Yarrowia Lipolytica

Yarrowia lipolytica is well known for its ability to decompose fatty acids, hydrocar-
bons, and alcohols via the glyoxylate pathway.

From: Bioprocessing for Value-Added Products from Renewable Resources, 2007

Related terms:

Nitrogen, Glycerols, Protein, Carbon Source, Crude Glycerol, Lipase, Cerevisiae,

Citric Acid, Microbial

View all Topics

White Biotechnology in Biosurfactants

Kuttuvan Valappil Sajna, ... Ashok Pandey, in Industrial Biorefineries & White
Biotechnology, 2015

5.2 Liposan
Candida lipolytica or Yarrowia lipolytica produce a water-soluble biopolymer com-
posed of 83% polysaccharide and 17% protein, which is called liposan. Liposan is
usually produced by fermentation of medium containing hydrophobic substrates
such as hexadecane. Liposan exhibits high emulsifying activity and emulsion sta-
bilizing properties.97 Because of its inert nature, liposan can be used as smokable
tobacco substitutes.98

Due to substrate specificity and low productivity of liposan, a thorough media

engineering and novel fermenter design are required to develop a cost-effective
technology for liposan production. Thiamine is very important nutrient for Y. lipoly-
tica fermentation for liposan production. Pinchuk et al.,99 proposed a self cycling
fermenter with continuous addition of hydrocarbon, which resulted in a yield of
0.08 g/L. During the final stage of fermentation, cycle-time delay was stopped during
which residual hydrocarbon was utilized and resulted in improved yield of 0.095 g/L.

> Read full chapter

A CRISPR Technology and Biomolecule
Production by Synthetic Biology Ap-
proach
Jitesh Kumar, ... Anshu Alok, in Current Developments in Biotechnology and Bio-
engineering, 2019

3.2 Genome Manipulations for the Production of High-Value

Biomolecules in Yeast
A number of yeast stains such as S. cerevisiae, Candida albicans, Kluyveromyces
lactis, Pichia pastoris, Ustilago maydis, and Yarrowia lipolytica were modified through
CRISPR/Cas and used as a common production strain for some valuable products.
S. cerevisiae [70] and K. lactis [73] are the successful examples of introducing biosyn-
thetic pathways in single transformation events using CRISPR/Cas technology.

A lactic acid producing S. cerevisiae strain was generated using single-step

CRISPR/Cas technology through inserting a heterologous gene (lactate dehydroge-
nase) and disrupting two endogenous genes (PDC genes PDC1 and PDC5). Recently,
Stovicek et al. demonstrated that CRISPR/Cas9 system is quite efficient for pathway
engineering of strains isolated from various industrial settings. In this study, they
also suggest that CRISPR/Cas9 genome editing technique is more suitable for easy
microbes engineering including gene/multiple alleles disruption or gene insertion.
For this a system was generated to test the gene insertion and deletion, in which
ADE2 gene was used as a disruption target and green fluorescent protein as insertion
target [74].

The genome of Baker's yeast S. cerevisiae was engineered through emerging

CRISPR/Cas technology and a strain was developed for production of an elevated
level of mevalonate, a key intermediate of industrially important terpenoid group
of compounds, synthesized through the isoprenoid biosynthesis pathway. This
CRISPR/Cas engineered S. cerevisiae strain, which is able to produce almost 40-fold
higher mevalonate content compared with wild type [42].

CRISPR/Cas technique is also considered as a time-saving approach because it takes

lesser time to modify the desired DNA fragment compared with other conventio-
nal methods. S. cerevisiae strain took approximately 6 weeks to create three point
mutations in the genome through standard methods, and multiplexed CRISPR/Cas
introduces the same within 1 week [73]. Using a single round of CRISPR/Cas
engineering, an increased ethanol tolerance stain S. cerevisiae CENPK2 was produced
by introducing four point mutations and a deletion mutation. A muconic acid
 biosynthesis pathway was integrated in native S. cerevisiae stain using CRISPR/Cas
engineering technique. In this process, total six DNA fragments, comprising 24-
 kb length, were transferred across three loci in a single transformation by using
CRISPR/Cas approach. Furthermore, a similar pathway was also introduced in K.
lactis for muconic acid production. These strains of S. cerevisiae and K. lactis could
be used for industrial scale production of muconic acid [73].

The commercial product (R, R)-2,3-BDO was produced from CRISPR/Cas9-en-

gineered S. cerevisiae using nonnative substrate xylose. In this strategy, a 24 kb
construct was constructed with six gene expression cassettes and integrated into
Ty transposon elements. Three genes from (R, R)-2,3-BDO biosynthesis and three
genes from xylose consumption pathway were selected for the construct preparation.
In this study, almost 10 copies of 24 kb DNA fragments were integrated, which
produces a significant yield of product (R, R)-2,3-BDO (0.31 g/L of product from
20 g/L xylose). Thus, longer length of DNA fragments (24 kb) with higher copy
number (10 copies) of integration was achieved, which resulted in enhanced yield of
desired product [75,76].

Recently, a system for genome editing, either deletion or insertion of gene(s), was
developed in actinomycetes by the use of emerging CRISPR/Cas technique. Two key
genes actIORF1 (SCO5087) and actVB (SCO5092) of actinorhodin biosynthesis from
Streptomyces coelicolor A3 were inactivated and thus, a significant decrement in acti-
norhodin yield was obtained. This strategy provides a genetic manipulation system
for actinomycetes group, which helps in metabolic engineering of actinomycetes
producing specific metabolites and it also provide a platform for heterologous
expression of biosynthetic gene/gene cluster [72].

