Metabolic engineering of Pichia pastoris for production of isobutanol and isobutyl acetate
© The Author(s) 2018
Received: 22 September 2017
Accepted: 21 December 2017
Published: 8 January 2018
Interests in renewable fuels have exploded in recent years as the serious effects of global climate change become apparent. Microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered Pichia pastoris, an industrial workhorse in heterologous enzyme production, to produce the biofuel isobutanol from two renewable carbon sources, glucose and glycerol. Our strategy exploited the yeast’s amino acid biosynthetic pathway and diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production. To further demonstrate the versatility of our yeast platform, we incorporated a broad-substrate-range alcohol-O-acyltransferase to generate a variety of volatile esters, including isobutyl acetate ester and isopentyl acetate ester.
The engineered strain overexpressing the keto-acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with 2-ketoisovalerate. To improve the production of isobutanol and eliminate the need to supplement the production media with the expensive 2-ketoisovalerate intermediate, we overexpressed a portion of the amino acid l-valine biosynthetic pathway in the engineered strain. While heterologous expression of the pathway genes from the yeast Saccharomyces cerevisiae did not lead to improvement in isobutanol production in the engineered P. pastoris, overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce 0.89 g/L of isobutanol. Fine-tuning the expression of bottleneck enzymes by employing an episomal plasmid-based expression system further improved the production titer of isobutanol to 2.22 g/L, a 43-fold improvement from the levels observed in the original strain. Finally, heterologous expression of a broad-substrate-range alcohol-O-acyltransferase led to the production of isobutyl acetate ester and isopentyl acetate ester at 51 and 24 mg/L, respectively.
In this study, we engineered high-level production of the biofuel isobutanol and the corresponding acetate ester by P. pastoris from readily available carbon sources. We envision that our work will provide an economic route to this important class of compounds and establish P. pastoris as a versatile production platform for fuels and chemicals.
Unsustainable demands and concerns over climate change have inspired interest in renewable fuels and chemicals [1–3]. In recent years, microbial production of high-energy fuels via environmentally sustainable and economically efficient bioprocesses has emerged as a viable alternative to the traditional production of transportation fuels . Although microbial fermentation of ethanol has played an important role for the transition to bio-based fuels, ethanol is not an ideal gasoline replacement due to its poor physicochemical properties. Specifically, ethanol has low energy density (~ 70% of the energy content of gasoline), high hygroscopicity and high vapor pressure . In contrast, branched-chain and higher alcohols, such as isobutanol and isoamyl alcohol, have superior energy density (at 90 and 110% of the energy content of gasoline, respectively) and are more compatible with the existing storage and distribution infrastructures. Moreover, branched-chain alcohols have higher octane numbers compared with their straight-chain counterparts and are therefore ideal gasoline substitutes for high performance petrol engines . A closely related class of compounds to branched-chain and higher alcohols is volatile esters. Importantly, because volatile esters typically contain either a floral or fruity scent, they are highly sought-after by the fragrance and cosmetic industries, which together account for a large global market of $16.6 billion in 2012 (http://www.ialconsultants.com/). Moreover, these esters can also be applied to solvents, coatings and paints.
Despite significant efforts in using metabolic engineering to improve natural producers of these alcohols and esters in the past, there has been little success in reaching commercially relevant titers and productivity . Indeed, commercial production of the vast majority of these alcohols in native organisms such as several Clostridium species is not economically feasible at present . Other disadvantages of using Clostridium species as a production host include their slow growth, their intolerance to oxygen and their production of the byproducts butyrate, acetone and ethanol . Therefore, development of a more efficient production platform in a non-native host for higher branched-chain alcohols and the corresponding acetate esters is urgently needed.
Overview of different microbial strains including Pichia pastoris (our work) engineered to produce isobutanol and isobutyl acetate
Strain name/host strain
Carbon source (g/L)
Specific growth rate (h−1)
Average titer (g/L)
Yield (mg/g carbon source)
Productivity (mg/L h)/maximum productivity (mg/L h)
LlkivD, ScADH2, BsalsS, ilvCD
350 (between 40 and 112 h)
Atsumi et al. 
