World Journal of Microbiology and Biotechnology (2020) 36:48

https://doi.org/10.1007/s11274-020-02828-z

REVIEW

Butanol production by Saccharomyces cerevisiae: perspectives,

strategies and challenges
Suéllen P. H. Azambuja1 · Rosana Goldbeck1 

Received: 10 December 2019 / Accepted: 3 March 2020 / Published online: 9 March 2020
© Springer Nature B.V. 2020

Abstract
The search for gasoline substitutes has grown in recent decades, leading to the increased production of ethanol as viable
alternative. However, research in recent years has shown that butanol exhibits various advantages over ethanol as a bio-
fuel. Furthermore, butanol can also be used as a chemical platform, serving as an intermediate product and as a solvent in
industrial reactions. This alcohol is naturally produced by some Clostridium species; however, Clostridial fermentation
processes still have inherent problems, which focuses the interest on Saccharomyces cerevisiae for butanol production, as
an alternative organism for the production of this alcohol. S. cerevisiae exhibits great adaptability to industrial conditions
and can be modified with a wide range of genetic tools. Although S. cerevisiae is known to naturally produce isobutanol,
the n-butanol synthesis pathway has not been well established in wild S. cerevisiae strains. Two strategies are most com-
monly used for of S. cerevisiae butanol production: the heterologous expression of the Clostridium pathway or the amino
acid uptake pathways. However, butanol yields produced from S. cerevisiae are lower than ethanol yield. Thus, there are still
many challenges needed to be overcome, which can be minimized through genetic and evolutive engineering, for butanol
production by yeast to become a reality.

Keywords  ABE fermentation · Amino acid pathway · Butanol production · Butanol tolerance · Saccharomyces cerevisiae

Introduction when compared to ethanol; such as, higher energy density,

lower hygroscopicity, lower vapor pressure, and being an
Biofuels, produced from microbial fermentation, represent important solvent (Sakuragi et al. 2015; Chen and Liao
an important and promising option for gasoline substitu- 2016).
tion. This lead to the demand for ethanol increasing rapidly; Clostridium species, especially C. acetobutylicum, C.
becoming the biofuel most produced today and the most beijerinckii, C. saccharoperbutylacetonicum and C. sac-
used as a substitute for gasoline (Atsumi et al. 2008; Choi charobutylicum are the most used for n-butanol production
et al. 2014). However, ethanol is still not considered the best (Kushwaha et al. 2019). However, this species is genetically
substitute for fossil fuels, with butanol being a more suitable complex and presents problems that directly interfere with
alternative due to its physical properties; which are superior industrial production, such as: low growth speed, forma-
to that of ethanol and comparable to those of gasoline (Choi tion of spores and several by-products, low tolerance to
et al. 2014; Sakuragi et al. 2015). n-butanol, and phage contamination, besides being strictly
Butanol is a four-carbon alcohol having four isomeric anaerobic. For these reasons, other organisms more com-
forms, with n-butanol being (one of the isomers) subject monly used on an industrial scale have been genetically
to many studies. Butanol has many advantages as a biofuel modified for the production of n-butanol, such as Escheri-
chia coli and Saccharomyces cerevisiae (Steen et al. 2008).
S. cerevisiae is a yeast widely used in the food industry
* Rosana Goldbeck and also for fuel production, and is the main microorgan-
rosana.goldbeck@gmail.com
ism producing first generation ethanol in Brazil and North
1
Laboratory of Bioprocesses and Metabolic Engineering, America (Beato et al. 2016). Due to the adaptability under
Department of Food Engineering, School of Food industrial conditions and the range of genetic tools available
Engineering, University of Campinas, Rua Monteiro Lobato,
80, Campinas, SP 13083‑862, Brazil

