Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism
Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.
KeywordsBiobutanol Biofuel Acetyl-coenzyme A Saccharomyces cerevisiae Metabolic engineering Synthetic biology
In recent years, biobutanol has generated attention as a potential gasoline additive. Butanol (1-butanol, 2-butanol, and isobutanol) is sufficiently similar to gasoline to be blended with it at any ratio. It is compatible with existing pipeline infrastructure and is superior to ethanol as a fuel because of its higher energy content and lower volatility, hygroscopicity, and corrosiveness [1, 8]. The biological pathway for 1-butanol production is through acetone–butanol–ethanol (ABE) fermentation in the Clostridia species of bacteria. However, difficulties associated with clostridial fermentation, such as the formation of by-products and requirement for strictly anaerobic conditions, have driven various research efforts to reconstruct the butanol production pathway in more commonly used industrial microorganisms. This includes engineering of various bacteria for butanol production either by introduction of the clostridial butanol pathway [14, 18, 22, 23] or intermediate genes of amino acid pathways [17, 23]. Despite high titers obtained from some of these studies, several major drawbacks exist with the use of bacteria for industrial biofuel production. These include a complex separation process from the fermentation media, narrow and neutral pH growth rate [9, 12], and susceptibility to phage infections when grown on a large scale .
The use of the yeast Saccharomyces cerevisiae as a cell factory for biofuel production could overcome these limitations. S. cerevisiae is a robust industrial organism that can grow under various industrial conditions, including low pH and less stringent nutritional requirements . In addition, the larger size (as well as higher mass) of S. cerevisiae makes it easier to separate it from the fermentation media than bacteria, reducing process costs. Furthermore, S. cerevisiae is very well characterized, with a wide variety of tools and resources available for its genetic manipulation [16, 19]. Much information is available on S. cerevisiae, including a complete genome sequence, as well as characterization of its metabolic pathways [7, 20]. A previous attempt to engineer S. cerevisiae for 1-butanol production involved the introduction of butanol-pathway genes together with overexpression of the native thiolase gene to obtain butanol titers of 2.5 mg/l . Therefore, significant improvement is required to further facilitate the use of S. cerevisiae for butanol production.
A possible limiting factor to butanol production by S. cerevisiae is the availability of the precursor acetyl-CoA. Acetyl-CoA metabolism is highly compartmentalized in yeast and occurs in the cytosol, mitochondria, peroxisomes, and the nucleus. Cytosolic acetyl-CoA is produced via the pyruvate dehydrogenase (PDH) bypass and is derived from acetaldehyde, which is formed by the decarboxylation of pyruvate. However, during growth on glucose, the majority of the glycolytic flux is directed toward ethanol because of the Crabtree effect , limiting the availability of acetyl-CoA in the cytosol. Previous studies have shown that engineering the PDH bypass in S. cerevisiae enhanced the cytosolic acetyl-CoA supply, resulting in increased production of acetyl-CoA derived products such as the isoprenoids amorphadiene  and α-santalene , as well as the polymer polyhydroxybutyrate . Therefore, a similar strategy could potentially be applied for butanol production.
In the present study, we aimed to increase biobutanol production by S. cerevisiae by increasing the pool of available cytosolic acetyl-CoA and by evaluating alternative enzymes of the 1-butanol pathway. This strategy resulted in increased butanol titers in S. cerevisiae, suggesting the availability of cytosolic acetyl-CoA to be rate-limiting in butanol production.
Materials and methods
Strains and media
Escherichia coli DH5α was used for general cloning procedures in this study. Lysogeny broth (LB) medium was used for routine culturing with 80 mg/l ampicillin added when needed.
Yeast strains used in this study and relevant genotypes
All restriction enzymes used in this study were from Thermo Fisher Scientific (Waltham, MA, USA).
Description of plasmids used in this study
PTEF1-acsL641PPPGK1-ALD6 PTEF1-ERG10 PHXT7-ADH2
PTEF1-adhE2 PPGK1-ter PTEF1-crt PPGK1-hbd
Butyraldehyde dehydrogenase/butanol dehydrogenase (adhE2), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) sequences were from Clostridium beijerinckii. The trans-enoyl-CoA reductase (ter) sequence was from Treponema denticola. All these genes were codon-optimized for high levels of expression in yeast and synthesized by DNA 2.0.
Adhe2 was cloned into pSP-GM1 using NotI and PacI under the control of a TEF1 promoter. Ter was then cloned into the same plasmid under the control of a PGK1 promoter using BamHI and NheI. This resulted in the plasmid pAK0.
Crt was cloned into pSP-GM1 using NotI and PacI under the control of a TEF1 promoter, and hbd was then cloned into the same plasmid under the control of a PGK1 promoter using BamHI and NheI. A cassette containing both of these genes and their promoters was then amplified from this plasmid using the primers Kpn-TCYC (GTTGTTTCCGGATGTTACATGCGTACACGCGTC) and Mre-TADH (GAAGAACGCCGGCGGAGCGACCTCATGCTATACCTG), which contained Kpn2I and MreI restriction sites. This cassette was then cloned into pAK0, yielding pAK01.
The plasmid pCS01 was constructed by cloning the native ERG10 gene into pIYC04 using the enzymes SpeI and SacI. This gene was cloned under the control of a TEF1 promoter.
