Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering

Metabolic Engineering 56 (2019) 17–27

Contents lists available at ScienceDirect

Metabolic Engineering journal homepage: www.elsevier.com/locate/meteng

Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering

T

Takahiro Bambaa, Takahiro Yukawaa, Gregory Guirimanda,b,1, Kentaro Inokumaa, Kengo Sasakia,b, Tomohisa Hasunumaa,b,∗∗, Akihiko Kondoa,b,c,∗ a

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan c Biomass Engineering Program, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Yeast cell factory Biomass utilization Metabolic engineering xylonate dehydratase 2-Ketoacid decarboxylase Fe–S cluster

1,2,4-Butanetriol can be used to produce energetic plasticizer as well as several pharmaceutical compounds. Although Saccharomyces cerevisiae has some attractive characters such as high robustness for industrial production of useful chemicals by fermentation, 1,2,4-butanetriol production by S. cerevisiae has not been reported. 1,2,4-butanteriotl is produced by an oxidative xylose metabolic pathway completely different from the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways conventionally used for xylose assimilation in S. cerevisiae. In the present study, S. cerevisiae was engineered to produce 1,2,4-butanetriol by overexpression of xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2-ketoacid decarboxylase. Further improvement of the recombinant strain was performed by the screening of optimal 2-ketoacid decarboxylase suitable for 1,2,4-butanetriol production and the enhancement of Fe uptake ability to improve the XylD enzymatic activity. Eventually, 1.7 g/L of 1,2,4-butanetriol was produced from 10 g/L xylose with a molar yield of 24.5%. Furthermore, 1.1 g/L of 1,2,4-butanetriol was successfully produced by direct fermentation of rice straw hydrolysate.

1. Introduction Lignocellulosic materials represent an abundant, inexpensive, and renewable resource with huge potential in the prospects of bioeconomy. Therefore, bio-production of fuels and commodity chemicals from lignocellulosic materials offers economically attractive and sustainable alternatives to their petroleum-based production (Hasunuma et al., 2015; Lane et al., 2018; Guirimand et al., 2016, 2019). Among a number of bio-based chemicals, 1,2,4-butanetriol is a particularly interesting target, as it is the precursor of 1,2,4-butanetriol trinitrate (BTTN) used for propellants and explosives, as well as a major building block for the synthesis of a number of drugs and other useful chemicals. BTTN has superior properties such as lower sensitivity to shock, higher thermal stability, and is less volatile than energetic plasticizer, nitroglycerin (Gouranlou and Kohsary, 2010). 1,2,4-Butanetriol is currently produced by chemical reduction of malic acid using

NaBH4 (Monteith et al., 1998), however, this chemical production process generates a large amount of borate salts as a disposal byproduct and is very polluting. Although other catalysts including copper chromite and rubidium are available for 1,2,4-butanetriol production (Niu et al., 2003), catalytic reduction demands harsh condition such as high pressure and high temperature. In addition, byproducts are inevitably generated, which reduces 1,2,4-butanetriol yield (Niu et al., 2003). Recently, microbial 1,2,4-butanetriol synthesis attracted attention for a cost-effective and safety approach. Although no natural microorganism able to produce 1,2,4-butanetriol has been described until now, the overexpression of heterologous xylose dehydrogenase, xylonolactonase, xylonate dehydratase, 2-ketoacid decarboxylase and alcohol dehydrogenase in Escherichia coli enabled 1,2,4-butanetriol production from xylose (Cao et al., 2015; Lu et al., 2016; Sun et al., 2016; Valdehuesa et al., 2014; Wang et al., 2018; Jing et al., 2018). However, E. coli is not highly resistant to fermentation inhibitors

∗

Corresponding author. Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. Corresponding author. Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. E-mail addresses: [email protected] (T. Bamba), [email protected] (T. Yukawa), [email protected] (G. Guirimand), [email protected] (K. Inokuma), [email protected] (K. Sasaki), [email protected] (T. Hasunuma), [email protected] (A. Kondo). 1 present address: Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, 37200 Tours, France. ∗∗

https://doi.org/10.1016/j.ymben.2019.08.012 Received 26 April 2019; Received in revised form 24 July 2019; Accepted 17 August 2019 Available online 18 August 2019 1096-7176/ © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

such as weak acids and furan derivatives generated by the pretreatment of lignocellulosic materials (Olsson and Hahn-Hägerdal, 1993). Wang et al. (2018) reported recombinant strains of E. coli able to produce 1,2,4-butanetriol from xylose, but these strains could not produce any 1,2,4-butanetriol by fermentation of corncob hydrolysate, due to the presence of fermentation inhibitors in the medium. In the present study, we used Saccharomyces cerevisiae as a host organism, in order to generate a microbial cell factory capable to produce 1,2,4-butanetriol from biomass and in the presence of fermentation inhibitors. Indeed, S. cerevisiae has attractive characters such as high robustness and stress tolerances, compatible with industrial applications such as bio-production of a variety of compounds (reviewed in Hong and Nielsen, 2012). Furthermore, a lot of fundamental studies on S. cerevisiae have been performed as a model of eukaryote, and a complete sequence of the genome is available, which is very helpful in solving problems in the production of useful chemicals by using this host microorganism for our system. On the other hand, as there is no report of 1,2,4-butanetriol production in recombinant S. cerevisiae strains, the choice of optimal enzymes candidates for the construction of 1,2,4-butanetriol biosynthesis required careful attention. Especially, selection of optimal 2-ketoacid decarboxylase suitable for the conversion of 2-keto-3-deoxy xylonate was indispensable for 1,2,4-butanetriol production. In addition, the functional expression of prokaryotic xylonate dehydratase containing iron-sulfur (Fe–S) cluster is regarded as a major key point to achieve the production of 1,2,4-butanetriol in yeast cells. Especially, it is known that bacterial Fe–S protein is not sufficiently formed in the cytosol of S. cerevisiae (Benisch and Boles, 2014; Carlsen et al., 2013; Partow et al., 2012), meanwhile iron metabolism is tightly regulated as this element is indispensable to the life of cells but has also potential toxicity when present in too high concentration (Martinez-Pastor et al., 2017; Encinar et al., 2015; Kaplan and Kaplan, 2009; Haussman et al., 2008; Courel et al., 2005). In the present study, Fe metabolism was modified in order to improve iron uptake and coupling of Fe–S cluster to proteins in order to ensure the formation of functional Fe–S enzymes. Heterologous overexpression 1,2,4-butanetriol biosynthesis pathway and improvement of xylonate dehydratase activity were conducted and the fermentation of rice straw hydrolysate was performed using the engineered yeast strain to produce 1,2,4-butanetriol from biomass.

Table 1 Plasmids used in this study. Plasmid

description

reference

pATP405 pIBG-SS

empty vector, Leu2 marker PCR template to amplify the PSED1 and TSAG1 empty vector, ADE2 marker PTDH3-xylB, PSED1-xylD, ADE2 marker PTDH3-xylB, PSED1-yjhG, ADE2 marker δ-integration vector, TRP1 marker δ-integration plasmid, PADH1-xylD empty vector, Leu2 marker PPGK1-mdlC, Leu2 maker PPGK1-ARO10, Leu2 maker PPGK1-kivD, Leu2 maker PPGK1-kdcA, Leu2 maker empty vector, PTDH3, LEU2 marker empty vector, PTDH3, URA3marker PTDH3-kivD, Leu2 maker PTDH3-kdcA, Leu2 maker empty vector, URA3 marker PTDH3-kivD, URA3 maker PTDH3-kdcA, URA3 maker empty vector, AUR1-C marker empty vector, PPGK1, AUR1-C marker PPGK1-CFD1, AUR1-C maker PPGK1-CIA2, AUR1-C maker PPGK1-TYW1, AUR1-C maker PPGK1-tTYW1, AUR1-C maker

Ishii et al. (2014) Inokuma et al. (2014) Ishii et al. (2009) This study

pGK402 pTS-A-xylBD pTS-A-xylByjhG pδW pδW-xylD pGK405 pIL-mdlC pIL-ARO10 pIL-kivD pIL-kdcA pIL pTDH3-tADH1 pIU pTDH3-tADH1 pIL-pTDH3-kivD pIL-pTDH3-kdcA pGK406 pIU-pTDH3-kivD pIU-pTDH3-kdcA pAUR101 pGK Aur pIAur-CFD1 pIAur-CIA2 pIAur-TYW1 pIAur-tTYW1