CRISPR/Cas approach is not only used for genome editing but also for efficient
promoter engineering. The combination of CRISPR/Cas9 and transformation-asso-
ciated recombination (TAR) was able to generate a promoter-engineering platform,
mCRISTAR (multiplexed CRISPR/TAR) based on yeast system. It was used for mul-
tiplexed promoter engineering in the gene clusters of natural product biosynthetic
pathway by refactoring the multiplex gene cluster. This strategy provides an effective
genetic method that could be used for replacement of multiple promoters at a time
in the valuable phytochemical biosynthetic gene clusters. Thus, mCRISTAR approach
opens the doors for discovery of silent natural products as it has ability to reframe the
transcription machinery and activate the expression of silent natural product [77].

Yeast can be also used as a host for introducing mutations in the genome of different
species. Thus, the efficiency of targeted mutagenesis in S. cerevisiae is reported
close to 100% using CRISPR/Cas9 [78]. Therefore, genome can be modified using
CRISPR/Cas tool for species in which any efficient genetic tool is unavailable.
Using this strategy the genome of M. mycoides subsp. capri GM12 (Mmc) was
first transformed in yeast as extrachromosomal genome. Furthermore, a mutation
in the gene encodes a glycerol-3-phosphate oxidase (glpO), which is involved in
the production of hydrogen peroxide using sgRNA specific to glpO. The mutated
genome was backtransplanted in Mmc genome and functional assay revealed the
absence of hydrogen peroxide production [79]. The various approaches of biomole-
cule production discussed above in yeast suggest the high potential of CRISPR/Cas9
for synthetic biology applications.

> Read full chapter

Acidogenic Biohydrogen Production

Integrated With Biorefinery Approach
S. Venkata Mohan, ... K. Swathi, in Biohydrogen (Second Edition), 2019

3 Yeast Integration
In addition to bacteria, yeasts can also be considered as promising candidates
for the use of acidogenic effluents and production of biodiesel and bioethanol.
Rhodosporidium toruloides, Cryptococcus curvatus, Lipomyces starkeyi, and Yarrowia
lipolytica are capable of accumulating oil to at least 20% of their dry cell mass [60].
Yeast is reported to grow on a variety of inexpensive feedstocks and exploiting these
feedstocks is absolutely necessary for commercial microbial lipid biofuel production.
Short-chain fatty acids (SCFA) produced from the AF of organic waste materials are
a potential alternate source of carbon for lipid production [61]. It is reported that
oleaginous yeast has the ability to transform short-chain fatty acids into acetyl-coen-
zyme A (acetyl-CoA), a central intermediate, by acetyl-CoA synthetase. Acetyl-CoA is
then used in the lipid biosynthesis pathway. Mixed volatile fatty acids or individual
SCFAs can be used for lipid synthesis. A two-stage fed-batch strategy for valorization
of SCFA into microbial lipids is usually followed, wherein initially the yeast is grown
on glucose or glycerol as carbon source, and then sequential additions of individual
SCFA is done to enhance the yield of intracellular lipids [59]. Acidogenic effluent
from biohydrogen production process rich in SCFA can also serve as a potential
feedstock for biodiesel production in a biorefinery approach. Yeasts are the most
common microorganisms used to ferment carbohydrates to produce ethanol under
anaerobic conditions. Biodiesel production by utilizing yeast as a biocatalyst is a
novel approach towards the production of sustainable fuels.

> Read full chapter

Innovation on Bioenergy
Francisco Gírio, in The Role of Bioenergy in the Bioeconomy, 2019

9.2.3 Long-chain fatty acids

Similar to LCFAls, the production of long-chain fatty acids (LCFAc) poses identical
metabolic problems for their biochemical production from sugars. The goal is to
carry out suitable metabolic engineering of oleaginous yeasts that already exhibit a
natural capacity for lipid accumulation, e.g., Yarrowia lipolytica, Trichosporon sp., and
Rhodosporidium sp. Oleaginous yeasts can produce up to 70% w/w dry cell weight
of lipids, and studies on their use as production hosts for various oleochemicals are
rapidly increasing (Adrio, 2017). Recently Y. lipolytica was engineered to reach yield of
0.27 g/g glucose (Qiao et al., 2017). This value is near the target (0.28 g fatty acids/g
lignocellulosic sugars) determined by the National Renewable Energy Laboratory
for the US Department of Energy’s 2017 cost goal of 5$/gallon gasoline equivalent
(Davis et al., 2013). Besides production yields, oleaginous strains need to show a
considerable tolerance behavior over harsh industrial media and not all yeasts are
suitable to attain an acceptable industrial performance. Yeast species, for example,
from the genera Trichosporon sp. and Rhodosporidium sp. are able to efficiently
utilize C5 sugars originating from hemicellulose. They are also more tolerant to
inhibitors. Koivuranta and co-workers did modify Trichosporon oleaginosus for LCFAc
production and reported good productivity on both glucose and xylose (Koivuranta
et al., 2011).

