerobic chemostat adaptive evolution of strain S. cerevisiae Ic in SMX medium containing 20 g/L xylose. Filled triangle, OD600; Solid line, dilution rate.
3. Results and discussion
adaptive evolution by twelve-time continuous transfer in SMX medium containing 20 g/L xylose (data not shown).
3.1. Expression and optimization of xylose fermentation and CCM biosynthetic pathway in S. cerevisiae INVSc1 3.2. Adaptive evolution of the recombinant yeast strain for enhanced xylose fermentation and CCM production
In S. cerevisiae, neither xylose fermentation pathway nor CCM biosynthetic pathway exists. In this study, we established heterologous xylose fermentation and CCM biosynthetic pathways in S. cerevisiae INVSc1. A three-step CCM biosynthetic pathway as described for E. coli (Niu et al., 2002) was imported into yeast S. cerevisiae INVSc1 by the heterologous expression of three new genes, i.e. AroZ-Neu, AroY-Com, and CatA-Cup. This is the first time to express these three genes in S. cerevisiae for muconic acid biosynthesis. In addition, XylA from B. vulgatus was imported to the same host yeast strain. In order to ensure xylose fermentation, the xylose metabolic pathway in the above background strain S. cerevisiae INVSc1 was optimized by overexpressing xylulokinase (XKS1) and the four enzymes from non-oxidative pentose phosphate pathway, transaldolase (TAL1), transketolase (TKL1) ribose 5-phosphate isomerase (RKI1), and ribulose 5-phosphate 3-epimerase (RPE1) according to our earlier report (Peng et al., 2015). Furthermore, to enhance the metabolic flux from 2-dehydro-3-deoxy-D-arabino-heptonate 7-P to 3,4-dihydroxybenzoate and reduce synthesis of amino acids that cause feedback inhibition to Aro4, a truncated Aro1 gene, Aro1AroEΔ, and a mutated Aro4 gene, Aro4opt, were overexpressed in the yeast strain. The arom multifunctional enzyme, Aro1, mediates the metabolic flux from 2-dehydro-3-deoxy-D-arabino-heptonate 7-P to 3,4dihydroxybenzoate (Fig. S1, Duncan et al., 1987). The Aro1AroEΔ gene contained the AroE gene with a termination codon to block the conversion of 3-DHS to shikimate (Fig. S1). This truncated Aro1 gene, Aro1AroEΔ, with a less well-expressed AroE established a balance between the 3-DHS net pathway flux and the flux into the remainder of the shikimate pathway (Leavitt et al., 2017). The Aro4opt encodes a mutated Aro4 enzyme with a single lysine-to-leucine substitution at position 229, which displayed alleviated amino acid feedback inhibition (Hartmann et al., 2003; Luttik et al., 2008). The generated recombinant yeast strain harbouring B. vulgatus XylA, N. crassa AroZ, C. intestini AroY and C. necator CatA was denoted as S. cerevisiae Ic (MATa/ α; XK; PPP; Aro1AroEΔ; Aro4opt; BvuXylA; AroZ-Neu; AroY-Com; CatACup; his3Δ; leu2Δ). It exhibited slow growth on xylose under aerobic conditions. Through HPLC analysis of the fermentation broth of strain S. cerevisiae Ic, CCM was detected in milligram per litre level after strain
Further strain adaptive evolution was conducted in a chemostat anaerobically (Fig. 2) and seven colonies were isolated according to their OD600 values (Fig. 2A and B). Anaerobic fermentation was conducted for these potential isolates and isolate Ic0.09-31 produced about 65 mg/L CCM at 96 h in SMX medium (50 g/L xylose), which was the highest compared to the rest isolates (Fig. 2C). To monitor the variation of exogenous enzyme activities in the yeast strain during adaptive evolution, XylA and AroZ enzyme activities of three isolated strains harvested at different time point of evolution were measured (Fig. 2D). The specific enzyme activities of XylA and AroZ of strain Ic0.09-31 were 0.0154 U/mg and 0.183 U/mg, respectively. They were the highest compared to those obtained for strains Ic and Ic-m, the initial and the intermediate strains. Enhanced XylA activity after strain adaptive evolution was reported earlier (Wang et al., 2013). It was not due to gene mutation or increased XylA expression levels, but due to the enhanced general metabolisms in the yeast strain or mutation of other genes (Wang et al., 2013). We sequenced both genes in strain Ic0.09-31 and no mutation was found. Therefore, the enhanced enzyme activity of XylA and AroZ could be due to the enhancement of overall metabolisms in this yeast strain by adaptive evolution in xylose medium (Wang et al., 2013). It could also be due to the mutation in other genes in the strain (Dos Santos et al., 2016). Strain directed evolution for enhanced CCM biosynthesis by recombinant S. cerevisiae was reported by Leavitt and his colleagues through EMS mutagenesis and strain selection based on a biosensor module that responds to the endogenous aromatic amino acid (AAA) biosynthesis (Leavitt et al., 2017). Such strain evolution improved AAA production, a surrogate target for the desired molecule of muconic acid. In our study, strain evolution was conducted based on the increased cell growth in xylose medium under oxygen limited conditions. Such strain evolution improved both xylose fermentation and CCM biosynthesis in xylose medium by the recombinant yeast (Fig. 2).
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Fig. 2. Strain adaptive evolution, isolation and characterization. Microplate strain screening (A); Optical density of isolates harvested at varying dilution rates (B); CCM production by seven potential isolates (C); XylA and CatA enzyme activity assay of the evolved strains (D). Ic- was the initial strain; Ic-m was the strain collected at dilution rate of 0.06 1/h; Ic0.09-31 was the best performing strain.
whereas under oxygen limited conditions, ethanol of 50.4 g/L (0.39 g ethanol/g sugar) was obtained and CCM titer reached 165.9 mg/L (1.30 mg CCM/g sugar) (Fig. 4). In addition, higher PCA titer was observed at 24 h for fermentation under aerobic conditions. Again, such accumulation of PCA seemed primarily due to glucose consumption and afterwards, PCA was gradually converted to catechol and CCM, consistent to the result shown in Fig. 3. Furthermore, lower level of catechol existed throughout aerobic fermentation processes as the conversion of catechol to CCM needs oxygen. The above results suggest that aerobic conditions are favourable to CCM biosynthesis, however, lower ethanol production (Fig. 4). In fact, aerobic fermentation resulted in higher titer of by products, such as xylitol and acetic acid (data now shown). Although aerobic condition favours CCM biosynthesis, for the co-production of ethanol and CCM, oxygen-limiting condition is preferred.
