5.

724

Review

Past, Present, and Future

Perspectives on Whey as a
Promising Feedstock for Bioethanol
Production by Yeast

Jing Zou and Xuedong Chang

Special Issue
Leveraging Yeast Biodiversity for Biotechnology
Edited by
Prof. Dr. Isabelle Masneuf-Pomarede and Dr. Cécile Neuvéglise

https://doi.org/10.3390/jof8040395
 Journal of
Fungi
Review
Past, Present, and Future Perspectives on Whey as a Promising
Feedstock for Bioethanol Production by Yeast
Jing Zou * and Xuedong Chang

College of Food Science and Technology, Hebei Normal University of Science and Technology,
Qinhuangdao 066600, China; [email protected]
* Correspondence: [email protected]; Tel.: +86-0335-2039074

Abstract: Concerns about fossil fuel depletion and the environmental effects of greenhouse gas
emissions have led to widespread fermentation-based production of bioethanol from corn starch
or sugarcane. However, competition for arable land with food production has led to the extensive
investigation of lignocellulosic sources and waste products of the food industry as alternative sources
of fermentable sugars. In particular, whey, a lactose-rich, inexpensive byproduct of dairy production,
is available in stable, high quantities worldwide. This review summarizes strategies and specific
factors essential for efficient lactose/whey fermentation to ethanol. In particular, we cover the
most commonly used strains and approaches for developing high-performance strains that tolerate
fermentation conditions. The relevant genes and regulatory systems controlling lactose utilization
and sources of new genes are also discussed in detail. Moreover, this review covers the optimal
conditions, various feedstocks that can be coupled with whey substrates, and enzyme supplements
for increasing efficiency and yield. In addition to the historical advances in bioethanol production
from whey, this review explores the future of yeast-based fermentation of lactose or whey products



for beverage or fuel ethanol as a fertile research area for advanced, environmentally friendly uses of


industrial waste products.
Citation: Zou, J.; Chang, X. Past,
Present, and Future Perspectives
on Whey as a Promising Feedstock
Keywords: whey; bioethanol; lactose; GAL gene; Kluyveromyces; Saccharomyces cerevisiae; metabolic
for Bioethanol Production by Yeast. engineering; optimization condition; evolutionary engineering
J. Fungi 2022, 8, 395. https://doi.org/
10.3390/jof8040395

Academic Editors:
1. Overview of Bioethanol Production and Whey as a Feedstock
Isabelle Masneuf-Pomarede and
Cécile Neuvéglise Energy security and environmental safety are two major issues currently faced by
the global population that have led to elevated demand for alternative and ecologically
Received: 7 March 2022 sustainable energy sources. By some estimates, fossil fuel reserves could be exhausted
Accepted: 11 April 2022
within the next 40–50 years due to the rapidly increasing consumption of these non-
Published: 12 April 2022
renewable fuels [1]. More importantly, the burning of fossil fuels contributes to the emission
Publisher’s Note: MDPI stays neutral of greenhouse gases and, consequently, global warming, which causes further climate
with regard to jurisdictional claims in change, rise in sea level, loss of biodiversity, and urban pollution [2–4]. In particular,
published maps and institutional affil- bioethanol refers to the alcohol produced from biological sources through the fermentation
iations. of starches, sugars, cellulose, or other waste by-products of industrial food or chemical
processing. Ethanol is distinct from gasoline in that it has both higher evaporation enthalpy
and laminar flame speed, which results in more efficient combustion with less greenhouse
gas emission. [5–8]. This higher evaporation temperature of ethanol results in higher
Copyright: © 2022 by the authors.
volumetric efficiency and thus more power generated by a gasoline/ethanol blend than
Licensee MDPI, Basel, Switzerland.
that provided by an equivalent unit of gasoline [9]. Specifically, bioethanol is more highly
This article is an open access article
oxygenated than gasoline (i.e., 34.7% oxygen) which improves its combustion efficiency
distributed under the terms and
by 15%.
conditions of the Creative Commons
Attribution (CC BY) license (https://
First- and second-generation bioethanol can be categorized according to the feedstocks
creativecommons.org/licenses/by/
used for production. The primary raw materials for first-generation bioethanol are starch
4.0/).
and sugars generally obtained from starchy cereal crops, especially corn, due to its global

J. Fungi 2022, 8, 395. https://doi.org/10.3390/jof8040395 https://www.mdpi.com/journal/jof

J. Fungi 2022, 8, 395 2 of 22

availability, relatively simple and high yield conversion, and amenability to long-term
storage. The United States is the dominant producer of corn ethanol, with stated goals
of 36 billion gallons of ethanol production annually by 2022 using corn and corn stover
as inputs [10]. Sugar-based feedstocks include some energy crops, such as sugarcane,
sugar beet, and sweet sorghum as well as sugar refinery wastes, namely cane and beet
molasses [11–14]. These sugar crops offer advantages over other feedstocks through greater
sugar yields and lower costs of ethanol conversion compared. However, the availability of
these crops can be limited by growing season [13].
Among second-generation bioethanol, lignocellulosic biomass is the most commonly
used feedstock since it represents the largest resource pool worldwide and can thus be
obtained for ethanol production without competing for arable land and agricultural in-
puts with crops for human or livestock consumption [15–17]. The chief components of
lignocellulosic biomass that are converted to ethanol include cellulose, hemicellulose, and
lignin [16,18–21]. However, the major drawback of these feedstocks is the recalcitrance to
degradation of the lignocellulosic matrix, which is comprised of covalently and hydrogen-
bonded cellulose and hemicellulose polymers that are further linked to lignin in its natural
state [21,22]. In addition, Saccharomyces cerevisiae, the most widely used ethanol-producing
species in bioethanol production, has difficulty utilizing β-D-xylose and α-L-arabinose, the
main pentoses in hemicellulose polymers [18,23–26]. These factors thus limit the applica-
tion of lignocellulosic biomass in ethanol production until the efficiency of its degradation
can be improved.
Apart from starch materials and lignocellulosic biomass, industrial waste, especially
from food production, offers a range of viable feedstock options for fuel ethanol production.
Among these by-products, whey generated by the dairy industry can be reliably substituted
for sugar.
In the process of cheese making or casein production, the water component of milk
is largely separated into the whey, while the solids aggregate in curds, which are further
processed. Whey can be categorized as either sweet whey, which results from casein coagu-
lation by rennet activity, or acid whey, produced by acid coagulation of milk. The former
is typical of aged cheeses, while the latter is commonly used in fresh cheese production,
such as cottage cheese or ricotta [27,28]. Table 1 shows the average composition of sweet
and acid wheys. In general, acid whey has a characteristically low pH, while the pH of
sweet whey is around 6.5 [29]. Whey accounts for approximately 85–90% of milk used to
make aged cheeses. In 2019, the FAO reported that a total of 116.9 million tons of fresh
whey were produced globally, compared to 2.8 million tons of dry whey (Figure 1), with
90% of fresh whey coming from facilities in Europe and the Americas (FAOSTAT, 2022).
Whey contains roughly 55% of the nutrients from milk, and it includes a lactose content of
6.8–8.5% and 0.8–2% mineral salts, depending on the production method [30–33].