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for this organism, S. cerevisiae has also become the subject Production of n-butanol can be accomplished through
of study for its ability to produce n-butanol. two methods, petrochemical or biological. When com-
However, the low tolerance of microorganisms to metabolic pared, the petrochemical method has the great advantage
products affects the recovery costs of the product and hinders of being carried out in only one step. From this, butanol
the industrial scale production of these metabolites (Liu and can be produced from ethanol, in the presence of catalysts,
Qureshi 2009). Once metabolic products begin to form, such as in a step involving three consecutive reactions, namely:
alcohols and acids, the microorganism must be able to tolerate dehydrogenation, aldol condensation and hydrogena-
the accumulation of these products in the medium, otherwise tion. In this sense, research involving the petrochemical
there may be a decrease in growth rate until cell death. route has focused on finding suitable catalysts, which can
S. cerevisiae is able to tolerate no more than 2% (v/v) increase the yield in butanol (Ndaba et al. 2015).
of n-butanol in the medium, as well as Clostridium strains. Through the biological method, butanol (also called
Thus, increased tolerance to butanol in S. cerevisiae strains bio-butanol) begins to be produced naturally by organ-
has been studied through genetic modification or evolution- isms of the Clostridiaceae family, through the so-called
ary engineering. Tools and methodologies have already acetone-butanol-ethanol (ABE) fermentation process,
been developed, in addition to the genetic improvement and with the production of these three compounds in the pro-
construction of strains capable of producing and tolerating portion of 3:6:1, respectively (Kuroda and Ueda 2015).
higher concentrations of butanol. The first report of biological butanol production was
Currently, the production of butanol by S. cerevisiae has published by the famous French scientist Louis Pasteur
been studied using two strategies: heterologous expression in 1861. He reported the production of C4-alcohol in a
of the Clostridium pathway and the pathways of amino culture named by him, Vidrion butyrique. However, it was
acid assimilation (Kuroda and Ueda 2015). However, while probably a mixed culture containing organisms similar to
promising, butanol production by S. cerevisiae still faces Clostridium (Dürre 2008; Sauer et al. 2016). In the early
many challenges. 1900s, there were companies and universities interested
in studying the production of synthetic rubber. Among
the researchers involved in these studies was the chem-
ist Chaim Weizmann, who concluded that synthetic rub-
Butanol as a biofuel ber should be produced from butanol or isoamyl alcohol
obtained by fermentation. Weizmann then isolated and
In the last decades, many studies have been carried out in studied an organism called BY, which was later named
the search to find for green and new energy to replace classi- Clostridium acetobutylicum (Jones and Woods 1986).
cal energy. In this way, many studies have achieved excellent From that moment, Clostridium species began to be more
results, with the development of technologies and microorgan- expressively studied and used for industrial production of
isms capable of producing large amounts of ethanol, mainly butanol, among other solvents.
from S. cerevisiae (Kuroda and Ueda 2015). In addition, there
is a growing search for microorganisms capable of using lig-
nocellulosic biomass for the production of this biofuel, called Butanol produced by Clostridium species
second generation ethanol (2G) (Brethauer and Studer 2015).
However, when compared to n-butanol, ethanol is still not the Few species of bacteria produce butanol as a major prod-
best substitute for gasoline (Hong and Nielsen 2012). uct, with the anaerobic bacterium Clostridium acetobutyli-
Butanol has advantages as biofuel compared to ethanol. cum being the most used species to obtain butanol (Dürre
The energy density of butanol is higher than that of ethanol, 2008). Since the first isolation of a Clostridium species,
and comparable to gasoline (Si et al. 2014). Butanol has
low hygroscopicity, making it less corrosive (Swidah et al. Table 1  Physicochemical properties of n-butanol, ethanol and gaso-
2015; Schadeweg and Boles 2016a), and therefore, can be line
transported through the pipeline infrastructure already in Propertie n-Butanol Ethanol Gasoline
place for gasoline (Si et al. 2014). Butanol has lower vapor
pressure and is also safer to handle. Ethanol can be mixed Energy density (MJ/L) 29.2a 21.2a 32.5a
with gasoline in up to 85% of the volume, while butanol Fusion point (°C)  − 89.3b  − 114c  − 40c
can be mixed at any ratio or used pure (Dürre 2007). Even Boiling point (°C) 117.7b 78° 27–225c
more, butanol is less water soluble compared to ethanol, Auto-ignition temperature (°C) 385d 423d 257c
making the butanol-gasoline mixture less susceptible to Density (g/mL) 0.8098b 0.79c 0.69–0.79c
phase separation (Amiri and Karimi 2019). Table 1 presents a
 Si et  al. (2014); bLee et  al. (2008); cYüksel and Yüksel (2004);
a comparison of butanol, ethanol and gasoline properties. d
Zhang et al. (2012)