LlkivD, ScADH2, BsalsS, ilvCD
Glucose (fed batch fermentation)
50.8 ± 1.1a
Baez et al. 
LlkivD, EcYqhD, BsalsS, EcilvCD Rubisco
Atsumi et al. 
ScIlv2_cytosolic, ScIlv3_cytosolic, ScIlv5_cytosolic, ScARO10, ScADH2
Brat et al. 
BY4741 × Y3929 (diploid)
ScIlv2, ScIlv3, ScIlv5, ScARO10_mitochondrial, adhA_mitochondrial
0.635 ± 0.023
6.7 ± 0.2
20.5 ± 1.2
Avalos et al. 
ScIlv2, ScIlv2_cytosolic, ScIlv3_cytosolic, ScIlv5_cytosolic, LlkivD, ScADH6, ScMAE1
Glucose (100) (Semi-anaerobic)
Matsuda et al. 
0.120 ± 0.002
0.037 ± 0.005
0.122 ± 0.001
0.049 ± 0.006
LlkivD, ScADH7, PpIlv5, PpIlv3
0.123 ± 0.001
0.048 ± 0.001
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized), PpIlv2 (codon optimized)
0.107 ± 0.002
0.885 ± 0.011
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized), PpIlv2 (codon optimized)
0.107 ± 0.001
0.200 ± 0.001
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized), PpIlv2 (codon optimized) and further overexpression of PpIlv6 and PpIlv2 by integration of another copy of the gene cassette
0.092 ± 0.002
1.699 ± 0.073
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized) and PpIlv2 (codon optimized) and further episomal-plasmid based expression of LlkivD and ScADH7
0.096 ± 0.001
1.716 ± 0.054
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized) and PpIlv2 (codon optimized) and further episomal-plasmid based expression of PpIlv5 and PpIlv3
0.108 ± 0.004
1.745 ± 0.090
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized) and PpIlv2 (codon optimized) and further episomal-plasmid based expression of PpIlv6 and PpIlv2
0.109 ± 0.002
2.221 ± 0.048
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized), PpIlv2 (codon optimized) and ScATF1
0.099 ± 0.001
0.006 ± 0.001
LlkivD, ScADH7, PpIlv5, PpIlv3, PpIlv6 (codon optimized), PpIlv2 (codon optimized) and episomal-plasmid based expression of ScATF1
0.091 ± 0.001
0.051 ± 0.007
Despite these considerable successes, E. coli and S. cerevisiae suffer from several disadvantages that make them unfavorable as production hosts for isobutanol and other higher alcohols. E. coli is susceptible to viral or bacteriophage infections . On the other hand, the Crabtree-positive S. cerevisiae suffers from low production yield due to the yeast’s inherent preference for ethanol production under high glucose conditions [18, 19]. While several strategies exist to minimize ethanol formation by engineered S. cerevisiae, most efforts only led to moderate increase in isobutanol yields . Given the shortcomings associated with higher alcohols production in E. coli and S. cerevisiae, we seek to establish Pichia pastoris, a Crabtree-negative methylotrophic yeast used industrially to produce recombinant proteins, as a production host for higher alcohols and the corresponding acetate esters.
For over a decade, P. pastoris has been extensively engineered to produce many industrially relevant enzymes such as xylanase, phytase, human serum albumin, hepatitis B surface antigen and glycoproteins with human-type N-glycosylation, many of which are already on the market [20, 21]. In particular, high-level heterologous expressions of various cellulase enzymes with comparable stability and activity to the native host have been achieved in P. pastoris [22–24]. In a recent example, Varnai and coworkers were able to obtain 3–5 g/L of secreted fungal endoglucanases using a recombinant P. pastoris system . We contend that the demonstrated robustness of P. pastoris in heterologous expression of cellulolytic and hemicellulolytic enzymes should make the organism an attractive host for consolidated bioprocessing of biofuels and other chemicals from biomass.