> Read full chapter

Production of fuels from microbial oil

using oleaginous microorganisms
E. Tsouko, ... A.A. Koutinas, in Handbook of Biofuels Production (Second Edition),
2016

8.3.3 Lipid production from fermentation of hydrophobic ma-

terials used as the sole carbon source
It is known that when microorganisms are cultivated on fat-type substrates (eg,
long-chain free-fatty acids, TAGs, fatty-esters), production of (intracellular, cell--
bounded, or extra-cellular) lipases is performed as a physiological response to the
presence of fatty materials into the growth medium (Fickers et al., 2005). This
secretion is obligatory in the case that TAGs or fatty-esters are used as substrates
(Fickers et al., 2005; Papanikolaou and Aggelis, 2010). In contrast, a large variety
of microorganisms are capable of utilizing soaps as well as free-fatty acids as sole
carbon and energy source, regardless of the lipolytic capacities of the microorgan-
isms used in order to break down fatty materials (Ratledge and Boulton, 1985;
Papanikolaou and Aggelis, 2010). Specifically, for the case of the yeast Yarrowia
lipolytica, its culture on TAG-type substrates is accompanied by secretion of an
extra-cellular lipase called Lip2p, encoded by the LIP2 gene (Pignede et al., 2000).
This gene encoded for the biosynthesis of a precursor premature protein with
Lys–Arg cleavage site. The secreted lipase was reported to be a 301-amino-acid
glycosylated polypeptide that belongs to the TAGs hydrolase family (EC 3.1.1.3)
(Pignède et al., 2000; Fickers et al., 2005). The Lip2p precursor protein was processed
by the KEX2-like endoprotease encoded by the gene XPR6, whereas deletion of
the above gene resulted in the secretion of an active but fewer stable pro-enzyme
(Pignède et al., 2000). Simultaneously, other intracellular lipases (Lip7p, Lip8p) may
also be produced and secreted into the culture medium, that present different fatty
acid specificities, with maximum activity being displayed against Δ9C18:1 (oleic acid),
C6:0 (capronic), and C10:0 (caprinic) fatty acids (Fickers et al., 2005).

The free-fatty acids (existed as initial substrate or produced after lipase hydrolysis of
the TAGs/fatty-esters) will be incorporated, with the aid of active transport, inside the
microbial cell. It is interesting to state that in the case of Yarrowia lipolytica yeast,
the various individual substrate fatty acids would be removed from the medium
(and hence incorporated inside the microbial cell) with different rates (Papanikolaou
et al., 2001, 2002a; Papanikolaou and Aggelis, 2003b). Specifically, regardless of
the initial concentrations of the extra-cellular fatty acids, the incorporation rate of
the lower aliphatic chain fatty acids, lauric acid (C12:0) and myristic acid (C14:0),
or the unsaturated fatty acids Δ9C18:1 and Δ9,12C18:2, is significantly higher than
that of stearic (C18:0) and to lesser extent palmitic (C16:0) acid (Papanikolaou et al.,
2001; Papanikolaou and Aggelis, 2003b). Moreover, the incorporated fatty acids will
be either dissimilated for growth needs or become a substrate for endo-cellular
biotransformations (synthesis of “new” fatty acid profiles which did not exist pre-
viously in the substrate) (Ratledge and Boulton, 1985; Koritala et al., 1987; Aggelis
and Sourdis, 1997; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al.,
2001, 2002a, 2007b; Papanikolaou and Aggelis, 2003a,b, 2010).

The intracellular dissimilation of the various catabolized fatty acids is performed by

reactions catalyzed by the various intracellular acyl-CoA oxidases (Aox). A significant
amount of experimental work has been performed in relation with the elucidation
of the above-mentioned reactions by using strains of the nonconventional yeast
Yarrowia lipolytica (Fickers et al., 2005). In fact, it has been revealed that the afore-
mentioned biochemical process is a multistep reaction requiring different enzymatic
activities of five acyl-CoA oxidase isozymes (Aox1p through Aox5p), encoded by
the POX1 through POX5 genes (Luo et al., 2002; Mličková et al., 2004a,b; Fickers
et al., 2005a). Aox3p is specific for short-chain acyl-CoAs, Aox2p preferentially oxi-
dizes long-chain acyl-CoAs, while Aox1p, Aox4p, and Aox5p do not appear of being
sensitive in the chain length of the aliphatic acyl-CoA chain (Mauersberger et al.,
2001; Luo et al., 2002; Fickers et al., 2005). It should also be noticed that genetically
modified strains of Y. lipolytica, namely JMY 798 (MTLY 36-2P) and JMY 794 (MTLY
40-2P), have been created from the wild-type W29 strain (Mličková et al., 2004a,b).
These strains were subjected to disruptions of the genes implicated in the encoding
of various intracellular Aox. The genetically engineered strains, hence, either un-
der—or did not at all express—several of the enzymes implicated in the catabolism
( -oxidation) of aliphatic chains. When cultures were performed on oleic acid utilized
as the sole substrate, although the genetically engineered strains showed almost
equivalent microbial growth compared with the wild strain (W29) from which they
derived, in contrast with W29 strain they presented significantly higher formation
of lipid bodies and, hence, increased lipid accumulation (Mličková et al., 2004a,b).
Therefore, the above-mentioned studies as well as various others reported in the
literature (Aggelis and Sourdis, 1997; Papanikolaou et al., 2003; Szczęsna-Antczak
et al., 2006; Mantzouridou and Tsimidou, 2007) indicate that external addition of
fat (ex novo lipid accumulation) can significantly enhance the bioprocess of SCO
production in various oleaginous microorganisms, but external utilization of fat
mainly serves for the “improvement” and “up-grade” of a fatty material utilized as
substrate (eg, valorization of low-cost or waste fats so as to produce specialty lipids
like cocoa-butter substitutes or substitutes of other high-added value lipids like illipé
butter, shea butter, sal fat; Papanikolaou and Aggelis, 2010), and not for the use of
the SCO produced in the manufacture of biodiesel.