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there has been a rise in solvent production through ABE Recent research has shown that it is possible to increase
fermentation, which is considered one of the oldest indus- the concentration of butanol obtained by Clostridium spe-
trial scale solvent productions (Jones and Woods 1986; cies through the integration of butanol recovery technologies
Schiel-Bengelsdorf et al. 2013). Clostridium species also during fermentation. An example of this was demonstrated
produces butyrate, in addition to acetone and ethanol, as by Chen et al. (2019), in which the authors used the Clostrid-
co-products, resulting in a lower final butanol concentra- ium sp. strain CT7 capable of using glycerol as a carbon
tion, mostly less than 20 g/L, in the traditional method source and producing butanol. The strain was cultured in a
of production. Which also adds to the maximum level of bioreactor initially with culture medium containing 60 g/L
toxicity to the bacteria. Due to low butanol yields, biologi- glycerol and three feeding operations with 20 g/L glycerol
cal production has a high cost for butanol recovery (Ndaba in fed-batch with membrane coupled pervaporation process
et al. 2015). In addition, Clostridiaceae family organisms and reached a final concentration of 41.9 g/L of butanol.
present low growth rates and spore formation, which causes In this way, the obstacles that still exist in the traditional
problems on an industrial scale (Atsumi et al. 2008). production of butanol by Clostridium species, have also
However, in addition to the limitations of using these spe- motivated the study of the use of industrially friendlier
cies to obtain butanol, the progression of the petrochemical organisms, which can be genetically modified for industrial
industry also led to the decline of the industrial production n-butanol production, as potential butanol producing organ-
of this alcohol via fermentation, between 1950 and 1960; isms. In the last decades, the interest in studying two poten-
whereas the production of butanol via microorganisms is not tially favorable and well-known organisms for the produc-
yet economically viable through the petrochemical method tion of this alcohol has increased, these are E. coli and the
(Gu et al. 2011; Jiang et al. 2015). On the other hand, due yeast S. cerevisiae (Schadeweg and Boles 2016a).
to the fluctuation of oil prices, ABE fermentation is still of
growing interest among researchers (Gu et al. 2011).
In the text published by Amiri and Karimi (2019), the Butanol production by Saccharomyces
authors describe and classify the existing obstacles to the cerevisiae
traditional manufacture of butanol into three categories:
substrate problems, process limitations, and strain shortcom- S. cerevisiae is a microorganism widely used as a model for
ings. When dealing with problems with substrate, one of the studies of other eukaryotic organisms. It is also called by
studied alternatives is the use of byproducts, lignocellulosic several authors as a microbial cell factory (Si et al. 2014) and
materials and syngas as low-cost substrates. In addition, in was the first eukaryotic organism to be fully sequenced. This
recent years the use of genetic engineering has grown, includ- cellular species has been the most used cellular organism
ing the use of CRISPR/Cas9 technique, to obtain more robust in the last decades for the industrial production of several
Clostridium strains, capable of producing butanol with higher bioproducts, as it is considered a robust organism and well
yields and productivity (Cheng et al. 2019), and with the adapted to industrial conditions. In addition, several spe-
development of specific tools for Clostridium species such as cific platforms for the S. cerevisiae species have been devel-
ClosTron (Heap et al. 2007) and flow-cytometric techniques oped to allow the production of new chemicals and fuels
(Tracy et al. 2008). In order to further minimize production (Hong and Nielsen 2012). Within this category of new fuels,
costs, the use of genetically modified Clostridium strains butanol has taken place in view of several research groups,
have been studied in conjunction with simultaneous butanol which are in search of strategies that make S. cerevisiae able
extraction technologies during cultivation (Lee et al. 2016). to produce large amounts of this alcohol.
In the work carried out by Huang et al. (2019), the authors It is known that, in addition to ethanol, S. cerevisiae
demonstrated the use of three strategies capable of solving the strains are capable of naturally producing isobutanol (one
obstacles described by Amiri and Karimi (2019), that is, use of the four isomers of butanol) by the synthesis pathway of
of low cost substrate (cassava bagasse hydrolysate), a geneti- 2-ketoisovalerate, an intermediate of the biosynthesis of the
cally modified strain (Clostridium tyrobutyricum overexpress- amino acid valine. Since 2-ketoisovalerate is synthesized by
ing an aldehyde/alcohol dehydrogenase gene, adhE2), and a the cell, it is converted to isobutanol via the Ehrlich pathway
different fermentation process (a repeated-batch fermentation and, for this reason, isobutanol is considered a byproduct of
with cells immobilized in a fibrous-bed bioreactor). In this valine synthesis (Generoso et al. 2015; Kuroda and Ueda
scenario, the strain was able to produce butanol with titer 2015). On the other hand, there was still some disagreement
greater than 15 g/L, yield of 0.30 g/g and productivity of among authors about the existence of a wild-type pathway of
0.3 g/L.h. In the end, the authors also carried out an economic n-butanol production in S. cerevisiae (Si et al. 2014).
analysis and showed that the use of cassava bagasse as a low- In view of the growing interest in the production of
cost substrate is economically competitive with traditional n-butanol by S. cerevisiae and the possibility of an endog-
food-based production. enous metabolism of this yeast that is capable of producing

