Production of d-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae

d-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d-glucose to d-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d-glucose to biomass, and to increase d-glucaric acid yield, the four step d-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of d-glucose to d-glucaric acid. In batch bioreactor cultures with pulsed d-fructose and ethanol provision 1.3 g d-glucaric acid L−1 was produced. The d-glucaric acid titer (0.71 g d-glucaric acid L−1) was lower in nitrogen limited conditions, but the yield, 0.23 g d-glucaric acid [g d-glucose consumed]−1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d-glucaric acid yeast strains.


Introduction
d-Glucaric acid (d-saccharic acid) is a di-carboxylic acid that can be used for example to produce furan dicarboxylic acid (van Strien et al. 2020) or various polyamides, and polyesters (Sakuta and Nakamura 2019).Biotechnical conversion of d-glucose to d-glucaric acid (or to the conjugate salt d-glucarate) can provide a selective and less energy intensive alternative to chemical production processes (Zhang et al. 2021).Moon et al. (2009) were the first to engineer Escherichia coli for production of d-glucaric acid.They introduced activities for myo-inositol-1-phosphate synthase, myo-inositol oxygenase and uronate dehydrogenase into E. coli for conversion of D-glucose via glucose-6-phosphate and 1L-myo-inositol-1-phosphate (1 d-myo-inositol 3-phosphate) to myoinositol, d-glucuronate and finally to d-glucaric acid, resulting in production of 1.1 g L −1 of d-glucaric Vol:.( 1234567890) acid (Table 1).By introducing a polypeptide scaffold to co-localize the pathway enzymes d-glucaric acid concentration was increased to ~ 2.5 g L −1 (Moon et al. 2010).The myo-inositol oxygenase (MIOX) with its di-iron center and low activity was suggested to be the rate-limiting step (Moon et al. 2010).The MIOX activity was subsequently improved by using an N-terminal fusion of small ubiquitin-related modifier (SUMO) to MIOX, showing 75% increase in myo-inositol to d-glucaric acid conversion (Shiue and Prather 2014).Overexpression of myo-inositol-1-phosphate phosphatase from E. coli was tested for further enhancement of the process and the flux of d-glucose from catabolism towards myo-inositol-1-phosphate was redirected by deletion of the phosphoglucose isomerase (Pgi) and glucose 6-phosphate dehydrogenase (Zwf) encoding genes in E. coli (Shiue et al. 2015).This resulted in an increased yield of d-glucaric acid from d-glucose (yield 0.73 g g −1 with titer of 1.19 g L −1 , d-xylose as supplementing carbon source).Another approach used to decrease the flux to glycolysis was altering Pfk activity (Brockman and Prather 2015;Gupta et al. 2017;Hou et al. 2020).The d-glucaric acid pathway has also been used as a demonstration pathway for different synthetic biology approaches including use of MAGE (Raman et al. 2014), and small molecule reporter (Rogers and Church 2016).Dynamic pathway regulation with a quorum sensing based system or myo-inositol biosensor (Doong et al. 2018;Verma et al. 2022), regulation of Pgi translation by a d-fructose dependent control system (Qu et al. 2018), and NAD + regeneration system (Su et al. 2020) have been applied for d-glucaric acid production in E. coli.d-Glucaric acid production has also been demonstrated by in vitro conversion (Lee et al. 2016;Petroll et al. 2020;Su et al. 2019).Without myo-inositol addition, volumetric titers have remained between 1 and 2.5 g L −1 , although a recent study reported 5.35 g L −1 for intra plus extracellular titer in E. coli (Su et al. 2020) (reviewed by (Chen et al. 2020)).