> Read full chapter

Single-Cell Biorefinery
Qingsheng Qi, Quanfeng Liang, in Industrial Biorefineries & White Biotechnology,
2015

3.3 Suitable and Value-Added Product Portfolio

Fermentative production of bulk chemicals has recently attracted a great deal of
attention owing to fossil deficiency. However, fermentative processes are still not
comparable to chemical processes because of the high cost of fermentation process-
es.77 Selection of suitable and value-added product portfolios could improve the
economics and facilitate application of fermentation processes. Cai et al.49 demon-
strated the possibility of an alternative approach for improving the economics of
ABE fermentation by coproducing butanol with a high-value compound, riboflavin,
which has been widely used as an additive to animal feed and food products and
in medicine.49 By overexpressing the C. acetobutylicum riboflavin operon, ribGBAH,
the recombinant strain accumulated more than 70 mg/l riboflavin together with
190 mM butanol in static cultures.

In addition to biofuels, organic acids have also been coproduced with high-value
products. A strain of Yarrowia lipolytica Wratislavia K1 has been reported to produce
citric acid and erythritol simultaneously from the crude glycerol.54 By optimization
during the fed-batch cultivation, Rymowicz et al.54 obtained a very high amount of
citric acid production (110 g/dm3) and erythritol accumulation (81 g/dm3). Since
erythritol is more expensive than citric acid, accumulation of erythritol provided
an additional cost compensator for citric acid production. A similar example was
the coproduction of propionic acid with trehalose in Propionibacterium freudenreichii
subsp. shermanii.55 Interestingly, the crude glycerol was more suitable for the copro-
duction than pure glycerol. This was likely because the presence of KCl in the crude
glycerol as an impurity enhanced the accumulation of trehalose in the microbe.
An osmotic sensitive mutant of this strain accumulated trehalose (391 mg/g of
biomass, 90 mg/g of substrate consumed) and propionic acid (0.42 g/g of substrate
consumed).56 Crude glycerol is a major by-product of the biodiesel manufacturing
industry, and coproduction from glycerol could be readily integrated into existing
biodiesel facilities, which could be real biorefineries and revolutionize the biodiesel
industry by dramatically improving its economics.78

Therefore, a highly efficient and value-added product portfolio is the major

consideration for coproduction. In this regard, the economics of the coproduction
system should be considered, selecting suitable coproducts, calculating the optimal
metabolic flux ratio and evaluating the final coproducts value. In the case of co-
producing butanol and riboflavin from clostridia, the coproduction of riboflavin in
amounts of 0.5–1 g/l together with solvent production would double the value of
the culture products on a per-liter basis.49 It was interesting to note that riboflavin
accumulated up to 70 mg/l without affecting solvent production. Several recent
reports have shown that the relative amounts of coproduct could be controlled
through strain- and process-based strategies.38,48,62

> Read full chapter

Bacterial and Yeast Cultures – Process

Characteristics, Products, and Applica-
tions
Wei-Cho Huang, I-Ching Tang, in Bioprocessing for Value-Added Products from
Renewable Resources, 2007

3 YEAST

3.1 General characteristics

Yeasts are one-celled fungi, 5~10 μm in size. Yeast cells are usually spherical, cylin-
drical, or oval and are important for their ability to ferment the carbohydrates within
various substances. They are widespread in nature, existing in soil and on plants.
Yeasts have been used since prehistoric times in the making of breads and wines, but
their cultivation and use in large quantities only started in the 19th century. Today,
they are used industrially in a wide range of fermentation processes, as feeds and
foodstuffs, as a source of vitamins, and to produce various antibiotics and steroid
hormones. Characteristics of some commonly used yeasts are list in Table 3. Yeasts
can grow in a wide range of pH. For instance, Candida spp., Torulopsis glabrata, and
Yarrowia lipolytica survive in the pH range from 3 to 8. Generally speaking, yeasts
prefer to live at temperatures between 25 and 35°C under aerobic conditions. Pure
yeast cultures are grown in a medium of sugars, nitrogen sources, minerals, and
water. In anaerobic environments, yeast transforms simple sugars, such as glucose
and sucrose, into ethanol and carbon dioxide.

Table 3. Characteristics of common yeasts [1–3]

Species Candida Kluyveromyces

Pichia
sp.@sp. Saccha- Torulopsis Yarrowia
sp. romyces glabrata lipolytica
cerevisiae
Generation 2h 3.5 4.5 h 2 3h 1.5 2 h 5 14 h 1.5 2 h
time
Growth me- n-Alkane, Glucose, Glucose, Glucose, n-Alkane, Sucrose,
dia, carbon methanol lactose, whey, maltose, polyols, n-decane,
source whey methanol molasses, methanol, alcohols,
sucrose glucose fatty acids
Growth tem- 0 48 20 28 37 42 0 40 23 43 (37) 16 38 (26)
perature (°C) (25 30)1 (28 35)
pH range 2.5 8 (5 7) 4.2 9 6 12 4.5 7.5 (6.0) 2 8 3.1 9
(4.2 5.8)
O2 demand Aerobic Aerobic Aerobic Aerobic Aerobic Aerobic
Genetic Easy Average Easy Very easy Average Easy
modification
GRAS Most Yes Yes Yes Some Yes
Others SCP* Recombi- Ethanol SCP* Preserva-
nant production tive-resistant
proteins

