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Mixed carbon substrates: a necessary nuisance or a
missed opportunity?
Nian Liu1, Suvi Santala1,2 and Gregory Stephanopoulos1
Although fermentation with single carbon sources is the
preferred mode of operation in current industrial biotechnology,
the use of multiple substrates has been continuously
investigated throughout the years. Generally, microbial
metabolism varies significantly when cells are presented with
mixed carbon substrates compared to a single carbon-energy
source, as different nutrients interact in complex ways within
the metabolic network. By exploiting these distinct modes of
interaction, researchers have identified unique opportunities to
optimize metabolism using mixed carbon sources. Here we
review situations where process yield and productivity are
markedly improved through the judicious introduction of
substrate mixtures. Our goal is to illustrate that with proper
design of the choice of substrates and the way they are
introduced to cultures, metabolic optimization with mixed
substrates can be a unique strategy that complements genetic
engineering techniques to enhance cell performance beyond
what is accomplished in single substrate fermentations.
Addresses
1
Department of Chemical Engineering, Massachusetts Institute of
Technology, 77 Massachusetts Ave, Cambridge, MA, USA
2
Faculty of Engineering and Natural Sciences, Tampere University,
Korkeakoulunkatu 10, FI-33720, Tampere, Finland
Corresponding author: Stephanopoulos, Gregory (gregstep@mit.edu)
Current Opinion in Biotechnology 2020, 62:15–21
This review comes from a themed issue on Energy biotechnology
Edited by Joe Shaw and Kirsten Benjamin
https://doi.org/10.1016/j.copbio.2019.07.003
0958-1669/ã 2019 Published by Elsevier Ltd.
Introduction
Industrial biotechnology uses cells as biocatalysts to con-
vert substrates into valuable products with high specific-
ity. In most cases, the carbon source is the most important
nutrient providing both energy and building blocks. Cur-
rently, the use of a single carbon source, most notably
sugars such as glucose, is prevalent in both laboratory and
industrial settings due to historical and practical reasons.
With rapidly developing metabolic engineering and syn-
thetic biology tools, microbes harboring rewired metabo-
lism can successfully transform a single sugar into a wide
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variety of products. However, when single substrates are
used, it naturally imposes several limitations on the
metabolism of cells. This is exemplified when the prod-
uct of interest requires long synthetic routes from the
starting substrate or when the product has distinct chem-
ical properties compared to the substrate, both of which
lead to low yield and productivity [1,2]. Providing
microbes with multiple carbon sources, contrastingly,
can potentially ease these constraints and mitigate the
issues since it adds an additional degree of freedom to
cellular systems that can be optimized for product forma-
tion. As such, this review aims to summarize recent
developments where mixed substrates are utilized to
enhance the performance of microbes. We will begin
by discussing carbon catabolite repression (CCR), the
most pressing issue hindering widespread adoption of
mixed substrate fermentation. Then, we will briefly touch
upon the various methods that alleviate CCR and their
importance in the efficient utilization of renewable feed-
stocks [3]. Finally, we will focus on the primary topic of
this review: a discussion of situations where multiple
carbon sources are provided to the cells by design for
significant enhancements in productivity and yield. We
summarize the benefits of mixed substrate metabolism in
four distinct categories, as it can either 1) better balance
the various biosynthetic components towards satisfying
the requirements of the product, 2) simultaneously acti-
vate multiple required metabolic pathways for improved
carbon conversion, 3) provide shortcut access to key
synthesis pathways reducing the overall number of enzy-
matic steps for product formation, or 4) serve as inexpen-
sive inducers for segregating growth and production
phases (Figure 1). By highlighting recent successes in
utilizing mixed substrates, we hope to illustrate the
significance of employing such strategies in biotechnol-
ogy and elucidate the circumstances under which this can
be an effective starting point for the biosynthesis of
products.
Challenges in mixed substrate utilization—
carbon catabolite repression
Bacteria and yeast can commonly utilize several carbon
sources, but this occurs under strict regulation. Most
catabolic pathways are subject to CCR, a global regulatory
system which is found in nearly all heterotrophic hosts
[4,5]. While CCR facilitates optimal growth in complex
environments, it imposes a great challenge for the effi-
cient utilization of multiple carbon substrates in biotech-
nological applications. Because of CCR, the uptake of a
secondary carbon source is inhibited in the presence of a
Current Opinion in Biotechnology 2020, 62:15–21
16 Energy biotechnology

