TIBTEC 1551 No.

of Pages 13

Review
The Impact of Systems
Biology on Bioprocessing
Kate Campbell,1 Jianye Xia,1,2 and Jens Nielsen1,3,*
Bioprocessing offers a sustainable and green approach to the production of
Trends
chemicals. However, a bottleneck in introducing bioprocesses is cell factory
Bioprocessing offers a green and sus-
development, which is costly and time-consuming. A systems biology tainable alternative to fossil fuel-based
approach can expedite cell factory design by using genome-wide analyses chemical production. However, cell
factory development is currently too
alongside mathematical modeling to characterize and predict cellular physiol- inefficient to allow bioprocessing to
ogy. This approach can drive cycles of design, build, test, and learn imple- become cost-competitive.
mented by metabolic engineers to optimize the cell factory performance.
Systems biology can streamline fac-
Streamlining of the design phase requires a clearer understanding of metabo- tory design by combining genome-
lism and its regulation, which can be achieved using quantitative and integrated wide datasets with computational
omic characterization[406_TD$IF], alongside more advanced analytical methods. We dis- models to characterize metabolism.

cuss here the current impact of systems biology and challenges [407_TD$IF]of closing the To increase the contribution of sys-
gap between bioprocessing and more traditional methods of chemical tems biology, large-scale omic data
analysis and interpretation need to
production. occur faster and more accurately,
and the predictive strength of in silico
metabolic models needs to be
The Growing Demand for Green and Sustainable Chemical Production
improved.
As the world population grows and non-renewable energy sources decline, there is an
increasing demand to produce chemicals in a sustainable and stable manner. Bioprocessing This can be achieved by (i) developing
offers an attractive alternative to traditional chemical production by using microbial cells as a new computational methods for data
analysis, such as machine learning,
factory for chemical synthesis (Table 1). These typically originate from cell factories used for the
and (ii) integrating into models regula-
production of classical products, but some have also served as model microorganisms used in tory structures that coordinate
basic research, whereby much of their metabolism has been characterized and understood. metabolism.
They include the baker’s yeast Saccharomyces cerevisiae, the Gram-negative bacterium
By increasing the predictive strength of
Escherichia coli, the Gram-positive bacteria Corynebacterium glutamicum and Bacillus subtilis,
models, the cost and time for cell fac-
and the filamentous fungus Aspergillus niger. To transform these microbes into cell factories, tory development can be reduced.
metabolic engineers rewire microbial metabolism to reroute metabolic flux towards chemical
production, a process which may include installation of heterologous genes and deliberate
deregulation of native pathways. If successful, this engineering would enable chemical pro-
duction to occur efficiently at large scale (Figure 1A) and [408_TD$IF]ideally at low cost, with such a cell
factory being referred to as having high titer, rate, and yield (TRY). 1
Department of Biology and Biological
Engineering, Chalmers University of
As well as being sustainable, bioprocessing is also considered to be a green approach owing to Technology, SE412 96 Gothenburg,
its environmentally favorable properties. For example, renewable feedstock can be leveraged Sweden
2
State Key Laboratory of Bioreactor
as an initial energy source (Figure 1B), as opposed to burning fossil fuels. Furthermore, Engineering, East China University of
chemicals produced using bio-based methods can possess ‘cleaner' attributes in comparison Science and Technology, Shanghai,
to their fossil-based equivalents, helping to reduce greenhouse gas emission and concomitant China
3
Novo Nordisk Foundation Center for
effects on global warming [1]. However, despite its environmental benefits, bioprocessing Biosustainability, Technical University
cannot currently compete with the fossil-based chemical industry because of the time and of Denmark, DK2800 Lyngby,
money required for cell factory development (Figure 2). This process typically takes more than 5 Denmark

years, and costs in excess of 50 million $US. For example, to enable opioid synthesis in yeast,
strain construction took 10 years, and [460_TD$IF]to scale up production of the antimalarial drug artemisinin[461_TD$IF] *Correspondence:
, it took 5 years of development and cost >50 million $US [2,3]. nielsenj@chalmers.se (J. Nielsen).