This study Yamada et al. (2010) This study Ishii et al. (2009) This study This study This study This study This study This study This study This study Ishii et al. (2009) This study This study Takara Bio This study This This This This

study study study study

USA) according to the manufacturer's instruction. C. crescentus xylB and xylD, P. putida mdlC, and L. lactis kivD were codon optimized for S. cerevisiae and synthesized by GenScript (Piscataway, NJ, USA). E. coli yjhG and L. lactis kdcA were codon optimized for S. cerevisiae and synthesized by Geneart (Thermo Fisher Scientific, Waltham, MA, USA). The xylB and xylD integrative plasmid (pTS-A-xylBD) was constructed as follows: the DNA fragment encoding xylB and xylD were amplified from synthetic xylB and xylD by polymerase chain reaction (PCR) using the xylB F and xylB R, and xylD F and xylD R primers. The xylB fragment was cloned into NotI digested pATP405 vector (Ishii et al., 2014) to connect TDH3 promoter (pTDH3) and TDH3 terminator (tTDH3). Then, pTDH3-xylB-tTDH3 fragment was amplified using the pTDH3-xylB F and pTDH3-xylB R primers by PCR. The xylD fragment was cloned into vector fragment which were amplified from pIBG-SS (Inokuma et al., 2014) by PCR using the SED1p-SAG1t F and SED1pSAG1t R, to connect SED1 promoter (pSED1) and SAG1 terminator (tSAG1). Then, pSED1-xylD-tSAG1 fragment was amplified using the pSED1-xylD F and pSED1-xylD R primers by PCR. pTDH3-xylB-tTDH3 and pSED1-xylD-tSAG1 fragments were simultaneously cloned into NotI and XhoI digested pGK402 vector (Ishii et al., 2009) to generate plasmid pTS-A-xylBD. The xylD δ-integration plasmid (pδW-xylD) was constructed as follows: the DNA fragment encoding xylD was amplified from synthetic xylD by PCR using the xylD 2F and xylD 2R primers. The xylD fragment was cloned into PmeI digested pATP405 vector to connect ADH1 promoter (pADH1) and ADH1 terminator (tADH1). Then, pADH1-xylDtADH1 fragment was amplified using the pADH1-xylD F and pADH1xylD R primers by PCR, and cloned into SmaI digested pδW vector (Yamada et al., 2010) to generate plasmid pδW-xylD. The integrative plasmids (pIL-mdlC, pIL-ARO10, pIL-kivD, and pILkdcA) for expression of 2-ketoacid decarboxylase under the PGK1 promoter were constructed as follows; the DNA fragment encoding mdlC, kivD, and kdcA were amplified from synthetic mdlC, kivD, and kdcA by PCR using the mdlC F and mdlC R, pgk1 kivD F and pgk1 kivD R, and pgk1 kdcA F and pgk1 kdcA R primers. The DNA fragment encoding ARO10 was amplified from S. cerevisiae YPH499 genome DNA

2. Materials and methods 2.1. Strains and media The NovaBlue strain of E. coli (Merck Millipore, Darmstadt, Germany) was used for plasmids construction and amplification. NovaBlue was routinely cultivated in LB medium (10 g/L tryptone, 5 g/ L yeast extract, and 5 g/L NaCl) containing 100 μg/mL ampicillin at 37 ΊC. YPH499 strain of S. cerevisiae [MATa ura3-52 lys2-801 ade2-101 trp1- 63 his3-Δ200 leu2-Δ1 (Stratagene, La Jolla, CA, USA)] was used as a yeast host strain. Selection of yeast transformants and pre-cultivation was conducted in synthetic dextrose (SD) medium [6.7 g/L of yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA) and 20 g/L of glucose] supplemented with appropriate amino acids and nucleic acids in a shaker incubator at 180 rpm for 24 h at 30 °C. The precultured cells were then inoculated into yeast extract peptone dextrose (YPD) medium [10 g/L yeast extract, 20 g/L Bactopeptone (Difco Laboratories), and 20 g/L glucose]. 2.2. Plasmids construction Table 1 and S1 show the genetic characteristics of all plasmids and primers used in this study, respectively. All plasmids were constructed by using In-fusion HD cloning Kit (Takara Bio USA, Mountain View, CA, 18

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

by PCR using the ARO10 F and ARO10 R primers. The mdlC, ARO10, kivD and kdcA fragment was cloned into smaI digested pGK405 (Ishii et al., 2009) vector to generate plasmids pIL-mdlC, pIL-ARO10, pILkivD, and pIL-kdcA. The kivD with TDH3 promoter integrative plasmids (pIL-pTDH3kivD and pIU-pTDH3-kivD) was constructed as follows; TDH3 promoter and ADH1 terminator fragment was amplified from S. cerevisiae YPH499 genome DNA by PCR using the tdh3p F and tdh3p R, and adh1t F and adh1t R. The two fragments were joined by overlap extension PCR using tdh3p F and adh1t R primers, and cloned into NotI and XhoI digested pGK405 and pGK406 (Ishii et al., 2009) vectors to generate plasmids pIL pTDH3-tADH1 and pIU pTDH3-tADH1. The DNA fragment encoding kivD were amplified from synthetic kivD by PCR using the tdh3 kivD F and tdh3 kivD R primers and cloned into smaI digested pILpTDH3-tADH1 and pIU-pTDH3-tADH1 vectors to generate plasmids pIL-pTDH3-kivD and pIU-pTDH3-kivD. The kdcA with TDH3 promoter integrative plasmids (pIL-pTDH3kdcA and pIU-pTDH3-kdcA) was constructed as follows; the DNA fragment encoding kdcA was amplified from synthetic kdcA by PCR using the tdh3 kdcA F and tdh3 kdcA R primers, and cloned into smaI digested pIL-pTDH3-tADH1 and pIU-pTDH3-tADH1 vectors to generate plasmids pIL-pTDH3-kdcA and pIU-pTDH3-kdcA. The integrative plasmids (pIAur-CFD1, pIAur-CIA2, pIAur-TYW1, and pIAur-tTYW1) for expression of iron metabolism genes under the PGK1 promoter were constructed as follows; PGK1 promoter-multicloning sites-PGK1 terminator fragment was amplified from pGK406 (Ishii et al., 2009) vector by PCR using the AUR-pgk F and AUR-pgk R. The fragment was cloned into SphI and SacI digested pAUR101 (Takara Bio, Shiga, Japan) vector to generate plasmid pGK Aur. the DNA fragment encoding CFD1, CIA2, TYW1, and tTYW1 were amplified from S. cerevisiae YPH499 genome DNA by PCR using the CFD1 F and CFD1 R, CIA2 F and CIA2 R, TYW1 F and TYW1 R, and TYW1 F and tTYW1 R primers and cloned into smaI digested pGK Aur vector to generate plasmids pIAur-CFD1, pIAur-CIA2, pIAur-TYW1, and pIAur-tTYW1.

Table 2 Strains of S. cerevisiae used in this study. Strains

description

YPH499

MATa ura3-52 lys2-801 ade2-101 trp1- 63 his3Δ200 leu2-Δ1 YPH499, gre3Δ::kanMX4 YPH499ΔGRE3, pTS-A-xylBD YPH499ΔGRE3, pTS-A-xylByjhG BD, pδW-xylD BDδD, pIL-mdlC BDδD, pIL-ARO10 BDδD, pIL-kivD BDδD, pIL-kdcA BDδD, pIL-tkivD BDδD-tkivD, pIU-tkivD BDδD, pIL-tkdcA BDδD-tkdcA, pIU-tkdcA BDδD-2tkdcA, grx3Δ::HIS3 BDδD-2tkdcA, bol2Δ::HIS3 BDδD-2tkdcA, yap5Δ::HIS3 BDδD-2tkdcA, tyw1Δ::HIS3 BDδD-2tkdcA, pIAur-CFD1 BDδD-2tkdcA, pIAur-CIA2 BDδD-2tkdcA, pIAur-TYW1 BDδD-2tkdcA, pIAur-tTYW1 BDδD-2tkdcA, bol2Δ::HIS3, pIAur-CFD1 BDδD-2tkdcA, bol2Δ::HIS3, pIAur-CIA2 BDδD-2tkdcA, bol2Δ::HIS3, pIAur-tTYW1 BDδD-2tkdcA, bol2Δ::HIS3, yap5Δ::AUR1-C

YPH499ΔGRE3 BD BG BDδD BDδD-mdlC BDδD-ARO10 BDδD-kivD BDδD-kdcA BDδD-tkivD BDδD-2tkivD BDδD-tkdcA BDδD-2tkdcA BDδD-2tkdcA-ΔGRX3 BDδD-2tkdcA-ΔBOL2 BDδD-2tkdcA-ΔYAP5 BDδD-2tkdcA-ΔTYW1 BDδD-2tkdcA-CFD1 BDδD-2tkdcA-CIA2 BDδD-2tkdcA-TYW1 BDδD-2tkdcA-tTYW1 BDδD-2tkdcA-ΔBOL2-CFD1 BDδD-2tkdcA-ΔBOL2-CIA2 BDδD-2tkdcA-ΔBOL2-tTYW1 BDδD-2tkdcA-ΔBOL2-ΔYAP5

All strains except YPH499 (Stratagene) were constructed in this study.