3.3. Co-fermentation of glucose and xylose by strain Ic0.09-31 Co-fermentation of 88 g/L glucose and 50 g/L xylose using the recombinant S. cerevisiae strain Ic0.09-31 resulted in 54 g/L ethanol (0.41 g ethanol/g sugar) and 248 mg/L CCM (1.88 mg CCM/g sugar) with 95% of total sugar consumption at 79 h (Fig. 3). A bottleneck in AroY activity was observed indicated by the excessive accumulation of precursors PCA in the broth before glucose was fully consumed. Such results are consistent with those in previous reports when glucose was used as the sole carbon source (Weber et al., 2012; Curran et al., 2013; Horwitz et al., 2015). Interestingly, slight conversion of PCA was observed in combination with xylose consumption. This suggests that the enhanced xylose-fermentation pathway is favourable to PCA conversion to CCM. In the end, the PCA concentration reached more than 2.5 times the level of CCM and 5.5 times the level of catechol, to 616.3 mg/L at 79 h, indicating that AroY is still limiting.
3.5. In vivo catechol feeding for enhanced CCM production 3.4. Fermentation under aerobic and oxygen-limited conditions High levels of the CCM precursor, PCA, were detected in almost all glucose and xylose co-fermentation experiments by S. cerevisiae Ic0.0931 (Figs. 3 and 4). This suggests that AroY is the bottleneck enzyme in CCM biosynthesis. Therefore, in vivo catechol feeding was examined to enhance CCM production according to an earlier report (Horwitz et al.,
Co-fermentation of glucose and xylose was performed under both aerobic and oxygen-limited conditions. Under aerobic conditions, 40.3 g/L ethanol (0.31 g ethanol/g sugar) was produced and CCM concentration reached 423.8 mg/L (3.26 mg CCM/g sugar) at 72 h; 5
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Fig. 3. Glucose and xylose co-fermentation in YP medium containing 88 g/L glucose and 50 g/L xylose by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported.
Fig. 4. Effect of oxygen exposure in glucose and xylose co-fermentation in YP medium containing 88 g/L glucose and 50 g/L xylose fermentation by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported.
CCM/g sugar, respectively. The concentration of acetic acid remained at 5.6 g/L throughout OPEFB hydrolysate fermentation. In addition, glucose was completely utilized at 25 h and 71.0% xylose was consumed at 79 h. Currently, glucose was the main carbon source used for CCM production though sometimes glucose and glycerol were also used (Averesch and Krömer, 2018). A summary of microbial strain engineering for CCM biosynthesis is shown in Table 2. Zhang and his colleagues reported the coculture of E. coli strains for the conversion of glucose and xylose mixture to CCM (Zhang et al., 2015a). However, our study is the first attempt to employ an engineered S. cerevisiae strain for the conversion glucose and xylose to ethanol and CCM (Table 2). Strain S. cerevisiae Ic0.09-31 presented lower titer of CCM than some earlier reported S. cerevisiae strains (Table 2). Co-fermentation of glucose and xylose to CCM is as challenging as CCM biosynthesis from glucose in
2015). Feeding 1 g/L catechol in the log phase of fermentation, CCM concentration reached 1025.1 mg/L at 25 h, about 101.8 times the result obtained for the negative control (Fig. 5). Using this catechol feeding strategy, strain Ic0.09–31 produced 1259.8 mg/L CCM at 79 h. No catechol was detected at the end of fermentation though low level of catechol was detected at 25 h. Almost equal amount of PCA was detected for fermentation carried out with or without catechol feeding. It was further confirmed that AroY is the most rate-limiting enzyme compared to AroZ and CatA.
3.6. OPEFB hydrolysate fermentation OPEFB hydrolysate fermentation resulted in 31.3 g/L ethanol and 53.4 mg/L CCM at 79 h (Fig. 6). About 89% total sugar were consumed with ethanol and CCM yields of 0.43 g ethanol/g sugar and 1.44 mg 6
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Fig. 5. Glucose and xylose co-fermentation in SM containing 42.4 g/L glucose and 25.5 g/L xylose supplemented with 1 g/L catechol at 8 h. Experiments were conducted in triplicates and average results are reported.
cerevisiae is more rigidly resistant to modifications. Co-production of ethanol and CCM could be an alternative given the fact that ethanol is still the primary product by fermentation using S. cerevisiae and it is an important commodity chemical product. In this case, downstream processing improvement could be essential to make the process costeffective. Much effort is still necessary to improve CCM yield to obtain an economically feasible lignocellulose bioconversion process using such engineered S. cerevisiae strains.
engineered yeast strains because AroY is still the bottleneck enzyme (Curran et al., 2013; Horwitz et al., 2015) although xylose conversion facilitated PCA conversion (Figs. 3 and 4). Improving AroY activity (Johnson et al., 2016) or further engineering of this yeast strain via manipulation of several other metabolic engineering targets is possible to further enhance the CCM titer (Pyne et al., 2018; Kildegaard et al., 2019). However, co-utilization of glucose and xylose makes it possible to convert lignocellulosic biomass effectively to ethanol and CCM. This study is the first demonstration on bioconversion of lignocellulosic biomass hydrolysate to CCM using an engineered xylose-fermenting S. cerevisiae strain. It is worthwhile noting that the CCM titer obtained in S. cerevisiae strains was lower than that obtained by engineered E. coli strains (Table 2). In general, metabolic engineering of S. cerevisiae seems much more difficult than that of E. coli as the metabolic network of S.
4. Conclusion In this study, we obtained a xylose-fermenting and CCM-biosynthesizing S. cerevisiae strain Ic0.09-31 through metabolic engineering and strain adaptive evolution. Strain Ic0.09-31 produced 424 mg/L of CCM and 50 g/L of ethanol in complex medium containing
Fig. 6. OPEFB hydrolysate fermentation by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported. 7
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Zhang et al. (2015b) 2 g/L
Johnson et al. (2016) Niu et al. (2002) Lin et al. (2014) Sun et al. (2013) Zhang et al. (2015a) 53.4 mg/L 4.92 g/L 36.8 g/L 1.5 g/L 389.96 mg/L 4.7 g/L
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement
Acknowledgments
Declaration of competing interest The authors declare no conflict of interest. Glycerol
Glucose + amino acids Glucose Xylose Glucose and Xylose Glucose and Xylose and 1 g/L catechol OPEFB hydrolysate Glucose Glucose Glucose, Glycerol Glucose, Glycerol Glucose, xylose
Tingting Liu: Conceptualization, Methodology, Investigation, Validation, Writing - original draft. Bingyin Peng: Conceptualization, Methodology. Shuangcheng Huang: Investigation, Validation. Anli Geng: Conceptualization, Project administration, Supervision, Writing review & editing.
The authors are grateful for the OPEFB samples provided by Teck Guan Holdings Sdn Bhd, Tawau, Malaysia.