Trends in Biotechnology, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tibtech.2017.08.011 1

© 2017 Elsevier Ltd. All rights reserved.
 TIBTEC 1551 No. of Pages 13

One reason that strain production was previously so cumbersome was because of the Glossary
metabolic engineering (ME) (see Glossary) [4] approach used. This process was usually Evolutionary engineering: a
untargeted and involved multiple rounds of random mutagenesis and screening, resulting in rational/random approach that is
only a subset of the genome of a microorganism being analyzed. As a result, any deleterious used alongside systems biology to
improve cell factory development,
side effects that arose could not easily be interpreted and were challenging to circumvent. one example of which is adaptive
Since then metabolic engineers have implemented a more structured approach to strain laboratory evolution (ALE). It involves
production. This involves performing iterative cycles of design, build, test, and learn (DBTL) controlled exposure of a candidate
strain to increasingly suboptimal
that integrate a systems biology [5–8] approach (Figure 3). This review aims to summaries the
conditions that may be encountered
most recent developments in systems biology within a bioprocessing context. Because this during industrial cultivation. As a
topic and those of a similar nature have been explored in the past, we direct the reader to such result, cells evolve genetic
reviews [410_TD$IF]for additional information [8–14]. adaptations in response to adverse
conditions based on the principles of
natural selection. Evolved strains can
Systems Biology and Bioprocessing then be deep-sequenced to identify
Systems biology uses a data-driven approach to acquire a comprehensive and holistic beneficial genetic mutations that may
understanding of cell metabolism. It allows cell physiology to be fully characterized by mea- have been too complex or non-
intuitive to obtain by rational
suring different components of a cell including mRNA, protein, and metabolites (Box 1).
approaches. Using genome editing,
Computational models can then be used as a framework for integrating these genome-wide causal mutations can then be
datasets, with the aim of representing cellular metabolism entirely in silico. The potential of introduced into a parent strain by
these models is considerable because they allow a large number of combinatorial gene reverse engineering to recapitulate
the desired phenotype. Depending
changes to be tested without experimentalists testing each option in the laboratory; further- on how broad the effect of the
more, they offer predictions for what the best metabolic route or pathway may be. However, for beneficial mutations is, these
these models to produce accurate predictions, quantitative and comprehensive datasets from engineered strains can be used as
different omic layers must first be acquired. platforms for other types of chemical
production as well.
Metabolic engineering (ME): the
Revolutionary developments in sequencing instruments have helped to acquire genome and goal of ME is to optimize the
transcriptome data, and, increasingly, quantitative mass spectrometry methods have enabled synthesis of a desired compound via
microbial metabolism. ME recruits
analysis of the proteome and metabolome. Metabolic flux, the final output of metabolism and its
tools from synthetic biology to
regulation, can also be traced using fluxomics [15]. This method uses 13[405_TD$IF]C-labeled glucose to genetically edit metabolism and
trace where carbon propagates through the metabolic network, enabling a sensitive ‘finger- reroute metabolic flux towards a
print' of flux distribution [16]. Advances in technical and analytical methods have also enabled given metabolic pathway. This may
include reducing the activity of
increasingly less time to be invested in omic measurements and analysis [17].
undesirable pathways that compete
for precursors or that degrade or
Metabolic engineers can also reduce time by (i) recruiting tools from synthetic biology [18,19] reimport the final product. ME may
and (ii) coupling systems biology with evolutionary engineering [20,21]. Since its emergence, also include the installation of
heterologous enzymes that are not
CRISPR/Cas technology has enabled candidate strains to be built in an unprecedentedly short
subject to native regulatory control.
timescale, leading to exponential advances in the initial stages of strain development [22,23]. As a result of metabolic deregulation,
The emergence of in vivo biosensors has also shown promise for expediting the DBTL cycle by the side effects of ME can include
helping to rebalance the build and test phases of strain development [19]. Via high-throughput promiscuous enzyme activity,
unwanted metabolic sinks, and the
fast screening and selection, biosensors increase efficiency by enriching for only a subset of production of toxic or unstable
strains with high performance. Therefore, fewer strains containing a greater proportion of biosynthetic intermediates, leading to
optimal candidates proceed to downstream in-depth physiological characterization [24]. If, poor cellular growth.
after iterative attempts at gene editing, cells show no further improvement in TRY, evolutionary Synthetic biology: because many
commercially attractive chemicals
engineering can also be applied. This approach exploits the natural ability of cells to evolve cannot be synthesized naturally or at
genetic mutations, particularly when they are grown under high selection pressure. When high levels within a host organism,
chemical production is coupled to growth, adaptive laboratory evolution (ALE) can produce tools from synthetic biology are used
to bridge this gap in metabolism. As
evolved strains whose causal mutations can be identified by sequencing and then reverse
well as gene editing via CRISPR/Cas,
engineered into the parental strain. When eukaryotes are used, chemical production can also synthetic biology tools include
be improved via spatial engineering, which aims to redirect [41_TD$IF]metabolic flux towards subcellular artificial codons, regulons, and
compartments where conditions for chemical synthesis may be more favorable [25]. synthetic genomes. This gene editing
toolkit subsequently allows tight
regulation of gene expression to
When impediments in cell metabolism still lead to less than optimal TRY, the open-ended coordinate metabolic output. As well
nature of systems biology analyses enable the identification of alternative microorganisms[412_TD$IF],