Yeast are considered advantageous for organic acid production because of their low pH tolerance and robustness (Abbott et al. 2009).Yeasts have also been engineered for production of d-glucaric acid, although more recently than E. coli (Table 1).In 2016 Gupta et al. first engineered Saccharomyces cerevisiae for production of d-glucaric acid by expressing the d-glucaric acid pathway genes coding for inositol monophosphatase, myo-inositol-1-phosphate synthase, myo-inositol oxygenase either from Mus musculus or from Arabidopsis thaliana and uronate dehydrogenase in a opi1 deletion background (Gupta et al. 2016).The engineered yeast produced a maximum titer of 0.56 g L −1 in batch culture, and 0.98 g L −1 in fed-batch.Liu et al. (2016) introduced the pathway into Pichia pastoris and produced 0.107 g L −1 d-glucaric acid from d-glucose.Since these pioneering studies further pathway engineering approaches by e.g.enzyme and expression optimization, use of scaffolds, or by improving viability, have increased d-glucaric acid titers up to 9.5 g L −1 (Fang et al. 2022(Fang et al. , 2023;;Li et al. 2021;Zhang et al. 2020).In general, co-feeding of myo-inositol has resulted in higher final d-glucaric acid titers, up to 12.96 g L −1 in S. cerevisiae (Chen et al. 2018;Fang et al. 2022;Guo et al. 2022;Gupta et al. 2016;Li et al. 2021;Marques et al. 2020;Zhao et al. 2021), and 6.61 g L −1 in P. pastoris (Liu et al. 2016).The reported yields on d-glucose using yeast are scarce, and typically below 0.1 g g −1 , although recently a yield of 0.216 g g −1 (Guo et al. 2022) was reported (Table 1).
In E. coli deletion of the phosphoglucose isomerase (Pgi) and glucose 6-phosphate dehydrogenase (Zwf) encoding genes resulted in 2.9-fold (Qu et al. 2018), or nearly 18-fold higher yield on d-glucose (Shiue et al. 2015), compared to when the genes were present.The phosphoglucose isomerase encoding gene has not been deleted from S. cerevisiae or P. pastoris strains engineered for d-glucaric acid production.The phosphoglucose isomerase (Pgi1p) -deficient S. cerevisiae strains metabolize d-glucose only poorly, whereas the Pgi-deficient strains of E. coli are able to grow on d-glucose (Vinopal et al. 1975).Even relatively low (< 2 g L −1 ) d-glucose concentrations inhibit or reduce the growth of S. cerevisiae Pgi1pdeficient strains, possibly because of ATP depletion or glucose-6-phosphate accumulation (Maitra 1971).The accumulation of glucose-6-phosphate, the first metabolite in the d-glucaric acid pathway, could be advantageous for directing d-glucose flux from glycolysis to d-glucaric acid and thus improve the yield of d-glucaric acid on d-glucose.We introduced the d-glucaric acid pathway into a Pgi1p-deficient S. cerevisiae strain (Fig. 1) and studied d-glucaric acid production from monomeric and polymeric d-glucose substrates in shaken flasks and controlled bioreactor conditions with varying nitrogen concentrations.The formation of myo-inositol and d-glucaric acid, and the absence of d-glucuronate was confirmed by using 13 C-labelled d-glucose and GC-MS analysis.

Strains and strain construction
E. coli strains DH5alpha or TOP10 were used for cloning steps, plasmid propagation and storage, and the Saccharomyces cerevisiae strain FY834 for recombination cloning.S. cerevisiae CEN.PK2-1D (VW-1B; MATα, leu2-3/112 ura3-52 trp1-289 his3∆1 MAL2-8 c SUC2) renamed H1346, and the pgi1-deficient strain H2493, described previously (Verho et al. 2002) were the parental strains in which the d-glu- caric acid biosynthetic pathway was expressed.Strain H2493 was cured from tryptophan and/ or leucine auxotrophies by transforming it with PCR fragments amplified with primers BCoreMT_ LEU2_F and BCoreMT_LEU2_R or BCoreMT_ TRP1_F and BCoreMT_TRP1_R (Table 2), using genomic DNA from S. cerevisiae strain S288C as a template, and selecting colonies on plates lacking leucine and/or tryptophan, as appropriate.
