TMB3043 was transformed with mutated XYL1(K270R), coding for aldose reductase (AR) and XYL2, coding for xylitol dehydrogenase (XDH), both inserted in the integrative plasmid YIpOB9 (Table 1), and the new strain was named TMB3662 (Table 1). The complete pentose utilizing pathway was introduced in S. cerevisiae by transforming TMB3662, carrying integrated AR and XDH, with the multicopy plasmid harboring the synthetic, codon optimized genes sLAD1 and sALX1, coding for L-arabitol dehydrogenase (LAD) and L-xylulose dehydrogenase (ALX), respectively (Table 1). This strain, containing a complete L-arabinose pathway, consisting of AR, LAD, ALX and XDH was named TMB3664. In addition, a D-xylose-only utilizing strain was constructed by transforming TMB3662 with plasmid p425GPD, 44 lacking the structural genes coding for LAD and ALX (Table 1). This strain, containing only AR and XDH, was named TMB3665 (Table 1).

The successful expression of the pentose metabolizing enzymes was verified by measuring the individual enzyme activities. The results are summarized in Table 2 and 3. Table 2 includes enzyme activity values for the reductases AR and ALX. Table 3 includes enzyme activity values for the dehydrogenases LAD and XDH, as well as an activity referred to as "polyol dehydrogenase", combining LAD, XDH and ALX. In fact, when LAD and XDH enzymes are co-present, NAD⁺ reduction coupled with L-arabitol oxidation could be the result of the combined activity of LAD and XDH (Figure 1). In addition, in strains where LAD, ALX and XDH are co-expressed, a further contribution is given by ALX activity with NAD⁺ as a cofactor when xylitol is the substrate (Figure 1, Table 3). For this reason L-arabitol and xylitol oxidizing activity with NAD⁺ as cofactor is referred to as "polyol dehydrogenase" 45.

First, AR activity of the mutant AR(K270R) with L-arabinose as substrate and NADH as cofactor was verified using cell extracts from TMB3662. The mutated AR(K270R) enzyme retained its catalytic activity with L-arabinose as substrate, with a specific activity of 0.82 ± 0.01 U/(mg total protein), compared with 0.70 ± 0.04 U/(mg total protein) when D-xylose was used as substrate (Table 2). The slightly higher reductase activity for L-arabinose is consistent with previously reported data for the natural enzyme 46.

Next, active expression of the codon optimized sLAD1 and sALX1 in S. cerevisiae was verified by measuring LAD activity and ALX activity (Table 2 and 3) in cell extracts from strains expressing these enzymes alone (TMB3659 and TMB3658, respectively, Table 1). LAD and ALX activity was also measured in cell extracts from analogous strains, expressing the natural lad1 and ALX1 genes individually in the same genetic background (TMB3657 and TMB3656, respectively, Table 1). The enzymatic activities were not significantly different, indicating that in this case codon optimization did not influence enzymatic activity (data not shown). Nevertheless, the synthetic gene versions were chosen for the continued work.

The LAD activity was 0.77 ± 0.1 U/(mg total protein) with L-arabitol as a substrate and 0.58 ± 0.07 U/(mg total protein) with xylitol (Table 3). The ALX activity was measured with NAD⁺ as cofactor (reverse reaction, see Methods section). The activity was 11.2 ± 1.4 U/(mg total protein) when xylitol was used as a substrate, while no activity was detected with L-arabitol (Table 2), in agreement with previous reports 2845. No significant activity (< 0.05 U/(mg total protein)) with either substrate was detected in the original host strain CEN.PK 113-16B 47 (data not shown).

Finally, all heterologous activities introduced in strains TMB3664, expressing AR, LAD, ALX and XDH and TMB3665, expressing AR and XDH, were measured. The AR activity in strain TMB3665 was essentially identical to that of the parental strain TMB3662 (Table 2), while the AR activity for TMB3664 was only about 65% of the parental strain. This could be due to lower expression owing to the metabolic burden of expressing two additional genes on a multicopy plasmid. Alternatively, L-arabitol and xylitol formed in the in vitro reaction could be re-oxidized by LAD and ALX, regenerating NADH and thus reducing the apparent activity of AR in cell extracts of TMB3664 (Figure 1). The difference in enzyme activity did, however, not influence D-xylose utilization (See below and Table 4).