Appendix A. Supplementary data
AroY, CatA, EcdB, EcdD, AsbF, pcaHGΔ, catRBCΔ TktA, AroZ, AroY, CatA, AroFFBR, AroB EntC, PchB, NahGopt, AroL, AroG, CatA, PpsA, TktA, PheAΔ, TyrAΔPpsA, TktA, AroG, AroE, AroB, AroL, TrpE, AntABC, CatA, GlnA, TrpDΔ AroD, AroZ, AroY, CatA, ShiA, YdiBΔ, AroEΔ
AroZ, AroY, CatA, ShiA, YdiBΔ, AroEΔ
Pseudomonas putida KT2440 E. coli E. coli E. coli E. coli
E. coli
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2020.100395. AroZ, AroZ, CatA, TKL1, ZWF1, Aro1AroEΔ, Aro4K229L, CRISPR-Cas9 and RNAi AroZ, AroZ, CatA, XylA, PPP, Aro1AroEΔ, Aro4K229L, adaptive laboratory evolution (ALE)
S. cerevisiae S. cerevisiae
Funding
S. cerevisiae S. cerevisiae
Glucose Glucose
Batch, shake flask Fed-batch, bioreactor Batch, shake flask Batch, shake flask Fed-batch, bioreactor Fed-batch, bioreactor Batch, shake flask Batch, shake flask Batch, shake flask Batch, shake flask TKL1, Aro1AroEΔ, AroZ, AroY, CatA, EMS mutation, adaptive laboratory evolution (ALE) and aromatic amino acid (AAA) biosensor TKL1, Aro4K229L, AroZ, AroY, CatA, Aro1Δ, ZWF1Δ AroZ, AroZ, CatA, Pad1, Aro1 degradation AroZ, AroZ, CatA, Pad1, Hqd2 Aro1 degradation, S. cerevisiae
both glucose and xylose. In addition, it generated 31.3 g/L ethanol and 53.4 mg/L CCM from OPEFB hydrolysate. This study demonstrated that AroZ Neurospora crassa, AroY from Commensalibacter intestini A911, and CatA from Cupriavidus necator were effective in CCM biosynthesis in S. cerevisiae. Strain Ic0.09-31 has high potential in cellulosic ethanol and CCM co-production using lignocellulosic biomass hydrolysate.
Batch, shake flask Fed-batch, bioreactor Fed-batch, bioreactor Batch, shake flask Batch, shake flask Fed-batch, bioreactor, coculture Batch, bioreactor, co-culture
Kildegaard et al. (2019) This study
Suástegui et al., 2017 Pyne et al., 2018
Leavitt et al., 2017
Weber et al. (2012) Curran et al. (2013) Horwitz et al., 2015
1.56 mg/L 141 mg/L 0 ~1 g/L 0.5 g/L 2.1 g/L 0.32 g/L 0.86 g/L 1.2 g/L 5.1 g/L 800 mg/L 65 mg/L 424 mg/L 1.286 g/L Batch, shake flask Batch, shake flask Batch, 96-well shake plate AroZ, AroZ and CatA, Aro1AroEΔ TKL1, AroZ, AroZ, CatA, Aro4K229L ZWF1Δ, Aro3Δ, Aro4Δ AroZ, AroZ, CatA, AroF, AroB, AroD, Aro1Δ, CRISPR-Cas9 S. cerevisiae S. cerevisiae S. cerevisiae
Glucose Glucose Glucose Glucose with 1 g/L Catechol Glucose
Detailed strategies Host strains
Table 2 Comparison of CCM production by engineered microorganisms.
Carbon source
Fermentation
CCM titer
References
T. Liu, et al.
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Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass Tingting Liu, Bingyin Peng, Shuangcheng Huang, Anli Geng
T
⁎
School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore
A R T I C LE I N FO
A B S T R A C T
Keywords: Saccharomyces cerevisiae Xylose-fermentation Ethanol Shikimate pathway Cis,cis-muconic acid Oil palm empty fruit bunch (OPEFB)
New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.
1. Introduction Muconic acid, also known as 2,4-hexadienedioic acid, is an unsaturated dicarboxylic acid. It can be converted to terepthalic acid, which is a precursor for the production of polyethylene terephthalate (PET) (Burk et al., 2011; Curran et al., 2013; Xie et al., 2014; Averesch and Krömer, 2018; Henard et al., 2019). In addition, muconic acid can also be converted to adipic acid, a chemical precursor used for the production of nylon, lubricants, coatings, plastics, and plasticizers (Niu et al., 2002; Weber et al., 2012; Curran et al., 2013; Xie et al., 2014; Bart and Cavallaro, 2015a, 2015b; Deng et al., 2016; Averesch and Krömer, 2018; Henard et al., 2019). Muconic acid is produced primarily from petroleum feedstock through chemical synthesis and it is environmentally unfriendly and non-renewable (Curran et al., 2013). As an alternative, metabolic engineering is able to solve this demand through the development of microorganisms producing a diverse variety of chemicals from renewable plant-based carbon sources, such as crops and lignocellulosic biomass (Jang et al., 2012; Curran et al., 2013; Lane et al., 2018; Pyne et al., 2018; Kwak et al., 2019). Aromatic compounds, such as benzoate, toluene, and benzene, are oxidized by some bacteria and produce catechol as the central aromatic intermediate (Harwood and Parales, 1996). The ortho-cleavage of catechol performed by catechol 1,2-dioxygenase (EC 3.1.11.1, CatA) yields cis,cis-muconic acid (CCM) (Vaillancourt et al., 2006; Wells Jr
⁎
and Ragauskas, 2012). Benzoate is stable, water soluble and non-volatile. It is therefore one of the most common raw materials for CCM production. Several bacteria, such as Arthrobacter and Pseudomonas species, are known to endogenously degrade aromatic compounds into CCM (Mizuno et al., 1988; Xie et al., 2014). However, in terms of using carbohydrates as the substrate, CCM is not endogenously produced by any known microorganisms. A common synthetic route using renewable resources such as glucose was established in Escherichia coli (Niu et al., 2002). This heterologous CCM biosynthetic pathway was based on the expression of three heterologous genes that encode 3-dehydroshikimate (3-DHS) dehydratase (AroZ) and protocatechuic acid (PCA) decarboxylase (AroY) from Klebsiella pneumoniae, and catechol 1,2-dioxygenase (CatA) from Acinetobacter calcoaceticus. Another novel artificial pathway in E. coli for CCM biosynthesis included the conversion of anthranilate, the first intermediate in the tryptophan biosynthetic branch, to catechol and subsequently to muconic acid by anthranilate 1,2-dioxygenase (AntABC) and catechol 1,2-dioxygenase (CatA), respectively (Sun et al., 2013). Even though these E. coli strains are able to generate considerable amounts of CCM under neutral-pH conditions, they do not allow a costcompetitive industrial production process because purification of CCM from the fermentation broth in its undissociated form is conducted at low pH and substantial acidification of the fermentation broth with subsequent recycling is necessary (Hsieh, 1984). On the other hand,
Corresponding author at: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, 599489, Singapore. E-mail address: [email protected] (A. Geng).
https://doi.org/10.1016/j.biteb.2020.100395 Received 1 January 2020; Received in revised form 28 January 2020; Accepted 28 January 2020 Available online 29 January 2020 2589-014X/ © 2020 Elsevier Ltd. All rights reserved.