homologous recombination to yield BDδD-2tkivD and BDδD-2tkdcA strains, respectively. GRX3, BOL2, YAP5 and TYW1 were disrupted by replacing with the HIS3 in BDδD-2tkdcA strain. The HIS3 fragment was amplified from the pGK403 plasmid (Ishii et al., 2009) by PCR using the primers HIS3 F and HIS3 R. To amplify 500 bp upstream of GRX3, BOL2, YAP5 and TYW1 ORF, PCR was performed using YPH499 genomic DNA as a template and primers dGRX3 up F and dGRX3 up R, dBOL2 up F and dBOL2 up R, dYAP5 up F and dYAP5 up R, and dTYW1 up F and dTYW1 up R, respectively. To amplify 500 bp downstream of GRX3, BOL2, YAP5 and TYW1 ORF, PCR was performed using YPH499 genomic DNA as a template and primers dGRX3 down F and dGRX3 down R, dBOL2 down F and dBOL2 down R, dYAP5 down F and dYAP5 down R, and dTYW1 down F and dTYW1 down R, respectively. To generate a fragment for GRX3, BOL2, YAP5 and TYW1 disruption, the three fragments (HIS3, ORF upstream 500 bp, ORF downstream 500 bp) were joined by overlap extension PCR using dGRX3 up F and dGRX3 down R, dBOL2 up F and dBOL2 down R, dYAP5 up F and dYAP5 down R, and dTYW1 up F and dTYW1 down R primers. The fragment for GRX3, BOL2, YAP5, and TYW1 disruption were transformed into BDδD2tkdcA strain to generate BDδD-2tkdcA-ΔGRX3, BDδD-2tkdcA-ΔBOL2, BDδD-2tkdcA-ΔYAP5, and BDδD-2tkdcA-ΔTYW1, respectively. pIAurCFD1, pIAur-CIA2, pIAur-TYW1, and pIAur-tTYW1 were digested with BsiWI within the AUR1-C. The linearized plasmids were then transformed into BDδD-2tkdcA, and integrated into the AUR1 locus of the chromosomal DNA by homologous recombination to yield BDδD2tkdcA-CFD1, BDδD-2tkdcA-CIA2, BDδD-2tkdcA-TYW1, and BDδD2tkdcA-tTYW1 strains, respectively. The linearized pIAur-CFD1, pIAurCIA2, pIAur-tTYW1 were also transformed into BDδD-2tkdcA-ΔBOL2, and integrated into the AUR1 locus of the chromosomal DNA by homologous recombination to yield BDδD-2tkdcA-ΔBOL2-CFD1, BDδD2tkdcA-ΔBOL2-CIA2, and BDδD-2tkdcA-ΔBOL2-tTYW1 strains, respectively. YAP5 was disrupted by replacing with the AUR1-C in BDδD2tkdcA-ΔBOL2 strain. The AUR1-C fragment was amplified from the pAUR101 plasmid (Takara Bio) by PCR using the primers AUR1-C F and AUR1-C R. To amplify 500 bp upstream and 500 bp downstream of YAP5 ORF, PCR was performed using YPH499 genomic DNA as a template and primers dYAP5 up F and dYAP5-Aur up R, and dYAP5-Aur

2.3. Yeast transformation The yeast transformation was conducted by the lithium acetate method (Chen et al., 1992). All yeast strains used in this study are summarized in Table 2. YPH499ΔGRE3 was constructed by replacing GRE3 with the G418 resistance gene (KanMX) in YPH499 strain of S. cerevisiae. The KanMX fragment for GRE3 disruption was amplified from the genomic DNA of S. cerevisiae BY4741ΔGRE3 obtained from the Yeast Deletion Mat-A Complete Set (Thermo Fisher Scientific, Waltham, Mass., USA) by PCR using the primers dGRE3 F and dGRE3 R. pTS-A-xylBD and pTS-AxylByjhG were digested with EcoRV within the ADE2. The linearized plasmids were then transformed into YPH499ΔGRE3, and integrated into the ADE2 locus of the chromosomal DNA by homologous recombination to yield BD and BG strains, respectively. In order to integrate multiple copies of xylD, AscI digested pδW-xylD was transformed into BD strain by homologous recombination to yield BDδD. pIL-ARO10, pIL-kivD, and pIL-kdcA were digested with EcoRV within the LEU2. pIL-mdlC was linearized by PCR using the primers PIL trans F and PIL trans R, as there was no suitable restriction enzyme to cut within the only LEU2 marker. The linearized plasmids were then transformed into BDδD, and integrated into the LEU2 locus of the chromosomal DNA by homologous recombination to yield BDδD-mdlC, BDδD-ARO10, BDδD-kivD and BDδD-kdcA strains. And also, pIL-tkivD and pIL-tkdcA were digested with EcoRV within the LEU2. The linearized plasmids were then transformed into BDδD, and integrated into the LEU2 locus of the chromosomal DNA by homologous recombination to yield BDδD-tkivD and BDδD-tkdcA strains, respectively. pIU-tkivD and pIU-tkdcA were digested with EcoRV within the URA3. The linearized plasmids were then transformed into BDδD-tkivD and BDδD-tkdcA, and integrated into the URA3 locus of the chromosomal DNA by 19

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

centrifuged at 3000 g for 5 min at room temperature, and 120 μL of the supernatant was transferred to a 150 μL glass insert, and analyzed using GC-MS (GCMS-QP 2010 Ultra; Shimadzu, Kyoto, Japan) equipped with a CP-Sil 8CB column (30 m length × 0.25 mm i.d., film thickness of 0.25 μm; Agilent Technologies, Palo Alto, CA, USA). The injection port was maintained at 230 °C. The injection volume was 1 μL and the split ratio was 1:25. Helium was used as the carrier gas, and the flow rate was held constant at 1.12 mL/min. The column temperature was held at 80 °C for 2 min, then raised by 15 °C/min to 330 °C, and held there for 6 min. Interface and ion source temperature was maintained at 250 °C and 200 °C, respectively. Electron impact ionization was conducted at 70 eV, and the mass spectra were recorded by scanning the range 100–650 m/z. The analysis was simultaneously performed in the scan mode (from 85 to 500 m/z) and a selected ion monitoring (SIM) mode (m/z 103, and 129 for 1,2,4-butanetriol, and m/z 103 for ribitol) by Shimadzu Fast Automated Scan/SIM (FASTT) mode.

down F and dYAP5 down R, respectively. To generate a fragment for YAP5 disruption, the three fragments (AUR1-C, ORF upstream 500 bp, ORF downstream 500 bp) were joined by overlap extension PCR using d dYAP5-Aur up F and dYAP5-Aur down R primers. The fragment for YAP5 disruption by AUR1-C marker was transformed into BDδD2tkdcA-ΔBOL2 strain to generate BDδD-2tkdcA-ΔBOL2-ΔYAP5, respectively. 2.4. Quantification of integrated copy numbers of xylD by real-time PCR The integrated copy number of the xylD in BDδD was quantified using real-time PCR. Template genome DNA was isolated from BD and BDδD strain cells cultivated in SD medium for 24 h at 30 °C using a Dr. GenTLE (yeast) high-recovery kit (Takara Bio). The PCR primers, qXylD F and qXylD R, and qAct1 F and qAct1 R were used to detect the xylD and ACT1 respectively. Quantitative real-time PCR was performed using an Mx3000P QPCR System (Agilent Technologies) with Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan). The normalized gene copy number was calculated by a 2−ΔΔCT method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008) using the housekeeping gene, ACT1.

2.7. Compositional analysis of rice straw hydrolysate In the liquid fraction of rice straw hydrolysate, acetic and formic acids were measured by GC–MS equipped with a DB-FFAP column (60 m length × 0.25 mm i.d., a film thickness of 0.50 μm; Agilent Technologies) as reported previously (Sakamoto et al., 2012). Furfural, vanillin, 5-hydroxymethylfurfural (5-HMF), and syringaldehyde were measured by GC–MS equipped with a CP-SIL 8 CB column (Agilent Technologies) as reported previously (Sakamoto et al., 2012). To determine the total amount of glucose and xylose, hydrolysis of polysaccharide in the liquid fraction of rice straw hydrolysate was performed with sulfuric acid according to the National Renewable Energy Laboratory (NREL) method (Sluiter et al., 2012). The glucose and xylose concentration was analyzed using the HPLC (Shimadzu) equipped with an Aminex HPX-87H column (Bio-Rad) as described in the Metabolite analysis section.

2.5. Fermentation method Precultured yeast cells were inoculated in 50 mL YPD medium at OD600 = 0.1 and cultivated at 30 °C, 150 rpm. After 24 h cultivation, yeast cells were collected by centrifugation at 2200g for 5 min at room temperature and washed twice with 10 mL distilled water. The cells were then inoculated (OD600 = 5) in fermentation medium [10 g/L yeast extract, 20 g/L Bactopeptone (Difco Laboratories), 10 g/L glucose and 10 g/L xylose] and total volume was adjusted to 20 mL in 200 mLbaffled Erlenmeyer Flask equipped with cap type plug (30 °C, 200 rpm). For the fermentation of rice straw hydrolysate, yeast cells were prepared following the same procedure. Liquid hot water treatment of rice straw hydrolysate at high temperature (130–300 °C) and high pressure (≤10 MPa) was purchased from Mitsubishi Heavy Industries, Ltd. (Tokyo, Japan). The liquid fraction of hydrolysate was separated from solid cellulose enriched fraction by centrifugation at 15,000g for 10 min at 4 °C. The obtained liquid fraction used in the fermentation broth was either undiluted or 2-fold diluted by distilled water. The cells were then inoculated (OD600 = 5) in the liquid fraction of hydrolysate supplemented with 10 g/L yeast extract and 20 g/L Bactopeptone as fermentation medium. 2% (w/v) mixture of hemicellulases G-Amano (Amano Enzyme, Nagoya, Japan) was added in hydrolysate just before fermentation. The fermentation was performed at 30 °C with an agitation speed of 200 rpm in 200 mL-baffled Erlenmeyer Flask.