Table 1. Composition of different types of whey [27,28].

Parameter Sweet Whey Acid Whey

Total solid (%) 6.21 5.70
Lactose (%) 4.82 4.60
Protein (%) 0.75 0.30
Fat (%) 0.05 <0.01
Ash (%) 0.60 0.80
pH 5.80–6.10 4.0–5.0

Despite its generally high biochemical and chemical oxygen demands (BOD and COD,
respectively), which are associated with environmental and health problems [34,35], in
many ways, whey represents an ideal substrate for fermentation into biofuel. Moreover,
whey can serve as an inexpensive resource for high-energy fuel gas production (e.g., hy-
drogen gas [36–38] or methane [39,40]) in anaerobic fermentation systems. In addition to
gaseous biofuels, whey can also be used as a feedstock in the fermentative production of
 –

J. Fungi 2022, 8, 395 3 of 22

liquid fuels such as bioethanol [41,42], bio-butanol [43,44], or other commercially valuable
chemicals (e.g., 2,3-butanediol [45,46] or organic acids such as lactic acid, caproic acid,
or citric acid [47–49]). Despite these numerous possible uses, bioethanol production has
emerged as the predominant product of industrial whey fermentation.

dry whey
1.2 × 108 fresh whey
Production of dry and fresh whey, tonnes

1.0 × 108

8.0 × 107

6.0 × 107

4.0 × 107

2.0 × 107

0.0
2010 2011 2012 2013 2014 2015 2016 2017 2018 2019
Years
Figure 1. Worldwide production of dry whey and fresh whey from 2010 to 2019 (FAOSTAT, 2022).

Therefore, whey is indeed an ideal alternative feedstock for fuel ethanol feedstock
because it can provide a remarkable 6–10 million tons of lactose annually. However,
despite the availability of this massive, underutilized resource, it faces challenges in being
adopted at a commercial scale since S. cerevisiae, the most common fermentative species for
transforming sugars to ethanol, lacks the enzyme required for lactose utilization and thus
cannot ferment whey into ethanol without further metabolic engineering. In this paper,
we review the past and future of S. cerevisiae and whey/lactose in bioethanol production
and introduce recent advances in whey/lactose utilization for biofuel fermentation, with
the broader goals of finding appropriate solutions to whey pollution and resolving the
shortage in raw materials for biofuel.

2. The Lactose/Galactose Metabolic Pathway and Its Regulation in Fungi

2.1. Lactose/Galactose Consumption in Fungi
Lactose is a disaccharide formed from galactose and glucose and chemically defined
as O-ß-D-galactopyranosyl-(1–4)-β-D-glucose. In addition to lactic acid bacteria and en-
terobacteria that are able to utilize lactose as a sole carbon through different transport
systems [50–53], some fungi, such as Aspergillus [54–56], Trichoderma reesei [57,58], and
members of the yeast genus Kluyveromyces [59–61] also display this metabolic capability.
In these fungi, lactose is utilized through two principal mechanisms: (1) the lactose is
 – –
– β –

J. Fungi 2022, 8, 395

– – 4 of 22
–

extracellularly hydrolyzed into D-glucose and D-galactose, which are subsequently taken
up by the fungi (Figure 2a); and (2) the disaccharide is first imported into the cytosol and
β then hydrolyzed into glucose and galactose (Figure 2b). β
β
β
β-D-galactoseext β-D-galactose

Lactose β-D-galactoseint Lactoseext

β-galactosidaseint glucose
glucoseext Lactoseint
glucoseint
Cell membrane Cell membrane
,β
(a) (b)
,β
Figure 2. Lactose metabolism in different microorganisms. (a) In Trichoderma reesei, β-galactosidase
(bga1) is secreted to the extracellular space, where it converts lactose into equimolar glucose and
) where it is catalyzed into galactose and glucose by cytosolic β
galactose monosaccharides, which are then imported into the cytoplasm. (b) In Aspergillus nidulans
and Kluyveromyces spp., lactose is first transported into the intracellular space by lactose permease
(LAC12)) whereuses
where it isthe
it is former
catalyzed
catalyzed into pathway
into galactose
galactose for
and
and lactose
glucose
glucose uptake,
by although
cytosolic
by cytosolic β
β-galactosidasethe extracellular
(LAC4). β

uses the
T. reesei uses the former
former pathway
pathwayfor forlactose
lactoseuptake,
uptake,although
although thethe
extracellular
extracellular β
β-galactosidase (encoded by bga1 gene) is a critical factor for lactose-induced cellulase
production [62,63]. A. nidulans and K. lactis, a model microorganism of Kluyveromyces
yeast, both utilize lactose through the latter strategy by lactose permease (encoded by
sequent
LAC12hydrolysis by βtransport of the disaccharide into the intracellular
gene)-mediated gene)compartment
into glucose and β
and
subsequent hydrolysis by β-galactosidase (encoded by the LAC4 gene) into glucose and
sequent hydrolysis
β-D-galactose. Thisby β does not discuss the catabolism of es,
review gene)
α into
D -glucose glucose
in detail and β
since
several current reviews already address this topic. In most eukaryotes, α-D-galactose
epimerase to the α
is converted by aldose 1-epimerase to the α-anomer before entering es, α the Leloir path-
way (Figure epimerase
3) [64,65], although filamentous
to the α fungi contain a second pathway, the oxido-
bolicreductive
pathway, for αpathway, for α-D-galactose catabolism [55,66]. However, some strains
catabolic
of K. marxianus have been found to hydrolyze lactose outside of the cell (Figure 2a) [67,68].
bolic pathway, for α

β-galactoseext

1P-glucose
1P-galactose
Galactokinase 6P-glucose

Cell membrane UDP-glucose UDP-galactose

ATP ADP

Figure 3. The Leloir pathway of D-galactose catabolism.