ALX specific activity was 3.01 U/(mg total protein) in TMB3664 and not detectable in TMB3665 (Table 2). In the presence of NADH, XDH can act as a D-xylulose reductase38, while no report on L-xylulose conversion by XDH is available. However, the absence of ALX activity in TMB3665 may indicate that this enzyme does not accept the L-xylulose stereoisomer as substrate, although a specific assay would be needed to confirm this speculation.

The polyol dehydrogenase activity was measured in strains TMB3664 and TMB3665 using L-arabitol and xylitol at different concentrations (Table 3). Consistent with the expression of sLAD1 in TMB3664, the dehydrogenase activity whith L-arabitol as a substrate was higher in this strain than in TMB3665. The activity difference increased when L-arabitol, a preferred substrate for LAD but not for XDH 273845, was used at a concentration of 100 mM instead of 330 mM (Table 3). The ratio between the activity in TMB3665 and TMB3664 was ~0.75 at 330 mM and ~0.55 at 100 mM, respectively (Table 3) 28. The co-existence of LAD, ALX and XDH activities in TMB3664 conferred three to four times higher xylitol dehydrogenase activity, 28.54 U/(mg total protein), compared with 7.2 U/(mg total protein) for TMB3665 (Table 3). Thus, the recombinant S. cerevisiae strain TMB3664 harbors enzymatic activities for L-arabinose and D-xylose utilization comparable to those previously measured in natural yeast species capable of fast pentose sugar consumption 45.

The ability of the recombinant S. cerevisiae strain TMB3664 to utilize L-arabinose and D-xylose as sole carbon source for growth was first assessed in aerobic culture in mineral media containing either of the pentose sugars as sole carbon source. TMB3664 was able to utilize both pentose sugars for growth. The maximum specific growth rate was 0.05 ± 0.003 h^-1 and 0.05 ± 0.002 h^-1 in L-arabinose and D-xylose medium, respectively. With L-arabinose, the strain reached a final cellular density, expressed as OD_{620 nm}, of 11.2 ± 0.6 after ~100 h. The final OD in D-xylose medium was 18.7 ± 0.9 and it was reached in 90 h. No growth was detected after more than 5 days in L-arabinose medium for the control strain TMB3665, while in D-xylose medium the growth rate 0.06 ± 0.002 h^-1, and final OD, 19.1 ± 0.8, were similar to TMB3664.

The anaerobic fermentation capacity of the L-arabinose and D-xylose utilizing strain TMB3664 was assessed in batch culture with a mixture of glucose, L-arabinose and D-xylose as a carbon source (Figure 2 and Table 4).

Glucose was utilized first and completely consumed after 30 hours (Figure 2). A minor fraction of pentose sugars was co-consumed with glucose; nonetheless L-arabinose and D-xylose consumption rates were higher after glucose depletion in the subsequent pentose fermentation phase. The specific growth rate during initial growth on glucose was μ = 0.109 ± 0.003 h^-1. Biomass formation continued in the pentose fermentation phase. It was, however, not possible to calculate the specific growth rate, although the biomass concentration increased reproducibly from ~1.4 g/l at the point of glucose depletion to ~1.8 g/l at the end of the fermentation. Specific substrate consumption and product formation rates (Table 4) were calculated only for the pentose phase, as described in the Materials and Methods section.

Besides ~20 g/l of glucose, the strain consumed 3.9 g/l of L-arabinose and 14.1 g/l of D-xylose. The consumption rate after glucose depletion was 0.020 g/(g cells)/h for L-arabinose and 0.080 g/(g cells)/h for D-xylose. Produced ethanol was in total 15.3 g/l (estimated based on the degree of reduction balance 48), with a specific productivity from pentose sugars of 0.035 g/(g cells)/h. Ethanol yield was 0.23 g/(g sugar) based on total sugars present at the beginning of the fermentation, 0.40 g/(g sugar) based on consumed sugars and 0.35 g/(g sugar) based on consumed pentose sugars, as calculated after glucose depletion.

The L-arabitol yield for TMB3664 was 0.48 g/(g consumed L-arabinose) demonstrating for the first time in an AR expressing strain, that L-arabinose was not only converted to L-arabitol 303149, but further channeled into the metabolism. The xylitol yield was only 0.09 g/(g consumed D-xylose), comparable to previously reported yield obtained with strains expressing mutant AR with increased preference for NADH 37424350.