Figure 1

(a) (b)

NAD(P)H

ATP

(c) (d)

Current Opinion in Biotechnology

Examples of strategies where mixed substrate fermentation is employed to optimize cellular metabolism, leading to increases in carbon yield and/
or productivity. (a) Well-designed mixed substrate co-utilization balances key biosynthetic components for more optimal product formation.
(b) Substrate mixtures simultaneously activate multiple pathways leading to increased yield or better consumption of substrates. (c) Using a
secondary substrate in addition to a growth-supporting primary substrate reduces overall enzymatic steps leading to the final product, thereby
enhancing productivity. (d) Automated transition from growth to production phase by adjusting the ratio of mixed substrates leads to optimal
redistribution of resources.

preferred substrate, forcing sequential utilization and
prolonging fermentation time. While some designs
exploit this phenomenon for the control of gene expres-
sion, CCR is generally undesirable in fermentation sys-
tems containing multiple substrates.
The phosphotransferase system (PTS) and cAMP-CRP
complex are the best studied mechanisms that lead to
CCR. They play major roles in the selective transport and
transcriptional regulation of catabolic genes in Escherichia
coli, giving rise to either diauxie or co-utilization depend-
ing on the substrate types [4,6]. Distinct mechanisms for
CCR have also been discovered in other model organ-
isms, such as Bacillus subtilis [7] and Saccharomyces cerevi-
siae [5,8], underlying similar effects. However, despite
numerous efforts to study CCR, the interaction between
two or more substrates is still not well understood in many
organisms. In Yarrowia lipolytica, for example, the inhibi-
tion of xylose uptake by glucose occurs presumably
through regulation of sugar transporters rather than gene
transcription [9], although strains adapted for xylose
utilization do not exhibit this effect [10]. CCR can also
be triggered by carbon sources other than glucose. For
instance, in Pseudomonas putida and Acinetobacter baylyi,
succinate primarily inhibits the pathways responsible for
hydrocarbon and aromatic compound degradation [11].
Overall, despite CCR being a widespread phenomenon
among microorganisms, the substrate pairs causing it as
well as the underlying mechanisms need to be analyzed
on a case-by-case basis, making it difficult to develop a
universal strategy that can overcome this undesirable
effect. A better fundamental understanding is still
required to enable smarter designs that can facilitate
multiple substrate utilization.
Efforts to enable substrate co-utilization for
better conversion of renewable feedstocks
Although CCR is still not well understood in many cases,
efforts have been made for alleviating its effects in order
to allow simultaneous consumption of multiple carbon
sources. Methods employed for substrate co-utilization
include engineering of sugar transporters [12], adaptive