2.8. Enzymes activity assay The total proteins from yeast cells collected from 5 mL YPD medium after 24 h cultivation, were prepared as previously described (Bamba et al., 2016). For the excess iron ion condition, 2 mM ammonium ferric citrate was added to YPD medium just before cultivation. Xylonate dehydratase (XylD) activity was measured using the thiobarbituric acid method (Kim and Lee, 2005). The assay mixture (400 μL) containing 50 mM Tris–HCl buffer (pH8.1), 5 mM MgCl2, 12 mM D-xylonate, and 20 μL of total protein solution was incubated at 30 °C for 10 min. The enzymatic reaction was terminated by adding 100 μl of 2.0 M HCl. The reaction mixture (50 μl) was oxidized by addition of 125 μl of 25 mM periodic acid in 0.125 M H2SO4 at 20 °C for 20 min. Oxidation was finished by the addition of 250 μl of 2% (w/v) sodium arsenite dissolved in 0.5 M HCl. Finally, 1 mL of 0.3% thiobarbituric acid was added and the red chromophore developed by heating at 100 °C for 10 min. After cooling to room temperature, an equal volume of DMSO was added in the reaction mixture to intensify the colour. The absorbance was measured at 549 nm, and the molar absorption coefficient of the chromophore was 6.78 × 104 M−1 cm−1 (Skoza and Mohos, 1976).

2.6. Metabolite analysis The glucose and xylose concentrations in the fermentation medium were analyzed using high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) equipped with a Aminex HPX-87H column (7.8 mm × 300 mm, particle size 9 μm; Bio-Rad, Hercules, CA, USA) and an RID-10A refractive index detector (Shimadzu). The HPLC system was operated at 65 °C, with 5mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min. The 1,2,4-butanetriol and xylonate concentration was analyzed using gas chromatography-mass spectrometry (GC-MS). The 5 μL fermentation supernatants with 2 μL of 10 g/L ribitol as internal standard were dried using the CentriVap Benchtop Vacuum Concentrators (Labconco, Kansas City, MO, USA). Derivatization of samples were conducted during 90 min in a shaker incubator (1200 rpm at 30 °C; MBR-022UP; Taitec, Saitama, Japan) with 100 μL of 20 mg/mL methoxyamine hydrochloride solution in pyridine, followed by 30 min reaction in a shaker incubator (1200 rpm at 37 °C; M-BR-022UP; Taitec) with 50 μL of N-methyl-n-TMS-trifluoroacetamide (MSTFA). Samples were

2.9. Quantification of transcript level by real-time PCR kivD and kdcA transcript levels were quantified by real-time quantitative PCR. Template total RNA was isolated from BDδD-kivD, BDδDtkivD, BDδD-kdcA, and BDδD-tkdA strain cells after 24 h fermentation using a NucleoSpin RNA (MACHEREY-NAGEL, Düren, Germany). Quantitative PCR was performed using an Mx3000P QPCR System (Agilent Technologies) with a KOD SYBR qPCR Mix (Toyobo). The PCR primers kivDq F and kivDq R, kdcAq F and kdcAq R, and TUB2q F and TUB2q R were used to quantify the transcript levels of kivD, kdcA, and TUB2, respectively. The normalized expression level was calculated by 20

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

a 2−ΔΔCT method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008) using the house-keeping gene, TUB2.

3. Results 3.1. Construction of a 1,2,4-butanetriol biosynthetic pathway in S. cerevisiae 1,2,4-Butanetriol can be biosynthesized from xylose by the introduction of several heterologous genes into S. cerevisiae (Fig. 1). First, xylose is converted to xylonolactone by C. crescentus xylose dehydrogenase (XylB) (Toivari et al., 2012). After the conversion of xylonolactone to xylonate by the spontaneous reaction, 2-keto-3-deoxy-Dxylonate (KDX) can be generated from xylonate by heterologous xylonate dehydratase. After the decarboxylation of KDX by 2-ketoacid decarboxylase, 3,4-dihydroxybutanal (DHB) is converted to 1,2,4-butanetriol by yeast endogenous alcohol dehydrogenase. In S. cerevisiae, xylose is converted to xylitol by endogenous nonspecific aldose reductase encoded by the GRE3 gene. In order to avoid the competition with xylose dehydrogenase, GRE3 was disrupted by replacement with the KanMX cassette in S. cerevisiae YPH499 to yield the recombinant strain YPH499ΔGRE3 (Table 2). Two cassette vectors containing C. crescentus xylB and xylonate dehydratase genes, xylD or yjhG, were constructed (Fig. 1; Table 2). For the expression of xylonate dehydratase gene, C. crescentus xylonate dehydratase (xylD) and E. coli xylonate dehydratase (yjhG) used in 1,2,4-butanetriol production in E. coli and Arabidopsis thaliana (AbdelGhany et al., 2013; Cao et al., 2015; Sun et al., 2016) were subcloned to yield plasmids pTS-A-xylBD and pTS-A-xylByjhG, respectively after the optimization of codon usage. YPH499ΔGRE3 was transformed with pTS-A-xylBD and pTS-A-xylByjhG to yield recombinant strains BD and BG, respectively. Fermentation was performed using BD and BG recombinant strains in a fermentation medium containing 10 g/L glucose and 10 g/L xylose. Glucose was totally consumed after 24 h by both strains (data not shown). In the present study, xylose was converted to xylonolactone by C. crescentus XylB recombinant enzyme. In previous study, Toivari et al. (2012) found that C. crescentus XylB recombinant strain had high xylose consumption ability. Our strains BD and BG were able to consume approximately 9 g/L of xylose in 96 h of fermentation (Fig. 2A). The BD strain demonstrated 0.6-fold lower intermediate xylonate secretion compared to the BG strain (Fig. 2B). On the other hand, 1,2,4-butanetriol production was observed only in the BD strain (Fig. 2C). 1,2,4butanetriol was found to be produced by C. crescentus XylB and XylD. However, 72% of consumed xylose was converted to xylonate in BD strain. In order to decrease the accumulation of xylonate as a byproduct and to increase 1,2,4-butanetriol production ability, additional copies of C. crescentus xylD gene were integrated into the genome by using δintegration method (Parekh et al., 1996). The xylD gene was integrated into the δ-sequence (Eichinger and Boeke, 1988) of the BD strain to construct the recombinant strain BDδD harboring three copies of xylD in its genome (Fig. 2D). The XylD enzymatic activity measured in the BDδD recombinant strain was 1.3-fold higher than in BD recombinant strain (Fig. 2E). The recombinant strain BDδD demonstrated more than 2.1-fold higher 1,2,4-butanetriol production ability (43.6 ± 2.9 mg/L) compared to the recombinant strain BD (Fig. 2C), while on the other hand, xylonate accumulation was almost the same between these two strains (Fig. 2B). The difference of 1,2,4-butanetriol amount of BD and BDδD was very small compared to the accumulated amount of xylonate. Therefore, the accumulated amount of xylonate was considered to have hardly changed. BD and BDδD indicated similar growth (Fig. S1). While, BG strain reached higher cell amount (OD600) than xylD integration strains. This data indicated that KDX or other compounds derived from KDX inhibit the yeast growth.

Fig. 1. Biosynthetic pathway of 1,2,4-butanetriol from xylose. The enzymes integrated into the genome of S. cerevisiae in this study are written in red. XylB; xylose dehydrogenase from C. crescentus, XylD; xylonate dehydratase from C. crescentus, YjhG; xylonate dehydratase from E. coli, MdlC; benzoylformate decarboxylase from P. putida, Aro10; phenylpyruvate decarboxylase from S. cerevisiae, KivD; 2-ketoisovalerate decarboxylase from L. lactis, KdcA; Branchedchain alpha-ketoacid decarboxylase from L. lactis.. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

21

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

Fig. 2. Xylose conversion to 1,2,4-butanetriol by recombinant strains of S. cerevisiae over-expressing xylose dehydrogenase (XylB) and xylonate dehydratase (XylD or yjhG). Time course monitoring of (A) xylose, (B) xylonate, and (C) 1,2,4-butanetriol concentrations during fermentation of YPDX medium (10 g/L glucose and 10 g/L xylose). (D) Determination of XylD gene copy number and (E) XylD enzymatic activity in single copy (BD) and multi-copy (BDδD) integration strains, respectively. BD = XylB/XylD over-expressing strain; BG = XylB/ yjhG over-expressing strain; BDδD = XylB/multicopy XylD over-expressing strain. Values represent averages ± standard deviation of the results from three independent experiments. * indicates significant difference at p-value < 0.01 as evaluated by paired comparisons using Student's t-test.

higher than BDδD strain, respectively. In our continuous effort to improve 1,2,4-butanetriol bio-production from xylose, we have investigated the importance of the promoter sequence controlling the overexpression of 2-ketoacid decarboxylase gene. According to Lu and Jeffries (2007), the TDH3 promoter is regarded as stronger than the PGK1 promoter in S. cerevisiae cells grown with xylose as a carbon source. In this context, we attempted to overexpress kivD and kdcA genes under the dependence of the TDH3 promoter. The resulting recombinant strains (TDH3 promoter; BDδD-tkivD and BDδD-tkdcA) exhibited higher 1,2,4-butanetriol production abilities (577.3 ± 3.6 mg/L and 722.0 ± 14.9 mg/L, respectively) compared to the previous strains (PGK1 promoter; BDδD-kivD and BDδDkdcA) (Fig. 3B). Moreover, one additional copy of each 2-ketoacid decarboxylase genes was integrated into each strain (BDδD-tkivD and BDδD-tkdcA, respectively) under the dependence of the TDH3 promoter, which increased 1,2,4-butanetriol production up to 767.9 ± 45.4 mg/L and 1151.4 ± 58.5 mg/L, respectively (Fig. 3B).