Fermentation with an identical set up was performed with the control strain TMB3665 (Table 1), in order to evaluate the advantage given to TMB3664 by capacity of metabolizing L-arabinose (Table 4). As shown in Figure 2, the overall behavior of the strain was similar to strain TMB3664. However, L-arabinose was taken up at a slower rate of 0.013 g/(g cells)/h and almost entirely excreted as L-arabitol (Table 4). The ethanol yield calculated on total consumed sugars was 0.39 g/(g sugar). Biomass and glycerol yields from to the pentose phase (Table 4) were slightly but reproducibly higher in the L-arabinose consuming strain TMB3664 than in the control strain TMB3665 (Table 4). In addition, the xylitol yield was about 10% higher in TMB3665, which could be ascribed to the unspecific substrate preference of LAD present in TMB3664 27.

Finally, anaerobic fermentation was performed with TMB3664 in batch containing only glucose and L-arabinose as a carbon source (Table 4). In this setup, cells grew anaerobically on glucose at μ = 0.113 ± 0.06 h^-1, co-consuming a small amount of L-arabinose. Biomass formation ceased after glucose depletion and no anaerobic growth was observed in the L-arabinose phase. Cells continued to consume L-arabinose at a rate of 0.011 g/(g cells)/h until the end of the fermentation at t = 120 hours (Table 4). The final ethanol concentration (estimated based on the degree of reduction balance 48) was 9.3 g/l, with a yield of 0.21 g/(g sugar) based on total sugars present at the beginning of the fermentation, 0.42 g/(g sugar) based on consumed sugars and 0.35 g/(g sugar) based on consumed L-arabinose (Table 4). By-product yields were lower than when D-xylose was also present in the medium. In fact, the xylitol concentration was below the detection limit and the L-arabitol yield was 0.39 g/(g of consumed L-arabinose) (Table 4).

Microbial strains and plasmids utilized for this work are summarized in Table 1. Escherichia coli DH5a (Life Technologies, Rockville, USA) was used as intermediate host for cloning steps and plasmid amplification and was routinely grown in LB medium 57 containing 100 mg/l ampicillin (Shelton scientific, Shelton, USA). S. cerevisiae strains were grown aerobically in Yeast Nitrogen Base medium (YNB, Difco Laboratories-Becton, Dickinson & Co., Sparks, USA), buffered at pH 5.5 with 50 mM potassium hydrogen phthalate (Merck, Darmstadt, Germany) 58 and formulated as it follows; YNBG: 20 g/l glucose, 6.7 g/l YNB; YNBA: 50 g/l L-arabinose, 13.4 g/l YNB; YNBX: 50 g/l D-xylose, 13.4 g/l YNB. According to strain requirements, the medium was supplemented with uracil and/or leucine at concentrations of 40 mg/l and 240 mg/l, respectively. Solid media were obtained by addition of 20 g/l agar (Merck, Darmstadt, Germany). Anaerobic mixed sugar batch fermentation was performed in defined mineral medium 50 supplemented with 0.4 g/l Tween 80 (Sigma-Aldrich, St. Louis, USA), 0.01 g/l ergosterol (Alfa Aesar, Karlsruhe, Germany), glucose (VWR International, Poole, UK), D-xylose (Acros Organics, Geel, Belgium) and L-arabinose (Sigma-Aldrich, St. Louis, USA) each at the concentration of 20 g/l or glucose and L-arabinose, each at the concentration of 20 g/l.

Standard molecular biology techniques were used 57. T. reesei and A. monospora chromosomal DNA was extracted using a bead-beater (Biospecs products, Bartlesville, OK, USA) and phenol/chloroform 57.

Plasmid DNA was purified from E. coli with Gene JET plasmid miniprep kit (Fermentas, Vilnius, Lithuania). Agarose gel DNA extractions were made using the QIAquick^® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). Purification of PCR products was made with the E.Z.N.A.^® Cycle-Pure Kit (Omega Bio-tek Inc, Doraville, GA, USA). Primers for PCR and sequencing service were purchased from Eurofins MWG Operon (Ebersberg, Germany). DNA modifying enzymes, restriction endonucleases and polymerases were purchased from Fermentas, Vilnius, Lithuania. Gene synthesis service was purchased from Genscript corp. (Piscataway, USA). Analytical PCR was performed with Taq DNA polymerase while preparative PCR was performed with High Fidelity PCR Enzyme Mix. Calf intestinal alkaline phosphatase and T4 DNA Ligase were used for DNA dephosphorylation and ligation, respectively. The lithium acetate/dimethyl sulfoxide protocol was used for yeast transformation 59.