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 Metabolic optimization using substrate mixtures Liu, Santala and Stephanopoulos 17
evolution [13], targeted genome engineering [14], and
introduction of non-native transporters or catabolic path-
ways that are not subject to CCR [15]. More recently,
engineered consortia with different population distribu-
tions have been applied to consume designated substrate
mixtures, which can be a promising alternative approach
[16,17]. The strategies for circumventing the effects
related to CCR are especially important when it comes to
the use of inexpensive and renewable feedstocks contain-
ing mixtures of organic carbon sources, such as lignocel-
lulosic hydrolysates and municipal waste streams. Many
detailed reviews can be found in literature that cover this
topic extensively [18,19–21]. In these cases, the separa-
tion of individual substrates is costly and impractical, and
thus the efficient utilization of substrate mixtures
becomes a necessity that requires additional consider-
ations and engineering efforts. Nonetheless, when con-
ducted properly, the switch from single substrates to
mixed substrates can benefit the cells in many different
ways. As such, the remainder of this review will focus on
the intentional application of mixed substrate metabolism
with an emphasis on how they improve product
biosynthesis.
Substrate co-utilization better balances
biosynthetic components
We begin the discussion by illustrating how co-utilizing
multiple carbon sources can balance key biosynthetic
components. Generally, product synthesis in cells is a
complex process involving the coordination of various types
of biological building blocks. Because of the nature of
metabolism, these are often generated with varying degrees
of efficiency. Thus, it can be difficult for cells to simulta-
neously satisfy all demands at the appropriate ratios using
only a single carbon substrate. For instance, one interesting
idea formulated by Babel et al. describes how different
substrates generate distinct carbon-to-energy ratios and
this ratio is rarely equal to the requirement of the product
[2]. Therefore, in single substrate bioconversions, the
surplus component, either carbon or energy, is wasted,
leading to suboptimal yield. This concept establishes that
for many products, glucose is an energy deficient sole
substrate (i.e. the carbon-to-energy ratio is too high). As
such, an additional energy-rich auxiliary substrate with a
lower carbon-to-energy ratio can be fed to the cells in
conjunction with glucose to compensate for the energetic
deficiency seen in glucose-to-product conversions [2]. Gen-
erally, organic C1 substrates are considered to be conve-
nient energy donors and can be readily supplied to organ-
isms. In particular, formate, formaldehyde, and methanol
have all been successfully demonstrated to improve yields
of various products when co-utilized with sugars for better
balancing of the carbon-to-energy ratio [22–26]. However,
the engineering of heterologous C1 pathways can be chal-
lenging for several industrial hosts and the toxicity induced
by these compounds can be problematic as well [24]. As
such, other carbon sources have also been explored to serve
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as energy-rich auxiliary substrates to be used in conjunction
with glucose, resulting in improvements in titer and yield
compared to glucose-only cultures [27,28].
Taking the auxiliary substrate concept one step further, a
recent publication argued that a balanced supply of three
principle biosynthetic components, carbon, ATP, and
reducing equivalents, are required in order to maximize
the performance of complex bioproduct synthesis [29].
The authors illustrated this in systems involving autotro-
phic CO2-fixation by Moorella thermoacetica, which has
surpluses in carbon and reducing electrons but is severely
limited by ATP. Similarly, the authors also noted that in
acetate-driven lipogenesis by Y. lipolytica, carbon and
ATP are generated in excess whereas reducing equiva-
lents are insufficient. To addresses these challenges,
‘dopant substrates’ providing facile access to the other-
wise limiting biosynthetic components were cofed to the
cells along with the primary substrates for co-utilization
and minimal feeding of the ‘dopant substrate’ (<5% of
total carbon source) was able to accelerate the productiv-
ity by more than twofold. Importantly, under controlled
cofeeding conditions, 13C tracing revealed that the two
substrates interacted synergistically, complementing
each other in their respective roles for generating the
three biosynthetic components. As a result, the measured
productivity exceeded that of the sum of the individual
substrates, a substantial improvement over previous
mixed substrate studies [29].
Substrate mixtures simultaneously activate
multiple critical metabolic pathways
In addition to balancing biosynthetic components, using
substrate mixtures can also simultaneously activate path-
ways that are isolated from each other in the metabolic
network. Doing so enables a unique flux distribution