3.2. Optimal 2-ketoacid decarboxylase gene candidate for 1,2,4-butanetriol production So far, a 2-ketoacid decarboxylase suitable for the conversion of KDX to DHB has never been reported in S. cerevisiae. In the present study, Pseudomonas putida benzoylformate decarboxylase (MdlC) (Cao et al., 2015; Niu et al., 2003; Valdehuesa et al., 2014), endogenous phenylpyruvate decarboxylase (Aro10) (Kneen et al., 2011), Lactococcus lactis 2-ketoisovalerate decarboxylase (KivD) (De La Plaza et al., 2004; Tai et al., 2016) and L. lactis branched-chain 2-ketoacid decarboxylase (KdcA) (Milne et al., 2015; Wang et al., 2018) were integrated into the genome of BDδD strain under the dependence of the S. cerevisiae PGK1 constitutive promoter to yield recombinant strains BDδD-mdlC, BDδD-ARO10, BDδD-kivD, and BDδD-kdcA, respectively. As shown in Fig. 3A, BDδD-kivD and BDδD-kdcA exhibited the highest 1,2,4-butanetriol production abilities (384.3 ± 26.3 mg/L and 213.2 ± 15.8 mg/L, respectively), which is 8.9-fold and 4.9-fold

Fig. 3. Comparison of 1,2,4-butanetriol production ability of recombinant strains of S. cerevisiae overexpressing different 2-ketoacid decarboxylases, after 96 h of fermentation. (A) Expression of different 2ketoacid decarboxylases (mdlC, ARO10, kivD and kdcA) under the dependence of the PGK1 promoter in BDδD strain. (B) Expression of kivD and kdcA under the dependence of the TDH3 promoter, and integration of 2 copies of each of these genes in BDδD strain. Error bars indicate the standard deviations of three independent experiments. mdlC; benzoylformate decarboxylase from P. putida, ARO10; phenylpyruvate decarboxylase from S. cerevisiae, kivD; 2-ketoisovalerate decarboxylase from L. lactis, kdcA; Branched-chain alpha-ketoacid decarboxylase from L. lactis. 22

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

but not least, in the flask-scale fermentation, the recombinant strains obtained by overexpression of tTYW1 as well as disruption of GRX3 or BOL2 in the BDδD-2tkdcA produced up to 1731.3 ± 7.6 mg/L, 1576.0 ± 70.1 mg/L and 1688.7 ± 13.3 mg/L 1,2,4-butanetriol, respectively (Fig. 4C). The effect of the combination of BOL2 deletion and other factors on XylD enzymatic activity was also investigated in the recombinant strain BDδD-2tkdcA. As shown in Fig. 4B, no further significant improvement of XylD enzymatic activity was observed by the additional CFD1 and CIA2 overexpression, or YAP5 deletion as well. The combination of BOL2 deletion and tTYW1 overexpression in the same recombinant strain demonstrated 2.3-fold higher XylD enzymatic activity compared to the strain obtained by BOL2 deletion only. The xylonate accumulation level was slightly lower in the recombinant strain combining BOL2 deletion and tTYW1 overexpression (Fig. 4C). Interestingly, although XylD enzymatic activity was improved in this strain, the final 1,2,4butanetriol production ability was not drastically increased, suggesting the potential accumulation of intermediates and/or byproducts.

The amount of accumulated xylonate was slightly decreased in TDH3p_kivD and TDH3p_kdcA recombinant strains (6.5 g/L) compared to BDδD (7.5 g/L), regardless to copy number and gene (kivD or kdcA) (Fig. S2). When PGK1 promoter was used for the expression of kivD and kdcA, the amount of 1,2,4-butanetriol produced was higher in the kivD integration strain than in the kdcA integration strain, whereas when TDH3 promoter was used, the production ability was higher in kdcA integration strain than kivD. In order to clarify this point, we investigated the expression levels of kivD and kdcA expressed under the dependence of the PGK1 and TDH3 promoter, respectively. The expression levels of kivD and kdcA were improved by 2.8 ± 1.0 and 2.0 ± 0.5 fold, respectively, when expressed by TDH3 promoter. (Fig. S3). 3.3. Engineering iron metabolism for the enhancement of XylD enzymatic activity C. crescents XylD belongs to IlvD/EDD family protein containing an iron-sulfur (Fe–S) cluster in its active centre (Andberg et al., 2016). Functional expression of prokaryotic Fe–S proteins has been so far difficult to achieve in S. cerevisiae (Benisch and Boles, 2014; Carlsen et al., 2013; Partow et al., 2012), conceivably due to the low ability of yeast enzymes to form Fe–S clusters. The precise mechanism of regulation of Fe–S cluster synthetic pathway in yeast cytosol remains unknown. However, recently, proteins involved in the synthesis and transport of Fe–S cluster in yeast cytosol have been gradually characterized. In the cytosol of S. cerevisiae, Cfd1 forms a complex with Nbp35 to act as a scaffold during Fe–S cluster formation (Netz et al., 2012; Pallesen et al., 2013) as shown in Fig. 4A. A cytosolic Fe–S cluster targeting complex consisting of Cia1, Cia2, and Met18 carries the Fe–S cluster to its apoprotein to form Fe–S protein (Lill, 2009; Vo et al., 2018). In this complex Cia2 was considered as the organizing centre. Tyw1 which encodes RNA 4-demethylwyosine synthase, involved in the synthesis of wybutosinemodified tRNA. Tyw1is considered to protects yeast cell from high-iron toxicity by binding with excess Fe–S clusters (Li et al., 2011). Aft1/Aft2 controls iron uptake as a transcription factor, which is negatively regulated by Bol2 (previously referred to as Fra2), Grx3 and Grx4 complex (Kumánovics et al., 2008; Courel et al., 2005; Kaplan and Kaplan, 2009). This complex has been considered to sensing the cytosolic iron level and transmit of signals to Aft1/Aft2 (Martínez;Pastor et al., 2017), however detail of mechanism is not revealed. Another iron-sensing transcription factor, Yap5, is involved in the sequestration of iron surplus in order to protect the cells against iron toxicity (Li et al., 2008; Encinar del Dedo et al., 2015; Martinez-Pastor et al., 2017). In the present study, overexpression of CFD1, CIA2 and TYW1 and deletion of BOL2, GRX3, YAP5 and TYW1 were performed in BDδD2tkdcA. As shown in Fig. 4B, overexpression of CFD1 and CIA2, as well as disruption of YAP5 and TYW1 had an only moderate effect on XylD enzymatic activity. On the contrary, recombinant strains obtained by overexpression of TYW1, or disruption of GRX3 or BOL2, showed significantly increased XylD enzymatic activity, of 1.5-fold, 2.3-fold, and 2.5-fold increase, respectively. We also overexpressed the truncated Tyw1 (tTyw1) in the BDδD2tkdcA strain. tTyw1 is the N-terminal hydrophobic transmembrane domain of Tyw1 and lacks its Fe–S cluster binding domain. It was reported that the expression of Aft1/2 Fe regulon is activated by overexpression of not only Tyw1 but also tTyw1 over expression (Li et al., 2011). Li et al. (2011) reported that overexpression of truncated Tyw1 (1–199 amino acids) enhanced the expression level of Aft1/Aft2 Fe regulon genes and Fe uptake stronger than full-length TYW1 overexpression in S. cerevisiae. Therefore, Additionally, tTYW1(a truncated version of TYW1) was also overexpressed in the BDδD-2tkdcA strain which led to a 1.6-fold improvement of XylD enzymatic activity. Last