1 Numbers in the parentheses are optimal temperature or pH range for growth.

* SCP: single-cell protein

Most cultivated yeasts belong to the genus Saccharomyces; those known as brew-
er's yeasts are strains of S. cerevisiae, which have been widely used for ethanol
production. S. cerevisiae is the eukaryotic model organism in molecular and cell
biology, similar to E. coli as the model prokaryote. Yeasts usually divide every few
hours, though they have longer generation times than bacteria. Most yeasts are
generally recognized as safe (GRAS), easy to be genetically modified, and easy to
separate in downstream processing because of their relatively large size. S. cerevisiae
is the most studied of simple eukaryotes. It is the first eukaryote with its genome
completely sequenced and its genetics and physiology thoroughly characterized. The
completion of the entire genome sequence of S. cerevisiae in 1996 was a milestone
in the fundamental understanding of its physiology and will undoubtedly accelerate
developments in the genetic improvement of S. cerevisiae and other yeasts.

Candida spp. are methanol-utilizing yeasts.

They produce lipases of commercial interest, can grow on paraffin oil, fatty acids,
triglycerides, and n-alkanes, and thus are widely used in bioprocessing and biore-
mediation. Kluyveromyces spp. can grow either as single cells or in filaments, which
provide larger surface area and thus increase the product yield for industrial appli-
cations. Pichia pastoris can be used in the production of enzymes and recombinant
proteins because it can grow on methanol to a high cell density. However, the heat
generated from its fermentation must be removed due to the highly exothermic
process. Torulopsis glabrata can decompose n-alkanes, polyols, and methanol. Its
cells as well as Candida spp. are used for SCP production. Yarrowia lipolytica is well
known for its ability to decompose fatty acids, hydrocarbons, and alcohols via the
glyoxylate pathway. It has been considered as a preservative-resistant yeast with
strong production of extracellular lipases and proteases.

Yeasts have the advantages of rapid growth and ease of genetic manipulation.
Other advantages of employing yeasts as hosts for fermentation are their abundance
of metabolic activities and safety. These characteristics have brought yeasts many
applications in chemical, food, and pharmaceutical industries.

3.2 Industrial applications

Yeasts have many applications in industrial food and beverage production (Table 4).
Industrial yeasts are suppliers of enzymes, proteins, and chemicals. The commercial
importance of yeasts extends to their application to the treatment of industrial
wastes and effluents. For example, Kluyveromyces marxianus can remove heavy
metals from waste stream; some Candida spp. can detoxify and remove pollutants
from wastewater.
Table 4. Industrial applications of yeasts

Applications Examples
Baking and brewing Bread, beer, wine, spirits
Bio-based fuels Bio-ethanol from sucrose, glucose, and xylose
Bioremediation Heavy metal removal, wastewater treatment
Chemicals Glycerol, bio-surfactants, enzymes, organic acids,
amino acids
Health-care Human therapeutic proteins, steroid hormones
Nutrition and animal feed Biomass, polysaccharides, vitamins, single cell
proteins

Alcoholic fermentation is the oldest known biological reaction. The German chemist
Eduard Buchner (1897) discovered that a cell-free extract of yeast can induce alco-
holic fermentation. Beer is an alcoholic beverage made from cereal grains, usually
barley but also corn, rice, wheat, and oats, by yeast fermentation that consumes
sugars in the grain and produces alcohol and CO2. Two yeasts, S. cerevisiae and
S. bayanus, are used to make wine by fermenting grapes. Yeast is responsible for
the presence of both positive and negative odors in wine. For example, yeast may
produce hydrogen sulfide when stressed. Adding nutrients to the fermentation
tank can avoid this undesirable quality. The time of fermentation also determines
wine character. Above all, subtle differences in ingredients determine the unique
characteristics of each brewing process. Yeast fermentation is also used to make
leavened breads. The main function of baker's yeast (S. cerevisiae) in bread dough is
to produce CO2 from sugars. The dough is placed in a warm and moist environment,
enabling the yeast to multiply, and CO2 produced during fermentation causes the
dough to rise. Alcohol produced during fermentation contributes to the aroma of
the bread. Secondary fermentation produces organic acids that also add to the flavor
of the bread. In the making of wines, beers, spirits, and industrial alcohol, the
fermented medium after separation and purification is the desired product, and the
yeast itself is used in animal feeds. Yeast biomass is a rich source of proteins, nucleic
acids, vitamins, and minerals.

Furthermore, yeasts contribute as hosts for expressing foreign genes not shared by
prokaryotic cells in modern recombinant DNA technology. They are used to produce
human proteins in spite of plasmid instability and the economic costs of providing
growth medium. Gene expression is better in S. cerevisiae than in E. coli because
S. cerevisiae is more capable of excreting and post-translationally modifying genetic
products [10]. Pichia pastoris has also been widely used with commercially available
expression systems. Yeast RNA polymerase recognizes many animal promoters, and
yeast utilizes inexpensive carbon sources. Recombinant yeasts take less time, reach
higher yields, and are more genetically stable and cheaper than the insect and
mammalian cell systems. Besides, yeast cultures are nonpathogenic, stable, and easy
to operate and scale-up. In addition, stable mutants exist that enhance productivity.
Yeasts are also widely used to produce fuels and chemicals from biomass, which is
discussed in the next section along with bacteria.