Unlike K. lactis, S. cerevisiae lacks the gene for β-galactosidase synthesis, although some
S. cerevisiae strains can secrete intracellular α-galactosidase, encoded by the MEL1 gene.
lacks the gene for β
These S. cerevisiae strains can thus utilize
lacks melibiose
the gene as
fora βsole carbon source [69,70]. In S.
strains can secrete intracellular α
cerevisiae and K. lactis, galactose is catabolized through the Leloir pathway, which includes
strains can secrete intracellular α
a four-step enzymatic reaction [64,65]. In S. cerevisiae, the galactokinase Gal1 mediates the
initial phosphorylation of galactose in an ATP-dependent manner, resulting in galactose-
1-phosphate. Then, uridine diphosphoglucose 4-epimerase (Gal10) exchanges glucose
in UDP-glucose with the phosphorylated galactose, thereby generating UDP-galactose,
J. Fungi 2022, 8, 395 5 of 22

changing the stereochemistry at C4 to create UDP-glucose. In the third step, galactose-1-

phosphate uridylyltransferase (Gal7) uses glucose moieties released in the previous step
to generate glucose-1-phosphate from galactose-1-phosphate. In the fourth step, glucose-
1-phosphate is converted into glucose-6-phosphate through phosphoglucomutase (Gal5)
activity. In S. cerevisiae, phosphoglucomutase is encoded by PGM2 (Table 2) [65,71–73].
Among the four catalytic enzymes required for the Leloir pathway, Gal1, Gal7, and Gal10,
which form a cluster of similarly regulated genes located on chromosome II, are specific
to this pathway, while phosphoglucomutase contributes to metabolic pathways for many
different carbon sources.

Table 2. Galactose/lactose catabolic and regulatory genes and their respective annotations in
S. cerevisiae and K. lactis.

S. cerevisiae K. lactis
Category
Gene Name Function Gene Name Function
MEL11 α-galactosidase LAC4 β-galactosidase
GAL2 Galactose permease LAC12 Lactose/galactose permease
Bifunctional Bifunctional galactokinase/sensor
GAL1 KlGAL1
Catabolic galactokinase/sensor inducer [74,75]
genes Galactose-1-phosphate Galactose-1-phosphate
GAL7 KlGAL7
uridylyltransferase uridylyltransferase
Uridine diphoshpoglucose Uridine diphoshpoglucose
GAL10 KlGAL10
4-epimerase 4-epimerase
GAL5(PGM2) Phosphoglucomutase KlGAL5 Phosphoglucomutase
GAL4 Transcriptional activator [75] KlGAL4(LAC9) Transcriptional activator [74,75]
Regulatory GAL80 Gal4p repressor [74,75] KlGAL80 Gal4p repressor
genes Gal80 repressor
GAL3
(sensor/inducer) [76]
1. Only some strains of S. cerevisiae carry the MEL1 gene.

2.2. GAL Gene Regulation in S. cerevisiae and K. lactis

In yeast, the expression of GAL genes is tightly regulated by different carbon sources
through three main GAL-specific regulatory proteins: a transcriptional activator, Gal4;
a repressor, Gal80; and a ligand sensor, Gal3 (Table 2). Furthermore, GAL regulation is
divided into three states depending on the carbon source. First, in the presence of aerobic
or respiratory carbon sources, such as glycerol or raffinose, GAL genes are in an inactive
or non-induced state that is expressed only at basal levels. In K. lactis, basal GAL gene
expression is higher than that in S. cerevisiae due to the higher endogenous levels of KlGal4
protein induced by an autoregulatory positive feedback loop [77–80].
In the second state, when galactose is present in the medium, GAL genes are in the
induced or activated state. In this state, galactose is imported into the cytosol by Gal2
permease, where it binds Gal3p, which also allosterically binds ATP, resulting in Gal3p
activation. The activated galactose-ATP-Gal3 complex interacts with Gal80 through a
cooperative network of hydrogen bonds [76,81]. As a result of this interaction, Gal80 disas-
sociates from Gal4, and Gal4 is released to transcriptionally activate GAL genes, ultimately
leading to >1000-fold increases in GAL2, GAL1, GAL7, and GAL10 expression in S. cere-
visiae [82]. Apart from GAL4 itself, the other GAL genes harbor specific upstream activating
sequences (UAS) in their promoter regions that are recognized by Gal4 homodimers to
induce their transcription. Notably, the GAL4 gene lacks a Gal4 binding site in S. cerevisiae,
while the LAC9 gene (i.e., KlGAL4) in K. lactis has a weak UAS bind site. Consequently,
Gal4 concentration in K. lactis is two- to three-fold higher than that in S. cerevisiae in the
non-induced state [74,75]. In addition, there is no GAL3 gene in K. lactis, and its function is
substituted by Gal1, a bi-functional protein that acts as a GAL gene inducer in addition to
its function as a galactokinase [75].
J. Fungi 2022, 8, 395 6 of 22

In the third regulatory state, GAL gene expression is actively repressed in the presence
of glucose, or so-called glucose repression. To induce glucose repression, the concentra-
tion of the transcriptional repressor Mig1, a Cys2-His2 Zinc-finger DNA-binding protein,
increases within minutes of yeast cell exposure to glucose, after which Gal80 enters the nu-
cleus and interacts with the general co-repressor complex Cyc8/Ssn6-Tup1, to form a com-
plex that targets a specific upstream repression sequence (URSG) present in the promoter
region of GAL genes [83–85]. For K. lactis, which is more adapted to lactose/galactose-rich
environments, glucose repression of GAL/LAC gene expression is absent in some K. lactis
strains, and in strains that do exhibit glucose repression, the repression is less pronounced
than in S. cerevisiae. This phenomenon is reflected by the fact that only the GAL1 gene
promoter in K. lactis carries a URSG, whereas the promoters of GAL1/2/3/4 all harbor
URSG motifs in S. cerevisiae [78,80].
In addition to this canonical model of GAL gene regulation, previously unrecognized
regulators of GAL gene function are still emerging. For instance, deletion of the cytochrome
c oxidase subunit COX9 results in a respiration-deficient strain that can rapidly and effi-
ciently ferment galactose [86]. Other work has shown that the SIP1 gene, which encodes a
component of the Snf1 kinase heterotrimer complex, is a regulator of preferential glucose
consumption in S. cerevisiae [87]. Deletion of SIP1 can destroy the glucose repression at 1:10
ratio of galactose-to-glucose [88]. In addition, the MIG1-related protein Imp2p has also
been reported to control GAL gene expression by positively affecting glucose de-repression
of maltose, galactose, and raffinose metabolic pathways. This protein was also found to
contribute to thermal, oxidative, or osmotic stress resistances in yeast [84].

