- Open Access
Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer
© The Author(s) 2018
- Received: 3 January 2018
- Accepted: 24 April 2018
- Published: 5 May 2018
Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway.
A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae.
In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae.
- Glucaric acid
- Metabolic engineering
- Saccharomyces cerevisiae
- Delta-sequence integration
Glucaric acid, which was one of the “top value-added chemicals from biomass” announced by the US Department of Energy in 2004 , has been studied for therapeutic uses in cholesterol reduction  and cancer therapy  as well as for use as a building block for polymers like nylon . Currently, glucaric acid is produced mainly by chemical oxidation of glucose, with nitric acid as solvent and oxidant , which has led to low yields and toxic byproducts. Therefore researchers have been showing more interest in microbial production of glucaric acid in an effective and environmentally friendly manner.
Moon et al.  constructed a biological pathway to produce glucaric acid from glucose in Escherichia coli by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae, myo-inositol oxygenase (MIOX) from Mus musculus, and uronate dehydrogenase (Udh) from Pseudomonas syringae. Analysis of this metabolic pathway revealed that MIOX was rate-limiting, leading to a low glucaric acid titer of 0.72 g/L. To increase d-glucaric acid titer in E. coli, novel synthetic tools like synthetic scaffolds and a N-terminal SUMO fusion were used to increase MIOX activity. These two engineered strains produced 2.5 and 4.75 g/L d-glucaric acid from 10 g/L glucose and 60 mM myo-inositol [6, 7]. However, it appeared that a d-glucaric acid titer above 5 g/L would inhibit further production in E. coli due to a pH-mediated effect. Gupta et al.  transferred the synthetic glucaric acid pathway from E. coli to S. cerevisiae and found that myo-inositol availability for the At strain (S. cerevisiae containing miox4 gene from Arabidopsis thaliana) and MIOX activity for the Mm strain (S. cerevisiae containing MIOX from M. musculus) were rate-limiting. Maximum titer in the At strain was 1.6 g/L glucaric acid from glucose supplemented with myo-inositol. Liu et al.  constructed a glucaric acid synthetic pathway in Pichia pastoris using MIOX from P. pastoris itself or mice and Udh from Pseudomonas putida, resulting in glucaric acid titers of 107.19 ± 11.91 and 785.4 ± 1.41 mg/L respectively.
Strains and culture conditions
Strains and plasmids used in this study
Strains and plasmids
E. coli JM109
Stored in author’s laboratory
S. cerevisiae BY4741
MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
Stored in author’s laboratory
MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, opi1∆0
BY474 carrying pY26GPD-TEF-miox4-udh
Stored in author’s laboratory
BY474 opi1∆ carrying pY26GPD-TEF-miox4-udh
BY474 opi1∆ carrying pY26GPD-TEF-miox4-histag
BY474 opi1∆ carrying miox4 and udh
E. coli pUG6
E. coli plasmid with segment LoxP-KanMX4-LoxP
Stored in author’s laboratory
E. coli pSH65
Shuttle plasmid for E. coli and S. cerevisiae, Cre gene under GAL2 regulative regulation
Integrating plasmid, URA3 marker, MCS derived from
pBLUESCRIPT (ColE1 (derivative) ori, f1 ori, AmpR)
pY26GPD-TEF carrying miox4 and udh
pY26GPD-TEF carrying miox4-histag
Stored in author’s laboratory
Stored in author’s laboratory
For shake flask cultivation, 50 mL of YPD medium with or without 10 g/L myo-inositol in a 250 mL shake flask was used as the initial fermentation medium. Cultures were first grown overnight at 30 °C and 250 rpm in YPD medium with myo-inositol and then inoculated to a density at 600 nm (OD600) of 0.05. Cultures were incubated at 30 °C and 250 rpm for about 7 days.