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 TIBTEC 1551 No. of Pages 13

which have a natural ability to produce compounds of value. For example, a recent analysis of as helping to build cell factories,
the genomic diversity of Penicillium species revealed that these fungi are an untapped source synthetic biology has also improved
the testing phase via use of
for new antibiotics and pharmaceuticals [26]. A systems biology approach was also applied to biosensors to screen for high-
the oleaginous yeast, Yarrowia lipolytica, which accumulates most of its biomass as lipids, production strains.
making it a promising cell factory for advanced biofuel production. By combining transcrip- Systems biology: this uses
tomics and metabolomics, lipid metabolism was found to have limited transcriptional regula- mathematical models for the analysis
of experimental data to predict the
tion, suggesting that metabolic engineering efforts could be more effective if post-translational behavior of biological systems. This
regulation was targeted instead [27]. data-driven approach can be used
synergistically with metabolic
engineering, evolutionary engineering,
Systems biology may also optimize the bioprocessing approach by helping to increase the
and synthetic biology to optimize cell
substrate range for a given cell factory. This can reduce the cost of microbial fermentation by factory development. Systems
utilizing low-cost and abundant sources of carbon for feedstock. For example, next-generation biology helps to characterize
bioprocessing proposes the use of non-food feedstock to mitigate resource competition microbial metabolism by using high-
throughput omics technology to
between food and fuel. Lignocellulose and its degradation constituents are one such sec-
quantify the entire functional system
ond-generation feedstock that is abundant, inexpensive, and renewable. A systems biology of a cell, including mRNA, proteins,
approach can help utilize this biomass by designing yeast strains that can metabolize hexose and metabolites. This is combined
and pentose sugars found in lignocellulose hydrolysates. Host strains can also be adapted to with computational modeling to
predict and test the activity of
resist toxic compounds arising from lignocellulose pretreatment and recalcitrant lignin removal[413_TD$IF], promising strains in cycles of DBTL.
which can inhibit growth and productivity [28]. For example, bacterial fermentation of switch- Systems biology thus permits more
grass, one of the dominant grasses in North America, was analyzed using multi-omics and accurate in silico representation of
revealed that cells respond to lignocellulose pretreatment by restructuring their cell membrane cell behavior, helping to reduce
human intervention during cell factory
and shifting metabolic flux towards the pentose phosphate pathway over time [29]. development.

Marine macroalgae such as seaweed are another abundant source of sugar, and have been
proposed as a third-generation feedstock. This type of biomass, unlike lignocellulose, does not
compete for arable land with edible crops, and has no or little lignin, reducing the costs and
complexity of biomass pretreatment [30]. However, despite its promise, pretreatment and
saccharification methods to access sugar monomers have yet to be established. Using tran-
scriptomics and metabolomics, however, recent work on red macroalgae led to the discovery
of two key genes and intermediate metabolites involved in carbohydrate metabolism, helping
progress towards future bioconversion of algal biomass into biofuel and chemicals [31].

Another way in which systems biology can enhance the function of cell factories is by
generating platform strains, also referred to as metabolic chassis [32]. These strains increase
metabolic flux towards a biosynthetic precursor which can then be used as a building block for
a wide array of chemicals. For example, much effort has been made in targeting central carbon
metabolism given the high flux that occurs [41_TD$IF]towards the precursor acetyl-coenzyme A (acetyl-
CoA). This metabolite is used in 34 compartmentalized reactions and can be used as a
chemical building block for a variety of commercially profitable compounds such as lipids
(biodiesel, pharmaceuticals), polyketides (antibiotics and anticancer drugs) and isoprenoids
(perfumes and food ingredients) [4,33]. However, central carbon metabolism has evolved to be
extensively regulated both transcriptionally and post-transcriptionally [34,35]. Therefore, to
redirect flux [415_TD$IF]towards these central pathways, metabolic regulation needs to be comprehen-
sively understood [36].

