- Open Access
Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe
© The Author(s) 2018
- Received: 17 April 2018
- Accepted: 7 November 2018
- Published: 13 November 2018
Economical production of value-added chemicals from renewable biomass is a promising path to sustainability. 3-Hydroxypropionic acid (3-HP) is an important chemical for building a bio-sustainable society. Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources.
Here, we describe metabolic engineering of the fission yeast Schizosaccharomyces pombe to enhance 3-HP production from glucose and cellobiose via the malonyl-CoA pathway. The mcr gene, encoding the malonyl-CoA reductase of Chloroflexus aurantiacus, was dissected into two functionally distinct fragments, and the activities of the encoded protein were balanced. To increase the cellular supply of malonyl-CoA and acetyl-CoA, we introduced genes encoding endogenous aldehyde dehydrogenase, acetyl-CoA synthase from Salmonella enterica, and endogenous pantothenate kinase. The resulting strain produced 3-HP at 1.0 g/L from a culture starting at a glucose concentration of 50 g/L. We also engineered the sugar supply by displaying beta-glucosidase (BGL) on the yeast cell surface. When grown on 50 g/L cellobiose, the beta-glucosidase-displaying strain consumed cellobiose efficiently and produced 3-HP at 3.5 g/L. Under fed-batch conditions starting from cellobiose, this strain produced 3-HP at up to 11.4 g/L, corresponding to a yield of 11.2% (g-3-HP/g-glucose; given that 1 g cellobiose corresponds to 1.1 g glucose upon digestion).
In this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.
- Schizosaccharomyces pombe
- 3-Hydroxypropionic acid
- CRISPR–Cas9 system
- Cell surface display
3-Hydoroxypropionic acid (3-HP) is a platform chemical that serves as a precursor to various valuable chemicals, including acrylic acid, acrylonitrile, and 1,3-propanediol. Acrylic acid, which is used as a super absorbent polymer and in plastic manufacturing, currently is produced primarily by catalytic oxidation of petroleum-derived propane. Acrylonitrile, a compound that is produced at more than 14 billion pounds per year, is used to generate plastics, resins, and fibers . Hence, the US Department of Energy has designated 3-HP as one of the most important substances that should be targeted for production from renewable resources . Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources.
Microbial bioproduction is one of the best solutions for building a sustainable economy. Four pathways (including those employing glycerol, lactate, malonyl-CoA, or β-alanine) have been defined for the production of 3-HP in vivo using microorganisms. Klebsiella pneumoniae and Lactobacillus reutei are known to produce 3-HP from glycerol . The mechanism of 3-HP production from glycerol consists of 2 steps. First, glycerol is converted to 3-hydroxypropionaldehyde (3-HPA) via a reaction catalyzed by a coenzyme vitamin B12-dependent glycerol dehydratase. Next, 3-HPA is converted to 3-HP via a reaction catalyzed by aldehyde dehydrogenase [4–7]. This route is not suitable for industrial use, because typical host production microorganisms [notably, E. coli and budding yeast (Saccharomyces cerevisiae)] cannot synthesize vitamin B12; such host organisms therefore would require a continuous exogenous supply of this cofactor [3, 8]. The 3-HP production pathway from lactic acid is thermodynamically unfavorable . Additionally, this reaction yields a mixture of lactate and 3-HP, a combination that is not suitable for industrial use . Both the malonyl-CoA and the β-alanine pathways for 3-HP synthesis employ thermodynamically favorable reactions that do not require coenzyme vitamin B12, making these pathways appealing for industrial use [10–13]. Notably, the NADPH-dependent malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus is widely used for 3-HP synthesis. Although MCR catalyzes a two-step reaction of malonyl-CoA to 3-HP via malonate semialdehyde , the imbalance of this cascade causes the accumulation of malonate semialdehyde, resulting in a low rate of conversion from malonyl-CoA to 3-HP. Separation of the MCR enzyme into two individual fragments [MCR-C (amino acids 550-1219) and MCR-N (amino acids 1-549)] has been shown to facilitate the rebalancing of the activities of MCR-C and MCR-N, yielding a drastic increase in 3-HP production (3.72 g/L in shaking flask cultivation) when expressed in E. coli .