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Table 1 Plasmids used in this work. Plasmid name
Characteristics
References
pUG6 pSH47 pJPPP-XK pJFE12 pGEMK14
E. coli plasmid containing KanMX gene with loxP sites Plasmid containing Cre recombinase under control of GAL1 promoter for removal of KanMX from integration cassettes pUC19, genomic integrative plasmid used to overexpress Saccharomyces cerevisiae endogenous genes RPE1, RKI1, TAL1, TKL1 and XKS1 pGBT9-based yeast shuttle plasmid, ADH1p-Gal4- ADH1t were replaced with TEF1p and CYC1t pUC19-based yeast integration plasmid, loxP–KanMX4–loxP, codon-optimized Aro4 from S. cerevisiae, Aro1 with AroE harbouring a stop codon from S. cerevisiae pUC19-based yeast integration plasmid with ura3-TDH3p-BvuXylA-PGK1t expression cassette pJFE12-based yeast shuttle plasmid containing AroZ-Neu, AroY-Com and CatA-Cup genes
Güldener et al., 1996 Güldener et al., 1996 Peng et al., 2015 This study This study
pXbUN pJAA9N
This study This study
fermenting S. cerevisiae for CCM biosynthesis.
Saccharomyces cerevisiae might be an ideal host microorganism for CCM production because fermentation can be performed at low pH, like lactic acid production (Lee et al., 2015; Nugroho et al., 2016). Moreover, it offers advantages of withstanding low temperature, tolerance to low pH and high osmatic pressure, and suitability in large-scale fermentation and industrial application (Liu et al., 2019). Significant advancement was achieved in engineering S. cerevisiae to produce ethanol from lignocellulosic sugars such as glucose and xylose (Lane et al., 2018; Kwak et al., 2019). Continuous efforts were made in engineering S. cerevisiae to produce muconic acid using glucose as the substrate and good progress was achieved (Weber et al., 2012; Curran et al., 2013; Horwitz et al., 2015; Leavitt et al., 2017; Pyne et al., 2018; Kildegaard et al., 2019). The first CCM-producing recombinant yeast was constructed by Weber and his colleagues by screening AroZ, AroY and CatA genes from a few microorganisms and 1.56 mg/L CCM was obtained (Weber et al., 2012). Up to 141 mg/L CCM were obtained in shake-flask fermentation using an engineered S. cerevisiae strain constructed by importing AroZ from Podospora anserina, AroY from Enterobacter cloacae, and CatA from Candida albicans, and overexpression of a feedback-resistant mutant of Aro4 (Curran et al., 2013). About 1 g/ L CCM was obtained with catechol feeding using a recombinant CCMproducing recombinant yeast strain constructed via CRISPR-Cas9 tools (Horwitz et al., 2015). By a combined biosensor-enabled strain directed evolution and overexpression of a truncated Aro1 gene, 0.5 g/L CCM was obtained by shake-flask batch fermentation and 2.1 g/L CCM was obtained by fed-batch fermentation in a bioreactor (Leavitt et al., 2017). Through the engineering of the Aro1 protein in the engineered yeast and overexpression of Pad1, which is responsible for activation of AroY, 1.2 g/L CCM was obtained under prototrophic conditions and 5.1 g/L CCM was obtained when amino acids were supplemented in a fed-batch bioreactor (Pyne et al., 2018). Recently, a few xylose-fermenting yeast strains were constructed in our research group and they were successfully applied in converting lignocellulosic biomass, such as oil palm empty fruit bunch (OPEFB), to ethanol (Peng et al., 2015; Liu et al., 2018; Huang et al., 2019). To further advance our research in converting lignocellulosic biomass to chemicals and fuels, here we established both heterologous CCM biosynthesis and xylose fermentation pathways in S. cerevisiae INVSc1. A three-step CCM biosynthetic pathway comprised of genes encoding AroZ from Neurospora crassa (AroZ-Neu, GenBank Accession Number KHE84566.1), AroY from Commensalibacter intestini A911 (AroY-Com, GenBank Accession Number EHD13070.1), and CatA from Cupriavidus necator (CatA-Cup, GenBank Accession Number WP_153957085.1), and xylose-fermenting pathway (Peng et al., 2015) were engineered in the yeast for CCM biosynthesis and ethanol production from both glucose and xylose (Fig. S1). To further optimize CCM production, the strain was modified by the overexpression of a truncated Aro1 gene, Aro1AroEΔ (Aro1 gene with AroE gene modified with a stop codon) (Leavitt et al., 2017). In addition, a feedback-resistant mutant of Aro4 gene from S. cerevisiae, Aro4opt, was overexpressed in the yeast, which caused a single lysine-to-leucine substitution at position 229 in the Aro4 enzyme (Hartmann et al., 2003; Luttik et al., 2008; Curran et al., 2013). To our knowledge, this is the first report on the construction of a xylose-
2. Materials and methods 2.1. Strains and media The commercial diploid S. cerevisiae strain INVSc1 (MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52) (Invitrogen, Carlsbad, CA, USA) was used as the host strain in this study. The yeast strain was routinely propagated at 30 °C in yeast extract peptone dextrose (YPD) medium or yeast synthetic minimal (SM) medium. YPD medium contained 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and/or was supplemented with 200 mg/L G418. SMG medium contained 6.7 g/L Difco yeast nitrogen base, 20 g/L glucose, and 76 mg/L of each individual amino acid, such as histidine (His), uracil (Ura), tryptophan (Trp) and leucine (Leu), to form SMG + His +Ura + Trp + Leu, SMG + His+Trp + Leu or SMG + His+Leu media, depending on the requirement for auxotrophy selection. SMX medium contained 6.7 g/L Difco yeast nitrogen base, defined concentration of xylose, 76 mg/L His and 76 mg/L Leu each. The pH of all yeast SM media was adjusted to 6.0. Strain E. coli DH5α was obtained from Life Technologies (Rockville, MD, USA) and used for all gene cloning and plasmid propagation. Luria–Bertani (LB) broth and agar (Thermo Fisher Scientific, Singapore) supplemented with 100 μg/mL ampicillin was used to select and culture positive E. coli transformants. All chemicals were of analytical grade and were obtained from SigmaAldrich (Singapore) unless otherwise stated. 