3.4. 1,2,4-Butanetriol production from lignocellulosic biomass The BDδD-2tkdcA-ΔBOL2-tTYW1 strain which indicated the highest XylD enzymatic activity was used to perform the direct fermentation of rice straw hydrolysate obtained after hydrothermal pretreatment (Sakamoto et al., 2012; Guirimand et al., 2016). This hydrolysate contains cellooligosaccharides, xylooligosaccharides, monosaccharides (2.69 g/L glucose and 1.43 g/L xylose) and fermentation inhibitors (2.43 g/L acetate, 1.04 g/L formate, 0.29 g/L furfural, 0.09 g/L vanillin, 0.03 g/L hydroxymethylfurfural, 0.02 g/L syringaldehyde). Total hydrolysis of this lignocellulosic biomass, by sulfuric acid treatment according to the NREL method (Sluiter et al., 2012), generated 11.5 g/L glucose and 15.7 g/L xylose. Bio-production of 1,2,4-butanetriol by direct fermentation of the rice straw hydrolysate was achieved using the BDδD-2tkdcA-ΔBOL2tTYW1 strain in the presence of 2% (w/v) of a commercially available mixture of hemicellulases, in order to saccharify oligosaccharides contained in the medium. When two-fold diluted rice straw hydrolysate containing 5.8 g/L of glucose and 7.9 g/L (52.3 mM) of xylose was used as substrate in fermentation broth, xylose accumulation was not observed through fermentation (Fig. 5A), and 1.1 g/L (10.7 mM) of 1,2,4butanetriol was produced along with 4.9 g/L xylonate, after 96 h (Fig. 5B and C) with a molar yield of 20.5%. On the other hand, when un-diluted rice straw hydrolysate containing 11.5 g/L of glucose and 15.7 g/L (104.6 mM) of xylose was used for the fermentation, more than 4 g/L xylose was remaining in the medium (Fig. 5A), and only 0.62 g/L (5.8 mM) and 5.8 g/L of 1,2,4-butanetriol and xylonate were produced, respectively (Fig. 5B and C), with a decreased 1,2,4-butanetriol molar yield of 5.5%. Glucose accumulation in fermentation medium was not observed through fermentation in both conditions. After 96 h fermentation, we determined the amount of remaining polysaccharide by acid hydrolysis of the fermentation residue. 4.65 g/L of xylose and 3.49 g/L of glucose was remained as polysaccharide from in non-diluted medium, and 1.71 g/L of xylose and 2.89 g/L of glucose was remained as polysaccharide from in two-fold diluted medium. Last but not least, cell growth was observed during the fermentation and diluted rice straw hydrolysate led to higher cell concentration (Fig. 5D). 4. Discussion Despite a number of studies attempting to biotechnologically produce 1,2,4-butanetriol (Cao et al., 2015; Lu et al., 2016; Sun et al., 2016; Valdehuesa et al., 2014; Wang et al., 2018; Jing et al., 2018), it is interesting to notice that yeast has never been used as a host microorganism to achieve this goal. Moreover, no success in the direct fermentation of lignocellulosic biomass has been reported yet. In the present study, the combined overexpression of C. crescentus XylB and 23

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

Fig. 4. Improvement of XylD activity and 1,2,4-butanetriol production ability by engineering Fe metabolism in recombinant strains of S. cerevisiae. (A) Different target enzymes involved in Fe metabolism in S. cerevisiae were overexpressed (Cfd1 and Cia2; red), deleted (Grx3, Bol2 and YAP5; blue), or either over-expressed or deleted ((t)Tyw1; purple) in the BDδD-2tkdcA platform strain. (B) Relative XylD enzymatic activity and (C) xylonate accumulation and 1,2,4-butanetriol production after 96 h were monitored in the resulting recombinant strains. Error bars indicate the standard deviations of three independent experiments. In Figure B, the XylD activity measured in the BDδD-2tkdcA recombinant strain was used as a standard value (=1) to normalize all the data set. * represent a statistically significant (p < 0.01) difference in XylD activity compared with BDδD-2tkdcA strain as evaluated by paired comparisons using Student's t-test. And also, statistically significant (p < 0.01) difference in XylD activity of the combination of BOL2 deletion and other recombination compared with BOL2 single deletion strain was evaluated. In Figure C, * represent a statistically significant (p < 0.05) difference in 1,2,4-butanetriol and xylonate amount compared with BDδD-2tkdcA strain as evaluated by paired comparisons using Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

converting xylose into 1,2,4-butanetriol, while the energy necessary to the cell growth and primary metabolism was supplied by glucose as a carbon source. To the best of our knowledge, this study is the first report of 1,2,4-butanetriol production by recombinant yeast cells. Moreover, the enhancement of XylD enzymatic activity is regarded as a major key point in 1,2,4-butanetriol bio-production, as a low XylD enzymatic activity is responsible for the accumulation and efflux of

XylD, L. lactis KdcA, and endogenous truncated Tyw1 (tTyw1), along with the deletion of Bol2 enabled us to efficiently produce 1,2,4-butanetriol from xylose and lignocellulosic hydrolysates in recombinant S. cerevisiae. In particular, the enhancement of XylD enzymatic activity along with the selection of optimal 2-ketoacid decarboxylase gene candidate greatly contributed to the improvement of 1,2,4-butanetriol production ability. Remarkably, our engineered cell factory was 24

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

Fig. 5. Fermentation of rice straw hydrolysate by BDδD-2tkdcA-ΔBOL2-tTYW1 recombinant strain of S. cerevisiae. Time course monitoring of (A) xylose, (B) xylonate, (C) 1,2,4-butanetriol, and (D) cell density (OD600). The rice straw hydrolysate used in the fermentation broth was either 2-fold diluted (orange) or un-diluted (blue). Error bars indicate the standard deviations of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

overexpression of TYW1. In particular, Tyw1 has an ER-tethering domain consisting of 199 amino acids at its N terminus (Li et al., 2011; Martinez-Pastor et al., 2017). tTyw1 (1–199 amino acids of Tyw1), is also known to be localized at the surface of the endoplasmic reticulum (Li et al., 2011). The previous study indicated that overexpression of tTyw1 strongly enhanced the expression level of Aft1/Aft2 Fe regulon genes than full-length Tyw1 overexpression (Li et al., 2011). There are very few studies on Tyw1 and it is not clear why overexpression of TYW1 activates Aft1/Aft2 Fe regulon. Interestingly, in tTyw1 there is no Fe–S cluster binding domain already. It is considered that a potential unknown factor would activate Aft1/Aft2 regulon in the ER-tethering domain itself. Consecutively to the uptake of iron ions, a scaffolding protein Cfd1 supports the formation of Fe–S clusters (Martinez-Pastor et al., 2017). As shown in Fig. 4B, overexpression of CFD1 did not improve XylD activity, suggesting that the scaffolding protein might be insufficient for overproduction of Fe–S cluster. After the formation of Fe–S cluster in the cytosol of yeast cells, the Fe–S cluster is transferred to Fe–S apoprotein, to make it functional. The transfer process requires carrier proteins, and a yeast cytosolic Fe–S cluster targeting complex including Cia2 different from bacterial carrier proteins (Lill, 2009). In this study, overexpression of CIA2 did not affect XylD enzymatic activity. This result indicates that the carrier protein might not be limiting in the Fe–S enzyme formation, or that another Fe–S cluster carrier component would be required. XylD enzymatic activity was dramatically increased by the combination of BOL2 deletion and tTYW1 overexpression (Fig. 4B). However, 1,2,4-butanetriol titer was almost the same between ΔBOL2 and ΔBOL2-tTYW1 recombinant strains (Fig. 4C). These data suggest the existence of (an) another potential bottleneck (s) in the biosynthetic

xylonate out of the cell. XylD is a Fe–S protein, and it has to be noticed that overexpression of functional forms of Fe–S cluster containing enzymes is regarded as difficult in S. cerevisiae, which led to the low activity of these particular enzymes (Partow et al., 2012; Carlsen et al., 2013; Benisch and Boles, 2014). It is notably suggested that the limitation of intracellular iron ion availability is a bottleneck for the Fe–S cluster formation, especially as iron uptake is strictly controlled due to the toxicity of this element (Haussman et al., 2008; Encinar del Dedo et al., 2015; Martinez-Pastor et al., 2017). Previously, disruption of BOL2 led to increasing in C. crescentus XylD activity in a recombinant xylose-utilizing yeast strain (Salusjärvi et al., 2017). In the present study, disruption of BOL2 or GRX3 improved not only XylD activity but also 1,2,4-butanetriol production ability. In addition, overexpression of TYW1 and truncated tTYW1 demonstrated increased XylD enzymatic activity and 1,2,4-butanetriol production ability. These data supported the assumption that iron ion import limits Fe–S protein activity. For further verification of the association between iron ion availability and Fe–S cluster formation, the effect of iron (2 mM ammonium ferric citrate) addition to the culture medium on XylD activity of BDδD-2tkdcA was examined. XylD activity was improved by 1.5-fold by the iron addition (Fig. S4), which is lower fold change than those observed by the disruption of BOL2 and GRX3 (2.5 and 2.3 folds, respectively) In S. cerevisiae, when iron is sufficiently present in cells, the expression of iron transporters are suppressed. Therefore, although further verification such as quantification of transcripts and proteins of XylD is necessary, it is considered that the improvement of XylD activity was greater when the iron uptake is constantly promoted by the disruption of BOL2 or GRX3 than add iron in medium. To the best of our knowledge, this study is the first report of improvement of heterologous Fe–S protein activity in S. cerevisiae by the 25