Using acid-tolerant host cells is one of the most important techniques for making 3-HP production an economical process. A bacterial host such as E. coli or K. pneumoniae need pH control during fermentation, and recovery of the desired product subsequently requires acidification of the culture. Acid-tolerant hosts, such as yeasts, have the advantage that the acid form of 3-HP can be produced directly . Notably, budding yeast has been engineered for 3-HP production . However, the growth of S. cerevisiae is severely impaired in the presence of 50 g/L 3-HP . Alternatively, Schizosaccharomyces pombe is naturally tolerant to 3-HP and can grow even in the presence of 50 g/L 3-HP . However, there are only a few reports of 3-HP production using S. pombe as a host .
Improvement of precursor supply is necessary for chemical production. Acetyl-CoA is a precursor of 3-HP and of a wide range of bioproducts, including isoprenoids, fatty acids and lipids, and butanol. Several strategies have been reported for enhancing the level of acetyl-CoA in the yeast cytosol, including introduction of a PDH bypass [10, 18], blocking the glyoxylate pathway to decrease acetyl-CoA consumption , and enhancing the supply of CoA . In addition, supplying of other key metabolites for bioproduct production can be used to optimize the cultivation process. For instance, the Iriana and Nielsen groups successfully demonstrated 3-HP production using S. cerevisiae as a host with defined medium or feed-in-time medium . This feed-in-time medium provides carbon-limited cultivation, possibly because the medium contains high levels of polysaccharides in combination with cellulolytic enzymes, which gradually degrade the polysaccharide and release glucose . The titer of 3-HP on feed-in-time medium was high; however, this S. cerevisiae medium has to be specially synthesized and so is not readily available. In contrast, we have developed cell surface-display techniques for this purpose [21–23]. One of our constructs consists of a beta-glucosidase (BGL)-displaying S. pombe strain; this strain degrades cellobiose and provides glucose as a carbon source, permitting direct growth on cellobiose without the need for enzymatic supplementation . However, there are no reports (to our knowledge) of the production of acetyl-CoA- and malonyl-CoA-derived chemicals using BGL-displaying S. pombe.
Strains and media
Strains and plasmids used in this study
h90 ura4-D18 ade6-M210 leu1-32
h90 ura4-D18 ade6-M210 leu1-32 ku70Δ
Ozaki et al. 
12804 ku70Δ, isp6Δ::Pcam1-acc, integration of mcr gene at leu
12804 ku70Δ, isp6Δ::Pcam1-acc, ppp16Δ::Pef1a-c-mcr-C*, adh8Δ::Pef1-a-c-mcr-C*-Phsp-mcr-N
SPO-02, atg4Δ::Pcam1-acsSE*, adh4Δ::Pcam1-atd1
SPO-03, integration of Pcam1-ptk1 at leu
SPO-05, aap1Δ::Pcam1-atd1, fma2Δ::Pcam1-acsSE*
Vector under cam1 promoter control
RIKEN BRC 
Vector under ef1-a-c promoter control
RIKEN BRC 
Vector under nmt1 promoter control
RIKEN BRC 
Vector for cell surface-display expression of protein fusion of BGL from Aspergillus aculeatus with SPBC359.04c anchor protein
Ozaki et al. 