3. Bioethanol Production from Whey/Lactose by Kluyveromyces

While the fermentation of alcohol from cheese whey or whey permeate represents a
relatively new approach to mitigating waste from the dairy industry, yeast-based fermen-
tation of lactose from whey to generate ethanol is first introduced as early as the 1940s,
or possibly earlier [89–91]. The separation of whey proteins generates whey permeate,
which contains the majority of lactose and other whey solids. Industrial facilities that use
whey permeate for ethanol production have been established in Ireland, New Zealand, the
United States, and Germany. Among these, the first plant to commercially produce ethanol
from whey permeate was built in 1978 in Ireland by Carbery Milk Products Ltd. to produce
alcohol for beverages. This company has also produced ethanol from whey for E85 and
E5 oil blends since 2005 [92]. The process first established at this plant is also employed at
fermentation facilities in the United States and New Zealand.
The private producer of ethanol from casein whey feedstock is Anchor Ethanol Ltd.,
a subsidiary of the Fonterra New Zealand dairy cooperative, which claims to produce
~5 million gallons of ethanol annually. In the United States, whey permeate is fermented at
two industrial-scale plants, both using the Carbery process, that are together responsible
for 8 million gallons of fuel ethanol annually [93]. The Carbery process uses batch fermen-
tations and continuous distillation in which the pH of the permeate is reduced to 5.0 with
sour whey or acid, then pasteurized by heating to 85 ◦ C for 15 s. The pasteurized permeate
is then chilled to 34 ◦ C, loaded into the bioreactor, and inoculated with K. marxianus. The
conditions have been optimized for efficient and rapid lactose conversion to ethanol in
12 h fermentations, with an additional 6 h of cooling in the chamber before distillation [94].
More recently, productivity has been increased through continuous fermentation and use
of whey concentrate as feedstock.
Now, the Kluyveromyces yeast can mediate lactose fermentation into ethanol because
they harbor genes for both lactose permease and β-galactosidase. Among these species, the
physiological and molecular characteristics of K. lactis have been widely studied as a model
for “non-conventional yeasts” in comparative analyses with S. cerevisiae [95,96]. K. lactis has
been used as a progenitor for other strains due to its ability to efficiently utilize the lactose
in concentrated cheese whey permeate as a raw material for producing ethanol in static
cultures [59]. Apart from applications in ethanol production, K. lactis strains are commonly
J. Fungi 2022, 8, 395 7 of 22

used to produce β-galactosidase [97–100]. However, this species is generally considered

suitable for industrial applications requiring high production and secretion of metabolites
or heterologously expressed proteins in a Crabtree-negative dependent manner [101–106].
K. marxianus, a closely related species to K. lactis, has been adopted by several in-
dustries due to some useful features that are absent in K. lactis [107,108]. Like S. cerevisae,
K. marxianus is a respiro-fermentative yeast that can generate energy either via oxidative
phosphorylation and the TCA cycle or by fermentation to ethanol. Thus, K. marixianus
is frequently used for ethanol production. Several studies have sought to optimize the
utilization of lactose in deproteinized whey, cheese whey powder (CWP), cheese whey
permeate, and cheese whey in batch and/or continuous mode fermentations [109–115].
In addition, K. marixianus can grow and ferment at elevated temperatures, enabling cost
savings in ethanol production bioprocesses [116,117]. Notably, some strains of K. marixianus
are reported to be highly thermotolerant, growing at 43 ◦ C under aerobic conditions with
lactose and/or whey permeate as the sole carbon source [116,118]. However, K. marxianus
is characteristically Crabtree-negative, and its ethanol yields are typically lower than those
of S. cerevisiae [118,119].

4. Strategies for Conferring Lactose Utilization to S. cerevisiae

S. cerevisiae is usually the first choice for industrial processes involving alcoholic fer-
mentation due to its high fermentative capacity and ethanol tolerance [120–122], GRAS
status, rapid growth under anaerobic conditions [123,124], well-established physiology
and genetics in industrial and laboratory applications [125], and the value of its biomass
as an animal feed [126]. However, wild or wild-type S. cerevisiae strains are unable to
metabolize lactose, and thus numerous strategies have been devised and tested for endow-
ing S. cerevisiae with the capacity for lactose hydrolysis to produce ethanol from whey or
cheese whey.

4.1. Pre-Hydrolysis of Extracellular Lactose for S. cerevisiae Utilization

Lactose hydrolysis in whey fermentation results in a mixture of glucose and galactose,
which can then be taken up by S. cerevisiae and fermented into ethanol. Three approaches
to extracellular lactose hydrolysis have been established:
First, β-galactosidase can be used to hydrolyze lactose into galactose and glucose,
which can then be utilized for ethanol production [127]. However, the presence of glucose
induces feedback repression of galactose utilization in S. cerevisiae, resulting in assimilation
of the produced ethanol, lower biomass, and diauxic growth.
Second, β-galactosidase and/or S. cerevisiae cells can be immobilized to produce
ethanol [128–130]. Through immobilization, the cell densities are higher and thus easier
to clear from the medium, thereby reducing the cost of removing cells before distillation.
Immobilization can also facilitate simplification of the fermentation process, thus further
saving equipment and operating costs. Similarly, strains of S. cerevisiae have been devel-
oped to ferment a wider range of substrates for combined fermentations, such as whey
mixed with cellulosic biomass, by engineering the expression of recombinant cellulolytic
proteins anchored or immobilized on the extracellular surface of the plasma membrane
along with β-galactosidase [131]. Substantial research efforts have also been committed to
developing the substrate for enzyme or cell immobilization and surfactants for improving
enzymatic activity, such as silicon dioxide-based nanoparticles, magnetic polysiloxane–
polyvinyl alcohol (mPOS–PVA), polymeric supports, and Triton X-100 [132–134]. However,
immobilization strategies are accompanied by some disadvantages, especially catabolite
repression by glucose after lactose hydrolysis and subsequent diauxic growth of the yeast.
This lag in lactose fermentation following glucose depletion extends the fermentation time
and increases the cost of fermentation.
Third, co-immobilization or co-culture of S. cerevisiae with other microorganisms,
which secrete extracellular β-galactosidase, can facilitate lactose fermentation by S. cerevisiae,
thus enhancing ethanol yields and shortening fermentation time. Co-immobilization of the
J. Fungi 2022, 8, 395 8 of 22

two yeasts has been demonstrated to increase the percentage of theoretical yield over that
of monocultures and enhance the overall volumetric productivity. In addition, immobilized
co-cultures are reportedly more effective than suspension cultures for high-temperature
ethanol fermentations [135–140].