Modeling Cell Behavior

To interpret how metabolism is regulated, large omic datasets need to be integrated and
interpreted easily. The use of genome-scale models (GEMs) can provide the necessary scaffold
for such data integration [37]. GEMs create a mathematical framework to assemble and sort
omic data. Subsequently, these data can be subjected to mathematical analysis and compu-
tational predictions. In addition, the open-ended nature of GEMs enables continual refinement
in their predictive power by recurrently adding constraints from new experimental data. GEMs

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 TIBTEC 1551 No. of Pages 13

Table 1. Commodity Chemicals Produced by Microbial Production

Chemical category Chemical Application [402_TD$IF]Refs

(Advanced) biofuels Isobutanol Next-generation biofuel. Gasoline additive [78]

Bioethanol Gasoline additive [79]

Butanol Gasoline substitute [80]

Biodiesel Petrodiesel additive or [4_TD$IF]substitute [81]

Isopropanol Drop-in fuel, gasoline [45_TD$IF]and diesel additive, possible gasoline [82]
substitute, solvent in the chemical industry

Polymers/commodity chemicals 1,4-Butanediol Building block for making spandex and automotive plastics [83]

2,3-Butanediol Antifreeze, precursor to 1,3-butanediene (synthetic rubber); [84]

derivatives are also used in the fuel and food industries

1,3-Propanediol Building block for textiles, thermoplastics, cosmetics, adhesives, [85]

engine coolants, detergents, insect repellents, fragrances, and
pharmaceuticals

3-Hydroxypropionic acid (3-HP) Building block for biodegradable polymers. Platform chemical for [86]
bulk chemicals such as acrylic acid that is used in manufacture of
plastics, coatings, adhesives, [46_TD$IF]rubber, and paint

Itaconic acid Building block for bulk chemicals. Precursor to polyesters, [87]
plastics, and artificial glass. Potential bioactive compound in
herbicides and pharmaceuticals

Isoprene Feedstock for synthetic rubber, adhesives, [47_TD$IF]paints and coatings. [88]
Potential fuel additive for gasoline, diesel, or jet fuel

Farnesene Precursor to emollients (moisturizer), surfactants, diesel, and [89]

industrial lubricants

Poly-3-hydroxybutyrate (P3HB) Biodegradable plastic [90]

2-Hydroxyisobutyric acid (2-HIBA) Precursor to poly-methyl methacrylate that is used in acrylic [91]
glass, coating materials, and ink

Polyamide (nylon) Fiber for textiles, carpets, and rubber reinforcements. Scaffold for [92]
tissue cultures; bone support for arthroplasty

Muconic acid Platform chemical for nylon and polyethylene terephthalate (PET) [93]

Terephthalic acid (TPA) Monomer precursor to PET that is used in fibers, films, and food [94]
and beverage containers

Hyaluronic acid (HA) Moisturizer used in cosmetics, oral medications, and treatments [95]
of eye and joint diseases

Lactams (butyrolactam, Precursor to biodegradable polyamides (nylon) [96]

valerolactam, caprolactam)

Food and feed additives L-Phenylalanine Raw material for aspartame and pharmaceuticals, a food and [97]
drink additive

[48_TD$IF]L-Tryptophan Building block for pharmaceuticals including antidepressants; [98]

precursor to antitumor drugs violacein and deoxyviolacein

[49_TD$IF]g-Aminobutyric acid (GABA) Bioactive compound in food and pharmaceuticals, precursor to [99]
biodegradable nylon

[450_TD$IF]Lactic acid Solubilizing agent in the pharmaceutical industry, acid-adjusting [100]

agent in the cosmetics industry, food preservative, building block
for polylactic acid (biodegradable plastic)

[451_TD$IF]Cinnamic acid (CA) and p- Taste enhancer, antibacterial agent, and precursor to [101]
hydroxycinnamic acid (pHCA) thermoplastics and cosmetics

Resveratrol Nutritional supplement, cosmetic ingredient, food, feed and [102]

fertilizer additive

Fine chemicals (nutrachemicals) Vanillin Taste enhancer [103]