Vector for expression of mcr from Chloroflexus aurantiacus
Vector for expression of mcr-C* from Chloroflexus aurantiacus
Vector for expression of mcr-N from Chloroflexus aurantiacus
Vector for expression of cut6 from Schizosaccharomyces pombe
Vector for expression of acsSE* from Salmonella enterica
Vector for expression of acs1 from Schizosaccharomyces pombe
Vector for expression of atd1 from Schizosaccharomyces pombe
Vector for expression of ptk1 from Schizosaccharomyces pombe
Vector for expressing adh1:cas9/rrk1:sgRNA in fission yeast: empty sgRNA target
pMZ374 derivative, aap1(1266) sgRNA target
pMZ374 derivative, atg4(46) sgRNA target
pMZ374 derivative, fma2(950) sgRNA target
pMZ374 derivative, isp6(1069) sgRNA target
pMZ374 derivative, ppp16(83) sgRNA target
pMZ374 derivative, sxa2(1436) sgRNA target
pMZ374 derivative, adh4(100) sgRNA target
pMZ374 derivative, adh8(434) sgRNA target
Ozaki et al. 
pMZ374 derivative, gut2(1749) sgRNA target
Ozaki et al. 
Plasmid and homologous recombination (HR) donor construction
Plasmids used in this study are listed in Table 1, and primers are listed in Additional file 1: Table S1. Cas9 and gRNA expression plasmid pMZ374  was purchased from Addgene. A 20-base seed sequence together with the NGG PAM sequence (N20NGG) in the S. pombe genome was selected using CRISPR direct (http://crispr.dbcls.jp/) . The HR donor sequences used as editing templates were designed to have about 500-bp homology arms to either side (upstream and downstream) flanking the Cas9 cutting site and the sites of the intended insertions.
A Cas9 expression plasmid targeting the protease-encoding aap gene (bp 1-1266) was constructed using the KOD-Plus-mutagenesis Kit (TOYOBO, Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. pMZ374 was used as a template with the primer pair F-aap1-1266 + R-aap1-1266. The resultant plasmid was named pMZ374-aap1-1266. Other plasmids for Cas9 targeting were constructed in a similar way and are summarized in Table 1.
To construct the MCR expression cassette, the MCR-encoding gene from Chloroflexus aurantiacus was amplified by PCR using codon-optimized synthetic oligonucleotide (Invitrogen) as a template with the primer pair For-MCR + Rev-MCR. The PCR product was cloned into the NheI and SalI sites of pDUAL-FFH1 (RIKEN BRC), and the resultant plasmid was named pDUAL-FFH1-MCR. Other plasmids for expression of mcr-c*, acc, acs from Salmonella enterica (acsSE*), atd1, and ptk1 were constructed similarly; these plasmids are summarized in Table 1. The PCR templates for fragments encoding mcr-c* and acs from S. enterica were obtained as codon-optimized synthetic oligonucleotides (Invitrogen). The PCR products encoding acc, acsSE*, atd1, and ptk1 were cloned (separately) into pDUAL-FFH-31(RIKEN BRC); the PCR product encoding mcr-c* was cloned into pDUAL-FFH-61(RIKEN BRC). The PCR product encoding mcr-n was cloned between the NcoI and XhoI sites of pDUAL-hsp-SPBC359.04c_BGL . The resulting plasmid carries mcr-N under the control of the hsp promoter .
To construct HR donor DNA for introduction of the acc expression cassette into the chromosomal isp6 region, the acc expression cassette (Pcam-acc-Tadh1) was amplified by PCR with the primer pair For-isp6-1069-3 + Rev-isp6-1069-4. Upstream and downstream regions were amplified with the primer pairs For-isp6-1069-1 + Rev-isp6-1069-2 and For-isp6-1069-5 + Rev-isp6-1069-6, respectively. The three amplified fragments were conjugated by overlap extension PCR with the primer pair For-isp6-1069-1 + Rev-isp6-1069-6, resulting in HR donor DNA for introduction of the acc expression cassette into the chromosomal isp6 region. Other HR donor DNAs for introduction of fragments encoding mcr-c*, mcr-n, acc, acsSE*, atd1, ptk1, and bgl into the respective genomic regions were constructed similarly; these plasmids are summarized in Table 1.
All mutagenesis experiments were carried out by co-transformation of 10 µL of the pMZ374 vector series with 20 μL of the respective PCR product being used as the HR donor DNA for the target locus, as described in a previous report . Briefly, strains were grown in 5 mL of YM medium at 30 °C with shaking at 180 rpm until the culture reached an optical density (OD) at 600 nm of 0.5; cells then were co-transformed by electroporation using a Gene Pulser Xcell II (Bio-Rad) and standard methodologies. Transformants were selected using EMM+Leu plate, then screened by colony PCR and DNA sequencing.