The myo-inositol oxygenase encoding gene Miox from Mouse was obtained as a synthetic gene, codon optimized for S. cerevisiae (Gene Art, Germany).The INO1-Miox-uro1 B5310 plasmid was introduced to the parental strain H1346 (with intact PGI1) with or without the INM1 plasmid B5154, resulting in strains H4254 and H4355, respectively.The INM1 was integrated to the GRE3 locus of H1346 as described for the pgi1 strains, and the plasmid B5310 was introduced, resulting in strain H5156.The INO1-Miox-uro1 plasmid B5310 was introduced to the pgi1-deficient strain cured with TRP1 and LEU2, with or without INM1 integration, resulting in strains H4350 and H4344, respectively.The B5310 plasmid was also introduced to the pgi1-deficient strain cured for TRP, together with the INM1 plasmid B5154, resulting in strain H4346 or to pgi1-deficient strain with KMX integrated into GRE3, resulting in strain H4351.Corresponding control strains with empty vectors B712 + B548 H4345, and H4349 were also created.The final strains used in the study are listed in Table 3.

Media and culture conditions
Yeast strains were cultured in 20 or 50 mL volume on modified synthetic complete medium (YSC, (Sherman et al. 1983)) without uracil and/or leucine, with 2% (w/v) d-glucose or the d-glucose and d-fructose concentrations indicated in the results, in 100 or 250 mL Erlenmeyer flasks, respectively, at 250 rpm, 30 °C.The pgi1-deficient strains were grown with 0.5 g d-glucose L −1 and 20 g d-fructose L −1 as carbon source for maintenance and biomass generation.The (NH 4 ) 2 SO 4 concentration in YSC was reduced from 5 to 1.5 g L −1 for nitrogen-restricted batch cultures.The flask cultures with pgi1-deficient strains were buffered by CaCO 3 (1%, w/v).For slow glucose release, EnBase B (Biosilta, Oulu, Finland) was prepared according to manufacturer's instructions, except pH was adjusted to pH 5.6 with HCl and enzyme (5 µL to 20 mL culture broth) added after 24 h incubation, because d-glucose (~ 1 g L −1 ) was present in the prepared medium.Cellulose (α-cellulose, Sigma) was provided as 16.6 g L −1 , with 4.5 mL Cellulast 1.5 L added to a volume of 250 mL in bioreactor culture.
For larger scale cultures, yeast were grown in 250 to 500 mL medium (YSC-ura, or YSC-ura-leu) in Multifors bioreactors (max.working volume 500 mL, Infors HT, Switzerland) at pH 5.5, 30 °C, 1 volume air [volume culture] −1 min −1 (vvm) and 500 rpm agitation with 2 marine impellors, as previously described (Toivari et al. 2010a, b).The pH was maintained constant by addition of 2 M NaOH or 1 M H 2 PO 4 .Clerol antifoaming agent (Cognis, France, 0.08-0.10µL −1 ) was added to prevent foam formation.d-Glucose concentration in the culture supernatant was monitored by HPLC and d-glucose, d-fructose and/or ethanol were added as pulses to keep the d-glucose concentration between 0.5 and 5 g L −1 , while providing d-fructose or ethanol as an energy and carbon source for growth.
Biomass was measured as optical density (OD) at 600 nm (OD 600 ) or as dry weight.For dry weight, samples were collected in 2 mL pre-dried, preweighed microcentrifuge tubes, washed twice with equal volume distilled water and dried at 105 °C.
The number of metabolically active (vital) cells was determined microscopically by methylene blue (0.25 g L −1 in 0.04 M NaCitrate buffer pH 8.3) staining.For the purpose of clarity the metabolically active cells will be referred to as viable and the inactive as metabolically inactive cells.Both empty and stained cell were counted as metabolically inactive cells.