4.2. Protoplast Fusions of S. cerevisiae and Kluyveromyces spp.

Protoplast fusion, a part of evolutionary engineering, has a great potential for genetic
analysis and strain improvement. It breaks down the barriers to genetic exchange imposed
by conventional mating systems. It can serve the purpose of developing a strain with
mixed substrate fermentation abilities [120,141,142]. This technique is generally applied
for developing inter specific, intra specific and inter generic, intra generic supra hybrids
with higher capability. It is a significant tool for genetic manipulation as it resolves the
barrier to genetic exchange imposed by conventional mating systems. It is particularly
useful for industrially important microorganisms [142]. Although S. cerevisiae cannot utilize
lactose, it is tolerant of ethanol and exhibits higher productivity than other fermentation
species, making it the most reliable option for ethanol production. In order to enhance
production by combining the characteristics of ethanol tolerance and lactose utilization
in a single strain, hybrid strains of S. cerevisiae and Kluyveromyces spp. can be generated
through protoplast fusion [143–145]. For example, Guo et al. obtained a stable hybrid
of K. marxianus with an S. cerevisiae, showing higher lactose utilization rates and ethanol
productivity than the parent strain [144]. Krishnamoorthy and colleagues constructed a
hybrid using a temperature-tolerant S. cerevisiae with K. marxianus that resulted in a 12.5%
increase in ethanol productivity at 42 ◦ C [143]. Similarly, Xin generated an S. cerevisiae–K.
marxianus fusion strain with higher lactose fermentation rates and ethanol tolerance than
that of the K. marxianus parental strain, most likely due to its elevated production of linoleic
acid and other unsaturated fatty acids [146]. It should be noted that hybridization can
increase the amount of chromosomal DNA in each cell [143,144].

4.3. Exogenous Expression of the Lactose Hydrolase Gene in S. cerevisiae

One robust approach for increasing the range of sugar substrates for S. cerevisiae
fermentation is through the transgenic expression of lactose hydrolysis enzymes from other
lactose-consuming microbes. S. cerevisiae can thus be metabolically engineered to consume
lactose through amplification, cloning, and the introduction of naturally occurring genes or
pathways that mediate its uptake and catabolism. The majority of lactose utilization genes
used in yeast have been obtained from either Escherichia coli, Kluyveromyces species, or the
filamentous fungus A. niger.

4.3.1. Lactose Metabolism Genes from E. coli

In E. coli, three genes are required for the uptake and metabolism of lactose and related
sugars, including lacZ, lacY, and lacA [147–149]. lacZ encodes β-galactosidase, which
converts lactose into allolactose and subsequent metabolic intermediates. LacY encodes the
lactose permease (LacY) transporter, which mediates lactose uptake into the cell. In order to
ensure that S. cerevisiae, a eukaryote, can successfully express the prokaryotic lacZ, the yeast
promoter CYC1 is typically fused to lacZ prior to its transformation into yeast. Since lacZ
expression is repressed by glucose, an additional ~300 nucleotide DNA fragment is also
fused to the expression construct upstream of CYC1 to drive β-galactosidase expression
in the presence of glucose in S. cerevisiae [150]. However, although lacZ expression was
not repressed by glucose in these yeast transformants harboring CYC1-lacZ, they were
still unable to utilize lactose because they lacked the E. coli lactose transport system and
therefore could not import lactose to the cytosol for access by β-galactosidase.
To overcome this obstacle, researchers sought to engineer the secretion of β-galactosidase
into the culture medium. Porro and his coworkers constructed a lactose-consuming S.
cerevisiae strain that overexpressed the lacZ gene from E. coli and then relied on lysis of the
older mother cells to release the recombinant β-galactosidase. Cell lysis was induced by
J. Fungi 2022, 8, 395 9 of 22

overexpression of the transcriptional activator GAL4. Characterization of the fermentation

properties of the transformed yeast strains indicated that β-galactosidase released in cell
lysates enabled growth on lactose as a sole carbon source or in medium containing whey
as growth substrate. Furthermore, these strains were found to efficiently produce ethanol
during the stationary culture phase following growth on lactose medium [151]. Martegani
and coworkers found that cell lysis was independent of heterologous LacZ expression
in the mother cells [152]. As suggested above, GAL4 overexpression was proposed as
the causative factor in mother cell lysis since the high accumulation of Gal4p can alter
regulatory pathways that affect the composition and structural integrity of cell walls,
ultimately leading to lysis in older cells. In addition, Gal4p participates in the repression
of galactose utilization in the presence of glucose, and excess Gal4p levels have been
shown to possibly mediate concurrent glucose and galactose consumption in the lacZ-
expressing yeast.

4.3.2. The Lactose Metabolism Genes from A. niger

A. niger can utilize an exceptionally broad spectrum of carbon substrates and is known
to secrete numerous glycoproteins, including β-galactosidase, which enables hydrolysis
of lactose in acid whey [153]. A lactose-consuming S. cerevisiae strain was constructed
through the expression of A. niger cDNA encoding secreted β-galactosidase. The lacA gene
(which encodes β-galactosidase in A. niger), including its N-terminal signal sequence that
directed its extracellular secretion, was inserted between the alcohol dehydrogenase (ADH1)
promoter and a transcriptional terminator to construct the pVK1.1 vector construct [154].
The pVK1.1 plasmid was then transformed into S. cerevisiae resulting in β-galactosidase
overexpression. Although the transformant could hydrolyze lactose, the generation time
was 8 h in lactose minimal medium. However, the plasmid exhibited high stability, and 84%
of the cells still contained the plasmid after nine doublings on whey permeate medium [154].
Ramakrishnan studied the fermentation properties of the transformants, GRF167(pVK1.1).
The transformant can hydrolyze the lactose in the medium, and the glucose and galactose
from lactose can be utilized at the same time [155]. In addition, a polyploidy distiller’s yeast
transformed the same plasmid to obtain a lactose-consuming strain, and the transformants
display a higher lactose hydrolysis rate with a simultaneous uptake of glucose and galactose.
In addition, the ethanol yield of the transformants is 90.16% the theoretical yield in lactose
YP medium; however, the stability of the plasmid is poor, as, during fermentation, only
about 10% of the cells retain the plasmid [155].
A lactose-consuming flocculent brewer’s yeast, W204-FLO1L(INT)/pLD1, was con-
structed through heterologous expression of lacA on the pVK1.1 plasmid, the CUP1 copper
resistance marker used to screen successful transformants. The transgenic yeast cells were
able to use lactose with no obvious impact on flocculability [156]. Domingues and cowork-
ers used a flocculent uracil auxotrophy mutant of S. cerevisiae carrying pVK1.1 as a parent
strain to construct a flocculent lactose-consuming strain, NCYC869-A3/pVK1.1 [157]. The
recombinant yeast exhibited high extracellular β-galactosidase activity. Although galactose
uptake was at least partially repressed by glucose in this strain, the strain showed a lactose
uptake rate close to 1.7 g/L/h and with ethanol contents close to the maximum theoretical
yield [157].