Cultivation and analytical methods
For 3-HP fermentation from glucose or cellobiose, strains were pre-cultured in 5 mL YM medium for 3 days at 30 °C with shaking in a 15-mL test tube, then washed twice with 1% NaCl and diluted to an initial OD of 3.0 in 5 mL EMM medium supplemented with appropriate amino acids and containing glucose (50 g/L) or cellobiose (50 g/L or 20 g/L). The resulting cultures were incubated at 30 °C with shaking, under aerobic conditions, with sampling at the indicated time points. Cell growth, glucose, and cellobiose levels were analyzed as described previously (Ozaki et al. ). 3-HP and acetic acid levels were analyzed using an HPLC equipped with a SCR-102H column (7 µm, 8.0 mm ID × 300 mm; Shimadzu) as described previously .
SPO-07 was pre-cultured in 5 mL YM medium for 1 day; the preculture then was inoculated into 100 mL YM medium in a 1-L baffled flask and cultured for approximately 24 h. Cells were collected by centrifugation at 900g for 10 min and used to inoculate 400 mL EMM medium containing cellobiose as a carbon source in a 1-L jar fermenter. Fermentation was performed as follows: agitation rate, 400 rpm; temperature, 30 °C; aeration, 1 L/min with sterile air; pH 5.0, maintained by automatic addition of NH3(aq); dissolved oxygen (DO), 40%, controlled by agitation. For fed-batch cultivations, the feed was started after 24 h of cultivation. The feed contained 200 g/L cellobiose as a carbon source. The feed rate was set to 5 mL/h; a total feed volume of 0.2 L was used per reactor. In the case of glucose was used as a carbon source, initial concentration was 20 g/L and the feed contained 200 g/L glucose as a carbon source. Other conditions were the same as described above. 3-HP and metabolite analyses were as described above.
Reconstruction of a pathway for 3-HP synthesis via malonyl-CoA by coexpression of MCR-C* and MCR-N
To develop a S. pombe strain capable of malonyl-CoA-mediated production of 3-HP, we introduced two genes: C. aurantiacus mcr, which encodes malonyl-CoA reductase (MCR); and the endogenous acc (cut6) gene, which encodes acetyl-CoA/biotin carboxylase. MCR converts malonyl-CoA to 3-HP via malonate semialdehyde. ACC has a critical role in both malonyl-CoA supply and fatty acid biosynthesis. Earlier work suggested that strains deficient for intracellular proteases may exhibit improved protein expression [26, 27], a property that might also enhance 3-HP production. In the present work, mcr was introduced by complementation of a Leu- auxotrophy with a leu marker and cut6 (expressed under the control of ef1a-c constitutive promoter) was introduced by gene replacement at the protease-encoding isp locus. After a 48-h cultivation in EMM medium supplemented with 50 g/L glucose, a wild-type S. pombe strain did not produce 3-HP to a detectable level, whereas the mcr acc strain (designated SPO-01) generated 3-HP to a titer of 0.016 g/L (Fig. 1a).