> Read full chapter

Remaining Challenges in the Metabolic

Engineering of Yeasts for Biofuels
Sun-Mi Lee, ... Hal S. Alper, in Direct Microbial Conversion of Biomass to Advanced
Biofuels, 2015

Beyond Saccharomyces cerevisiae

S. cerevisiae is the most well-studied model yeast for biofuel production. However,
there are many other yeasts that also hold promise. Not surprisingly, there is
ongoing research to engineer strains of yeast isolated from lignocellulose-degrading
ecosystems, or yeasts that produce superior precursors to fuels. Some yeasts with
attractive bioprocessing traits include (but are not limited to) S. stipitis (formerly
Pichia stipitis158) with its pentose sugar fermentation, Yarrowia lipolytica with its lipid
production, and Trichoderma reesei with its cellulosic biomass utilization. Of these,
the progress in research with filamentous fungi T. reesei has been covered in earlier
chapters in this book (Chapters 10 and 11Chapter 10Chapter 11). Therefore, this
chapter will briefly discuss the recent studies with other representative yeast strains.

S. stipitis is a native xylose fermenting yeast. The capability of xylose fermentation

in S. cerevisiae was originally conferred by introducing the genes involved in xylose
metabolic pathway of S. stipitis.76 In addition to xylose fermentation, cellulosic
ethanol fermentation has been reported by native S. stipitis with high titer of
41 g/L.159 Recently, improved ethanol production from cellulolytic hydrolysate has
been demonstrated via genome shuffling of S. stipitis and achieved titers of up to
140 g/L.160 Although the genome sequence of S. stipitis has been available since
2007,161 the lack of metabolic engineering tools for S. stipitis has slowed progress in
this host. Most biofuel production studies have been reported by using either native
or randomly modified strains of S. stipitis rather than rationally engineered strains.

Y. lipolytica, one of oleaginous yeasts that produce lipids in wild type state, has also
been demonstrated as a promising microbial host for advanced biofuels, specif-
ically biodiesel alternative fuels. Used extensively in industrial applications such
as citric acid, protease, and lipase production, the whole genome of Y. lipolytica
is fully sequenced and metabolic engineering tools for Y. lipolytica are available.2
However, the recent development of hybrid promoters162 and a gene overexpression
platform163 enable high expression of native and heterologous genes in Y. lipolytica,
and thus enable metabolic engineering tools that were previously unavailable in this
host. Y. lipolytica is of interest chiefly for its free fatty acid, precursor to biodiesel,
accumulation properties. As an example, a recent study reports improved lipid
production with a lipid content of up to 61.7% of dry cell weight.163 Furthermore,
the specific composition of lipid produced in Y. lipolytica can be adjusted for more
energy-efficient biofuel production. So far, the composition of lipid produced
in Y. lipolytica has been predominately modified by changing culture conditions
rather than by engineering metabolic pathways2 such as desaturases. However, lipid
extraction issues remain, because the lipids accumulate in liposomes, requiring cell
lysis to enable extraction. Additionally, Y. lipolytica is not suited for consuming many
of the sugars present in lignocellulosic biomass. Even so, the ability to accumulate
long-chain lipids makes this organism an attractive host.

Beyond these two strains, there are a great number of xylose-fermenting yeasts.
Significant organisms include Neurospora crassa,164 Hansenula polymorpha,165,166
Pachysolen tannophilus,167 Candida arabinofermentans,168 and Pichia guillermondii.168
A recent report describes the progress of and interest in the yeast Spathaspora pas-
salidarum as a unique host for fuels production.169 This yeast could potentially serve
as a host for lignocellulosic ethanol production, because it has been recently shown
to be tolerant to toxins and can conferment several components of lignocellulosic
derivatives.170,171

It is unclear whether these yeasts will be better hosts for advanced biofuels pro-
duction from lignocellulosic biomass over the model organism of S. cerevisiae. The
lack of metabolic engineering tools for these yeasts prevents replicating approaches
taken in S. cerevisiae to compensate for its shortcomings. By extending current
metabolic engineering tools to these organisms, and potentially developing novel
tools, more complex rewiring will become possible. Eventually, the ability to evaluate
optimized strains of many species of yeast will be within reach, and the optimal strain
for fuel production selected. In the end, it may be that a different organism is suited
for production of each different biofuel. However, creating new synthetic tools for
rapid testing in nonconventional organisms remains a challenge in the field.

> Read full chapter

Identification of Genetic Targets to

Improve Lignocellulosic Hydrocarbon
Production in Trichoderma reesei Us-
ing Public Genomic and Transcriptom-
ic Datasets
Shihui Yang, ... Min Zhang, in Direct Microbial Conversion of Biomass to Advanced
Biofuels, 2015

Background
Bacteria, yeasts and fungi can naturally synthesize fatty acids, isoprenoids, or
polyalkanoates for energy storage. These compounds have high energy densities
and are compatible with current fuel infrastructure, permitting their exploitation
for hydrocarbon fuel production.1–4 Many microorganisms are being developed as
potential biofuel production strains, yeast strains are currently the leading indus-
trial biocatalyst microorganisms,5 and engineered bacteria such as Escherichia coli,
Zymomonas mobilis, Corynebacterium glutamicum, and Bacillus subtilis are also being
developed and deployed to address commercially important inoculum require-
ments.6–10 However, all have limitations for economic advanced biofuel production
in terms of robustness, substrate use, productivity, and yield.