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this alcohol, many researchers have spared no effort to cerevisiae strains with a clean background. The first authors
understand the cellular metabolism of this species in terms to report the construction of this pathway were Steen et al.
of fermentative capacity for n-butanol production. A consen- (2008). In this work, the authors tested the insertion of sev-
sus exists among researchers in saying that it is possible to eral isoenzymes (Table 2) that catalyzed different reactions
explore two metabolic pathways for n-butanol production by in the metabolic pathway for the production of n-butanol in
S. cerevisiae. The first of these is the heterologous expres- S. cerevisiae BY4742 (strain derived from S288C). Using
sion of the Clostridium n-butanol pathway and the second this strain as background, the authors observed a concentra-
through amino acid assimilation pathways (Si et al. 2014; tion of 2.5 mg/L n-butanol, with the best modified strain,
Schadeweg and Boles 2016a); or even, the combination of from galactose (2%) as the sole carbon source (Table 3).
the two strategies (Fig. 1). From this work, others came up with the proposal to use
the same strategy, but in different strains and culture media,
besides the insertion and deletion of different enzymes to
Heterologous expression of the Clostridium increase n-butanol concentrations (Tables 2, 3).
n‑butanol pathway in S. cerevisiae In the study by Krivoruchko et al. (2013), the authors
demonstrated that, in addition to the pathway reconstruc-
The heterologous expression pathway is based on the idea of tion, the improved flux towards cytosolic acetyl-CoA (the
reconstructing a pathway by inserting the enzymes responsi- precursor metabolite for 1-butanol biosynthesis) is of
ble for the Clostridium n-butanol production pathway into S. utmost importance for increasing the final concentration of

Fig. 1  Simplifed endogenous and exogenous metabolic pathways for oruchko et  al. 2013; Lian et  al. 2014; Si et  al. 2014; Sakuragi et  al.
butanol production in S. cerevisiae. Only genes from the relevant 2015; Swidah et al. 2015, 2018; Schadeweg and Boles 2016a, b; Shi
steps for butanol production were shown, including heterologous et al. 2016). Sequential arrows indicate contraction of the glycolysis
genes (gray background box). Co-factors are omitted for simplic- pathway. Dotted lines refer to the strategy used by Shi et  al. (2016)
ity. The information of biochemical pathways and enzyme locations with the introduction of a citramalate synthase (CimA) gene
is from literature (Steen et  al. 2008; Branduardi et  al. 2013; Kriv-

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Table 2  List of genes inserted in S. cerevisiae strains for n-butanol production

Gene Host Function Reference

Aad C. acetobutylicum Aldehyde-alcohol dehydrogenase Sakuragi et al. (2015)

