Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae
© The Author(s) 2018
Received: 4 July 2017
Accepted: 26 February 2018
Published: 9 March 2018
To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels.
We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose.
These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.
As a means of mitigating environmental issues, such as global warming and the depletion of fossil fuels, biofuels and products from sustainable biomass resources have received significant attention in recent years. In particular, alcohols have been extensively studied, since they are already available as next-generation fuels and represent the building blocks of other chemicals. In this study, we focused on 1-propanol, which is generically used as a solvent and as a food additive, is found in paint and cosmetics, and is a chemical intermediate in the production of n-propylamine . However, a natural microbial producer of 1-propanol has yet to be identified.
The budding yeast Saccharomyces cerevisiae is likely to be a good candidate for the production of 1-propanol. As S. cerevisiae has been used to produce ethanol, it is clear that the strain has tolerance to high concentrations of alcohols and other stresses during fermentation [2, 3], properties that should be useful for the industrial production of 1-propanol. Furthermore, S. cerevisiae strains that utilize not only glucose, but also xylose, as a sugar substrate have been developed to expand the range of applications of this biomass . For these reasons, S. cerevisiae may be more suitable for the production of 1-propanol than other microbial hosts.
Furthermore, others have reported that deleting the acetolactate synthase gene (ILV2), which directs a competing biochemical pathway, results in the production of 60 mg/L of 1-propanol in yeast . Therefore, in the present study, we aimed to develop a metabolically engineered S. cerevisiae strain suitable for the production of 1-propanol utilizing a combination of these approaches, specifically the overexpression of genes for 1-propanol biosynthesis, together with deletion of the competing metabolic pathway.
Strains, plasmids, and primers
Yeast strains constructed in this study
MATa URA3-52 LYS2-801 ADE2-101 TRP1-Δ63 HIS3-Δ200 LEU2-Δ1
MATa HIS3Δ1 LEU2Δ0 MET15Δ0 URA3Δ0
Plasmids used in this study
Source of reference
Yeast expression vector containing PGK1 promoter, 2 μ origin, HIS3 marker, no expression (control plasmid)
Ishii et al. 
Yeast expression vector containing PGK1 promoter, 2 μ origin, URA3 marker, no expression (control plasmid)
Ishii et al. 
Yeast integration vector containing PGK1 promoter, URA3 maker
Ishii et al. 
Yeast three gene expression vector containing ADH1, TDH3, and PGK1 promoters, 2 μ origin, LEU2 marker, no expression (control plasmid)
Yeast three gene expression vector containing ADH1, TDH3 and PGK1 promoters, 2 μ origin, TRP1 marker, no expression (control plasmid)
Yeast three gene expression vector containing ADH1, TDH3 and PGK1 promoters, 2 μ origin, HIS3 marker, no expression (control plasmid)
HIS3, expression of L. lactis kivd gene
Kondo et al. 
HIS3, expression of S. cerevisiae ARO10 gene
Kondo et al. 
HIS3, expression of S. cerevisiae THI3 gene
Kondo et al. 
URA3, expression of S. cerevisiae ADH1 gene
Kondo et al. 
URA3, expression of S. cerevisiae ADH2 gene
Kondo et al. 
URA3, expression of S. cerevisiae ADH5 gene
Kondo et al. 
URA3, expression of S. cerevisiae ADH6 gene
Kondo et al. 
URA3, expression of S. cerevisiae ADH7 gene
Kondo et al. 
URA3, expression of S. cerevisiae SFA1 gene
Kondo et al. 
URA3, expression of M. jannaschii cimA gene
URA3, genomic integration of M. jannaschii cimA gene
LEU2, co-expression of C. beijerinckii leuC and leuD genes
LEU2, co-expression of E. coli leuC and leuD genes
LEU2, co-expression of M. jannaschii leuC and leuD genes
LEU2, co-expression of M. jannaschii cimA, C. beijerinckii leuC and leuD genes
LEU2, co-expression of M. jannaschii cimA, E. coli leuC and leuD genes
LEU2, co-expression of M. jannaschii cimA, leuC and leuD genes
TRP1, expression of S. cerevisiae ILV1 gene
TRP1, expression of E. coli tdcB gene
TRP1, expression of E. coli ilvA gene
TRP1, co-expression of E. coli tdcB and asd genes
HIS3, co-expression of E. coli thrA, thrB and thrC genes
Primers used in this study
Deletion of competing pathway
GLY1 was disrupted according to the method of Akada et al. . Briefly, 300 bp of the 5′-flank of GLY1 was PCR amplified with a standard forward primer, and a reverse primer containing a 20 bp sequence of the 5′-flank followed by 40 bp sequence of the 3′-flank of GLY1. Separately, the URA3 marker cassette of pGK426 was PCR amplified with a forward primer containing a 20 bp overlap of the former PCR product and a reverse primer containing a 70 bp sequence of the 3′-flank of GLY1. Both amplified fragments were mixed and combined by PCR. The final PCR product was introduced into YPH499 using the lithium acetate method and the correctly integrated transformant was selected. The URA3 marker was then eliminated by counter selection with 5-fluoroorotic acid. Disruption of GLY1 and elimination of URA3 was confirmed by diagnostic PCR to check fragment sizes. The constructed strain, which has ΔGLY1 allele, was designated YPH499 ΔGLY1. Subsequently, double deletion strains with ΔGLY1 and other (ΔARO4, ΔALT1, ΔILV6, ΔCIT1 or ΔMET2) were constructed in common with deletion of GLY1.
