Acetoin

Sodium pyruvate,
citric acid, sodium citrate, thiamine pyrophosphate (TPP), and magnesium
chloride were obtained from Sigma Chemical Co. (St. Louis, USA) for
the determination of enzyme activity and reaction media. Ethanol,
potassium phosphate, sodium acetate, and sodium bicarbonate were obtained
from Scharlab, S.L. (Barcelona, Spain) for the determination of enzyme
activity and reaction media. Acetoin and lactic acid standards were
obtained from Sigma Chemical Co. (St. Louis, USA) and used as analytical
standards for gas chromatography and LC–MS analysis, respectively.
NADH and NAD⁺ were obtained from Bontac Bioengineering
Co. (Shenzhen, China) as reaction cofactors. Carbon dioxide gas as
well as gas mixture mimicking real blast furnace off-gases composition
[24.5% CO₂, 46.6% N₂, 23.9% CO, 1.2% O₂, and 3.8% H₂] was obtained from Carburos Metalicos (Barcelona,
Spain). PDC from Zymobacter palmae (ZpPDC) was produced
according to Alcover et al.^{27 (link)} PDC from Saccharomyces cerevisiae (ScPDC), ADH
from Saccharomyces cerevisiae (ScADH) and Thermotoga maritima LDH
(TmLDH) were produced according to Benito et al. (see Supporting Information).^{28 (link)}

Analytical grade reagents NaCl and NaOH were obtained from Guangnuo Chemical Technology (Shanghai, China); chromatographic grade reagents including ethanol, methanol, dichloromethane; polyvinylpolypyrrolidone (PVPP) and d-gluconic acid lactone with ≥99% purity were purchased from Aladdin Reagent (Shanghai, China); standards of C8–C40 n-alkanes, IBMP, Isopropyl-methoxypyrazine (IPMP), sec-butyl-methoxypyrazine (SBMP) with ≥99% purity and nonanal, acetoin, 1-octanol, isopentyl acetate, linalool with ≥98% purity were from Sigma-Aldrich (St. Louis, MO, USA); standard of 4-methyl-2-pentanol with ≥98% purity from Tokyo Chemical Industry (Tokyo, Japan). Cleanert PEP-SPE columns (200 mg/6 mL) were from Bonna-Agela Technologies (Tianjin, China). Rapidase AR 2000 glucosidase was supplied by Creative Enzymes (New York, NY, USA).

Sulfuric acid for HPLC analysis was employed (30,743 Honeywell Fluka 95–97%). MEthanol for RP-HPLC was purchased from Merck (34,860, ≥99.9%).
Standard solutions for HPLC (TraceCERT^®, 1,000 mg/L in water) were purchased from Sigma-Aldrich (Milan, Italy). Analyte stock solutions were prepared by dissolving a weighed amount of the pure compound in deionized water and stored at 4°C up to 1 month. Ethanol, ¹³C-labeled Ethanol, acetoin, acetone, butanal, 2-methylbutanal, 3-methylbutanal, butanedione, butanol, 2-methyl-butanol, 3-methyl-butanol, butanone, hexanal, mEthanol, methylacetate, 2-pentanol, propanal, 2-methylpropanal, propanol, 2-methyl-propanol, and sec-butanol were purchased from Sigma-Aldrich (Italy). All chemicals, having purity higher than 99%, were used without any further purification.
Solid-phase microextraction fiber based on 85 um carboxen/polydimethylsiloxane (CAR/PDMS) was employed for the preconcentration of volatile compounds in the HS.
Helium 5.6 IP was purchased from SOL Group Spa (Italy) and was further purified with a super clean filter purchased from Agilent Technologies (United States) to remove water, oxygen, and hydrocarbon contaminants.
Preparation/dilution of samples and solutions was performed gravimetrically using ultrapure water (Milli-Q; 18.2 MΩ cm⁻¹ at 25°C, Millipore, Bedford, MA, United States).

The following reagents were obtained from Sigma-Aldrich: glucose, 1,3-propanediol, phenylboronic acid, cycloheximide, 23BD, acetoin, pyruvate, PEP, 6PG, G6P, F6P, Ru5P, R15P, ATP, dithiothreitol (DTT), NADH, NADPH, NADP⁺, phosphocreatine, RuBisCO, pyruvate kinase, lactate dehydrogenase, G6P dehydrogenase, 3PGA kinase, GAPDH and creatine kinase. U-¹³C glucose and ¹³C-NaHCO₃ were obtained from Cambridge Isotope Laboratories. IPTG and chloramphenicol were obtained from Fischer Scientific. Gentamycin was purchased from Teknova. Spectinomycin was purchased from MP Biomedicals. Kanamycin was purchased from IBI Scientific. Phusion polymerase was purchased from New England Biolabs. All oligonucleotide synthesis and DNA sequencing were performed by Eurofins MWG Operon Inc.