Consolidated bioprocessing (CBP) is a promising strategy for economic lignocel-

lulosic biofuel production, which integrates all steps of enzyme production, sac-
charification, and fermentation biologically. A CBP microbial biocatalyst should
therefore produce and secret cellulytic enzymes to solubilize lignocellulosic biomass
substrates into simple sugars and, at the same time, produce desired chemicals
efficiently with high yield and titer. Despite recent exploration on developing
microbial consortia as CBP biocatalysts,11–13 it will still be challenging to overcome
the complexity of CBP consortia to meet the needs for commercial production
of biofuels at an acceptable cost. The classical CBP strain development strategies
focusing on a single microorganism are still the better approach for industrial
biotechnology applications, which include two strategies termed “native strategy”
to increase productivity of a native cellulolytic microorganism and “recombinant
strategy” to enable lignocellulosic biomass use capability of a microbial biocatalyst
with excellent productivity.14,15 Despite a lot of efforts being devoted to developing
a promising CBP strain in the direction of recombinant strategy to enable lig-
nocellulosic assimilation capability of classical microbial biocatalysts such as yeast
Saccharomyces cerevisiae, oleaginous yeast Yarrowia lipolytica, and bacterial species
of E. coli and Z. mobilis, no obvious champion could be developed yet. Clostridium
species are the models for native strategy, receiving intensive attention, but a
lot of barriers still need to be overcome to increase productivity for commercial
production.16–19 Fungal CBP is a novel concept proposed by National Renewable
Energy Laboratory scientists recently, and the feasibility and strategies to develop a
model fungal cellulase producer, T. reesei, as the fungal CBP platform was discussed,
which opens a new direction toward CBP development for industrial application.20

The filamentous fungus T. reesei is one of the main producers of cellulases and
hemicellulases for commercial lignocellulosic bioethanol production, with efficient
systems for cellulase induction and nutrient transportation.21 It possesses at least
three classical enzymes of exoglucanases (syn. cellobiohydrolases), endoglucanases,
and -glucosidases, with new players involved in cellulose degradation identified
recently, such as GH61 polysaccharide monooxygenases (PMOs), expansin-like
proteins swollenin (SWOI), and expansin/family 45 endoglucanase-like proteins
(EEL1, EEL2, and EEL3).22–24 The main cellulase production in T. reesei is regulated
sophisticatedly, and various transcriptional factors (TFs) controlling cellulase gene
expression have been discovered, such as XYR1, CRE1, ACE1, ACE2, AreA, BglR, and
HAP2/3/5 complex.23,25–32 In addition, the environmental conditions for cellulase
induction have also been investigated, with several inducers identified, including
cellulose, disaccharides of cellobiose, lactose and sophorose, and low-molecular
weight compounds such as l-arabitol and l-sorbose.33,34

Recent technical breakthroughs in next-generation sequencing (NGS), systems bi-

ology, and synthetic biology35–38 have significantly increased the amount of data
available on the metabolism and regulation of biofuel-producing organisms, leading
to a potential paradigm shift in industrial biocatalyst development.3,4,38–50 As one
of the major producers of cellulases and hemicellulases at the commercial scale, T.
reesei has also been widely studied using genomic and systems biology approaches.
For example, the genome sequence of T. reesei has been reported and annotated.24
Comparative genomic studies between wild type and mutant strains with improved
cellulase production (e.g., QM9414, RUT C30) were carried out, and the genetic loci
associated with excellent cellulase production were identified and characterized.51,52
In addition, quite a few studies using transcriptomic and proteomic approaches
have been reported.23,53–58 Although nearly all of them were focused on cellulase
induction, and there is no systems biology study reported yet for T. reesei as a CBP
candidate for biofuel production, these systems biology datasets still can help us
understand the global transcriptional profiles of T. reesei in different environmental
conditions. In this chapter, we will show the promise of using public genomic and
transcriptomic datasets to help us identify genetic targets and to guide metabolic
engineering practice of improving hydrocarbon production in T. reesei.

> Read full chapter

Applications and Future Perspectives of

Synthetic Biology Systems
Manisha Chownk, ... Sudesh Kumar, in Current Developments in Biotechnology and
Bioengineering, 2019

2 Genetic Manipulations in Synthetic Biology for Value-Added

Products
Talking about creating, editing, and manipulating DNA fragments, apart from the
technology of transferring small gene segments and operons into host cells to
express a certain product, there have been techniques that utilize the vectors capable
of transfer and express several hundreds to thousands of kilobases of DNA into
eukaryotic organisms such as yeasts, plants, and mouse. These are BACs (Bacterial
artificial chromosomes) and YACs (yeast artificial chromosomes) [16,17]. Although
they have been introduced and used for last 30–40 years, they have gained usage
for the expression of large-sized DNA fragments in recent years due to growth in
the field of synthetic biology. Later, the integration of sequence homologous to
amyE integration target locus in Bacillus chromosome into BAC as well as genetic
circuits ven and cat was described [18]. They reported the transfer of 10 Kb of
DNA fragment from E. coli into B. subtilis and claimed that the engineered BAC
has the ability to transfer and express much larger DNA fragments and has direct
applications in the field of synthetic biology. Also, the use of YAC in the production
of monoclonal antibodies has been extensively studied. The rearrangement and
expression of human IgL loci by arranging microgene segments on plasmids and
much larger region on YAC successfully was also reported [19].