2.2. Plasmid construction Plasmids and primers used in this work are listed in Tables 1 and S1. Plasmid maps are provided in Fig. S2. The polymerase chain reactions (PCRs) were conducted using Phusion High-Fidelity DNA Polymerase from New England Biolabs (Ipswich, MA, USA) following manufacturer's instructions. Primers were synthesized from Integrated DNA Technologies (Singapore). The DNA fragments, ADH1p, PGK1t, FBA1p, TPI1t, CYC1t, AroEΔ, Trp1u (-571,-226) and Trp1d (1365) were amplified from the genomic DNA of S. cerevisiae strain INVSc1. The loxPKanMX4-loxP cassette was amplified from the plasmid pUG6 (Güldener et al., 1996). Gene Aro4 encoding 3-deoxy-D-arabino-heptulosonate-7phosphate synthase was amplified from S. cerevisiae INVSc1. It was fused with a DNA fragment encoding a single lysine-to-leucine substitution at position 229 of Aro4 enzyme through overlapping extension PCR (OE-PCR) to obtain Aro4opt. Through OE-PCR of the above DNA fragments, gene cassettes of Trp1u-loxP-KanMX4-loxP, ADH1p-Aro4opt-PGK1t and FBA1p-Aro1AroEΔ-TPI1t-Trp1d were sequentially cloned into plasmid pUC19, resulting in plasmid pGEKA14. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The pJFE12 vector was constructed by replacing the ADH1p-Gal4ADH1t cassette with TEF1p-CYC1t into SphI-digested plasmid pGBT9 (Fields and Song, 1989). Genes AroZ-Neu, AroY-Com, and CatA-Cup were codon-optimized for their expression in S. cerevisiae and 2
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conducted in 100-mL shaking flasks containing 50 mL SMX medium (50 g/L xylose) with an initial OD600 of 2.0 (0.48 g/L cell dry weight). The flasks were capped with rubber stoppers to maintain oxygen-limited conditions and incubated at 200 rpm and 30 °C.
synthesized from GenScript (Singapore). TDH3p, PGK1p and CYC1t were amplified from the genomic DNA of S. cerevisiae. Gene fragments, AroZ-CYC1t, TDH3p-AroY-CYC1t and PGK1p-CatA, were fused through OE-PCR and sequentially cloned into plasmid pJFE12, resulting in plasmid pJAA9N. TDH3p-BvuXylA-PGK1t cassette encoding xylose isomerase (XylA) from B. vulgatus was amplified from pJFX11 (Peng et al., 2015) and DNA fragment ura3-pNTS was amplified from plasmid XXUN (Liu et al., 2018). DNA fragments of ura3-pNTS and TDH3p-BvuXylA-PGK1t were respectively cloned into BamHI/HindIII and SacI/BamHI sites of pUC19, resulting in plasmid pXbUN. URA3 was selected as an auxotrophic marker and pNTS was used as recombinant arm targeting to NTS2-2 following SwaI digestion in yeast.
2.5.2. Co-fermentation of glucose and xylose by strain Ic0.09-31 The preculture of the evolved recombinant S. cerevisiae strain was grown in SMX medium (50 g/L xylose). Cells in the exponential phase were centrifuged, washed twice, and subsequently used to inoculate 50 mL YP medium, which was supplemented with 88 g/L glucose and 50 g/L xylose. Fermentation was conducted in 100-mL shake flasks capped with rubber stoppers under oxygen-limited conditions at 200 rpm and 30 °C. To test effects of oxygen exposure on ethanol production and CCM biosynthesis, co-fermentation of glucose and xylose was performed in YP medium containing 88 g/L glucose and 50 g/L xylose as described above at an initial OD600 of 1.0 under both aerobic and oxygen-limited conditions. To examine the catechol feeding effects, fermentation was conducted in 50 mL SM medium containing 42.4 g/L glucose and 25.5 g/L xylose in 100-mL shake flasks capped with rubber stoppers under oxygen-limited conditions at 200 rpm and 30 °C. One gram per liter catechol was added to log-phase culture. A culture under the same conditions without catechol feeding was used as the negative control.
2.3. Strain construction Xylulokinase and non-oxidative pentose phosphate pathway were overexpressed by integrating SwaI-digested pJPPP-XK into S. cerevisiae INVSc1 (Peng et al., 2015). Sequentially, genes Aro1AroEΔ and Aro4opt were introduced into the yeast strain by transforming them both to SbfIdigested pGEKA14. The KanMX4 marker for the integrated pJPPP-XK and pGEKA14 was individually removed by transforming a Cre plasmid pSH47 containing Ura3. Later on, pSH47 was lost by streaking the yeast on YPD agar plates containing 1 g/L 5-Fluoroorotic acid (5-FOA) (Güldener et al., 1996). Genes BvuXylA, AroZ-Neu, AroY-Com, and CatA-Cup were introduced into the yeast strain by co-transforming SwaI-digested pXbUN and the episomal plasmid pJAA9N.
An Agilent 1200 series high performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) was used to analyse the concentration of CCM, ethanol and pathway intermediates in S. cerevisiae cultures. The metabolites were separated by HPLC using an Aminex HPX-87H ion exchange column (Bio-Rad Laboratories, Woodinville, WA, USA) eluted at 65 °C using 5 mM of sulfuric acid at a flow rate of 0.6 mL/min. A 1200 series diode array detector was used for the detection of PCA (220 nm), catechol (220 nm), and CCM (250 nm). The detection of ethanol and other intermediates was performed using a 1200 series refractive index detector. Cell density was measured using a UV–visible spectrophotometer (Shimadzu, Tokyo, Japan) at the wavelength of 600 nm (OD600). One OD600 is equivalent to about 0.24 g/L dry cell weight (DCW) (Liu et al., 2018).
2.5.3. Biomass hydrolysate fermentation Oil palm empty fruit bunch (OPEFB) was collected from Teck Guan Holdings Sdn Bhd (Tawau, Malaysia). OPEFB hydrolysate was prepared using crude cellulase from Trichoderma reesei Rut-C30 according to the protocols described in our earlier report (Wang et al., 2020). OPEFB hydrolysate was sterilized using 0.22 μm filter membrane (Merck Millipore, Singapore). It contained 22 g/L glucose, 15 g/L xylose and 5.6 g/ L acetic acid. It was supplemented with 7 g/L yeast extract 2 g/L peptone, 2 g/L (NH4)2SO4, 2.05 g/L KH2PO4, and 0.25 g/L Na2HPO4 to make the OPEFB hydrolysate fermentation medium as described in our earlier report (Liu et al., 2018). Fermentation experiments were conducted as described in the previous session under oxygen-limited conditions in 100-mL shake flasks containing 50 mL OPEFB hydrolysate medium. For all fermentation experiments, the initial cell density was 0.48 g/ L DCW (OD600 of 2.0) unless otherwise stated. Samples were taken at time intervals for OD600 measurement and metabolite content analysis. Experiments were conducted in triplicates and average results are reported.