Chemical analyses
For determination of intracellular d-glucaric acid concentration, cells were collected from 10 mL culture.Cell pellets were washed with 1.8 mL of 0.9% w/v NaCl solution (9 g L −1 ), and then 1.8 mL deionised water, and frozen at − 20 °C to disrupt membranes.The frozen pellets were freeze-dried using a Christ Alpha 2-4 lyophiliser (Biotech international, Belgium), removing all excess moisture.Intracellular d-glucaric acid was extracted from the lyophilized pellets (6 to 45 mg biomass) in 5 mM H 2 SO 4 (0.5 mL) as described by Nygård et al. (2011) for extraction of d-xylonate.Cell debris was removed by centrifugation and the supernatant analysed by GC-MS.Intracellular concentrations are given as mg per g dry biomass.For a conservative estimate of intracellular concentration, assume that 1 g dry cell weight corresponds to 2 mL cell volume (de Koning and van Dam 1992; Gancedo and Serrano 1989).This estimate is conservative since it does not take into account the volume of intracellular organelles, variation in cell wall thickness, or the contribution of dead cells to the dry biomass.Concentrations of d-glucose and d-fructose, ethanol, glycerol, and acetate, were analysed by HPLC using a Fast Acid Analysis Column (100 mm × 7.8 mm, BioRad Laboratories, Hercules, CA) linked to an Aminex HPX-87H column (Bio-Rad Labs, USA) with 5 mM H 2 SO 4 as eluent and a flow rate of 0.3 mL min −1 .The column was maintained at 55 °C.Peaks were detected using a Waters 410 differential refractometer and a Waters 2487 dual wavelength UV (210 nm) detector.The ability of d-glucaric acid and its dissociated form d-glucarate to form glucaric acid 1,4-lactone or dilactone needs to be considered in the analytics.d-Glucaric acid, glucaric acid 1,4-lactone, d-glucuronate and myo-inositol were quantified with GC-MS.The d-glucaric acid concentration is presented as the sum of d-glucaric acid and glucaric acid 1,4-lactone.Samples (100 µL), with arabitol as internal standard, were evaporated to dryness and silylated by adding 100 µL pyridine, 100 µL chlorotrimethylsilane and 100 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA).Derivatisation was performed at + 70 °C for 60 min.Derivatised samples (1 µL) were subjected to GC-MS analysis (Agilent 6890 Series, USA combined with Agilent, 5973 Network MSD, USA and Combipal injector, Varian Inc., USA).Analytes were injected on split mode (30:1) (200 °C) and separated on a ZB-1HT INFERNO capillary column (30 m × 0.25 mm) with a phase thickness 0.25 µm (Phenomenex, Denmark).Helium (0.9 mL min −1 ) was used as carrier gas in constant flow mode.The temperature program started at 70 °C with 3 min holding time and then increased 10 °C min −1 up to 320 °C.Mass selective detector (MSD) was operated in electron-impact mode at 70 eV, in the full scan m/z 40-550.The ion source temperature was 250 °C and the interface was 280 °C.Compounds were identified according to corresponding standards and with the Palisade Complete 600 K Mass spectral library (Palisade Mass Spectrometry, USA).
d-Glucaric acid concentrations were also measured as the lactone using the hydroxymate method (Lien 1959) as described by Toivari et al. (2010b).The lactone assay was used for analysing samples from cultures grown with cellulose and although it correlated well with GC-MS results (Fig. 2), the assay would also measure d-gluconic acid, which was probably present in the sample.Thus, lactone measurements should be considered as indicative, but may be over-estimates.plasmid and INM1 either as the native genomic copy (H4344), from an additional integrated copy under a constitutive promoter (H4350), or from a multicopy plasmid (H4346) (Table 3).Strains H4349 or H4345 without the glucarate pathway were used as controls.Similar strains, with the d-glucaric acid pathway, but with intact PGI1 gene (H4354, H4355, H4356), were also created (Table 3).