4.3.3. The Lactose Metabolism Genes from Kluyveromyces spp.

Different from A. niger, which breaks down lactose in the extracellular space, yeast
in the genus Kluyveromyces transports lactose into the cell for metabolism (Figure 2b).
Therefore, a lactose permease is needed to access the substrate in addition to the lactose
hydrolase. The lactose hydrolase, i.e., β-galactosidase, is encoded by the LAC4 gene, while
the lactose permease is encoded by the LAC12 gene. Since Kluyveromyces is relatively close
to S. cerevisiae, phylogenetically, the eukaryotic lactose transport system from Kluyveromyces
is more compatible with S. cerevisiae regulatory mechanisms. Thus, S. cerevisiae can be
J. Fungi 2022, 8, 395 10 of 22

engineered to grow on lactose through the simultaneous expression of lactose permease

and non-secreted β-galactosidase from Kluyveromyces.
Sreekrishna was the first to construct Lac+ S. cerevisiae strains using a shuttle vector
carrying a 13 kb region of the K. lactis genome [158]. The 13 kb region contained LAC4
and a flanking upstream sequence from K. lactis. Despite the low lactose uptake rate in
transformants, these uptake assays confirmed that the lactose permease gene, LAC12, was
contained in that flanking sequence from K. lactis, between 2.0 and 8.6 kb upstream of
LAC4. That study represented the first report of heterologous expression of a eukaryotic
membrane-bound permease, which laid a foundation for the subsequent cloning of lactose
permease and lactose hydrolase genes from K. lactis [158]. In order to improve the lactose
uptake rate and increase the mitotic stability of the heterologous construct, researchers
then created the MRY276 yeast strain by inserting the LAC4 and LAC12 genes from K. lactis,
under the control of the CYC1-GAL galactose-inducible promoter, into the RDN1 (ribosomal
DNA) locus [159]. MRY276, which harbored multiple copies of both LAC4 and LAC12,
retained the Lac+ phenotype for longer than 60 generations in growth on non-selective
medium, although it exhibited slow growth. The suboptimal growth characteristics of this
strain were likely attributable to the burden imposed by overexpression of these transgenes.
To resolve this issue, transgenic MRY276 was crossed with wild-type strains to yield meiotic
segregants with faster growth and the capacity for lactose assimilation, such as the diploid
strain MRY286 [159]. However, this strain showed low ethanol production but high biomass
production on lactose minimal medium.
Domingues constructed the Lac+ flocculent S. cerevisiae strain, T1, that expressed
LAC4 and LAC12 from K. marxianus rather than K. lactis [160]. The T1 strain showed a
lower capacity for flocculation than that of the parent strain but showed a doubling time
of 5 h, which was substantially faster than the 6.7 h reported for the original Lac+ strain
constructed by Sreekrishna and Dickson [158], but slower than the K. marxianus donor of
the LAC4 and LAC12 genes. However, after an adaptation period, where the strain was
kept in liquid lactose medium, refreshed periodically, fermentation assays indicated that
T1 could metabolize ~90 g/l lactose in liquid medium, thus highlighting increases in rates
of both biomass accumulation and ethanol production from lactose.
In general, the use of a lactose-consuming strain for direct fermentation of whey
to produce ethanol is not economically feasible because the low lactose content in whey
results in low ethanol titer (i.e., 2–3% v/v). Therefore, fermentation should begin with
concentrated whey to obtain high ethanol yields at the end of the process, which also
requires high tolerance to ethanol and osmotic pressure. In S. cerevisiae, the accumulation of
trehalose is widely considered a critical determinant for the improvement of stress tolerance
in yeast, and the deletion of the trehalose degrading enzyme gene can significantly increase
intracellular trehalose content [161].
In particular, a neutral cytosolic trehalase (NTH1) and an acidic vacuolar trehalase
(ATH1) have been identified as the main trehalose hydrolases in S. cerevisiae [162,163]. In
order to obtain a Lac+ S. cerevisiae strain that tolerates high sugar and ethanol conditions
associated with concentrated whey fermentation, the ∆ath∆nth Lac+ strain AY-51024A
was generated, which expressed the permease and β-galactosidase genes from K. marx-
ianus [164]. In this strain, the ATH1 and NTH1 loci were used as the target regions for
integrating the LAC4 and LAC12 genes driven by the PGK1 promoter. AY-51024A showed
roughly equal β-galactosidase activity during growth on different carbon sources and
exhibited high tolerance to ethanol, and could withstand higher osmotic pressure than
the parent strain. In addition, it showed an almost equal rate of glucose uptake and a
higher rate of galactose uptake than its parent strain, AY-5. However, this transgenic
strain was subject to glucose repression, and lactose uptake rates were very slow in lactose
medium (0.98 g lactose/g cell/h). Further analysis confirmed that glucose repression of
lactose/galactose metabolism was responsible for the slower lactose uptake rather than the
result of low copy number of LAC4 and LAC12 [164].
J. Fungi 2022, 8, 395 11 of 22

5. Approaches for Enhancing Lactose Utilization Rate

Considerable efforts have been undertaken to increase lactose uptake rates in order
to improve the efficiency, and hence economic viability, of whey, lactose, or cheese whey
as substrates for bioethanol production. These efforts largely follow three approaches,
discussed below.

5.1. Optimizing Culture Conditions and Media Components

While selecting or developing a strain with appropriate metabolic capabilities may
be an essential step in bioprocessing, low yield rates from lactose to ethanol and ethanol
production rates pose the largest obstacle to efficient whey fermentation. The performance
of fermentation systems is strongly affected by culture conditions such as temperature,
pH, initial inoculum, substrate and nutrient concentrations, trace element availability, and
other factors. Therefore, optimizing culture conditions represents an essential step, and
often underestimated challenge, in fermenting whey products or lactose to bioethanol.
Several approaches have been developed for evaluating and optimizing factors that affect
fermentation time and ethanol production, such as response surface methodology (RSM),
central composite design (CCD), and orthogonal design [112,127,165–167]. In addition,
electro-activation has also been used to enhance both biomass and ethanol production [49].
In addition to culture conditions, optimizing media components is also necessary
for effective ethanol production. Sufficient availability of nitrogen, phosphorus, trace
elements, vitamins, and co-factors, are all important to ensure that fermentation proceeds to
completion as fast as metabolically possible. Thus, inexpensive and nutritionally complete
inputs are needed to supplement industrial whey fermentation [115]. One widely reported,
nutrient-rich input for whey fermentation is corn steep liquor (CSL), which is the primary
by-product of corn starch production. CSL contains abundant nutritional resources, such as
proteins, amino acids, vitamins, minerals, and trace elements, and is relatively inexpensive
and easily obtained [168–170]. In fermentations using the recombinant T1-E strain for
ethanol production from cheese whey powder solutions (CWPS), the addition of 10 g/L
CLS resulted in significantly increased yields of 7.4% (v/v) ethanol from 150 g/L initial
lactose, with a productivity rate of 1.20 g/L/h, whereas 0.75 g/L/h ethanol was obtained
in the absence of CLS. Notably, this approach also stimulated lactose uptake [171].