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

pathway, between the formation of KDX intermediate and the final 1,2,4-butanetriol product. To the best of our knowledge, no comparative study on the conversion of KDX and optimal 2-ketoacid decarboxylase in yeast has been reported in the literature. Therefore, we have evaluated several 2-ketoacid decarboxylases gene candidates in order to identify the optimal enzyme for our system. As shown in Fig. 3A, overexpression of L. lactis kivD and kdcA led to the more efficient production of 1,2,4-butanetriol, compared to P. putida MdlC and endogenous Aro10 genes. According to Milne et al., 2015, L. lactis KivD and KdcA demonstrated higher Vmax/Km ratio for various linear and branched chain 2-ketoacids such as α-ketoisovalerate, α-ketoisocaproate, 2-oxopentanoate than Aro10 in a recombinant S. cerevisiae. Although enzymes involved in the decarboxylation of ketoacids with hydroxyl group have not been explored, this study indicated that the KivD and KdcA enzymes were found to be more suitable to utilize KDX as a substrate than the MdlC and Aro10 enzymes. After having identified the optimal 2-ketoacid decarboxylase gene candidate, we evaluated the importance of the promoter sequence for the over-expression of this gene in our recombinant strain. Especially, the higher promoter activity of TDH3 in glucose and/or xylose fermentation compared to PGK1 promoter has been regarded as an advantageous option in the improvement of our system (Lu and Jeffries, 2007; Xiong et al., 2018). In particular, when kivD and kdcA were over-expressed under the dependence of TDH3 promoter the 1,2,4-butanetriol production ability was improved by 1.5-fold and 3.4-fold, respectively. However, the increase in expression level under the dependence of the TDH3 promoter was higher in kivD than in kdcA (Fig. S3). This result suggested that KdcA may have superior kinetic parameters (such as Km and Vmax) for KDX than KivD. Additionally, there were almost no difference regarding the amount of accumulated xylonate among the kivD and kdcA integration strains (BDδD-kivD, BDδD-tkivD, BDδD-kdcA, and BDδD-tkdcA) (Fig. S1). There is a possibility that the only intracellular pooled KDX was converted to 1,2,4-butanetriol by these strains. Therefore, Improvement of 2-ketoacid decarboxylase activity along with XylD activity would be important to facilitate the carbon flux from xylose to 1,2,4-butantriol and to prevent the xylonate accumulation. Finally, we conducted the direct fermentation of rice straw hydrolysate to produce 1,2,4-butanetriol by our recombinant strain of S. cerevisiae (Fig. 5). In particular, the BDδD-2tkdcA-ΔBOL2-tTYW1 recombinant strain was able to efficiently produce 1,2,4-butanetriol with a molar yield of 20.5% from a two-fold diluted rice straw hydrolysate as a carbon source (Fig. 5C). This yield was comparable to the yield obtained when pure xylose was used for fermentation (23.1%) (Table S2). However, the 1,2,4-butanetriol yield was drastically decreased to 5.5% when undiluted hydrolysate was used as feedstock, and a decrease of growth was observed as well (Fig. 5C and D). These data suggest that fermentation inhibitors present in the rice straw hydrolysate negatively affect xylose consumption and 1,2,4-butanetriol production as well. In the present study, a commercially available mixture of hemicellulases (2% w/v) was added to rice straw hydrolysate, however complete saccharification was not achieved in our experimental condition. In previous our study have shown that expressing hemicellulase in the yeast cell surface can effectively degrade the hemicellulose in the hydrolysate during fermentation, even without addition of commercial enzyme (Guirimand et al., 2016). Therefore, similar approach is considered effective to produce 1,2,4-butanetriol from xylose enrich materials from lignocellulosic biomass. In this study, only xylose was converted to 1,2,4-butanetriol. In order to make more effective use of lignocellulosic biomass, glucose should be co-utilized, not only for growth, but also for the bio-production of useful chemicals in the future. To the best of our knowledge, this study is the first report of lignocellulosic biomass direct fermentation to produce 1,2,4-butanetriol. Improvement of tolerance to fermentation inhibitors or utilization of industrial strains would be required to meet high yield of 1,2,4-butanetriol production from the lignocellulosic hydrolysate.

Acknowledgements This work was funded by project P16009, Development of Production Techniques for Highly Functional Biomaterials Using Smart Cells of Plants and Other Organisms (Smart Cell Project), from the New Energy and Industrial Technology Development Organization (NEDO). This work was partly supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ymben.2019.08.012. References Abdel-Ghany, S.E., Day, I., Heuberger, A.L., Broeckling, C.D., Reddy, A.S.N., 2013. Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes. Metab. Eng. 20, 109–120. https://doi.org/10.1016/j.ymben.2013.10.003. Andberg, M., Aro-Kärkkäinen, N., Carlson, P., Oja, M., Bozonnet, S., Toivari, M., Hakulinen, N., O'Donohue, M., Penttilä, M., Koivula, A., 2016. Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases. Appl. Microbiol. Biotechnol. 100, 7549–7563. https://doi.org/10.1007/s00253-0167530-8. Bamba, T., Hasunuma, T., Kondo, A., 2016. Disruption of PHO13 improves ethanol production via the xylose isomerase pathway. Amb. Express 6, 4. https://doi.org/10. 1186/s13568-015-0175-7. Benisch, F., Boles, E., 2014. The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase. J. Biotechnol. 171, 45–55. https://doi.org/ 10.1016/j.jbiotec.2013.11.025. Cao, Y., Niu, W., Guo, J., Xian, M., Liu, H., 2015. Biotechnological production of 1,2,4butanetriol: an efficient process to synthesize energetic material precursor from renewable biomass. Sci. Rep. 5, 18149. https://doi.org/10.1038/srep18149. Carlsen, S., Ajikumar, P.K., Formenti, L.R., Zhou, K., Phon, T.H., Nielsen, M.L., Lantz, A.E., Kielland-Brandt, M.C., Stephanopoulos, G., 2013. Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 97, 5753–5769. https://doi. org/10.1007/s00253-013-4877-y. Chen, D.C., Yang, B.C., Kuo, T.T., 1992. One-step transformation of yeast in stationary phase. Curr. Genet. 21, 83–84. https://doi.org/10.1007/BF00318659. Courel, M., Lallet, S., Camadro, J.-M., Blaiseau, P.-L., 2005. Direct activation of genes involved in intracellular iron use by the yeast iron-responsive transcription factor Aft2 without its paralog Aft1. Mol. Cell. Biol. 25, 6760–6771. https://doi.org/10. 1128/mcb.25.15.6760-6771.2005. De La Plaza, M., Fernández De Palencia, P., Peláez, C., Requena, T., 2004. Biochemical and molecular characterization of α-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. FEMS Microbiol. Lett. 238, 367–374. https://doi.org/10.1016/j.femsle.2004.07.057. Encinar del Dedo, J., Gabrielli, N., Carmona, M., Ayte, J., Hidalgo, E., 2015. A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast. PLoS Genet. 11, e1005106. https:// doi.org/10.1371/journal.pgen.1005106. Eichinger, D.J., Boeke, J.D., 1988. The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell 54, 955–966. https://doi.org/10.1016/0092-8674(88)90110-9. Gouranlou, F., Kohsary, I., 2010. Synthesis and characterization of 1,2,4-butanetrioltrinitrate. Asian J. Chem. 22, 4221–4228. https://doi.org/10.1142/ S1793604711001804. Guirimand, G., Sasaki, K., Inokuma, K., Bamba, T., et al., 2016. Cell-surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate. Appl. Microbiol. Biotechnol. 100 (8), 3477–3487. Guirimand, G., Inokuma, K., Bamba, T., Matsuda, M., Morita, K., Sasaki, K., Ogino, C., Berrin, J.-G., Hasunuma, T., Kondo, A., 2019. Cell-surface display technology and metabolic engineering of Saccharomyces cerevisiae for enhancing xylitol production from woody biomass. Green Chem. 1795–1808. https://doi.org/10.1039/ c8gc03864c. Hasunuma, T., Ishii, J., Kondo, A., 2015. Rational design and evolutional fine tuning of Saccharomyces cerevisiae for biomass breakdown. Curr. Opin. Chem. Biol. 29, 1–9. https://doi.org/10.1016/j.cbpa.2015.06.004. Hausmann, A., Samans, B., Lill, R., Mühlenhoff, U., 2008. Cellular and mitochondrial remodeling upon defects in iron-sulfur protein biogenesis. J. Biol. Chem. 283, 8318–8330. https://doi.org/10.1074/jbc.M705570200. Hong, K.K., Nielsen, J., 2012. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell. Mol. Life Sci. 69, 2671–2690. https://doi.org/10.1007/s00018-012-0945-1.