2.5. CCM production using the recombinant yeast
2.6. Enzyme activity assays
2.5.1. Adaptive evolution of the recombinant yeast strain for enhanced xylose fermentation and CCM production Adaptive laboratory evolution was shown to be effective to reinforce the desired flux network in vivo from xylose to ethanol or chemicals in xylose selective medium (Dragosits and Mattanovich, 2013; Reyes et al., 2014; Peng et al., 2015). To enhance xylose fermentation and CCM biosynthesis, the recombinant S. cerevisiae strain was anaerobically evolved in a chemostat by continuous diluting cultivation according to the protocols described in our earlier report (Peng et al., 2015). Its dilution rate was increased from 0.01 to 0.1 1/h in SMX medium containing 20 g/L xylose at 30 °C and pH 6.0 (Fig. 1). Samples were taken periodically at varying dilution rates of 0.06–0.1 1/h for OD600 measurement and colony isolation. These colonies were then cultivated aerobically in 24-well plates containing 600 μL SMX medium (50 g/L xylose) in each well at 218 rpm and 30 °C. OD600 at 30 h was monitored using a microplate reader (TECAN Group, AG, Männedorf, Switzerland) (Fig. 2A) and seven colonies with higher absorbance at 15 h indicated in red dots were chosen for further investigation (Fig. 2B). Fermentation of these seven colonies was subsequently
Cells were grown to the exponential phase in the SMX medium containing 20 g/L xylose. Enzyme activities of XylA, and AroZ were assayed using total cell protein extract, which was obtained using the Pierce Y-PER Yeast Protein Extraction Reagent (Thermo Fisher Scientific, Singapore) and EDTA-free Halt Protease Inhibitor Cocktail Set V (Merck, Singapore). Protein concentration of the cell extracts was determined using a NanoDrop™ 2000/2000c Spectrophotometer (Thermo Fisher Scientific, Singapore). Extraction of raw protein from the yeast strains was performed in duplicate. Spectrophotometric assays of enzyme activity were then performed at an ambient temperature below 30 °C (Curran et al., 2013). For the measurement of AroZ activity, 300 mg of the crude protein was added to a cuvette containing excessive Y-PER reagent and 0.1–0.75 mM 3-DHS in a total volume of 1 mL. The absorbance at 290 nm was recorded every 10 s for 3 min. For the measurement of XylA activity, the assay mixture (1 mL) contained extraction buffer, 0.15 mM nicotinamide adenine dinucleotide (NADH), 1 U sorbitol dehydrogenase (SDH), and 300 mg of the crude protein (Peng et al., 2015). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of the substrate per min.
2.4. Metabolite content analysis and cell density measurement
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Fig. 1. Anaerobic chemostat adaptive evolution of strain S. cerevisiae Ic in SMX medium containing 20 g/L xylose. Filled triangle, OD600; Solid line, dilution rate.
3. Results and discussion
adaptive evolution by twelve-time continuous transfer in SMX medium containing 20 g/L xylose (data not shown).
3.1. Expression and optimization of xylose fermentation and CCM biosynthetic pathway in S. cerevisiae INVSc1 3.2. Adaptive evolution of the recombinant yeast strain for enhanced xylose fermentation and CCM production
In S. cerevisiae, neither xylose fermentation pathway nor CCM biosynthetic pathway exists. In this study, we established heterologous xylose fermentation and CCM biosynthetic pathways in S. cerevisiae INVSc1. A three-step CCM biosynthetic pathway as described for E. coli (Niu et al., 2002) was imported into yeast S. cerevisiae INVSc1 by the heterologous expression of three new genes, i.e. AroZ-Neu, AroY-Com, and CatA-Cup. This is the first time to express these three genes in S. cerevisiae for muconic acid biosynthesis. In addition, XylA from B. vulgatus was imported to the same host yeast strain. In order to ensure xylose fermentation, the xylose metabolic pathway in the above background strain S. cerevisiae INVSc1 was optimized by overexpressing xylulokinase (XKS1) and the four enzymes from non-oxidative pentose phosphate pathway, transaldolase (TAL1), transketolase (TKL1) ribose 5-phosphate isomerase (RKI1), and ribulose 5-phosphate 3-epimerase (RPE1) according to our earlier report (Peng et al., 2015). Furthermore, to enhance the metabolic flux from 2-dehydro-3-deoxy-D-arabino-heptonate 7-P to 3,4-dihydroxybenzoate and reduce synthesis of amino acids that cause feedback inhibition to Aro4, a truncated Aro1 gene, Aro1AroEΔ, and a mutated Aro4 gene, Aro4opt, were overexpressed in the yeast strain. The arom multifunctional enzyme, Aro1, mediates the metabolic flux from 2-dehydro-3-deoxy-D-arabino-heptonate 7-P to 3,4dihydroxybenzoate (Fig. S1, Duncan et al., 1987). The Aro1AroEΔ gene contained the AroE gene with a termination codon to block the conversion of 3-DHS to shikimate (Fig. S1). This truncated Aro1 gene, Aro1AroEΔ, with a less well-expressed AroE established a balance between the 3-DHS net pathway flux and the flux into the remainder of the shikimate pathway (Leavitt et al., 2017). The Aro4opt encodes a mutated Aro4 enzyme with a single lysine-to-leucine substitution at position 229, which displayed alleviated amino acid feedback inhibition (Hartmann et al., 2003; Luttik et al., 2008). The generated recombinant yeast strain harbouring B. vulgatus XylA, N. crassa AroZ, C. intestini AroY and C. necator CatA was denoted as S. cerevisiae Ic (MATa/ α; XK; PPP; Aro1AroEΔ; Aro4opt; BvuXylA; AroZ-Neu; AroY-Com; CatACup; his3Δ; leu2Δ). It exhibited slow growth on xylose under aerobic conditions. Through HPLC analysis of the fermentation broth of strain S. cerevisiae Ic, CCM was detected in milligram per litre level after strain
Further strain adaptive evolution was conducted in a chemostat anaerobically (Fig. 2) and seven colonies were isolated according to their OD600 values (Fig. 2A and B). Anaerobic fermentation was conducted for these potential isolates and isolate Ic0.09-31 produced about 65 mg/L CCM at 96 h in SMX medium (50 g/L xylose), which was the highest compared to the rest isolates (Fig. 2C). To monitor the variation of exogenous enzyme activities in the yeast strain during adaptive evolution, XylA and AroZ enzyme activities of three isolated strains harvested at different time point of evolution were measured (Fig. 2D). The specific enzyme activities of XylA and AroZ of strain Ic0.09-31 were 0.0154 U/mg and 0.183 U/mg, respectively. They were the highest compared to those obtained for strains Ic and Ic-m, the initial and the intermediate strains. Enhanced XylA activity after strain adaptive evolution was reported earlier (Wang et al., 2013). It was not due to gene mutation or increased XylA expression levels, but due to the enhanced general metabolisms in the yeast strain or mutation of other genes (Wang et al., 2013). We sequenced both genes in strain Ic0.09-31 and no mutation was found. Therefore, the enhanced enzyme activity of XylA and AroZ could be due to the enhancement of overall metabolisms in this yeast strain by adaptive evolution in xylose medium (Wang et al., 2013). It could also be due to the mutation in other genes in the strain (Dos Santos et al., 2016). Strain directed evolution for enhanced CCM biosynthesis by recombinant S. cerevisiae was reported by Leavitt and his colleagues through EMS mutagenesis and strain selection based on a biosensor module that responds to the endogenous aromatic amino acid (AAA) biosynthesis (Leavitt et al., 2017). Such strain evolution improved AAA production, a surrogate target for the desired molecule of muconic acid. In our study, strain evolution was conducted based on the increased cell growth in xylose medium under oxygen limited conditions. Such strain evolution improved both xylose fermentation and CCM biosynthesis in xylose medium by the recombinant yeast (Fig. 2).