5.2. Metabolic Engineering

Since it was first defined as ‘the improvement of cellular activities by manipulation
of enzymatic, transport, and regulatory functions of the cell with the use of recombinant
DNA technology’ [172], metabolic engineering has been broadly applied to restructure
metabolic networks by altering the reaction rates and distribution of (carbon) resources in
specific pathways in order to improve metabolite and protein production. As previously
mentioned, although S. cerevisiae cannot utilize lactose, it can consume the lactose metabo-
lite, galactose. Galactose metabolism is subject to dual control by glucose and GAL genes in
S. cerevisiae. Research has revealed that glucose repression of galactose metabolism affects
the overall lactose metabolic process in Lac+ strains [164]. The transcription of GAL genes
is regulated Gal4, which is repressed by the Ssn6-Tup1-Mig1 complex in the presence of
glucose. Mig1, in particular, recognizes a GAL4 promoter motif, and complex binding to
this motif represses GAL4 transcription. In addition to glucose repression, GAL genes are
also negatively regulated by Gal80 and Gal6.
Since eliminating glucose repression can improve the rate of galactose catabolism [173–175],
and the rate of galactose metabolism impacts lactose uptake [164], deletion of MIG1 in Lac+
S. cerevisiae, such as reported in strain AY-51024M, should decoupling the co-regulation of
glucose and galactose of glucose repression. The ∆mig1 strain showed a 2.55-fold increase
in the rate of lactose uptake, and ethanol production increased by 1.75-fold compared with
the control strain, AY-51024A, which harbored intact MIG1 gene, in anaerobic shaken flask
fermentations of lactose medium. In CWPS medium, AY-51024M could produce 63.3 g/L
ethanol from a 150 g/L lactose starting concentration in 120 h fermentations. By contrast,
J. Fungi 2022, 8, 395 12 of 22

under glucose repression, AY-51024A produced only 35.9 g/L ethanol, consuming 63.7% of
the lactose input [164].
Apart from glucose repression, the regulation of GAL genes also impacts the rate
of galactose uptake. For example, the deletion of GAL6, GAL80, and MIG1, which all
downregulate GAL genes, can increase galactose uptake rates by 41% while overexpressing
the transcriptional activator GAL4 increases uptake rates by 26% [174]. Other studies
reported similar effects of manipulating GAL regulators [174,175]. Rønnow and colleagues
showed that deletion of MIG1 and GAL80, combined with transgenic expression of MEL1,
which encoded melibiase, resulted in a melibiose-consuming strain that can metabolize
galactose, even in the presence of glucose. This strain could produce ethanol through the
utilization of melibiose in molasses substrate [176].
Similarly based on the deletion of MIG1, the ∆mig1∆gal80 Lac+ S. cerevisiae strain AY-
GM, ∆mig1∆gal6 strain AYG6M, and ∆gal6∆gal80∆mig1 strain AYG68M were all constructed
using the loxp-KanMX-loxp resistance cassette [177]. However, AYG6M and AYG68M
performed the same or significantly worse than AY-GM and/or AY-51024M in preliminary
assays for carbon uptake and utilization, stress tolerance, and ethanol production [177].
Compared with the ∆mig1 strain, AY-51024M, diauxic growth and glucose repression were
both abolished in AY-GM, leading to significantly higher galactose uptake rates and final
ethanol concentrations. Moreover, tests of AY-GM in three repeated 5L batches using CWPS
medium with 100 or 150 g/L lactose revealed significantly higher rates of lactose uptake
and ethanol production compared to AY-51024M, with lower fermentation times.

5.3. Evolutionary Engineering

In contrast with metabolic engineering, directed evolution or evolutionary engineer-
ing is another main strategy for obtaining high-performance strains for industrial ethanol
fermentation. Evolutionary engineering can be used as an alternative to metabolic engineer-
ing strategies to circumvent obstacles in cloning and gene expression that result from the
incomplete characterization of metabolic networks or a lack of sufficient genetic/metabolic
information about strains of interest. Directed evolution-based strategies thus represent
an inverse approach to that of metabolic engineering in that they can be used to generate
diversity or explore mechanistic differences in performance as a starting point for metabolic
engineering. In S. cerevisiae, evolutionary engineering is generally applied (1) to modify
substrate utilization and product formation or (2) to alter stress resistance [178–181].
Thus, evolutionary engineering can be used to generate lactose-consuming S. cerevisiae
strains. For example, this strategy was used to generate the T1 strain, a Lac+ strain that
can grow on lactose but slowly [160]. Guimarães and coworkers subsequently built on
this work, using evolutionary engineering to obtain a highly effective lactose-utilizing S.
cerevisiae strain, T1-E [182]. After batch selection by serial dilution of cells and transfer to
medium with gradually increasing lactose content, the evolved T1-E exhibited two-fold
higher rates of lactose consumption, 30% greater ethanol production, and the ability to
efficiently ferment concentrated cheese whey [182]. These effects on improved lactose
fermentation were the result of two molecular events. First, the plasmid copy number
was decreased by 10-fold that of T1, and second, a deletion was introduced in the inter-
genic region between LAC4 and LAC12 [182]. Further transcriptomic analysis revealed
that the expression levels of 173 genes differed by more than 1.5-fold in the engineered
strain, roughly half of which were related to RNA-mediated transposition [183]. Although
evolutionary engineering requires more time and does not show the molecular mechanism
underlying new phenotypes, which are disadvantages compared to metabolic engineering,
evolution-based strategies can be an efficient means of inducing metabolism of unfavorable
carbon sources.