Fermentation of engineered strains
The transformants were cultured for 48 h at 30 °C in 5 mL of SD minimal medium (6.7 g/L yeast nitrogen base without amino acids and 20 g/L glucose) containing the required amino acids. Following centrifugation at 3000 rpm for 5 min and removal of the supernatant, yeast cells were cultured in 5 mL of fresh SD minimal medium containing the required amino acids with/without 8 g/L 2 KB. The concentration of 1-propanol in the medium 72 h after the start of fermentation was determined using GC–MS (GCMS-QP2010 Plus; Shimadzu) following a previously described procedure .
For oxygen-limited fermentation, yeast transformants were anaerobically cultivated in SD minimal medium containing the required amino acids for 48 h at 30 °C. The cells were collected by centrifugation at 1000g for 5 min at 4 °C and washed twice with sterile water. The cells were then placed in 50 mL of SD minimal medium. The initial cell concentration was adjusted to OD600 = 20. All fermentations were performed at 30 °C with mild agitation in 100 mL closed bottles equipped with a bubbling CO2 outlet.
Results and discussion
Overexpression of 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase
The genes encoding these KDC and ADH enzymes were co-introduced into the YPH499 yeast strain in all possible combinations, and the transformants were fermented in SD selective media containing 8 g/L of 2 KB for 72 h. We found that all transformants showed similar productivities for 1-propanol (approx. 400 mg/L) (Fig. 2). The fact that overexpression of KDC and ADH in S. cerevisiae provided no advantage for the production of 1-propanol, indicates either that the selected enzymes did not have specific activity for the conversion of 2 KB into 1-propanol, or that endogenous yeast KDC and ADH enzymes already provide sufficient activity for this purpose. Given that the negative control strain (YA0K0; exogenously overexpressing neither KDC nor ADH) also produced 1-propanol, the latter is most probable. Indeed, engineered E. coli overexpressing ARO10 (from S. cerevisiae) or Kivd (from L. lactis) with ADH2 (from S. cerevisiae) have been shown to exhibit the activity required to convert 2 KB into 1-propanol .
Construction of a 2-ketobutyrate biosynthesis pathway via citramalate
Since S. cerevisiae appears to have sufficient KDC and ADH activity to convert 2 KB into 1-propanol, we next tried engineering yeast metabolic pathways to increase levels of 2 KB, the precursor of 1-propanol, using glucose as a carbon source. In E. coli, 2 KB is normally produced through the enzymatic conversion of threonine by threonine dehydratase. Engineered E. coli with increased 1-propanol productivity have been developed by introducing an artificial pathway via citramalate, which can convert pyruvate into 2 KB, in addition to the original threonine-mediated pathway (Fig. 1b) [15, 16]. Since E. coli has endogenous genes encoding citramalate hydrolyase (leuC and leuD) and 3-isopropylmalate dehydrogenase (leuB), the citramalate-mediated pathway can been completed by artificially expressing the citramalate synthase (cimA) gene derived from Methanococcus jannaschii (Mj). In S. cerevisiae, 2 KB is also produced endogenously via threonine (Fig. 1a), however, it does not carry the corresponding genes for citramalate synthase (cimA) or citramalate hydrolyase (leuC and leuD). Drawing on the experience from E. coli, we therefore constructed an artificial citramalate-mediated pathway to overproduce 2 KB from pyruvate and examined the productivity of 1-propanol in S. cerevisiae (Fig. 1b).