Engineered yeast strain generation and characterization
Strains AKY1, AKY2, and AKY3 were constructed by co-transforming CEN.PK113-11C, SCIYC33 and SCIYC32 (from Chen et al. ) with pIYC08 and pAK01. Strain AKY0 was constructed by transforming CEN.PK113-11C with pIYC04 and pAK01. Strain AKY4 was constructed by co-transforming CEN.PK113-11C with pCS01 and pAK01. Strains were selected on SD-URA-HIS plates.
Shake flask cultivation and analysis of butanol production
To test for butanol production from different strains, 20-ml cultures were started in 100-ml unbaffled flasks by inoculating an amount of pre-culture that resulted in a final optical density of 0.02 at 600 nm (OD600). The strains were grown at 30 °C with 180 rpm orbital shaking in defined minimal medium with the following composition: 7.5 g/l (NH4)2SO4; 14.4 g/l KH2PO4; 0.5 g/l MgSO4·7H2O; 2 ml/l trace metal solution [per liter, pH 4.0: EDTA (sodium salt), 15.0 g; ZnSO4·7H2O, 4.5 g; MnCl2·2H2O, 0.84 g; CoCl2·6H2O, 0.3 g; CuSO4·5H2O, 0.3 g; Na2MoO4·2H2O, 0.4 g; CaCl2·2H2O, 4.5 g; FeSO4·7H2O, 3 g; H3BO3, 1 g and KI, 0.1 g]. The pH of the mineral medium was adjusted to 6.5 by adding 2 M NaOH and autoclaved separately from the carbon source solution. Glucose was added at a concentration of 20 g/l. Vitamin solution (per liter, pH 6.5: biotin, 0.05 g; p-amino benzoic acid, 0.2 g; nicotinic acid, 1 g; Ca-pantothenate, 1 g; pyridoxine-HCl, 1 g; thiamine-HCl, 1 g and myo-inositol, 25 g) was filter sterilized and aseptically added to the medium after autoclaving at a concentration of 1 ml/l. To prepare the pre-culture, culture tubes containing 5 ml of defined medium (as described above) were inoculated with a single colony of strains of interest. These inocula were cultured at 30 °C with 200 rpm orbital shaking to an OD600 between 1 and 2.
To quantify 1-butanol levels, samples at different time points were collected, centrifuged, and filtered. Samples were then analyzed by high-pressure liquid chromatography (Dionex-HPLC, Sunnyvale, CA) equipped with an Aminex HPX-87H ion exclusion column (300 × 7.8 mm; Bio-Rad, Hercules, CA) and RI detector. Commercially available 1-butanol (Sigma-Aldrich, St, Louis, MO) was used as a standard. The HPLC was operated at 45 °C and a flow rate of 0.6 ml/min of 5 mM H2SO4.
Results and discussion
Maximal ethanol titers observed for the strains at the end of the glucose phase
6.2 ± 0.2
6 ± 0.1
5.5 ± 0.2
5.8 ± 0.1
6.4 ± 0.1
pAK01 does not contain a thiolase gene. This gene is necessary for the conversion of acetyl-CoA to acetoacetyl-CoA and represents the first step in the butanol pathway. We therefore co-transformed pAK01 with pCS01, a plasmid that encodes the native thiolase gene, ERG10. The resulting strain (AKY4) produced 6.6 mg/l of butanol, representing a 3.1-fold increase over the strain containing the heterologous genes only, suggesting levels of acetoacetyl-CoA to be limiting in AKY0.
We then further engineered S. cerevisiae for increased levels of cytosolic acetyl-CoA, which serves as a precursor for butanol production. This involved the use of plasmid pIYC08, which ensures overexpression of endogenous ADH2 encoding alcohol dehydrogenase, ALD6 encoding NADP-dependent aldehyde dehydrogenase, and a codon-optimized acs variant (L641P) from Salmonella enterica (acsL641P), encoding acetyl-CoA synthetase. While the native ACS enzymes Acs1 and Acs2 are subject to regulation via acetylation, acsL641P contains a point mutation that prevents the enzyme from being inhibited by acetylation, bypassing this regulation. Furthermore, the use of this variant was previously demonstrated to successfully redirect flux from acetaldehyde to acetyl-CoA in the cytosol to increase production of isoprenoids in yeast . In addition, this plasmid also leads to overexpression of ERG10. Co-expression of the butanol pathway with plasmid pIYC08 (strain AKY1) resulted in butanol titers of 10.3 mg/l, which represents a 4.9-fold increase in 1-butanol titers compared to AKY0 and a 1.6-fold increase compared to AKY4 (Fig. 2). These results demonstrate the importance of an adequate acetyl-CoA supply for 1-butanol production.
Clonal variation in butanol production
Max. butanol titers
The maximal titers obtained in the present study represent a 6.5-fold improvement in butanol production over previous values reported in yeast , and this therefore is an important proof of principle. However, further optimization is necessary to make yeast a commercially competitive host for butanol production. Steen et al.  have pointed to the step catalyzed by AdhE2 as a potential bottleneck. Further engineering efforts might therefore benefit from testing different AdhE2 variants that might have improved activity/solubility in the yeast cytosol. Another approach that has benefited isobutanol production in yeast involved the targeting of pathways to the mitochondria , and this approach might also be beneficial for 1-butanol production. Finally, another strategy that could be of potential interest is the deletion of ADH1 in butanol-producing strains to increase carbon flux toward butanol and away from ethanol production.
This work has been funded in part by the Chalmers Foundation, the Knut and Alice Wallenberg Foundation, and the European Research Council. C.S.A. is the recipient of an FPU predoctoral fellowship from the Spanish Ministerio de Educación. A. K. is a recipient of an Ångpanneföreningens Forskningsstiftelse project grant.
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