The field of synthetic biology has changed the face of biofuel industry too. The pro-
duction of ethanol from hexoses has been commercially done by utilizing microbes.
But by the use of synthetic biology approach, pentoses utilizing microbes has been
able to produce ethanol at higher rates. In similar approaches, xylose isomerase,
xylulokinase, arabinose isomerase, transketolase, and transaldolase from E. coli were
expressed in Zea mobilis, which produced about 89% and 98% more ethanol than
wild-type on xylose- and arabinose-containing media [20,21]. Butanol has gained
popularity as the alternative to ethanol as a potential biofuel. To increase the pro-
duction of butanol in E. coli, the essential genes required for the transformation
of acetyl-CoA to butanol (thl, hbd, crt, bcd, etfAB, and adhE2) were introduced into
E. coli and that has increased the production of butanol to 1.4 mg/L of glucose [22].
More recently, the use of engineered yeasts such as Yarrowia lipolytica, Hansenula
polymorpha, Pichia pastoris, and Kluyveromyces lactis have been increased because
they can tolerate the harsh temperatures, usually required during the fermentation
procedures [23]. Similarly, the productions of fatty acids have been done using
S. cerevisiae and E. coli. However, S. cerevisiae has been proven more suitable for the
production than E. coli due to its capability for posttranslational modifications. The
production yields of some fatty acids have also been recorded more in E. coli than
S. cerevisiae where posttranslational modifications are not required [24]. Therefore,
the microbes have been genetically altered to produce more amounts of fatty acids.
Fatty acid methyl esters and fatty acid ethyl esters (FAEEs) have been used as biofuels
in recent past [25]. There are several examples of metabolic engineering describing
the potential of microbes to produce the desired fatty acids. It was reported that
production of FAEEs in final concentration was 8.2 mg/L by expressing wax-synthe-
sizing genes from Marinobacter hydrocarbonoclasticus DSM 8798 and upregulating
the E. coli endogenous acetyl-CoA genes [26]. To further improve the production of
FAEEs, researchers overexpressed the endogenous acyl-CoA–binding protein, and
NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus
mutans in the parent strain. With the later, final concentration of FAEEs production
was achieved as high as 47.6 mg/L [27].

Synthetic biology has also revolutionized the field of plant-based pharmaceuticals by

reinventing the procedures through which the plant metabolites are synthesized and
modified in plant systems [28]. The plant products or plant secondary metabolites are
usually not essential in the plant growth and functioning, rather help in the survival
of the plant by being UV protectant, antigerminant to other plants, protect the plants
from insects and pathogens, etc. [29]. But these secondary metabolites are highly
useful to humans as they can be used as drugs, food colorant, flavors, and fragrances.
They can be classified into phenolics, terpenes and steroids, and alkaloids. However,
the production of these metabolites in plants is limited. Because of this, the use
of microbial systems seems a promising approach to produce metabolites in large
quantities [30].

Besides these, the most studied use of synthetic biology remains the production
of enzymes in large amounts through strain improvement of microbes, genetic
engineering, random mutagenesis, site-directed mutagenesis with the help of di-
verse synthetic biology tools or areas such as bioinformatics, molecular biology,
microbiology, genetic engineering, and recombinant DNA to manipulate the final
product or the whole pathway altogether.

Recently, the improvement in the production of human recombinant enzyme

N-acetyl galactosamine-6-sulfatase (rhGALNS) in E. coli has been demonstrated
[31]. The protein was usually produced in aggregated forms that lead to lower activity.
The authors described the use of different promoters, overexpression of native
chaperon proteins, osmotic shock, and enhancement of cytosolic disulfide bond
formation. It was concluded that the expression of protein under the regulation of
s promoters or expression of protein with proUmod as promoter has enhanced
the protein production effectively and also increased the solubility of the protein.
Another interesting study proposed the increase in the metabolite production by
colocalization of enzymes on synthetic lipid-containing scaffolds (SLSs). In this
technique, lipid-encapsulated bacteriophage 6 was used as SLSs and colocalized
two fluorescent proteins to check the diffusion pattern of the molecules. The size
of the protein-SLSs complex was less than 20 nm. Also, the scaffolds could be used
for carrying out the enzymatic reactions that they confirmed by attaching SLSs to
two indigo biosynthesizing enzymes and showing an increase in the production of
indigo in the host cells [32].

Engineered polyhydroxyl alkanoate synthase from Aeromonas punctata in E. coli

mutant strain XL-1 red has been reported to overproduce the polyhydroxyalkanoate
[33]. There was 126% increase in the production of PHA due to the increased
enzymatic activity by four strains. The resultant activity was approximately five folds
higher than wild-type by all the five strains, which were screened from about 200,000
mutant colonies. In another application of synthetic biology, the substrate prefer-
ence of E. coli glutamate was changed from 2-ketoglutarate to 2-ketobutyrate from
which an expensive drug precursor l-homoalanine was produced. The result of the
study showed that from a feedstock of 30 g/L glucose, 5.4 g/L of l-homoalanine could
be produced [34]. Changing substrate preferences to change the end product of an
enzyme, synthetic biology has endless possibilities. This was also shown by the work
where homology modeling has been used to change the end product of the enzyme
humulene synthase from Abies grandis. This was achieved by changing the 19 target
amino acids out of which 4 had a profound effect on the catalysis of the enzyme that
used farnesyl diphosphate as a sole substrate to produce 52 different sesquiterpenes.
After the mutagenesis, the resultant mutants could produce different but only one
specific product [35]. These studies discussed above showed the importance of
synthetic biology and its use in generating synthetic pathways in host cells. Possible
applications of synthetic biology in diverse fields are shown in Fig. 16.1.
 Figure 16.1. Applications of synthetic biology in various fields.

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