Despite the proven versatility of P. pastoris in industrial settings, biotechnological applications of the yeast have been mostly limited to recombinant enzyme expression. Indeed, examples of harnessing the yeast to produce industrially relevant biofuels and chemicals are scarce when compared to other industrial hosts such as S. cerevisiae and E. coli. In a recent example, P. pastoris was engineered to produce nootkatone, a sesquiterpene in great commercial demand for its aroma, by overexpressing the sesquiterpenoid pathway . However, despite this success, the best-engineered P. pastoris strain only produces ~ 200 mg/L of nootkatone in bioreactor cultivations. This titer is still an order of magnitude below a commercially relevant level.
In this work, we engineered P. pastoris to produce the advanced biofuel isobutanol and its corresponding ester, isobutyl acetate ester. We exploited P. pastoris’ endogenous amino acid biosynthetic pathway and channeled the amino acid intermediate, 2-ketoisovalerate, to the 2-keto acid degradation pathway for isobutanol production. Overexpression of the endogenous l-valine biosynthetic pathway genes led to a strain that is able to produce approximately 0.89 g/L of isobutanol directly from glucose. Further improvement of the production titer to more than 2.2 g/L was achieved by employing an episomal-plasmid based expression system to fine-tune the expression of all pathway enzymes. Finally, to demonstrate the versatility of our yeast platform, we further incorporated a broad-substrate-range alcohol O-acyltransferase to generate isobutyl acetate ester. We envision that our yeast platform will pave the way for an economic route to biofuels and chemicals.
Yeast strain, media and transformation
The yeast strains used in this study were constructed from P. pastoris KM71 (Invitrogen) (Table 1; Additional file 1: Table S1). The plasmids used in this study were generated from the pGAPZ_A and pPIC3.5K vectors (Invitrogen). Detailed description of plasmid construction is provided in Additional file 1. Yeast transformation was performed as previously described using an electroporator . The following parameters were used for the electroporation: 1.5 kV, 25 μF, 200 Ω. Colony PCR and DNA sequencing were used to verify strain construction. Yeast and bacterial strains were stored in 25% glycerol at − 80 °C. E. coli was grown in Luria–Bertani medium supplemented with ampicillin (at 100 μg/L), hygromycin (at 100 μg/L), kanamycin (at 50 μg/L), or zeocin (at 100 μg/L) when required. P. pastoris was grown in YPD medium (10 g/L yeast extract, 20 g/L Bacto Peptone and 20 g/L glucose) supplemented with hygromycin (at 200 μg/L), G418 (at 100 μg/L), or zeocin (at 100 μg/L) when required. Selection of P. pastoris transformants with HIS4 was done on a yeast minimal medium [MGY (pH 6.0) containing: 13.6 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.1 M phosphate buffer, 0.4 mg/L d-biotin, 133.3 mg/L thiamine-hydrochloride].
Screening of KDC and ADH candidate enzymes
Characterization of engineered P. pastoris strains overexpressing various KDC and ADH enzymes was carried out in a minimal medium [MGYH (pH 6.0) containing: 13.6 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.1 M phosphate buffer, 0.4 mg/L d-biotin, 133.3 mg/L thiaminchloride–hydrochloride, and 20 mg/L l-histidine]. Briefly, engineered strains were pre-cultured in 5-mL aliquots in MGYH medium overnight and used to inoculate 10 mL MGYH medium (either with or without 4 g/L 2-ketoisovalerate) in 50 mL Corning tubes to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken at 24 and 48 h to determine OD600, extracellular metabolites and production of higher alcohols.