The miox4  gene encoding MIOX4 enzyme in A. thaliana and the udh gene encoding Udh in P. syringae  were codon-optimized and synthesized by Genewiz (Suzhou, China). To construct the pY26-miox4 plasmid, the miox4 gene was first digested with BglII/NotI and then ligated to the pY26-GPD-TEF plasmid , which was digested with BglII/NotI to generate the pY26-miox4 plasmid. The udh gene was ligated to pY26-miox4 using the Gibson Assembly method to construct the pY26-miox4-udh plasmid. The miox4 gene with 6× his-tag was PCR-amplified, digested with EcoRI/XhoI, and ligated to EcoRI/XhoI-digested pY26-GPD-TEF to generate the pY26-miox4-6×His plasmid expressing the MIOX4-6×His fusion protein.
To knock out the OPI1 [20, 21] gene, which was functional in the negative regulation of Inositol-1-phosphate synthase (Ino1), the loxP-kan-loxP cassette  containing the upstream and downstream 50 bp of the OPI1 gene was amplified by PUG 6F and PUG 6R primers from the pUG6 plasmid and transformed into S. cerevisiae BY4741-competent cells through the Li–Ac method . The kanMX selection cassette was cured from successful deletion mutants using Cre recombinase expressed by the pSH47  plasmid. Then the deletion mutant was subcultured continuously for loss of pSH47, generating the BY4741 opi1∆ strain. To integrate exogenous genes into the genome, delta1 and delta2 [25, 26] fragments were amplified from the S. cerevisiae genome using genF-2/genR-2 and genF-5/genR-5 primers respectively, and the miox4 and udh expression cassettes TMA and GUC were amplified from the pY26-miox4-udh plasmid by genF-3/genR-3 and genF-4/genR-4 primers respectively. In addition, two parts of the selection marker HIS3, HIS3-1 and HIS3-2, were amplified from pRS313 using genF-1/genR-1 and genF-6/genR-6. The above PCR products were gel-extracted, and touchdown PCR was used to assemble two homologous arms containing delta1, TMA, HIS3-1 and HIS3-2, GUC, and delta2. Then the two homologous arms were transformed into a solid medium lacking histidine, and the high glucaric acid-producing strain was screened.
MIOX4 activity assay and immunoblot analysis
Bga-2 containing pY26-miox4-6×His plasmid was cultured in SD-Ura medium at 30 °C in a rotary shaker at 250 rpm up to OD600 of 0.6–0.8, then inoculated into 50 mL of SD-Ura medium using S. cerevisiae BY4741 containing pY26-GPD-TEF plasmid as a control. After 24 h cultivation, cell pellets were harvested and re-suspended in 50 mM Tris–HCl buffer (pH 7.0), then disrupted by ultrasonication and centrifuged at 13,000×g for 10 min. The supernatant was used for the enzymatic activity assay. The reaction system (200 μL) contained 50 mM Tris–HCl buffer (pH 7.0), 2.0 mM l-cysteine, 1.0 mM ammonium ferrous sulfate, 60 mM (10.8 g/L) myo-inositol, and a certain amount of enzyme. The reaction system without myo-inositol was pre-heated at 30 °C for 10 min and then proceeded at 30 °C, 200 rpm for 1 h after addition of myo-inositol. Then the reaction was stopped by boiling for 15 min, and the supernatant was extracted after centrifugation at 12,000×g for 10 min. The glucuronic acid concentration in the supernatant was determined by a chromogenic method using orcinol reagent (100 mL of 37% HCl, 0.1 g of orcinol, and 0.1 g of ferric trichloride). The supernatant and two times the volume of orcinol reagent were boiled together for 30 min and cooled to room temperature, after which OD660 was measured to quantify the glucuronic acid concentration . The reaction system without myo-inositol was set as a control to determine MIOX4 activity. The protein concentration was determined by the Bradford method using BSA as a standard. One unit of MIOX4 was defined as the enzyme content needed to convert 1 μmol myo-inositol in 1 min [28–30].