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Fig. 2. Strain adaptive evolution, isolation and characterization. Microplate strain screening (A); Optical density of isolates harvested at varying dilution rates (B); CCM production by seven potential isolates (C); XylA and CatA enzyme activity assay of the evolved strains (D). Ic- was the initial strain; Ic-m was the strain collected at dilution rate of 0.06 1/h; Ic0.09-31 was the best performing strain.
whereas under oxygen limited conditions, ethanol of 50.4 g/L (0.39 g ethanol/g sugar) was obtained and CCM titer reached 165.9 mg/L (1.30 mg CCM/g sugar) (Fig. 4). In addition, higher PCA titer was observed at 24 h for fermentation under aerobic conditions. Again, such accumulation of PCA seemed primarily due to glucose consumption and afterwards, PCA was gradually converted to catechol and CCM, consistent to the result shown in Fig. 3. Furthermore, lower level of catechol existed throughout aerobic fermentation processes as the conversion of catechol to CCM needs oxygen. The above results suggest that aerobic conditions are favourable to CCM biosynthesis, however, lower ethanol production (Fig. 4). In fact, aerobic fermentation resulted in higher titer of by products, such as xylitol and acetic acid (data now shown). Although aerobic condition favours CCM biosynthesis, for the co-production of ethanol and CCM, oxygen-limiting condition is preferred.
3.3. Co-fermentation of glucose and xylose by strain Ic0.09-31 Co-fermentation of 88 g/L glucose and 50 g/L xylose using the recombinant S. cerevisiae strain Ic0.09-31 resulted in 54 g/L ethanol (0.41 g ethanol/g sugar) and 248 mg/L CCM (1.88 mg CCM/g sugar) with 95% of total sugar consumption at 79 h (Fig. 3). A bottleneck in AroY activity was observed indicated by the excessive accumulation of precursors PCA in the broth before glucose was fully consumed. Such results are consistent with those in previous reports when glucose was used as the sole carbon source (Weber et al., 2012; Curran et al., 2013; Horwitz et al., 2015). Interestingly, slight conversion of PCA was observed in combination with xylose consumption. This suggests that the enhanced xylose-fermentation pathway is favourable to PCA conversion to CCM. In the end, the PCA concentration reached more than 2.5 times the level of CCM and 5.5 times the level of catechol, to 616.3 mg/L at 79 h, indicating that AroY is still limiting.
3.5. In vivo catechol feeding for enhanced CCM production 3.4. Fermentation under aerobic and oxygen-limited conditions High levels of the CCM precursor, PCA, were detected in almost all glucose and xylose co-fermentation experiments by S. cerevisiae Ic0.0931 (Figs. 3 and 4). This suggests that AroY is the bottleneck enzyme in CCM biosynthesis. Therefore, in vivo catechol feeding was examined to enhance CCM production according to an earlier report (Horwitz et al.,
Co-fermentation of glucose and xylose was performed under both aerobic and oxygen-limited conditions. Under aerobic conditions, 40.3 g/L ethanol (0.31 g ethanol/g sugar) was produced and CCM concentration reached 423.8 mg/L (3.26 mg CCM/g sugar) at 72 h; 5
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Fig. 3. Glucose and xylose co-fermentation in YP medium containing 88 g/L glucose and 50 g/L xylose by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported.
Fig. 4. Effect of oxygen exposure in glucose and xylose co-fermentation in YP medium containing 88 g/L glucose and 50 g/L xylose fermentation by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported.
CCM/g sugar, respectively. The concentration of acetic acid remained at 5.6 g/L throughout OPEFB hydrolysate fermentation. In addition, glucose was completely utilized at 25 h and 71.0% xylose was consumed at 79 h. Currently, glucose was the main carbon source used for CCM production though sometimes glucose and glycerol were also used (Averesch and Krömer, 2018). A summary of microbial strain engineering for CCM biosynthesis is shown in Table 2. Zhang and his colleagues reported the coculture of E. coli strains for the conversion of glucose and xylose mixture to CCM (Zhang et al., 2015a). However, our study is the first attempt to employ an engineered S. cerevisiae strain for the conversion glucose and xylose to ethanol and CCM (Table 2). Strain S. cerevisiae Ic0.09-31 presented lower titer of CCM than some earlier reported S. cerevisiae strains (Table 2). Co-fermentation of glucose and xylose to CCM is as challenging as CCM biosynthesis from glucose in
2015). Feeding 1 g/L catechol in the log phase of fermentation, CCM concentration reached 1025.1 mg/L at 25 h, about 101.8 times the result obtained for the negative control (Fig. 5). Using this catechol feeding strategy, strain Ic0.09–31 produced 1259.8 mg/L CCM at 79 h. No catechol was detected at the end of fermentation though low level of catechol was detected at 25 h. Almost equal amount of PCA was detected for fermentation carried out with or without catechol feeding. It was further confirmed that AroY is the most rate-limiting enzyme compared to AroZ and CatA.