The strains with intact PGI1 gene were grown in flask cultures with 2% (w/v) d-glucose as a sole carbon source.In 48 h at most 35 mg d-glucaric acid L −1 was produced (data not shown).
Although d-glucose inhibits the growth of pgi1deficient strains at concentrations above 2 g L −1 , concentrations up to 4 or 5 g L −1 could be tolerated in cultures with sufficient biomass (OD600 12).With 4 g d-glucose L −1 and 20 g d-fructose L −1 , strain H4344, with the d-glucaric acid pathway with endogenous INM1, produced 240 ± 11 mg d-glucaric acid L −1 in 98 h, whereas the control strain H4345 did not produce any d-glucaric acid.In addition, 370 ± 11 mg L −1 of myo-inositol was detected from strain H4344 compared to 100 ± 26 mg L −1 from H4345.All d-glucose and d-fructose were consumed, biomass, ethanol, acetate and glycerol were formed and partly consumed.
The other pgi1-deficient S. cerevisiae strains H4346, with the d-glucaric acid pathway with INM1 on a multicopy plasmid, and the control strain H4349 were cultivated in the presence of 3.4 g L −1 uniformly labelled d-glucose.To decrease biomass formation, lower d-fructose (12 g L −1 ) and ammonium sulphate concentrations were used with initial high biomass (OD600 13).In 73 h, 280 mg L −1 of labelled d-glucaric acid and 330 mg L −1 labelled myo-inositol was detected.Thus, altogether, 610 mg L −1 of 13 C-labelled d-glucose was directed to the d-glucaric acid pathway confirming formation of d-glucaric acid from d-glucose via myo-inositol.The d-glucaric acid pathway intermediate d-glucuronate was not detected.With the control strain H4349 no d-glucaric acid, or d-glucuronate, was detected.With higher d-glucose concentration the pgi1-deficient strains lost viability (data not shown).

Evaluation of different feeding strategies for production of d-glucaric acid in bioreactor cultures
The pgi1-deficient strains were further studied in bioreactor cultures with d-glucose pulses in nitrogen sufficient or restricted conditions.When pgi1deficient S. cerevisiae strain H4346 expressing the d-glucaric acid pathway was grown in pH regulated batch cultures at pH 5.5, 1.3 g d-glucaric acid L −1 (yield 0.12 ± 0.01 g d-glucaric acid [g d-glucose consumed] −1 ) were produced (H4346, Figs. 3, 4a).d-Glucaric acid was produced at a rate of approximately 7 mg L −1 h −1 .Myo-inositol was detectable in the culture supernatant after 45 h (0.06 g L −1 ) and accumulated in proportion (10 ± 1%) to d-glucaric acid to a final concentration of 0.15 g L −1 .Up to 0.7 g glycerol L −1 and 2.5 g acetate L −1 were also produced, but acetate was consumed when ethanol was not available as a carbon source.Only 4.7 ± 0.1 g biomass L −1 was produced from the 15.9 g d-fructose L −1 , 14.7 g d-glucose L −1 and 6.1 g ethanol L −1 which were provided (yield of biomass on substrate ~ 0.17 g [g substrate consumed] −1 ).
The intracellular concentration of d-glucaric acid was constant at 10 ± 1 mg [g biomass] −1 .Intracellular myo-inositol concentrations (6 ± 1 mg [g Fig. 4 S. cerevisiae pgi1-deficient strain with a pathway for conversion of d-glucose to d-glucaric acid (pathway genes expressed on multicopy plasmids, H4346), was grown in SC -ura -leu a nitrogen sufficient or b nitrogen-restricted medium at pH 5.5 with d-glucose, d-fructose and ethanol as carbon sources (upper graphs), added initially and as pulses at intervals, as shown.Lower graphs show the concentra-tions of d-glucaric acid, myo-inositol and biomass produced.Data on product formation by a strain with INM1 integrated (H4350) are also shown (small symbols) for nitrogen-restricted medium.Substrate concentrations and feeding were similar for H4350 and H4346 and are only shown for H4346 on the upper graph.Error bars (biomass) represent ± sem (n = 3) and where not visible were smaller than the symbol biomass] −1 ) were similar to those of d-glucaric acid, and the ratio of myo-inositol to d-glucaric acid in the cytoplasm was much higher than in the supernatant.