To analyze the expression level of MIOX4 protein, the Bga-2 recombinant strain was cultured in YPD medium to OD600 = 0.8, then inoculated into 50 mL of YPD medium and cultured for 48 h. The whole cell protein was extracted to perform a Western blot, after which 15 μg of total protein from each lysate was separated by 10% SDS-PAGE and transformed onto PVDF membranes (Sangon Biotech, Shanghai, China). After blotting, the membranes were incubated overnight in a 1:2000 dilution of anti-HIS antibody according to the manufacturer’s instructions. Western blot analysis was carried out as described previously .
Relative quantification of gene expression levels
To quantify gene expression levels, samples of approximately 108 cells were harvested after inoculation for 16 h. Total RNA was extracted from each of the samples using the RNAiso Plus RNA isolation kit (TaKaRa, Dalian, China) after liquid nitrogen grinding according to the manufacturer’s instructions. Following genomic DNA eraser treatment to remove DNA, cDNA was synthesized from 500 ng of total RNA using random primers with the PrimerScript RT reagent kit. The synthesized cDNA was then amplified in a quantitative PCR (qPCR) with primers Miox-a/Miox-s, Udh-a/Udh-s, and Pgk-a/Pgk-s respectively for relative quantification of mRNA levels of miox4 and udh, using the phosphoglucokinase gene (PGK1) as an internal control. qPCR reactions containing SYBR Premix Ex Taq were performed in a Thermo Scientific CFX96 instrument. Each reaction was carried out in triplicate, and the reported Ct value was the average of the triplicate samples. Transcript levels were calculated using the − ΔΔCt method .
Fed-batch fermentation and metabolite analysis
Slant culture was inoculated into 60 mL YPD medium and cultured at 30 °C, 250 rpm for 24 h to the exponential growth phase. Before fermentation, the rotation speed, ventilation volume, and DO were set to 600 rpm, 3 L/min, and 100% respectively. Then 60 mL seed culture was inoculated into a 5-L bioreactor containing 3 L fermentation medium, where pH was controlled at 4 ± 0.1 using 2.0 M HCl and 2.0 M NaOH and DO was controlled at 50% at 30 °C.
Fermentation samples were taken, filtered through a 0.22-μm membrane, and subjected to high-performance liquid chromatography (HPLC). Glucaric acid, lactic acid, ethanol, acetic acid, and glucose concentration were quantified by a Hitachi Chromaster using an Aminex HPX-87H column (300 mm × 7.8 mm; Bio-Rad Laboratories, Hercules, CA). Sulfuric acid (5 mM) was used as the mobile phase at 30 °C and a flow rate of 0.6 mL/min in isocratic mode. Compounds were detected from 30-µL injections using a refractive index detector.
Construction of the biosynthetic pathway to produce glucaric acid in Saccharomyces cerevisiae
miox4 derived from Arabidopsis thaliana
Increased glucaric acid titer through delta-sequence-based integrative expression
Increased MIOX4 activity and gene expression level by multi-copy integrative expression
Cell factories created by metabolic engineering and synthetic biology to produce fine chemicals have emerged as an increasingly popular alternative to chemical synthesis . Ever since the glucaric acid biosynthetic pathway was constructed in E. coli , great efforts have been expended to increase target product titer. Gupta et al.  reported that MIOX activity and myo-inositol availability were rate-limiting in glucaric acid production, not only in E. coli, but in S. cerevisiae. Therefore, the strategy to increase glucaric acid production was to improve MIOX activity and stability while increasing the myo-inositol flux to glucaric acid.