[452_TD$IF]Santalene Perfume ingredient [104]

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 TIBTEC 1551 No. of Pages 13

Table 1. (continued)
Chemical category Chemical Application [402_TD$IF]Refs

[453_TD$IF]Patchoulol Perfume ingredient. Drug precursor to chemotherapeutic drug [105]

paclitaxel (Taxol)

Acetoin Taste and fragrance enhancer [106]

[40_TD$IF]Active pharmaceutical ingredients (APIs) Artemisinic acid Antimalarial agent [2]

[45_TD$IF]Human insulin Diabetes treatment [107]

[45_TD$IF]Hepatitis B virus Hepatitis B vaccine [108]

[456_TD$IF]Human papillomavirus (HPV) HPV vaccine [108]

[456_TD$IF]Hydrocortisone steroid (cortisol) Anti-inflammatory drug. Also used to treat a variety of cancers [109]
and autoimmune diseases

[457_TD$IF]Codeine Opioid with antitussive (cough-suppressing), antidiarrheal, and [3]

analgesic activities

[458_TD$IF]Thebaine Drug precursor to hydrocodone, oxycodone, hydromorphone, [3]

morphine, and codeine

[459_TD$IF]b-Lactams Antibiotic [67,110]

can be used to perform flux balance analysis (FBA), which uses stoichiometric coefficients of
each metabolic reaction and an objective function, such as biomass, to predict cellular growth
or production rate for a compound of interest [38]. By integrating disparate data types across
functional levels, GEMs therefore help to discover novel correlations and non-intuitive regula-
tory features in metabolism [39].

The aim of in silico predictions is to find an optimal solution space for cell factory designs that
produce optimal chemical TRY. Predictive power, however, is currently handicapped by an
incomplete understanding of the metabolic network. To improve estimates of an optimal ME
strategy, both the physical limitations of metabolic reactions, such as enzyme Kcat and Km
values and the regulatory features that govern metabolic output, such as feedback and
feedforward loops, need to acknowledged [40]. Defining these parameters and incorporating
them into constraint-based modeling with GEMs can subsequently improve quantitative
analyses for how cells respond to changes in either genetic or environmental conditions
[41,42]. By adding additional constraints such as enzyme dependence on cofactors, as well
as pH, thermodynamics, energy coupling, and enzyme promiscuity, it would then be possible
to generate more accurate simulations [43].

Genome-wide data in particular for metabolites is lacking [44]. This immaturity in data analysis is
predominantly due to (i) the chemical nature and complexity of metabolites, (ii) their ability to
change in abundance over sub-second timescales[416_TD$IF], as a result of their interactivity with proteins
and fast turnover, and (iii) their concentrations varying by several orders of magnitude. As a
result, it is inherently challenging to quantify metabolite levels accurately and reproducibly [45].
Despite these setbacks, new insight into gene–metabolome associations have been discov-
ered in unprecedented detail via systematic metabolomic profiling of single-gene knockouts for
S. cerevisiae and E. coli [46,47]. This integration of genome-wide associations with metab-
olomics can then be used to map which genes are linked directly and indirectly with which
metabolites.

Alongside reconciling biological networks, efforts are being made to integrate spatial limitation
into models to understand how a cell may trade between space availability and enzyme

Trends in Biotechnology, Month Year, Vol. xx, No. yy 5

 TIBTEC 1551 No. of Pages 13

(A) Culvaon volume (ml) in orders of magnitude

Shake flask Chemostat Industrial bioreactor
101–102 102–103 > 106

(B) A sustainable carbon cycle

Atmospheric
Carbon fixaon
CO2
CO2
rereleased

Bio-based chemicals
Feedstock e.g., e.g., biofuel, pharmaceucals
lignocellulose or macroalgae and bioplascs

Bioprocessing via
microbial fermentaon
Compound
biosynthesis

Figure 1. The Scale and Impact of Bioprocesses. (A) Cultivation scaling up for the industrial production of valuable
chemicals via microbial fermentation (adapted from [9]). During the development of microbial cell factories culture volumes
can vary by orders of magnitude. Initial metabolic engineering efforts begin by using cultures within Erlenmeyer shake
flasks, which, if successful, progress to optimization in chemostats that can help to simulate industrial bioreactor
conditions. However, larger culture vessels can introduce heterogeneity into the local environment during microbial
fermentation, leading to possible [43_TD$IF]suboptimality in cell performance. (B) A sustainable carbon cycle as a result of clean and
green energy production via bioprocessing. CO2 in the atmosphere can be fixed by photosynthetic organisms such as
plants or algae, which then serve as abundant and renewable sources of carbon for bioprocessing. Microbial fermentation
can then convert this biomass feedstock into value-added chemicals which, after combustion, return carbon back to the
atmosphere.