The amount of isobutanol and other extracellular metabolites were determined using high-performance liquid chromatography (HPLC). Briefly, 1 mL of culture was centrifuged at 18,000g for 5 min and the supernatant was filtered through 0.45 μm nylon syringe filter (Filtrex). The purified sample was then applied to an Agilent 1100 series HPLC equipped with an Aminex HPX-87H ion exchange column (Biorad). The LC program was performed using 5 mM H2SO4 as the solvent at a flow rate of 0.72 mL/min for 42 min. The column was maintained at 55 °C. All metabolites were detected with Agilent 1200 series DAD and RID detectors.
Quantification of isobutanol production in engineered strains
Characterization of engineered P. pastoris strains was carried out in a minimal medium as described in “Screening of KDC and ADH candidate enzymes” section. l-Histidine was omitted from the medium when HIS4 was used as the selection marker; this medium is designated MGY. In the study comparing the different carbon sources, glycerol was replaced with glucose, resulting in a modified MGYH with glucose as a sole carbon source (designated MGYH-glu). In the study comparing different concentrations of glucose, the media were prepared with either 2 or 10% glucose and were designated MGYH-glu (2% glucose) and MGYH-glu (10% glucose), respectively.
Shake flask fermentation was also carried out in selected strains (KM71, PP100, PP200, PP300, PP310, PP302, PP303 and PP304). For this, engineered strains were pre-cultured in 5-mL aliquots in MGYH medium overnight and used to inoculate 50 mL MGYH-glu medium (at either 2 or 10% glucose as indicated) in 250 mL Erlenmeyer flasks closed with cotton wool plugs to achieve an initial optical density of 0.05 at 600 nm (OD600). The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken to determine OD600, biomass, extracellular metabolites and production of higher alcohols. The amount of isobutanol and other extracellular metabolites were determined using HPLC as described above.
Quantification of isobutyl acetate production in engineered strains
Quantification of isobutyl acetate ester in engineered strains was performed as previously described with some modifications . Engineered strains were pre-cultured in 5-mL aliquots in MGYH overnight and used to inoculate 10 mL modified MGY-glu medium in 50 mL Corning tubes as described in “Screening of KDC and ADH candidate enzymes” section. The yeast cultures were overlaid with 10 mL hexadecane (Sigma) to reduce evaporation of the acetate esters. The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken to determine biomass, extracellular metabolites and production of higher alcohols and acetate esters. The amount of acetate esters dissolved in the hexadecane layer was determined using gas chromatography–mass spectrometry (GC–MS). The GC program was as follows: an initial temperature of 40 °C was maintained for 4 min, followed by ramping to 300 °C at a rate of 45 °C per min, where the temperature was held for 3 min. The injector temperature was held at 250 °C. The injection volume was 5 μL, injected at a 10:1 split ratio. Hydrogen was used as the carrier gas. The MS is a GCMS-QP2010S (Shimadzu). The ion source temperature was 200 °C, and the interface temperature was 250 °C. The solvent cut time was 2 min. The start m/z was 40, and the end m/z was 500. Mass spectra and retention times from samples were compared with authentic standards (Sigma).
RNA isolation and transcript quantification
Strains were pre-cultured in 5-mL aliquots in MGYH medium overnight and used to inoculate 10 mL modified MGYH-glu medium as described in “Screening of KDC and ADH candidate enzymes” section. After 24 and 48 h, a 5-mL aliquot of each culture was collected and centrifuged for 5 min at 3000g. The pellets were washed with 5 mL of distilled water. Total RNA was extracted using the QIAgen RNeasy Kit under the manufacturer’s protocol. Contaminating genomic DNA was removed from the RNA samples by DNaseI (NEB) digestion using the manufacturer’s protocol. The RNA quantity was analyzed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and samples were stored at − 80 °C until RT-PCR analysis. cDNA was obtained using RevertAid Reverse Transcriptase (Thermo Fisher Scientific) using the manufacturer’s protocol. Relative expression levels of PpIlv6_CodOpt, PpIlv2_CodOpt, PpIlv5, PpIlv3, LlkivD_CodOpt, ScADH7 and ScATF1 were quantified using iQ SYBR Green Kit (Biorad) on CFX96 Touch Real-time PCR Detection System (Biorad). Real-time PCR was performed in triplicates, and PpACT1, a gene that encodes actin, was used to normalize the amount of the total mRNA in all samples.