distinct from single-substrate metabolism that can benefit
the cells in various ways. Mixotrophic fermentation,
where organic (e.g. glucose) and inorganic carbon sources
(e.g. CO2) are co-utilized in CO2 assimilating cells, best
illustrates this idea [30,31]. Under these conditions, both
glycolysis and carbon fixation pathways operate concur-
rently, with the latter re-assimilating the CO2 evolved
from the former, thus allowing for near 100% carbon yield
to the key metabolic intermediate acetyl-CoA [32]. This
has been applied to address the carbon conversion chal-
lenge of traditional heterotrophic sugar-based cultures
where the carbon yield is constrained to at most 67%
acetyl-CoA yield due to decarboxylation of pyruvate.
Jones et al. applied this concept where they engineered
Clostridium ljungdahlii for the production of acetone under
fructose and CO2 mixotrophic conditions [33]. In their
experiments, a product yield higher than the theoretical
maximum heterotrophic yield was achieved due to re-
fixing of CO2. More recent developments in mixotrophy
have demonstrated its utility in producing other
complex products such as mevalonate and isoprene
Current Opinion in Biotechnology 2020, 62:15–21
18 Energy biotechnology
[34]. Furthermore, it can also be applied to various non-
model acetogens [33,35] as well as photosynthetic bac-
teria [36], each with their unique strengths in forming
specific products. Using substrate mixtures to activate
key metabolic pathways is not limited to mixotrophy
however [37,38]. Uranukul et al., for instance, demon-
strated that supplementing limited quantities of glucose
to xylose-based S. cerevisiae cultures allows for efficient
operation of both glycolysis and the monoethylene glycol
(MEG) synthesis pathway. This helps maintain cell via-
bility while allowing for exclusive diversion of xylose
assimilation flux into MEG formation, resulting in
4 g/L of MEG, a near 50% increase compared to
xylose-only cultures [39].
Mixed substrate metabolism provides
shortcut access to key synthesis pathways
Another area that mixed substrate fermentation has
achieved considerable success is in the production of
natural products (e.g. terpenes, polyketides, etc.). The
synthesis of these compounds occurs through long and
complex pathways that are heavily regulated by the cells,
leading to many bottlenecks when using sugars as the sole
carbon source [1]. A strategy to circumvent this issue is to
provide another substrate, in addition to sugars, that can
be more readily converted to the final product in fewer
steps [40]. Under these conditions, a sugar substrate is still
required to sustain cell growth while the added secondary
substrate can bypass decarboxylation steps [41,42], the
need to express heterologous pathways [43], or native
regulations and cofactor requirements [44,45] to boost
flux and carbon conversion towards product formation. To
illustrate this, recent research has engineered an alterna-
tive two-step synthetic pathway that can efficiently phos-
phorylate isopentenols into isopentenyl pyrophosphate
(IPP), the common precursor for nearly all isoprenoids
[44,45]. This reduces the number of enzymatic steps
considerably compared to using glucose as the carbon
source. As a result, when carrying out fermentations using
a mixture of glucose and an isopentenol, the flux through
the isoprenoid synthesis pathway was elevated signifi-
cantly, thereby supporting rapid accumulation of lyco-
pene and other terpenoids [44,45]. In contrast, cell
growth through catabolism of glucose remained mostly
unhindered as the engineered route is completely orthog-
onal to central carbon metabolism [45].
To further expand on this concept, researchers have also
used a secondary substrate as a precursor analog to incor-
porate various functional groups not found in nature into a
final product. Typically, introducing non-native func-
tional groups to metabolites relies on the promiscuity
of all enzymes present in the pathway and hence the
amount of functionalized product decreases dramatically
with longer synthesis steps. By employing functionalized
precursor substrates with fewer steps to the final product,
the chances of obtaining new products can be improved
Current Opinion in Biotechnology 2020, 62:15–21
significantly with the added benefit of detailed control
over functionalization location and fraction. However, a
mixture of substrates is required in this case since the
functionalized secondary substrate generally does not
support growth. A recent publication by Li et al. success-
fully demonstrated this strategy where the authors
achieved alkaloid halogenation at the target location by
feeding halogenated tyrosine derivatives to glucose-
based cultures [46]. Other studies have also employed
this idea for the microbial synthesis of new compounds
with antibiotic functions [47,48]. Overall, the flexibility in
chemical transformations enabled by such a mixed sub-
strate approach can greatly expand the properties of
various natural products, aiding the discovery of novel
applications.
Multi-substrate enabled metabolic control
enhances fermentation performance