allocation. Because many ME strategies overexpress membrane enzymes, new predictions on

membrane spatial limitation could benefit these strategies greatly [48]. For example, flux
balance analysis with membrane economics (FBAME) has already been developed to examine
membrane composition of bacterial cells [49]. This method is based on the theory that
transmembrane proteins, such as substrate transporters and metabolic enzymes, compete
for membrane space. For example, although glycolytic enzymes have lower efficiency than
respiratory enzymes, their higher turnover and ability to occupy relatively less space [418_TD$IF]may enable
more room for substrate transporters and therefore could promote higher catabolic activity in
nutrient-rich environments. Based on relative membrane costs and enzyme efficiency, FBAME[417_TD$IF]
can [462_TD$IF]therefore predict more accurately gene expression and cell function [49]. Using protein-
constrained FBA to simulate yeast metabolism, it was also shown that cells may [463_TD$IF]trade off long
energy efficient pathways, such as the electron transport chain, with less energy-efficient but
smaller enzymes used in fermentation[46_TD$IF], to maximize growth using a finite protein pool [50].

6 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1551 No. of Pages 13

Target for industrial implementaon:

Final
strain
Titer, rate, yield (TRY)

Novel
technologies

Exisng
technologies

Proof-of-principle strain

Time

5–10 years
(>200 person years)

Figure 2. Cost and Time Required for Current Cell Factory Development Versus Industrial Targets (adapted
from [4]). Currently, bioprocessing requires heavy investment in time and money to develop cell factories because of
inefficiencies in their optimization. Optimal titer (chemical concentration), rate (chemical production over time), and yield
(chemical synthesized relative to raw material consumed), known as TRY, can be achieved faster if novel technologies are
implemented. Such technologies can include advances in genome-wide data analysis and more predictive models to
simulate metabolism.

Metabolic network New gene funcons Metabolic regulaon Compound Microorganism Enzyme Pathway Synthec Strain
interacvity selecon selecon and predicon pathway opmizaon
pathway design
idenficaon
Gene expression
cluster:

Annotated
Unknown TRY
P

Des
n i
gn
r
Lea

Bu
il

Next- Genome d Cas PAM

generaon t
sequencing Tes F1
F2
RNA-seq Transcriptome F3 ...

LC-MS Target DNA

(phospho) Proteome
gRNA
proteomics Time
LC-MS Metabolome
metabolomics

CRISPR/Cas Adapve laboratory Synthec codons,

High-throughput screening Mul-omic analyses genome eding evoluon regulons, and
chromosomes

Figure 3. T [39_TD$IF] he Design, Build, Test, and Learn (DBTL) Cycle Implemented by Metabolic Engineers for Cell Factory Development. DBTL cycles leverage
tools and technologies from systems biology, metabolic engineering, evolutionary engineering, and synthetic biology to develop efficient cell factories. Gene editing
tools have allowed a large number of candidate strains to be built in short timescales, leading to rate-limiting steps in testing and learning. To balance this cycle, synthetic
biology tools such as biosensors can help to screen and select for top-performing strains. This allows less time to be spent on in-depth physiological characterization via
genome-wide analyses. However, considerable time must still be invested in analyzing and learning from these large datasets. Once a cycle is completed, knowledge
acquired from characterizing the cell factory can be integrated into [40_TD$IF]in silico metabolic models. These models can then evolve to predict with better accuracy how the
next cell factory should be designed. Abbreviations: gRNA, guide RNA; LC, liquid chromatography; MS, mass spectrometry; PAM, protospacer adjacent motif.