Although S. cerevisiae has an endogenous LEU2 gene that encodes 3-isopropylmalate dehydrogenase (encoded as leuB in E. coli), the laboratory yeast strains (YPH499 and BY4741) used in this study lack the functional LEU2 gene, as they are auxotrophs for the purposes of selection after gene transfection. Therefore, we used an expression plasmid carrying the LEU2 auxotrophic marker to compensate for 3-isopropylmalate dehydrogenase activity. For citramalate hydrolyase, we selected leuC (citramalate hydrolyase, large subunit) and leuD (small subunit) genes from three different sources: thermophilic methanogenic archaea, M. jannaschii (Mj), gram-negative and facultative anaerobic bacteria, E. coli (Ec), and the gram-positive and obligate anaerobe Clostridium beijerinckii (Cb). These gene pairs were introduced into the autonomously-replicating plasmid harboring the LEU2 marker. For citramalate synthase, which catalyzes 2 KB biosynthesis via citramalate from the central metabolite pyruvate, we used the cimA gene derived from M. jannaschii, which was successfully used in E. coli in the previous study [15, 16]. Two methods of expressing cimA were tested, the first being a single-copy genomic integration into the ura3 locus to stabilize gene replication, and the second being co-integration into the LEU2 marker plasmid along with leuC and leuD, in order to increase overall expression.
Overexpression of threonine dehydratase
Deletion of competing biochemical pathways
Next, we attempted to increase the production of 1-propanol by decreasing carbon flux into competing pathways for 2 KB and amino-acid metabolism. To do this, we used yeast strains with deletion of specific biochemical pathways from a single gene deletion library of BY4741. As shown in Fig. 1a, ILV2, ILV3, ILV5 and BAT1 are candidate target genes for the knockout of the biosynthetic pathway for valine, leucine and isoleucine, however there was no strain with deletion of these genes in the library. This indicates that the deletion of each of these genes is either lethal or results in poor growth, ruling out these genes as candidates for deletion in our study.
Comparing Fig. 5b with Fig. 3, it is clear that production of 1-propanol in YPH499 was higher than that of BY4741. Therefore, we subsequently constructed a YPH499ΔGLY1 strain to enhance 2 KB biosynthesis via both the citramalate and threonine pathways. As shown in Fig. 5c, YPH499ΔGLY1 (YG5040) demonstrated higher production of 1-propanol than wildtype YPH499 (Y5040). Furthermore, YPH499ΔGLY1 with expression of cimA, leuC, leuD, and tdcB (YG5C42) produced 68.3 mg/L of 1-propanol (Fig. 5c), whereas YPH499 harboring the same genes (Y5C42) produced 42.6 mg/L (Fig. 4). Thus, just as in BY4741, the deletion of GLY1 enhanced the production of 1-propanol in YPH499 yeast strains with modifications of both the citramalate and threonine pathways.
Overexpression of threonine synthase
Oxygen-limited fermentation of engineered strains
In the present study, we modified metabolic pathways of S. cerevisiae to engineer yeast strains producing 1-propanol. Firstly, we observed that the activity of endogenous yeast KDC and ADH is sufficient to convert 2 KB to 1-propanol. Secondly, we found that 1-propanol production could be increased by constructing an artificial 2 KB biosynthetic pathway from pyruvate via citramalate, with the introduction of cimA and leuC/leuD genes from M. jannaschii and C. beijerinckii, respectively. Furthermore, in addition to the overexpression of threonine dehydratase (with the introduction of tdcB), and enhancement of threonine biosynthesis from aspartate (with the introduction of thrA, thrB and thrC), 1-propanol production was greatly increased by deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. While the control YPH499 strain (Y5040) produced only 12 mg/L of 1-propanol in test tubes, the engineered strain YG5C4231 produced 99 mg/L. Moreover, in the context of high-density anaerobic fermentation, we succeeded in producing 179 mg/L of 1-propanol using this strain. These results demonstrate that construction of a citramalate-mediated pathway as the production method of 1-propanol in S. cerevisiae is effective. For yet further improvement of 1-propanol production in S. cerevisiae, it may be necessary to engineer the carbon flux from ethanol to 2 KB and oxidoreduction balance due to coenzyme. For example, as shown in Additional file 2 using yeast strains of Additional file 3, double deletion of competing pathway have the potential to increase 1-propanol production.
Conceived and designed the experiments: YN, JI, AK. Performed the experiments: YN and TM. Analyzed the data: YN. Wrote the paper: YN and JI. Supervised the whole work: AK. All authors read and approved the final manuscript.
This work was supported in part by a Special Coordination Fund 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; to AK), and by Science Research Grants from the Ministry of Health, Labor and Welfare, Japan (to AK).
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting the conclusions of this article are included with the article. Strains examined are available from the corresponding author.
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