26

Metabolic Engineering 56 (2019) 17–27

T. Bamba, et al.

Biol. Chem. 287, 12365–12378. https://doi.org/10.1074/jbc.M111.328914. Niu, W., Molefe, M.N., Frost, J.W., 2003. Microbial synthesis of the energetic material precursor 1,2,4-butanetriol. J. Am. Chem. Soc. 125, 12998–12999. https://doi.org/ 10.1021/ja036391+. Olsson, L., Hahn-Hägerdal, B., 1993. Fermentative performance of bacteria and yeasts in lignocellulose hydrolysates. Process Biochem. 28, 249–257. https://doi.org/10. 1016/0032-9592(93)80041-E. Pallesen, L.J., Solodovnikova, N., Sharma, A.K., Walden, W.E., 2013. Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold. J. Biol. Chem. 288, 23358–23367. https://doi.org/10.1074/jbc.M113.486878. Parekh, R.N., Shaw, M.R., Wittrup, K.D., 1996. An integrating vector for tunable, high copy, stable integration into the dispersed Ty delta sites of Saccharomyces cerevisiae. Biotechnol. Prog. 12, 16–21. https://doi.org/10.1021/bp9500627. Partow, S., Siewers, V., Daviet, L., Schalk, M., Nielsen, J., 2012. Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae. PLoS One 7, 1–12. https://doi.org/10.1371/journal.pone.0052498. Sakamoto, T., Hasunuma, T., Hori, Y., Yamada, R., Kondo, A., 2012. Direct ethanol production from hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of hemicellulolytic enzymes on the surface of xyloseutilizing Saccharomyces cerevisiae cells. J. Biotechnol. 158, 203–210. https://doi.org/ 10.1016/j.jbiotec.2011.06.025. Salusjärvi, L., Toivari, M., Vehkomäki, M.-L., Koivistoinen, O., Mojzita, D., Niemelä, K., Penttilä, M., Ruohonen, L., 2017. Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 101, 8151–8163. https://doi.org/10.1007/s00253-017-8547-3. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108. https://doi.org/10.1038/nprot.2008.73. Skoza, L., Mohos, S., 1976. Stable thiobarbituric acid chromophore with dimethyl sulphoxide. J. Biol. Chem. 159, 457–462. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of Structural Carbohydrates and Lignin in Biomass. National Renewable Energy Laboratory. Technical Report NREL/TP- 510-4268. Sun, L., Yang, F., Sun, H., Zhu, T., Li, X., Li, Y., Xu, Z., Zhang, Y., 2016. Synthetic pathway optimization for improved 1,2,4-butanetriol production. J. Ind. Microbiol. Biotechnol. 43, 67–78. https://doi.org/10.1007/s10295-015-1693-7. Tai, Y., Xiong, M., Jambunathan, P., Wang, J.J., Wang, J.J., Stapleton, C., Zhang, K., 2016. Engineering nonphosphorylative metabolism to generate lignocellulose-derived products. Nat. Chem. Biol. 12, 247–253. https://doi.org/10.1038/nchembio. 2020. Toivari, M., Nygård, Y., Kumpula, E., Vehkomäki, M.-L., Benčina, M., Valkonen, M., Maaheimo, H., Andberg, M., Koivula, A., Ruohonen, L., Penttilä, M., Wiebe, M.G., 2012. Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate. Metab. Eng. 14, 427–436. https://doi.org/10.1016/j.ymben. 2012.03.002. Valdehuesa, K., Liu, H., Ramos, K., 2014. Direct bioconversion of d-xylose to 1, 2, 4butanetriol in an engineered Escherichia coli. Process Biochem. 49, 25–32. https://doi. org/10.1016/j.procbio.2013.10.002. Vo, A., Fleischman, N.M., Froehlich, M.J., Lee, C.Y., Cosman, J.A., Glynn, C.A., Hassan, Z.O., Perlstein, D.L., 2018. Identifying the protein interactions of the cytosolic ironsulfur cluster targeting complex essential for its assembly and recognition of apotargets. Biochemistry 57, 2349–2358. https://doi.org/10.1021/acs.biochem. 7b00072. Wang, X., Xu, N., Hu, S., Yang, J., Gao, Q., Xu, S., Chen, K., Ouyang, P., 2018. D-1,2,4Butanetriol production from renewable biomass with optimization of synthetic pathway in engineered Escherichia coli. Bioresour. Technol. 250, 406–412. https:// doi.org/10.1016/j.biortech.2017.11.062. Xiong, L., Zeng, Y., Tang, R.Q., Alper, H.S., Bai, F.W., Zhao, X.Q., 2018. Condition-specific promoter activities in Saccharomyces cerevisiae. Microb. Cell Factories 17, 1–15. https://doi.org/10.1186/s12934-018-0899-6. Yamada, R., Tanaka, T., Ogino, C., Fukuda, H., Kondo, A., 2010. Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch. Appl. Microbiol. Biotechnol. 85, 1491–1498. https://doi.org/10.1007/ s00253-009-2198-y.

Inokuma, K., Hasunuma, T., Kondo, A., 2014. Efficient yeast cell-surface display of exoand endo-cellulase using the SED1 anchoring region and its original promoter. Biotechnol. Biofuels 7, 8. https://doi.org/10.1186/1754-6834-7-8. Ishii, J., Izawa, K., Matsumura, S., Wakamura, K., Tanino, T., Tanaka, T., Ogino, C., Fukuda, H., Kondo, A., 2009. A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast. J. Biochem. 145, 701–708. https://doi.org/10.1093/jb/mvp028. Ishii, J., Kondo, T., Makino, H., Ogura, A., Matsuda, F., Kondo, A., 2014. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 14, 399–411. https://doi.org/10.1111/1567-1364. 12138. Jing, P., Cao, X., Lu, X., Zong, H., Zhuge, B., 2018. Modification of an engineered Escherichia coli by a combined strategy of deleting branch pathway, fine-tuning xylose isomerase expression, and substituting decarboxylase to improve 1,2,4-butanetriol production. J. Biosci. Bioeng. 126, 547–552. https://doi.org/10.1016/j.jbiosc.2018. 05.019. Kaplan, C.D., Kaplan, J., 2009. Iron acquisition and transcriptional regulation. Chem. Rev. 109, 4536–4552. https://doi.org/10.1021/cr9001676. Kim, S., Lee, S.B., 2005. Identification and characterization of Sulfolobus solfataricus Dgluconate dehydratase: a key enzyme in the non-phosphorylated Entner-Doudoroff pathway. Biochem. J. 387, 271–280. https://doi.org/10.1042/BJ20041053. Kneen, M.M., Stan, R., Yep, A., Tyler, R.P., Saehuan, C., McLeish, M.J., 2011. Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae. FEBS J. 278, 1842–1853. https://doi.org/10.1111/j. 1742-4658.2011.08103.x. Kumánovics, A., Chen, O.S., Li, L., Bagley, D., Adkins, E.M., Lin, H., Dingra, N.N., Outten, C.E., Keller, G., Winge, D., Ward, D.M., Kaplan, J., 2008. Identification of FRA1 and FRA2 as genes involved in regulating the yeast iron regulon in response to decreased mitochondrial iron-sulfur cluster synthesis. J. Biol. Chem. 283, 10276–10286. https://doi.org/10.1074/jbc.M801160200. Lane, S., Dong, J., Jin, Y.S., 2018. Value-added biotransformation of cellulosic sugars by engineered Saccharomyces cerevisiae. Bioresour. Technol. 260, 380–394. https://doi. org/10.1016/j.biortech.2018.04.013. Li, A., Jia, X., Ward, D.M., Kaplan, J., 2011. Yap5 protein-regulated transcription of the TYW1 gene protects yeast from high iron toxicity. J. Biol. Chem. 286, 38488–38497. https://doi.org/10.1074/jbc.M111.286666. Li, L., Bagley, D., Ward, D.M., Kaplan, J., 2008. Yap5 is an iron-responsive transcriptional activator that regulates vacuolar iron storage in yeast. Mol. Cell. Biol. 28, 1326–1337. https://doi.org/10.1128/MCB.01219-07. Lill, R., 2009. Function and biogenesis of iron–sulphur proteins. Nature 460, 831–838. https://doi.org/10.1038/nature08301. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408. https://doi.org/ 10.1006/meth.2001.1262. Lu, C., Jeffries, T., 2007. Shuffling of promoters for multiple genes to optimize xylose fermentation in an engineered Saccharomyces cerevisiae strain. Appl. Environ. Microbiol. 73, 6072–6077. https://doi.org/10.1128/AEM.00955-07. Lu, X., He, S., Zong, H., Song, J., Chen, W., Zhuge, B., 2016. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins. World J. Microbiol. Biotechnol. 32, 1–9. https://doi.org/10.1007/ s11274-016-2085-5. Martínez-Pastor, M.T., Perea-García, A., Puig, S., 2017. Mechanisms of iron sensing and regulation in the yeast Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 33, 75. https://doi.org/10.1007/s11274-017-2215-8. Milne, N., van Maris, A.J.A., Pronk, J.T., Daran, J.M., 2015. Comparative assessment of native and heterologous 2‑oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae. Biotechnol. Biofuels 8, 1–15. https://doi.org/ 10.1016/j.meteno.2016.01.002. Monteith, M.J., Schoefield, D., Bailey, M., 1998. Process for the Preparation of Butanetriols. WO1998008793. Netz, D.J.A., Pierik, A.J., Stümpfig, M., Bill, E., Sharma, A.K., Pallesen, L.J., Walden, W.E., Lill, R., 2012. A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation. J.

27