Acs Salmonella enterica Acetyl-CoA synthetase Krivoruchko et al. (2013) and Lian et al. (2014)
Ad C. acetobutylicum Aldehyde dehydrogenase Sakuragi et al. (2015)
Adh2 S. cerevisiae Alcohol dehydrogenase Krivoruchko et al. (2013)
AdhE2 C. acetobutylicum Alcohol dehydrogenase Schadeweg and Boles (2016a, b) and Schadeweg and Boles
(2016b)
AdhE2 C. beijerinckii Alcohol dehydrogenase Steen et al. (2008), Krivoruchko et al. (2013) and Swidah et al.
(2015)
Ald6 S. cerevisiae NADP-dependent aldehyde dehydrogenase Krivoruchko et al. (2013)
BdhB C. acetobutylicum NADH-dependent butanol dehydrogenase B Lian et al. (2014)
Ccr Streptomyces collinus Crotonyl-CoA reductase Steen et al. (2008) and Krivoruchko et al. (2013)
Crt C. acetobutylicum 3-hydroxybutyryl-CoA dehydratase Sakuragi et al. (2015), Schadeweg and Boles (2016a, b) and
Schadeweg and Boles (2016b)
Crt C. beijerinckii 3-hydroxybutyryl-CoA dehydratase Steen et al. (2008), Krivoruchko et al. (2013), Lian et al. (2014)
and Swidah et al. (2015)
Erg10 S. cerevisiae Acetyl-CoA C-acetyltransferase Steen et al. (2008), Krivoruchko et al. (2013), Swidah et al.
(2015), Schadeweg and Boles (2016a, b) and Schadeweg and
Boles (2016b)
EutE E. coli Butyraldehyde dehydrogenase Lian et al. (2014), Schadeweg and Boles (2016a, b) and
Schadeweg and Boles (2016b)
Hbd C. acetobutylicum 3-hydroxybutyryl-CoA dehydrogenase Lian et al. (2014), Sakuragi et al. (2015), Schadeweg and Boles
(2016a, b) and Schadeweg and Boles (2016b)
Hbd C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase Steen et al. (2008), Krivoruchko et al. (2013) and Swidah et al.
(2015)
Ter Treponema denticola Trans-enoyl-CoA reductase Krivoruchko et al. (2013), Lian et al. (2014), Sakuragi et al.
(2015), Swidah et al. (2015), Schadeweg and Boles (2016a, b)
and Schadeweg and Boles (2016b)
Thl Candida tropicalis Thiolase Sakuragi et al. (2015)
Thl C. acetobutylicum Thiolase Lian et al. (2014)

Table 3  S. cerevisiae strains constructed for n-butanol production by heterologous expression of the Clostridium pathway
Strain Characteristics Butanol Reference
production
(mg/L)

ESY7 MATα-his3Δ1-leu2Δ0-lys2Δ0-ura3Δ0: pESC-ERG10-hbd-crt + pESC-ccr- 2.5 Steen et al. (2008)

adhe2
AKY3 MATa-SUC2-MAL2-8c-ura3-52-his3-D1-cit2D: pAK01-adhE2-ter-crt- 16.3 Krivoruchko et al. (2013)
hbd + pIYC08-acsL641P-ALD6-ERG10-ADH2
JL0534 MATa-his3D1-leu2-3–112-ura3-52-trp1-289-MAL2-8c-SUC2-ΔGPD1– 120 Lian et al. (2014)
ΔGPD2–ΔADH1–ΔADH4: pRS426-CaThl-CaHbd-CbCrt-TdTer-EcEutE-
CaBdhB + pRS414-EcLpdA-EcAceE-EcAceF + pRS425-SeAcsL641POpt
Strain #4 MATa-ade2-1-his3-11,15-leu2-3,112-trp1-1-ura3-1-can1-100: pRS406-thl- 14.1 Sakuragi et al. (2015)
hbd-crt + pRS403-ter + pRS405-ad-aad
A6A2 ­BR MATa-ade2-1-his3-11,15-leu2-3,112-trp1-1-ura3-1-can1-100: TRP1-Acs2- 300 Swidah et al. (2015)
adh1Δ + 5 g Ald6-Erg10-TRP1 + Integ-Adhe2-Bcd-Hbd-Crt-Ter-Integ*
VSY10 MATa-ura3-52-trp1-289-leu2-3,112-his3Δ1-MAL2-8C-SUC2 adh1::loxP; 130 Schadeweg and Boles (2016a, b)
adh3::loxP; adh5::loxP; adh4Δ::loxP; adh2Δ::LEU2; sfa1Δ:adhEA267T/E568K;
adh6Δ:coaA; gpd2Δ: ERG10-hbd-crt-ter-adhE2-eutE
VSY19 MATa-ura3-52-trp1-289-leu2-3,112-his3Δ1-MAL2-8C-SUC2 adh1::loxP; 859.05 Schadeweg and Boles (2016b)
adh3::loxP; adh5::loxP; adh4Δ::loxP; adh2Δ::LEU2; adh6Δ::coaA, loxP;
sfa1Δ::adhEA267T/E568K/R577S, loxP; pFMS1Δ::HIS3, pADH1; ald6Δ; gpd2Δ:
ERG10-hbd-crt-ter-adhE2-eutE + pRS62H_ter