Methylene blue staining showed that 63 ± 2% of the cells were metabolically inactive after 70 h (Fig. 3).A high proportion of metabolically inactive cells was also observed for the pgi1-deficient parent strain without the d-glucaric acid pathway in flask cultures (58 ± 2%, after 43 h on SC medium with 12 g d-fructose L −1 and 0.5 g d-glucose L −1 and 92 ± 1% metabolically inactive cells with 12 g d-fructose L −1 and 3.4 g d-glucose L −1 ), indicating that the high level of metabolically inactive cells was a response to the relatively high concentration of d-glucose in the medium, not to d-glucaric acid production.Some cells adapted to the presence of d-glucose in the medium after ~ 90 h in the bioreactor and the proportion of metabolically inactive cells did not increase much after 70 h.
To lower the d-glucose toxicity by fed-batch cultivation the pgi1-deficient d-glucaric acid strains, H4350 and H4346 were grown with d-glucose and d-fructose in the feed.d-Glucose concentration was maintained at less than 2.2 g L −1 during the first 120 h feeding, with d-fructose concentrations less than 1.5 g L −1 The strains produced 0.32-0.34g d-glucaric acid L −1 after 117 h, with yield of 0.12 g d-glucaric acid [g d-glucose consumed] −1 , but no further production after 117 h.The d-glucose concentration increased after 117 h, reaching concentrations of 3.7-4.5 g L −1 by 160 h (data not shown).Cell viability was measured at 138 h, with 63 ± 4% and 71 ± 4% metabolically inactive cells for strains H4350 and H4346, respectively.
Polymeric substrates for production of d-glucaric acid To evaluate use of cheap polymeric substrates for production of d-glucaric acid by the pgi1-deficient S. cerevisiae strains a commercial polysaccharide and α-cellulose were used as carbon sources and hydrolytic enzymes used for d-glucose release during d-glucaric acid production.
The d-glucose slowly released from the commercial polysaccharide was rapidly consumed and no d-glucose was detected in the culture media in shake flask cultures.In 100 h 12 g L −1 of d-glucose (as approximated from medium without cells) was released.To decrease production of biomass the amount of nitrogen provided was reduced.d-Fructose provided as a carbon source was consumed during the first 20 h.The pgi1-deficient, d-glucaric acidpathway-expressing strains H4346 and H4351 produced 0.79 and 0.51 g L −1 d-glucaric acid within 100 h, respectively.Myo-inositol (~ 0.2 g L −1 ) and biomass were also formed.In bioreactors a maximum ~ 0.65 g d-glucaric acid L −1 was produced from the commercial polysaccharide, and 0.32 (pH 5.5) and 0.35 (pH 7) g d-glucaric acid L −1 was produced from α-cellulose within less than 250 h (Fig. 5a, lactone assay).In the cellulose pH 5.5 culture d-glucose started to accumulate from the beginning of the culture, and in the culture with the commercial polysaccharide after 50 h, whereas with cellulose pH 7 culture d-glucose remained low throughout the culture (Fig. 5b).