3.6. OPEFB hydrolysate fermentation OPEFB hydrolysate fermentation resulted in 31.3 g/L ethanol and 53.4 mg/L CCM at 79 h (Fig. 6). About 89% total sugar were consumed with ethanol and CCM yields of 0.43 g ethanol/g sugar and 1.44 mg 6
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Fig. 5. Glucose and xylose co-fermentation in SM containing 42.4 g/L glucose and 25.5 g/L xylose supplemented with 1 g/L catechol at 8 h. Experiments were conducted in triplicates and average results are reported.
cerevisiae is more rigidly resistant to modifications. Co-production of ethanol and CCM could be an alternative given the fact that ethanol is still the primary product by fermentation using S. cerevisiae and it is an important commodity chemical product. In this case, downstream processing improvement could be essential to make the process costeffective. Much effort is still necessary to improve CCM yield to obtain an economically feasible lignocellulose bioconversion process using such engineered S. cerevisiae strains.
engineered yeast strains because AroY is still the bottleneck enzyme (Curran et al., 2013; Horwitz et al., 2015) although xylose conversion facilitated PCA conversion (Figs. 3 and 4). Improving AroY activity (Johnson et al., 2016) or further engineering of this yeast strain via manipulation of several other metabolic engineering targets is possible to further enhance the CCM titer (Pyne et al., 2018; Kildegaard et al., 2019). However, co-utilization of glucose and xylose makes it possible to convert lignocellulosic biomass effectively to ethanol and CCM. This study is the first demonstration on bioconversion of lignocellulosic biomass hydrolysate to CCM using an engineered xylose-fermenting S. cerevisiae strain. It is worthwhile noting that the CCM titer obtained in S. cerevisiae strains was lower than that obtained by engineered E. coli strains (Table 2). In general, metabolic engineering of S. cerevisiae seems much more difficult than that of E. coli as the metabolic network of S.
4. Conclusion In this study, we obtained a xylose-fermenting and CCM-biosynthesizing S. cerevisiae strain Ic0.09-31 through metabolic engineering and strain adaptive evolution. Strain Ic0.09-31 produced 424 mg/L of CCM and 50 g/L of ethanol in complex medium containing
Fig. 6. OPEFB hydrolysate fermentation by strain Ic0.09-31. Experiments were conducted in triplicates and average results are reported. 7
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Zhang et al. (2015b) 2 g/L
Johnson et al. (2016) Niu et al. (2002) Lin et al. (2014) Sun et al. (2013) Zhang et al. (2015a) 53.4 mg/L 4.92 g/L 36.8 g/L 1.5 g/L 389.96 mg/L 4.7 g/L
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement
Acknowledgments
Declaration of competing interest The authors declare no conflict of interest. Glycerol
Glucose + amino acids Glucose Xylose Glucose and Xylose Glucose and Xylose and 1 g/L catechol OPEFB hydrolysate Glucose Glucose Glucose, Glycerol Glucose, Glycerol Glucose, xylose
Tingting Liu: Conceptualization, Methodology, Investigation, Validation, Writing - original draft. Bingyin Peng: Conceptualization, Methodology. Shuangcheng Huang: Investigation, Validation. Anli Geng: Conceptualization, Project administration, Supervision, Writing review & editing.
The authors are grateful for the OPEFB samples provided by Teck Guan Holdings Sdn Bhd, Tawau, Malaysia.
Appendix A. Supplementary data
AroY, CatA, EcdB, EcdD, AsbF, pcaHGΔ, catRBCΔ TktA, AroZ, AroY, CatA, AroFFBR, AroB EntC, PchB, NahGopt, AroL, AroG, CatA, PpsA, TktA, PheAΔ, TyrAΔPpsA, TktA, AroG, AroE, AroB, AroL, TrpE, AntABC, CatA, GlnA, TrpDΔ AroD, AroZ, AroY, CatA, ShiA, YdiBΔ, AroEΔ
AroZ, AroY, CatA, ShiA, YdiBΔ, AroEΔ
Pseudomonas putida KT2440 E. coli E. coli E. coli E. coli
E. coli
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2020.100395. AroZ, AroZ, CatA, TKL1, ZWF1, Aro1AroEΔ, Aro4K229L, CRISPR-Cas9 and RNAi AroZ, AroZ, CatA, XylA, PPP, Aro1AroEΔ, Aro4K229L, adaptive laboratory evolution (ALE)
S. cerevisiae S. cerevisiae
Funding
S. cerevisiae S. cerevisiae
Glucose Glucose
Batch, shake flask Fed-batch, bioreactor Batch, shake flask Batch, shake flask Fed-batch, bioreactor Fed-batch, bioreactor Batch, shake flask Batch, shake flask Batch, shake flask Batch, shake flask TKL1, Aro1AroEΔ, AroZ, AroY, CatA, EMS mutation, adaptive laboratory evolution (ALE) and aromatic amino acid (AAA) biosensor TKL1, Aro4K229L, AroZ, AroY, CatA, Aro1Δ, ZWF1Δ AroZ, AroZ, CatA, Pad1, Aro1 degradation AroZ, AroZ, CatA, Pad1, Hqd2 Aro1 degradation, S. cerevisiae
both glucose and xylose. In addition, it generated 31.3 g/L ethanol and 53.4 mg/L CCM from OPEFB hydrolysate. This study demonstrated that AroZ Neurospora crassa, AroY from Commensalibacter intestini A911, and CatA from Cupriavidus necator were effective in CCM biosynthesis in S. cerevisiae. Strain Ic0.09-31 has high potential in cellulosic ethanol and CCM co-production using lignocellulosic biomass hydrolysate.
Batch, shake flask Fed-batch, bioreactor Fed-batch, bioreactor Batch, shake flask Batch, shake flask Fed-batch, bioreactor, coculture Batch, bioreactor, co-culture
Kildegaard et al. (2019) This study
Suástegui et al., 2017 Pyne et al., 2018
Leavitt et al., 2017
Weber et al. (2012) Curran et al. (2013) Horwitz et al., 2015
1.56 mg/L 141 mg/L 0 ~1 g/L 0.5 g/L 2.1 g/L 0.32 g/L 0.86 g/L 1.2 g/L 5.1 g/L 800 mg/L 65 mg/L 424 mg/L 1.286 g/L Batch, shake flask Batch, shake flask Batch, 96-well shake plate AroZ, AroZ and CatA, Aro1AroEΔ TKL1, AroZ, AroZ, CatA, Aro4K229L ZWF1Δ, Aro3Δ, Aro4Δ AroZ, AroZ, CatA, AroF, AroB, AroD, Aro1Δ, CRISPR-Cas9 S. cerevisiae S. cerevisiae S. cerevisiae
Glucose Glucose Glucose Glucose with 1 g/L Catechol Glucose
Detailed strategies Host strains
Table 2 Comparison of CCM production by engineered microorganisms.
Carbon source
Fermentation
CCM titer
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