The XYL1(K270R) gene fragment was excised from plasmid YIpOB5 37 and inserted into YIpOB7 [Bengtsson O, Bettiga M, Garcia-Sanchez R, Gorwa-Grauslund MF: Differential behaviour of two commonly used promoters in xylose utilizing recombinant Saccharomyces cerevisiae, unpublished] using the XbaI restriction sites, creating plasmid YIpOB9 (Table 1). The constructed plasmid was analyzed with restriction analysis and PCR to confirm correct insertion. The inserted part was sequenced to verify that no mutations were introduced.

The T. reesei (H. jecorina) gene lad1 codes for a NAD⁺-dependent L-arabitol dehydrogenase 27, while A. monospora ALX1 codes for a NADH-dependent L-xylulose reductase 2845 (Figure 1). T. reesei lad1 contains an intron of 69 base pairs 27. First, the two exons were separately PCR amplified using T. reesei chromosomal DNA as template. Exon N.1 was PCR amplified with primers containing the restriction site BamHI (LAD-BamHI-FW: 5'-TAGAGGATCCATGTCGCCTTCCGCAGTCGATGAC) and a 24-base region overlapping exon N.2 (LAD-Ex1-R: 5'-GGACATCTGAACCACAGATACCAGTGCTGCGGAC). Exon N.2 was PCR amplified with primers containing a 24-base region overlapping exon N.1 (LAD-Ex2-F: 5'-CTGGTATCTGTGGTTCAGATGTCCATTTCTGGCACG) and the restriction site HindIII (LAD-HindIII-RV: 5'-CAGCAAGCTTTCTAGATCAATCCAGGCTCTGAATCATG). The two PCR products were purified and pooled in a new PCR reaction, carried out with primers LAD-BamHI-FW and LAD-HindIII-RV and yielding the complete coding sequence of lad1. The PCR product was inserted into the vector p425GPD 44 creating the plasmid p425GPD_LAD1 (Table 1).

ALX1 was PCR-amplified using A. monospora chromosomal DNA as template with primers containing the restriction sites for SpeI (ALX-SpeI-FW: 5'-GCTACTAGTAGATCTATGACTGACTACATTCCAACTT) and XhoI (ALX-Xho-RV: 5'-TTACTCGAGAGATCTTTACCAAGAAGTGAAACCACCAT). The PCR product was inserted into the vector p425GPD 44 creating plasmid p425GPD_ALX1 (Table 1).

Based on the published amino acid sequence of Lad1p and Alx1p (Genebank accession numbers AF355628 and AJ583159, respectively), the corresponding optimized DNA sequences were synthesized and the synthetic genes were designated sLAD1 and sALX1, respectively. Synthetic sequences were designed taking into account S. cerevisiae codon usage and the possible presence of secondary structure generating sequences, according to a proprietary algorithm of Genscript Corp (Genscript Corp., Piscataway, USA). A fragment containing sLAD1 was excised from the vector pUCsLAD1 (Table 1) by digestion with BamHI and HindIII and inserted into the vector p425GPD 44 creating plasmid p425GPD_sLAD1 (Table 1). A fragment containing sALX1 was excised from the vector pUCsALX1 (Table 1) by digestion with SpeI and XhoI and inserted into the vector p425GPD 44 creating plasmid p425GPD_sALX1 (Table 1). The cassette consisting of TDH3 promoter, sALX1 and CYC1 terminator was PCR-amplified from plasmid p425GPD_sALX1 with primers containing the restriction site SapI (Cass-Sap-FV: 5'-GCTCTTCCGCTTGGTACCGGCCGCAAATTAAAG and Cass-Sap-RV: 5'-AAGCGGAAGAGCCAGTTTATCATTATCAATACTCGCC). The PCR product was inserted into plasmid p425GPD_sLAD1 creating plasmid p425GPD_sLAD1_sALX1 (Table 1). Each new construct was sequenced to verify the absence of mutations.