Discussion
In S. cerevisiae the major flux of d-glucose is through glycolysis, and only a small fraction, approximately 1-4%, is channelled to the PPP, depending on strain and culture conditions (Blank et al. 2005;Fiaux et al. 2003;Gancedo and Lagunas 1973;Maaheimo et al. 2001;Nidelet et al. 2016).In the pgi1-deficient S. cerevisiae strains, the block to glycolysis leads to accumulation of glucose-6-phosphate (e.g.Ciriacy and Breitenbach 1979;Heux et al. 2008;Maitra 1971).Possibly, this glucose-6-phosphate accumulation could increase the flux of glucose-6-phosphate to myo-inositol and further to d-glucaric acid.Indeed, the yield of d-glucaric acid on d-glucose (0.12 g g −1 ) in the bioreactor with pulsed d-glucose was four-fold higher in our pgi1-deficient strains compared to the value in a strain with intact Pgi1p reported by Gupta et al. (2016) and was further increased to 0.23 g g −1 (seven fold improvement over Gupta et al. (2016)) by restricting the nitrogen supply.Recently, Guo et al. reported a yield of 0.216 g g −1 (Guo et al. 2022), close to our best yield.However, considering that our strains accumulated inositol at 12-17% of the d-glu- caric acid concentration, it is clear that the yield could be even higher if all myo-inositol would be converted to d-glucaric acid.In S. cerevisiae, the PGI1 deletion alone increased the yield, compared to E. coli where the zwf deletion was also needed (Shiue et al. 2015).In yeast, deletion or downregulation of both PGI1 and glucose-6-phosphate dehydrogenase encoding gene ZWF1 has not been reported, but during preparation of this work Zhao et al. (2023) reported that downregulation of ZWF1 in S. cerevisiae improved d-glucaric acid titer by 22.4%.
In volumetric terms our pgi1-deficient strains produced about half of the d-glucaric acid concentration reported by Gupta et al. (2016) in shake flasks (average 0.26 compared to 0.54 g L −1 ), as confirmed by using 13 C-labelled d-glucose.The titer was improved to 1.3 g L −1 in the bioreactor by providing the d-glucose in pulses, which was higher than reported by Gupta et al. (2016), but lower compared to recently reported volumetric titers of up to 9.5 g L −1 (Table 1).Also, the highest yield was observed with restricted nitrogen supply, and in this condition the volumetric concentration was only 0.71 g L −1 .Interestingly, our parent strains with intact Pgi1p produced only ~ 30 mg L −1 of d-glucaric acid as measured with GC-MS.This in line with the results of Liu et al. (2016) who found that P. pastoris produced 108 mg L −1 d-glucaric acid on d-glucose, but much lower compared to other studies (Table 1).In our strains the OPI1 gene was intact.We hypothesized that expression of INO1 under a constitutive promoter is comparable to the effect of OPI1 deletion because Opi1p is reported to regulate INO1 at the transcriptional level (Henry et al. 2014;Ye et al. 2013).However, possibly the OPI1 deletion could improve d-glucaric acid production also in the pgi1-deficient strains by a still unknown mechanism.In addition, differences in the strain background, pathway genes, and culture conditions, e.g.aeration and medium composition, may contribute to the differences in D-glucaric acid amounts produced.
The S. cerevisiae pgi1-deficient strains do not tolerate d-glucose concentration above 2 g L −1 , potentially decreasing their viability, and suitability to larger scale processes.Our fed-batch cultures did not drastically improve viability and more optimised feeding strategies would be needed.The viability was better on polymeric substrate α-cellulose where hydrolytic enzymes were used to release d-glucose, but the rate of d-glucose release would require further optimization to increase d-glucaric acid production.Recently, consolidated bioprocess (CBP) with concomitant sugar release and conversion to products has been developed for d-glucaric acid production (Fang et al. 2022(Fang et al. , 2023;;Li et al. 2021).This could be an option for the pgi1-deficient strains.
d-Glucaric acid production with S. cerevisiae has developed rapidly during recent years, especially considering volumetric titers (Table1).New myoinositol oxygenases (Marques et al. 2020), and ways for improved viability (Guo et al. 2022) would be highly interesting to test in the pgi1-deficient S. cerevisiae.Also, uncoupling growth and production, or regulating the Pgi1p amount by synthetic promoters, gene switches, and/or degradation approaches like those implemented in E. coli (Brockman and Prather 2015;Gupta et al. 2017;Hou et al. 2020;Qu et al. 2018) could improve d-glucaric acid production and viability in the pgi1-deficient S. cerevisiae.