Improvement of 3-HP production by increasing the supply of acetyl-CoA
To increase the malonyl-CoA supply, we sought to enhance the acetyl-CoA supply. Notably, engineering of the availability of acetyl-CoA has been reported to yield a positive effect on 3-HP production, presumably via the increase in the supply of malonyl-CoA. This strategy has been employed successfully in S. cerevisiae to improve the production of 3-HP [11, 28], butanol [29, 30], and isoprenoids [31, 32]. Specifically, these laboratories enhanced the flux towards acetyl-CoA by co-expression of a Salmonella enterica gene encoding acetyl-CoA synthase harboring a point mutation (referred to here as acsSE*) and a gene encoding an intact aldehyde dehydrogenase (S. cerevisiae ald6). To apply this approach in S. pombe, we introduced acsSE* (under the control of the cam1 promoter) into the protease-encoding atg4 locus of strain SPO-02. We then introduced a copy of the endogenous atd1 gene (a S. pombe homolog of the S. cerevisiae ald6 aldehyde dehydrogenase; also under the control of the cam1 promoter) into the adh4 locus (which encodes an alcohol dehydrogenase) of the resulting strain. We chose to target an alcohol dehydrogenase-encoding gene based on the observation that the loss of alcohol dehydrogenase activity permits accumulation of acetoaldehyde, a substrate of aldehyde dehydrogenase. However, given a previous report  that disruption of the major S. pombe alcohol dehydrogenase locus adh1 significantly impaired growth, we chose to target the adh4 locus, which encodes a minor alcohol dehydrogenase.
Engineering the CoA supply is an alternative to for improving the acetyl-CoA supply. Pantothenate kinase (PTK) is a key enzyme for improving CoA production . For example, the overexpression of mpanK1b (encoding a mouse PTK homolog) yielded a 13-fold increase of the intracellular CoA concentration in mammalian cells . Similarly, a fivefold increase of acetyl-CoA levels (with tenfold higher CoA production) was obtained by overexpression of the endogenous PTK in E. coli . In S. cerevisiae, overexpression of the endogenous PTK in combination with the introduction of acs and ald6 was reported to yield ~3.6-fold increased production of naringenin (a flavonoid) compared to the level obtained upon introduction of acs and ald6 alone . To implement this strategy, we introduced a copy of the endogenous gene encoding pantothenate kinase (ptk1) into SPO-03 by leu auxotrophic marker. The resultant strain (SPO-04) produced 3-HP of 1.1 g/L in after 48 h cultivation (Fig. 3a), a doubling of titer compared to SPO-03. These results indicated that improving the CoA supply is an effective approach for enhancing the production of malonyl-CoA-derived chemicals. Although intracellular amount of acetyl-CoA could not be detected by acetyl-CoA kit (data not shown), acetate accumulation at 48 h was increased from 2.4 g/L (SPO-03) to 4.7 g/L (SPO-04) (Fig. 3b; yellow) in the new strain. The introduction of additional copies of ptk1 did not further improve 3-HP production (data not shown), suggesting that acetyl-CoA is sufficient in strain SPO-04.
Improvement of 3-HP production by controlling the carbon supply
In an attempt to further improve 3-HP production, we introduced additional copies of acc and acsSE* into SPO-05. The resultant strain (SPO-06) produced 3-HP at 1.8 g/L of after 48 h of cultivation, a slight improvement compared to SPO-05. Although SPO-06 exhibited decreased acetate accumulation (1.6 g/L in 48 h cultivation), 3-HP production was not improved. This observation suggested that an improved malonyl-CoA supply was failed to enhance 3-HP production. Therefore, further improvement of MCR activity is desirable.
Re-tuning of MCR-C expression level
Fed-batch cultivation using cellobiose as a carbon source
In this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. 3-HP production was significantly improved through combinatorial strategies including coordination of the expression of MCR-N and MCR-C, overexpression of the acetyl-CoA synthetic pathway, and enhancement of the CoA synthesis pathway. The additional incorporation of BGL display and the use of cellobiose as a carbon source improved 3-HP titer to 11.2 ± 0.6 g/L, corresponding to a yield of 12 ± 0.07% (g-3-HP/g-glucose; 1 g cellobiose is equivalent to 1.1 g glucose) using fed-batch cultivation with cellobiose feeding. These results were superior to the peak 3-HP production previously reported in budding yeast using the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.
ST, AO and TT designed and performed the experiments. RK, CO, MK, and YH. performed the experiments. ST, AO, TM and TT wrote the paper. AK supervised the research. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional information files.
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Ethics approval and consent to participate
This work was supported primarily by a Grant-in-Aid for Young Scientists (A) (Grant Number 16H06132 to TT), and supported in part by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.
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