*The site of integration was not specified in the reference

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n-butanol. Initially, the authors inserted the same enzymes showed that the VSY10 strain was able to produce n-butanol,
as the study carried out by Steen et al. (2008), however they but only half of the glucose was consumed, due to the inef-
were unable to detect butanol production in this strain. Then, ficiency of NADH re-oxidation. Thus, in the next study,
the authors proposed replacing the crotonyl-CoA reductase Schadeweg and Boles (2016b) repeated the cultivation of
(ccr) gene with a NADH-dependent crotonyl-CoA-specific this same strain, however under more aerobic conditions,
trans-enoyl-CoA reductase (ter) from Treponema denticola, reaching final concentration of 235 mg/L n-butanol. From
resulting in a strain capable of producing 2.1 mg/L butanol, this, different strategies were used to construct a strain capa-
a concentration comparable to the previous work. From this, ble of producing the highest concentration among the pub-
the authors also proposed deletions in the CIT2 and MLS1 lished works, up until that time by S. cerevisiae, the VSY19
genes to reduce the use of acetyl-CoA via the glyoxylate strain (Table 3) with 859.05 mg/L n-butanol, via a synthetic
pathway and noted that these deletions contributed to cyto- ABE pathway.
solic acetyl-CoA supply. After several modifications, the In summary, the authors have observed that one of the
best producer strain (Table 3) had a final n-butanol concen- remaining challenges is that the mechanisms of n-butanol
tration of 16.3 mg/L, an increase of 6.5-fold when compared production still compete strongly with the formation of other
to the strain of work performed by Steen et al. (2008). primary metabolites, such as ethanol and glycerol. There-
Lian et al. (2014) developed a S. cerevisiae strain capable fore, in addition to the insertion of different enzymes from
of increasing levels of acetyl-CoA by combining the inser- the Clostridium pathway, researchers still need to delete
tion of heterologous genes and deletion of competitive path- pathways responsible for the production of these primary
ways (ADH1 and ADH4 for ethanol production and GPD1 metabolites and further increase the availability of coenzyme
and GPD2 for glycerol production) (Table 2). After different A and cytosolic acetyl‑CoA.
combinations, the optimized strain (Table 3) was able to
produce nearly 120 mg/L butanol.
In the work conducted by Sakuragi et  al. (2015) the Amino acid assimilation pathway in S.
authors also confirmed that the deletion of the GPD1 and cerevisiae
GPD2 genes reduces the production of glycerol and con-
sequently increases the final concentration of butanol. Production of n-butanol via the amino acid uptake path-
Furthermore, in this same work the authors demonstrated way (Fig. 1) is based on the idea that the degradation of
that the use of the trans-enoyl-CoA reductase gene, in the intermediates, such as keto acids in biosynthesis and amino
construction of the Clostridium pathway in S. cerevisiae, acid degradation pathways, may result in the formation of
increases the production of n-butanol, as well as performed alcohols by S. cerevisiae yeast. In a study by Villas-Bôas
by by Krivoruchko et al. (2013). For this construction, the et al. (2005), the authors confirmed the existence of a meta-
strain was able to produce 14.1 mg/L n-butanol after 48 h of bolic pathway in S. cerevisiae for glyoxylate synthesis from
cultivation (Table 3). However, the authors did not construct glycine, which has not yet been fully described. In addi-
a strain containing the trans-enoyl-CoA reductase gene in tion, the authors detected α-ketovalerate formation as one
conjunction with the deletion of genes from the glycerol of the intermediates of this pathway, which is the precursor
pathway. In this sense, it is worth mentioning the strain con- of n-butanol (Shen and Liao 2008). Knowing this informa-
structed by Swidah et al. (2015), which was able to produce tion, Branduardi et al. (2013) hypothesized and biochemi-
300 mg/L n-butanol, in complex medium containing 2% cally demonstrated the production of n-butanol through the
glucose (Table 3). degradation of the amino acid glycine.
Schadeweg and Boles (2016a, b) and Schadeweg and The authors (Branduardi et al. 2013) used the S. cerevi-
Boles (2016b) published two complementary works on the siae CEN.PK102-5B strain as background and, to verify if
insertion of the Clostridium pathway in S. cerevisiae. In the cell was capable of producing n-butanol. The strain was
the first one, the authors started by testing several genes cultivated in synthetic medium and observed good growth,
and obtained a production of 15 mg/L n-butanol. Then, but did no n-butanol production when ammonium sulfate
the implementation of different strategies led to the final was used as a source of nitrogen. On the other hand, when
production of 120 mg/L n-butanol, under anaerobic condi- the same strain was cultivated with glycine as the only
tions (Table 2 and 3), which are: increased CoA synthesis by nitrogen source, they observed the production of 92 mg/L
overexpression of the pantothenate kinase coaA gene; pan- n-butanol. In this experiment the authors used glucose
tothenate supplementation in the culture medium; deletion (20 g/L) and glycine (15 g/L) and observed the consump-
of the ADH1-6 and GPD2 genes to reduce the formation of tion of both substrates, proving that S. cerevisiae is able to
ethanol and glycerol; and expression of an ATP independ- produce n-butanol from these substrates.
ent acetylating acetaldehyde dehydrogenase to converting To confirm the hypothesized metabolic pathway, Bran-
acetaldehyde into acetyl-CoA. In this first study, the authors duardi et al. (2013) performed a step by step study, verifying