6. The Future of Ethanol Production from Whey or Lactose by Yeast

Given the high abundance of organic compounds in whey or cheese whey, combined
with the increasing amounts of these by-products generated by dairy production facilities,
J. Fungi 2022, 8, 395 13 of 22

the utilization or disposal of whey and cheese-whey pose a non-trivial problem as well as
a threat to the ecosystem [34,184,185]. In future solutions to this issue, whey utilization
may be categorized by application. From the perspective of maximizing the value of
this industrial by-product, whey could be used in several different fields, such as the
food industry [184,186–188], animal or livestock feed production [186,189,190], as a raw
substrate for industrial biosynthesis of recombinant proteins, lactic acid bacteria culture,
or for synthesizing metabolites such as L (+) lactic acid and medium-chain carboxylic
acids [191–194], or in the production of alcoholic beverages [195–197], or whey-based fruit-
flavored beverages [198–200]. Alternatively, from the perspective of solving the feedstock
shortage for bioethanol, future research will likely focus on whey-based raw materials and
development of efficient strains.

6.1. Whey/Cheese Whey Mixed with Other High Carbon Feedstocks for Bioethanol
As the lactose content is relatively low, direct fermentation of whey/cheese whey to
ethanol is generally not economically viable and results in low ethanol titre (2–3% v/v),
making distillation costs prohibitively expensive. Thus, concentrated whey powder (CWP)
represents an effective option for fermentation but requires yeast with a high tolerance
for lactose and ethanol. Alternatively, the sugar concentration can be increased by mixing
native whey with other easily-obtained or sustainable materials. Lignocellulosic feedstock
is considered the primary input material for second-generation bioethanol [201]. Ligno-
cellulosic plant matter is the most abundant form of biomass for ethanol fermentation
and can thus serve as a sustainable and cost-effective substrate for bioethanol production.
Approximately 650 billion tonnes of carbon in lignocellulose are estimated to be stored
in forests worldwide, and the forestry or timber industries generate staggering amounts
of sawdust and thinned trees, representing a completely unused lignocellulose resource,
accompanied by increased greenhouse gas emissions. Among harvested trees, Eucalyptus
is the most widely planted hardwood (approximately 18 million ha) and includes over
700 species across tropical, subtropical, and temperate growing regions. Therefore, Eucalyp-
tus waste is a strong candidate raw material for bioethanol production [202,203]. In order to
use these wood by-products for fermentation, they are first subjected to autohydrolysis or
hydrothermal treatment in hot water. This process is purported to have low environmental
impacts and enables simultaneous saccharification and fermentation (SSF) of bioethanol
from lignocellulose in hardwoods or agro-industrial waste. In order to increase sugar con-
centration, the hydrothermally pretreated E. globulus wood (EGW) can also be mixed with
cheese whey powder to increase the available carbon and subsequently obtain high ethanol
concentrations [42]. Whey can also be mixed with other cellulosic biomass than E. globulus,
such as sugarcane bagasse [204,205], spent brewer’s grains [206], or corn cob [131], all of
which improve ethanol yields and result in high ethanol titers when mixed with whey to
increase nitrogen and micronutrient bioavailability.

6.2. Approaches for Efficient Generation of Lactose-Consuming Strains

Obtaining robust yeast strains with strong industrial potential is equally important
for economical bioethanol production as using a high-concentration carbon source to
intensify the ethanol titre. In particular, high-performance yeast should exhibit rapid
conversion of sugar substrate to ethanol, as well as tolerance to high ethanol concentrations
and high temperatures. Advances in metabolic, evolutionary, and genetic engineering
have facilitated the development of research methods for improving lactose/galactose
metabolism. For instance, Chunha used genetic engineering to construct a S. cerevisae strain
that can simultaneously utilize the sugars from cheese whey powder and a zymolytic
solution of hydrothermally treated E. globulus in a multi-waste valorization [42]. In another
good example of inverse metabolic engineering, Lee and coworkers screened a genome-
wide perturbation library to identify three galactose utilization-enhancing genes, including
the phosphomannomutase, SEC53, a small nuclear RNA, SNR84, and a general repressor of
transcription, TUP1 [207]. While SNR84 overexpression improved both growth and ethanol
J. Fungi 2022, 8, 395 14 of 22

production from galactose, the overexpression of a truncated TUP1 variant, tTUP1, resulted
in strikingly increased capability for galactose fermentation, 250% higher in both the rate
of galactose consumption and ethanol productivity compared to that of the control strain.
In addition, overexpression of tTUP1 significantly shortened the lag periods accompanying
the change in carbon source from glucose to galactose [207]. The mechanism for improving
galactose metabolism through SEC53 overexpression may be similar to that of PGM2
overexpression, which encodes a phosphoglucomutase [208].
Adaptive evolution may also serve as an effective strategy for improving galactose
metabolism, especially in combination with metabolic engineering, as illustrated in the
following examples. In a screen for unique strategies for improving galactose uptake in
yeast, Hong et al. used integrated, systems-level analyses to determine the mechanisms
responsible for enhancing galactose metabolism over ≈400 generations of serial culture
on galactose [209]. They found that isolates that evolved the most efficient galactose
consumption harbored mutations in Ras/PKA pathway proteins, which mediate global
carbon sensing, and that these strains had upregulated reserve carbohydrate metabolism
and ergosterol biosynthesis. In particular, the RAS2Tyr112 mutation resulted in a higher
specific growth rate on galactose, indicating that adaptive evolution can lead to increased
galactose flux through cellular exploitation of unexpected pathways, whereas rationally
engineered strains rely on pathways with known effects. This demonstration of the effec-
tiveness of adaptive evolution suggests its potential as a viable alternative approach to
bioengineering metabolically-enhanced strains compared to rational design. In conjunction
with systems biology analyses, genes harboring mutations that strongly affect metabolism
can be rapidly identified and used as targets of further metabolic engineering to enhance
microbial production of biofuels and other valuable metabolites [209].

7. Conclusions
The research focused on using biodegradable wastes, such as whey, for ethanol produc-
tion by combining biochemical pathways from various organisms is very important for both
reducing environmental impacts of food production while improving, cost-effectiveness
and sustainability for other industrial sectors (such as commercial polymers and fuel pro-
duction). Despite the numerous innovations described here, the potential for renewable
energy production and other applications of whey fermentation are largely untapped. Thus,
the research aimed at improving production efficiency and energy consumption through
fermentation of agricultural by-products such as cheese whey will provide substantial eco-
nomic benefits while decreasing energy dependence and mitigating the effects of pollution
as developing nations adopt these advanced technologies.

Author Contributions: J.Z. contributed to the conception and literature survey of the study and to the
first draft and revision of the manuscript. X.C. contributed to the critical revision of the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: This research funded by Hebei Province Innovation Ability Promotion Project, grant num-
ber 20537101D and National Sustainable Development Innovation Demonstration Zone Construction
Science and Technology Special Project (Chengde), grant number 202104F025.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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