Total DNA isolation and gene copy quantification
Strains were pre-cultured in 5-mL aliquots in MGY medium overnight in 50 mL Corning tubes as described in “Screening of KDC and ADH candidate enzymes” section. Total DNA was isolated using Wizard Genomic DNA purification kit (Promega) using the manufacturer’s protocol. The DNA quantity was assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and samples were stored at − 20 °C until real-time PCR analysis. Gene copy numbers of PpIlv6_CodOpt, PpIlv2_CodOpt, PpIlv5, PpIlv3, LlkivD_CodOpt, ScADH7 and ScATF1 were quantified using iQ SYBR Green Kit (Biorad) on CFX96 Touch Real-time PCR Detection System (Biorad) as previously reported with some modifications (Marx 2009). Real-time PCR was performed in triplicate, and normalization of the data was achieved using actin as a reference (i.e. PpACT1 gene copy number = 1). Primers used for RT-PCR are listed in Additional file 1.
Results and discussion
Overexpression of the 2-keto acid degradation pathway genes to enhance alcohol production from 2-keto acids
To metabolically engineer P. pastoris to produce higher alcohols, we employed a strategy that had been used successfully in E. coli and S. cerevisiae [8, 12–14]. Specifically, we exploited P. pastoris’ endogenous amino acid biosynthetic pathway and channeled the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production (Fig. 1). In the 2-keto acid degradation pathway (also known as the Ehrlich pathway), 2-keto acids are converted into higher alcohols in two enzymatic steps: decarboxylation of 2-keto acids to aldehydes by 2-keto acid decarboxylase (KDC), and subsequent reduction of aldehydes to alcohols by alcohol dehydrogenase (ADH).
We first searched for suitable KDC and ADH enzymes to enhance the endogenous activity of the Ehrlich pathway and to improve the higher alcohol production. Specifically, we screened three different candidates of KDC that have demonstrated high activity in either E. coli or S. cerevisiae. These are: KIVD from Lactococcus lactis, and ARO10 and THI3 from S. cerevisiae . For ADH, we screened two enzymes—both from S. cerevisiae—that have different substrate specificities and catalytic properties. These are: ADH6 and ADH7 . To assess the in vivo activities of these KDC and ADH candidates in P. pastoris, we supplemented the culture medium with 2-ketoisovalerate and quantified the production of isobutanol.
Overexpression of l-valine biosynthetic pathway genes to further increase the pool of 2-ketoisovalerate and subsequently the production titer of isobutanol
Analysis of the effects of carbon sources on isobutanol production
Improvement of isobutanol production by fine-tuning the expression of isobutanol biosynthetic pathway genes
To broaden the genetic toolbox for engineering the yeast P. pastoris, we created a set of episomal expression vectors by utilizing the recently discovered autonomously replicating sequence (panARS) from Kluyveromyces lactis (Additional file 1: Figure S4) [31, 32]. Camattari et al. demonstrated that vectors containing this 452-nt element outperformed the traditional integrative plasmids in heterologous enzyme expression, both in productivity as well as clonal homogeneity . We expanded this previous work by introducing the panARS sequence into four different expression plasmids: pGAPz (for zeocin selection), pGAPhyg (for hygromycin selection), pGAPneo (for G418 selection) and pGAPHis4 (for histidine auxotrophy). All of these plasmids use the strong constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) to express recombinant proteins.
Using PP300 as the parental strain, we introduced additional copies of the PpIlv5_T2A_PpIlv3, PpIlv6_T2A_PpIlv2, and LlkivD_T2A_ScADH7 expression cassettes by placing them on the episomal expression vector pGAPHisKlARS and transforming the individual plasmids into PP300. The resulting strains, PP302, PP303 and PP304, showed superior performance to the parental strain. In particular, PP304, which harbors the episomal plasmid containing the PpIlv6_T2A_PpIlv2 expression cassette, produced the highest titer of isobutanol at 2.22 ± 0.05 g/L (isobutanol yield of 22.2 ± 0.1 mg/g glucose, specific isobutanol production titer of 153 ± 9 mg/L/OD600). To the best of our knowledge, this is the highest reported isobutanol production titer and yield in a yeast system.