S. cerevisiae was grown aerobically in Erlenmeyer baffled flasks filled to maximum 1/10 of the volume with medium, incubated at 30°C in a rotary shake-incubator (INR-200 shake incubator, Gallenkamp, Leicester, UK) at 200 rpm. Cultures were inoculated at an initial O.D._{620 nm} of 0.20 ± 0.02 with sterile H₂O-washed cells from a late-exponential YNBG pre-culture. The maximum specific growth rate, μ, was calculated from exponential fitting of growth curves from at least two biological duplicates. Anaerobic mixed-sugar batch fermentation was performed in 1.5 l working volume bioreactors (Sartorius Stedim Biotech S.A., Aubagne, France), for 120 h, at 30°C, 200 rpm and at pH 5.5 automatically controlled by addition of 3 M KOH. Anaerobic conditions were established prior to inoculation by sparging the medium for at least 3 hours with nitrogen gas (< 5 ppm O₂, AGA, Malmö, Sweden) at 0.2 l/min flow rate. A water-lock was placed at the fermentor gas outlet. Cells were pre-grown aerobically in shake flasks in defined mineral medium 50, harvested by centrifugation, resuspended in ~10 ml sterile medium and inoculated into the fermentor at an initial O.D._{620 nm} of 0.20 ± 0.02. Fermentation experiments were performed in duplicate.

For crude protein extract preparation, cells were grown in 100 ml YNBG medium until O.D._{620 nm} of 1, harvested and stored at -80°C. Proteins were extracted with Yeast Protein Extraction Reagent (YPER, Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Protein concentration was determined with Coomassie Protein Assay Reagent (Pierce, Rockford, IL, USA). All activity measurements were performed on fresh extracts. Aldose reductase activity 4660 was measured essentially as previously described. Where indicated, L-arabinose was used as a substrate instead of D-xylose. L-arabitol dehydrogenase activity 2038 was measured as previously described. L-xylulose reductase 28, xylitol dehydrogenase 38 and global polyol dehydrogenase activities were measured as previously described. Where indicated, a substrate concentration of 100 mM instead of 330 mM and L-arabitol instead of xylitol were used. Only NAD⁺ and NADH were used as cofactors. Enzyme activity measurements were performed in triplicate at least on extracts from two independent cultivations. For all the activities measured, 1 U is defined as the amount of enzyme converting 1 μmol of substrate per minute in the conditions of the assay.

Samples were drawn from the fermentors after discharging the sample tubing dead-volume, cells were separated by centrifugation and the supernatant was filtered through 0.20 μm membrane filters (Toyo Roshi Kaish, Tokyo, Japan) and stored at 4°C until further analysis.

Concentration of glucose, D-xylose, acetate and glycerol and ethanol was determined by HPLC (Beckman Instruments, Fullerton, USA). The compounds were separated with three Aminex HPX-87H resin-based columns (Bio-Rad, Hercules, USA) connected in series and preceded by a Micro-Guard Cation-H guard column (Bio-Rad, Hercules, USA). Separation was performed at 45°C, with 5 mM H₂SO₄ at a flow rate of 0.6 ml/min as mobile phase. Concentration of L-arabinose, L-arabitol and xylitol was determined by HPLC (Waters, Milford, USA) using two HPX-87P resin-based column (Bio-Rad, Hercules, USA) preceded by a Micro-Guard Carbo-P guard column (Bio-Rad, Hercules, USA). Separation was performed at 80°C, with H₂O at a flow rate of 0.5 ml/min as mobile phase. All compounds were quantified by refractive index detection (Shimadzu, Kyoto, Japan). For each HPLC run, a 7-point calibration curve was made for each compound to calculate concentrations. Each sample was analyzed at least in duplicate and a maximum of 10% difference between replicate analyzes was accepted.

For each fermentation experiment, cell dry weight was determined at least in three points, in triplicate for each point. The end point of the fermentation (t = 120 h) was always included. For dry weight determination, a known volume of cell culture was filtered through dry pre-weighed 0.45 μm nitrocellulose filters, which were subsequently dried in a microwave oven and weighed. Because of evaporation, ethanol concentration was calculated from the degree of reduction balance of the overall carbon stoichiometry of the fermentation 48. Evaporated ethanol was always <10% of the total ethanol present at the end of the fermentation. Substrate consumption and product formation rates during the pentose phase (Table 4) were calculated as average rates. Total consumed D-xylose and L-arabinose and average biomass concentration between the point of glucose depletion and the end of the fermentation were used for the calculation.