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the influence and presence of each enzyme and intermediates these combined strategies is a promising step for synthetic
of the pathway. The major obstacle in this study was the first biotechnology.
reaction of the pathway, i.e., conversion of glycine to gly- Although S. cerevisiae is still capable of producing a
oxylate. However, a gene coding for this enzyme has not yet much lower titer of n-butanol, the production of this alco-
been described in the metabolism of S. cerevisiae. To prove hol by Clostridium species becomes industrially complicated
the method proposed by them and to assume the existence due to the characteristic of these organisms to be strictly
of a native gene in S. cerevisiae, the authors suggested the anaerobic. In addition, ABE fermentation generates other
use of the goxB gene of Bacillus subtilis that codes for a byproducts, reducing yields in butanol, since this type of
glycine oxidase, and that can catalyze the same reaction. In fermentation occurs in two phases (acidogenesis and sol-
conclusion, they described as existing the proposed pathway ventogenesis). As well as ethanol production by S. cerevi-
for n-butanol production from glycine as a nitrogen source siae still going through to changes and improvements, even
in S. cerevisiae. In addition, they speculated that n-butanol though it is a very well consolidated process, the butanol
is derived from butyryl-CoA and that glycine acts as a meta- production by this yeast is a process with the potential
bolic flux driver, and is called a co-substrate of the reaction. to become economically viable. However, as it is a topic
After this work, other studies were carried out to verify recently addressed in the literature, it still requires a lot of
the production of n-butanol by S. cerevisiae from the amino study, with the results obtained until today being the begin-
acid degradation pathway. Si et al. (2014) studied the pro- ning of a long work.
duction of n-butanol via the degradation pathway of the
threonine amino acid from overexpression of the proposed
enzymes and elimination of the ethanol, glycerol, valine, Butanol tolerance by S. cerevisiae
leucine and isoleucine production pathways. Using S. cer-
evisiae YSG50 as background, the modified strain was capa- Once an organism begins to form metabolic products,
ble of producing 242.8 mg/L n-butanol. In this work, the the cells must be able to tolerate these compounds in the
authors studied carbon marked in glycine (L-glycine-2-13C) medium. The mechanism of microorganism tolerance to
and glucose (D-glucose-13C6) and observed that all carbons butanol is very similar to ethanol tolerance (Liu and Qureshi
of n-butanol formed were derived only from glucose and 2009). S. cerevisiae is an organism capable of tolerating
not from glycine. This confirms the hypothesis raised by up to 18% of ethanol in media, depending on the condi-
Branduardi et al. (2013) of glycine as a co-substrate. tions of cultivation (Pereira et al. 2011; Della-Bianca and
In another study on the role of the amino acid degra- Gombert 2013; Ishmayana et al. 2017), however, this yeast
dation pathway for butanol production, Shi et al. (2016) is not able to tolerate more than 2% butanol (Knoshaug and
implemented a synergistic pathway with the endogenous Zhang 2009). Ishmayana et al. (2017) reported that, although
threonine pathway and the introduced citramalate pathway butanol tolerance is related to membrane fluidity, different