S. cerevisiae genomic DNA (from S288C or CEN.PK2-1D) was used to amplify the INO1 (YJL153C) and INM1 (YHR046C) genes (resulting amino acid sequences are the same in both genomes).The uronate a Originally reported in mol/mol b Reported only per steam exploded corn stover Results d-Glucose conversion to d-glucaric acid with pgi1-deficient S. cerevisiae The pgi1-deficient S. cerevisiae CEN.PK2-1D strains expressed INO1, Miox and uro1 from a multicopy

Fig. 2
Fig. 2 Correlation between d-glucaric acid measured by GC-MS and the lactone measured using the hydroxylamate assay, with background subtracted

Fig. 3
Fig. 3 d-Glucaric acid and myo-inositol concentrations (left), d-glucaric acid yield on glucose (middle) and the proportion of viable cells (right) in the culture after 70 h cultivation for two d-glucaric acid producing strains (H4346 and H4350).Cultures were grown in bioreactors at pH 5.5 with pulsed addition of d-glucose (with d-fructose or ethanol for energy) or in

Fig. 5
Fig. 5 d-Glucaric acid production in bioreactor cultures with polymeric substrates.a d-Glucaric acid production.b d-Glucose release and consumption.Cellulose with hydrolytic

Table 1
(Chen et al. 2020)ucaric acid by E. coli, yeast S. cerevisiae or P. pastoris in vivo.The highest extracellular volumetric titer and corresponding yield of a study are presented.Partly adapted from(Chen et al. 2020) Vol:. (1234567890)

Table 1
(continued) (Gueldener et al. 2002)92 al. 2010yo-inositol, while d-fructose and ethanol would allow growth and support the production of biomass.Glucose-6-phosphate can also be channeled to pentose phosphate pathway or to storage carbohydrates Vol.: (0123456789) dehydrogenase (Udh) gene of Agrobacterium tumefaciens (uro1, GI:223,717,948)(Yoon et al. 2009;Boer et al. 2010) was obtained from the plasmid described byBoer et al. (2010).Miox, INO1 and uro1 were cloned into pRS426 based (B712,(Christianson et al. 1992)) plasmid B5054(Salusjärvi et al. 2017) that has three promoter-terminator pairs pTEF1 and tADH1, pTPI1 and tCYC1, pPGK1 and tPGK1 by homologous recombination using primers BcoreMT_MIOX_F and BcoreMT_MIOX_R for Miox, BcoreMT_INO1_F and BcoreMT_INO1_R for INO1, and BcoreMT_ URO1_F and BcoreMT_URO1_R for uro1 (Table2), resulting in plasmid B5310.The INM1 gene was expressed either as a single copy genomic integrant or from a multicopy plasmid.INM1 dIII and ligating it to the HindIII site of YEplac181 resulting in plasmid B5154.For genomic integration the INM1 was targeted to GRE3 locus.The 315 bp and and 290 bp regions of GRE3 gene were amplified from the H1346 genomic DNA with primer pairs S.c GRE3 nt-5frw and S.c GRE3 nt310rev and S.c GRE3 nt677frw and S.c GRE3 nt966rev, respectively, where the numbers are relative to nucleotide A in ATG of the GRE3 gene.The BamHI site for cloning the gene expression cassette was included in the 315 bp region.The 315 bp region was ligated into a PvuII and BsiWI linearised plasmid pUG6(Gueldener et al. 2002), and the plasmid obtained was then cut with EcoRV and SpeI for introducing the 290 bp region.The resulting plasmid, containing the KanMX cassette flanked by the S.cerevisiae GRE3 regions, was named pMLV84.

Table 2
Primers used for strain construction