Finally, engineering cells to segregate native (e.g. growth)
and non-native (e.g. product overaccumulation) functions
in response to the ratio of various substrates present in the
media represents the last class of mixed substrate fer-
mentation applications that we will discuss [49,50]. This
is typically achieved by coupling the expression of genes
to regulatory elements responsive to specific substrates
such that the substrates themselves serve as inducers for
the transition from growth to production phase. Such a
transition can facilitate optimal resource allocation within
the cells, where the expression of enzymes can be tailored
to biomass production during the initial growth phase and
product accumulation during the later production phase
[51,52]. Additionally, using substrate mixtures to achieve
this goal offers some key advantages. Most notably,
carbon substrates (sugars, organic acids, etc.) are much
cheaper and less toxic than dedicated chemical inducers
and thus should provide a better incentive for applications
in large scale industrial fermentations. The time of tran-
sition between the two phases can be also automated and
controlled through adjusting the initial concentrations of
the different carbon sources present in the mixture.
Here we highlight two recent publications from literature
to serve as examples illustrating how this concept
improves product accumulation. The first example
involves A. baylyi ADP1 engineered to decouple cell
growth and wax ester synthesis using two common con-
stituents of lignocellulose hydrolysates, acetate and
arabinose [53]. The shift from growth-mode to synthe-
sis-mode was achieved by placing a growth-essential gene
under an arabinose-inducible promoter. Initially when
both substrates were present, the cells could replicate.
However, as arabinose was progressively consumed
throughout fermentation, cell growth gradually decreased
and eventually ceased when arabinose was no longer
present. Accordingly, excess acetate was instead chan-
neled through the wax ester synthesis pathway towards
the final product. Tuning the initial ratio of arabinose to
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 Metabolic optimization using substrate mixtures Liu, Santala and Stephanopoulos 19
acetate led to various relative lengths of growth and
production phases. The optimal strain and bioprocess
conditions gave rise to accumulation of nearly 20% of
dry cell weight as wax esters, a fourfold improvement
compared to wild type cells. Similarly, Lo et al. also
developed a system for the automated transition from
cell growth to bioproduct synthesis through the sequen-
tial utilization of substrate pairs [54]. Using their
approach, product biosynthesis was strategically delayed
to occur only after sufficient biomass has accumulated.
The results showed lowered metabolic stress, as well as
increases in both the growth rate during the growth phase
and the productivity during the production phase, illus-
trating the utility of this strategy.
Conclusions and future outlook
The synthesis of bioproducts using a single carbon source
has historically been the preferred mode of operation. By
contrast, using multiple substrates for cell growth and
product formation adds a layer of complexity to the
system, which can be discouraging. Nevertheless, mixed
substrate fermentation introduces an additional degree of
freedom metabolically that facilitates enhancements in
yield and productivity. This is achieved by the judicious
choice of the co-substrates such that they work in
conjunction to either optimally balance biosynthetic com-
ponents, flux distributions, and cellular resources, or
efficiently supply the rate limiting components in fewer
enzymatic steps. Many studies listed in this review have
demonstrated that having multiple substrates can achieve
a similar effect to metabolic rewiring without the need for
extensive genetic engineering. This is essential to
improving the performance of unconventional organisms
that lack the necessary genetic tools. Even in model
organisms, the utilization of more than one substrate
can provide a shortcut to accomplishing the desired goal.
Overall, metabolic optimizations enabled by the interac-
tions among mixed carbon substrates have been success-
fully employed in cultures with different organisms under
various conditions. As this concept is currently attracting
increasing attention from researchers in the field, we
anticipate that the use of mixed substrates, in combina-
tion with current metabolic engineering methods, can be
an opportunity to discover novel designs that further
increase performance of microbes compared to the
state-of-the-art systems.
Conflict of interest statement
Nothing declared.
Acknowledgements
The authors would like to acknowledge the DiSTAP Center of the
Singapore-MIT Alliance for Research and Technology (SMART) and the
US Department of Energy (grant no. DE-SC0008744) for their generous
support. Suvi Santala was also supported by the Academy of Finland (grant
no. 310135, 310188).
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Current Opinion in Biotechnology 2020, 62:15–21