in S. cerevisiae, besides overexpression of keto-acid decar- strains may present different behaviors due to the intrinsic
boxylases (KDC) and alcohol dehydrogenase (ADH), and properties of each strain.
co-expression of LEU genes. The final strain was able to González-Ramos et al. (2013) analyzed the butanol toler-
produce 835 mg/L n-butanol in anaerobic glass tubes. When ance of S. cerevisiae BY4741 and CEN.PK 113-7D strains
cultivated in bioreactor, under micro-anaerobic condition, in synthetic medium containing different concentrations of
the same strain produced a final concentration of 1.05 g/L n-butanol (0 to 1.9%) in sealed 96-well plates to prevent
n-butanol. transfer and oxidation of butanol. The authors observed that
Recently, a study by Swidah et al. (2018) demonstrated the strains grew 50% and 30%, respectively, slower in 1% of
the contributions of the two strategies (heterologous expres- n-butanol and the growth of both was drastically reduced;
sion and amino acid pathways) in the production of butanol not being able to grow in concentrations of n-butanol above
by S. cerevisiae, called the combination of endogenous and 1.45% (BY4741) and 1.57% (CEN.PK 113-7D). From
exogenous pathways (Fig. 1). For this, they used a strain genomic-scale analyses, the authors identified mutations
already constructed in a previous study containing the ABE in three genes that encode transcription factors, showing
pathway and the ADH1 deletion of the ethanol production that n-butanol tolerance in S. cerevisiae is related to protein
pathway (Swidah et al. 2015). At the end of this study, the degradation.
authors suggested that the exogenous pathway is responsible Knoshaug and Zhang (2009) performed a screening for
for most of the butanol produced. In addition, the presence tolerance at different concentrations of butanol in non-Sac-
of the amino acid glycine and the deletion of ADH1 dem- charomyces and S. cerevisiae strains in microplates con-
onstrated that the endogenous pathway is also responsible taining YPD (yeast-peptone-dextrose) media. Among the
for part of the production of butanol, and the optimization of 10 strains evaluated, only one was not able to grow in 1% of
n-butanol, while the others presented relative growth (RG%)

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Page 8 of 9 World Journal of Microbiology and Biotechnology (2020) 36:48

of around 60%. However, in the presence of 2% n-butanol, Acknowledgements  The authors thank the São Paulo State Research
only three S. cerevisiae strains (ATCC26602, ATCC20252 Foundation (FAPESP, Grants Nos. 2015/20630-4 and 2019/08542-
3) for their financial support. This study was financed in part by
and Fali) were able to grow, with RG% between 10 and 20%. the Coordenação de Aperfeiçoamento de Pessoa de Nível Superior
Genetic and evolutionary engineering are the most widely (CAPES, Brazil, Finance Code 001).
used laboratory strategies to develop both the consumption
ability and tolerance of a certain substrate or product and, Compliance with ethical standards 
when combined, can yield very efficient results (Mans et al.
2018). Tolerance to a particular product, such as butanol, Conflict of interest  The authors declare that they have no conflict of
laboratory evolution has been very useful, especially for a interest.
better understanding of the mechanisms that perform toler-
ance by identification of gene targets that improve alcohol
tolerance through inverse metabolic engineering (Hong et al.
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