In S. cerevisiae, myo-inositol can be produced by converting d-glucose-6-phosphate by native inositol-1-phosphate synthase (Ino1), which is tightly regulated by Opi1 . In this study, the OPI1 gene was deleted to remove its negative regulation of myo-inositol synthesis. Although more myo-inositol was produced by S. cerevisiae, exogenous supplementation of myo-inositol was still necessary to increase MIOX4 activity (Fig. 2a) and glucaric acid accumulation. In addition, the constructed strain supplemented with 60 mM (10.8 g/L) myo-inositol apparently produced more glucaric acid than 10 mM (1.8 g/L) myo-inositol (Fig. 5a), which indicated that efficient production of glucaric acid at high titer needs a high concentration of myo-inositol might because the low affinity activity of the Itr1/2 myo-inositol transporter in S. cerevisiae . However, only a small proportion of the supplemented myo-inositol was transported into the cell (Additional file 1: Figure S2). When the myo-inositol residue was fed 60 mM (10.8 g/L) myo-inositol to a shake flask culture, only about 20% (2.16 ± 0.11 g/L) of myo-inositol was consumed (Additional file 1: Figure S2A). At least 3.26 g/L myo-inositol was required to produce 3.8 g/L glucaric acid in the shake flask culture. This means that at least 1.1 g/L myo-inositol was endogenously synthesized from glucose; the yields were 1.76 ± 0.08 g glucaric acid/g myo-inositol and 0.037 ± 0.003 g myo-inositol/g glucose. In the fed-batch fermentation of glucaric acid, about 3.6 ± 0.18 g myo-inositol was consumed (Additional file 1: Figure S2B), and about 5.14 g/L myo-inositol was required to produce 6.0 g/L glucaric acid. Therefore, at least 1.54 g/L myo-inositol came from glucose, and the yield was 1.67 ± 0.08 g glucaric acid/g myo-inositol, or 0.051 ± 0.006 g myo-inositol/g glucose. Robinson reported that the uptake activity of the Itr1p myo-inositol transporter was affected by growth phase in S. cerevisiae because it would reach a maximum at exponential and then decrease rapidly to minimum in the stationary phase, and this regulation was independent of OPI1 . Improving the transport ability of Itr1p through protein engineering may be very important.
Besides myo-inositol availability, low activity or instability of myo-inositol oxygenase is the key bottleneck in the biosynthetic pathway to glucaric acid. Given that the codon-optimized mouse-derived MIOX gene had low activity and instability according to Moon et al. , the miox4 gene from A. thaliana was selected for this study. MIOX4 showed relatively high stability compared with mouse MIOX, but the specific activity of episomal plasmid expression of miox4 was still very slow. To overcome this problem, delta-sequence-based integrative expression of miox4 and udh genes was carried out. A delta sequence is a kind of long-end repeated DNA sequence located in the reverse transcription transposon Ty of chromosome DNA in S. cerevisiae. Because there are approximately 425 delta sequences, delta sequence-based homologous recombination is more efficient than traditional methods for exogenous DNA integration into the S. cerevisiae genome, and the target gene was expressed more stably in the delta sequence because it avoided plasmid loss in the episomal expression strain . The miox4 transcription level as determined by quantitative real-time PCR was an indirect characterization of gene copies compared with that of the episomal plasmid. The transcription level of miox4 in integrative expression was 5.43 times that of episomal expression (Fig. 6). These results indicated that this multi-copy integrative expression method is efficient for overexpression of miox4 genes, resulting in obviously enhanced MIOX4 activity and stability and high glucaric acid titer. The authors believe that the delta-sequence integration method can be used in a wide range of low-copy genes to remove the bottleneck in metabolic engineering of S. cerevisiae.
In this study, glucaric acid titer in S. cerevisiae was increased by expressing a more stable MIOX4 from A. thaliana and integrating the target genes into the delta sequence of the genomes. Delta-sequence-based constitutive expression increased both the number of target gene copies and their stability and can be used for a wide range of metabolic pathway engineering projects in S. cerevisiae. The final strain produced 6.0 g/L (28.6 mM) glucaric acid, which is the highest titer reported in S. cerevisiae.
YD and YZ contributed to designing the experiments, analyzing the data, and writing the manuscript. NC and JW performed construction of plasmids and yeast strains and fermentation and enzyme assays. All authors read and approved the final manuscript.
We thank the Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University for LC–MS analysis. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.
The authors declare that they have no competing interests.
Consent for publication
The manuscript is original, has not already been published, and is not currently under consideration by another journal.
Ethics approval and consent to participate
This work was supported by Grants from the National Natural Science Foundation of China (31500070), the Natural Science Foundation of Jiangsu Province (BK20150136, BK20150151), and the Fundamental Research Funds for the Central Universities (JUSRP51705A).
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