As was also the case for strain PP310, all strains harboring the episomal plasmids that contain additional copies of isobutanol biosynthetic pathway genes exhibited improved glucose consumption compared to strain PP300 (Figs. 7b, 8). Additionally, we observed elevated levels of the competing side-product ethanol (more than 10 g/L) in strains PP302, PP303 and PP304 when compared to the control strain KM71 (~ 3 g/L). The isobutanol pathway produces excess NADH (from glycolysis) and requires additional NADPH (from the reduction of isobutanal to isobutanol). Large accumulation of ethanol indicates that isobutanol production potentially caused redox cofactor imbalances, which ultimately forced the cells to use up the excess NADH by producing ethanol. Similar results have been observed in other microbial systems, particularly the Crabtree-positive S. cerevisiae. For example, Matsuda and coworkers have engineered S. cerevisiae to produce 1.6 g/L of isobutanol (16 mg/g glucose) by re-localizing the upper portion of valine metabolism to the cytosol and overexpressing the keto acid degradation pathway . The authors observed that the strain also accumulated high levels of ethanol, at approximately 40 g/L, 25-folds the level of the desired product isobutanol. Several strategies have led to lower ethanol accumulation and improvement in isobutanol production in S. cerevisiae. These include: (1) downregulating the pyruvate decarboxylase enzyme ; (2) overexpression of a transhydrogenase or constructing a transhydrogenase-like shunt to convert excess NADH to NADPH ; and (3) replacing the alcohol dehydrogenase with an NADH-dependent engineered mutant . These strategies could potentially improve isobutanol production in P. pastoris.
Overexpression of a broad-substrate-range alcohol O-acyltransferase to produce acetate esters of isobutanol and other higher branched-chain alcohols
In this work, we engineered P. pastoris, an industrial powerhouse in enzyme production, to produce the biofuel isobutanol and the volatile isobutyl acetate from glucose and glycerol, both of which are simple and renewable carbon sources. Our strategy exploited the yeast’s endogenous amino acid biosynthetic pathway and diverted the amino acid intermediates to the 2-keto acid degradation pathway for higher alcohol production. The engineered strain overexpressing the keto acid degradation pathway was able to produce 284 mg/L of isobutanol when supplemented with the pathway intermediate KIV. Further yield improvement was accomplished by overexpressing a portion of the amino acid l-valine biosynthetic pathway, leading to the engineered strain PP304 that is able to produce 2.22 g/L of isobutanol directly from glucose without the addition of 2-KIV to the culture medium. Finally, to demonstrate the versatility of our yeast platform, we further incorporated a broad-substrate-range alcohol O-acyltransferase to generate isobutyl acetate along with smaller quantities of other acetate esters. Episomal plasmid-based expression of the alcohol O-acyltransferase improved the production titer of isobutyl acetate to 51.2 mg/L, a ninefold improvement over the integrative plasmid-based expression. We envision that our work will pave the way for an economic route to this important class of compounds and establish P. pastoris as a versatile production platform for fuels and chemicals.
WR and ST conceived the study. WP, PW, TPK, JJD and WR constructed and characterized the engineered strains and performed the fermentation studies. WR and KK wrote the manuscript. All authors read and approved the final manuscript.
We thank NSTDA Characterization and Testing Center for their assistance with the GC–MS analysis.
The authors declare that they have no competing interests.
Availability of data and materials
Additional file 1 provides additional data.
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This work conducted by the National Center for Genetic Engineering and Biotechnolgy was supported by the Thailand Research Fund (TRF) under Contract No. TRG5880073.
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