Trends in Biotechnology, Month Year, Vol. xx, No. yy 7

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Box 1. Omics Integration

Since the 1990s, omics has enabled high-throughput analysis of cells, and increasingly these technologies have
become accessible for global pathway analyses, enabling interrogation of cellular activity at different functional levels
such as the transcriptome [71], proteome [72], and metabolome [73]. Previously, omics technologies were dominated
by transcriptomic analysis, and the current open-platform method of RNA sequencing enables almost limitless
quantification of most RNA species in the cell [74]. Although single omic analyses are prevalent, an increasing number
of multi-omic analyses have been employed to obtain complementary coverage of metabolism [75,76]. By combining
different omics approaches, solid conclusions can be drawn about metabolic activity and regulation which may be hard
to confirm when a single omic level is analyzed. So far these analyses have mostly been between the transcriptome and
proteome, enabling insight into how post-transcriptional regulation may occur [76,77]. Although these studies are
increasing in prevalence, improvements will still be necessary to enhance the accuracy of the biological data produced
because variation in data quality can often be introduced owing to differences in sample preparation, biases from the
instrument used, and the data analysis approach used, to name but a few. Nonetheless, by integrating different omic
data into computational models, metabolic reactions can be constrained and their predictive accuracy improved [43].

Predictions could also be improved by incorporating protein structure and thermodynamic

properties[420_TD$IF], which may reveal structural effects on enzyme promiscuity, rate of catalysis, and
allosteric regulation [42,51]. For example, GEMs that include structural protein data (GEM-
PROs) can use protein melting temperature to determine how temperature may affect the
growth rate of a given organism [52]. Because many cell factories are not thermotolerant,
insight into protein stability could improve the development of these biocatalysts by pinpointing
the most limiting metabolic processes. For example, using the GEM-PRO platform in E. coli, it
was possible to determine that the cofactor metabolic processes, in particular involving CoA
and biotin, were most growth-limiting under heat stress, therefore their exogenous supple-
mentation could potentially improve thermotolerance [53].

As computational models are developed further, the in silico role of systems biology will
increasingly contribute to the cell factory design process. However, despite expanded models
now containing thousands of reactions and metabolites, metabolic flux analysis can only be
accurately determined for well-known pathways such as central carbon metabolism where
intracellular fluxes are best understood. Similarly, precise kinetic modeling, which usually
requires absolute metabolite concentrations, can only be used on well-defined subcompo-
nents of the entire system [54]. Because these models do not yet integrate the thousands of
metabolites present within a cell, it remains a challenge to predict metabolite and reaction
interactivity on a global scale. Before cell metabolism can be dynamically modeled and
accurately predicted, it is therefore necessary to bridge the gap between model scale and
model precision [44].

Systems Biology and Recombinant Protein Production

Biopharmaceutical proteins such as antibodies, vaccines, blood factors, and hormones (e.g.,
insulin) currently make up 25% of total pharmaceuticals and 40% of sales, and, after
mammalian cells, E. coli and S. cerevisiae are the predominant host organisms for their
production [55]. One way in which systems biology could guide production efforts is through
GEMs for protein secretory machinery. The cell wall of S. cerevisiae can be a significant
deterrent when trying to access intracellular chemical end-products, therefore information on
its secretome could guide efforts towards end-product secretion [56]. Microfluidic [465_TD$IF]screening
can also be used to screen mutant libraries, and this [46_TD$IF]enabled the isolation of S. cerevisiae
strains with more than fivefold improved production of recombinant proteins [57]. Genome
sequencing could identify causal mutations in these strains[42_TD$IF], furthermore, RNA-seq analysis
[423_TD$IF]could identify the underlying physiological changes in these mutated strains that govern the
improved production of recombinant protein [58].

Proteomics can also be applied to determine how genetic editing affects the host protein pool.
Understanding proteome allocation would reveal whether a cell is expressing non-essential
proteins during the bioprocess, such as proteins modulating nutrient adaptation or stress

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resistance, that might hinder recombinant protein production [59]. By removing the expression
burden of such proteins, and ‘streamlining’ the proteome, ribosomes could have a greater
capacity to translate target polypeptides [60]. Decreasing the abundance of non-essential
proteins with high expression costs could lead to the largest improvements in bioprocess
performance [61]. This could, for example, improve growth rate, which may then enhance
chemical production if productivity is growth-coupled. For example, in a recent study that
combined E. coli global absolute proteomic data with genome-scale metabolic modeling, it was
revealed that under many environmental conditions a large fraction of the proteome is unused
[59]. Subsequent proteomic data from hosts re-engineered with a reduced and optimized
proteome could then be used to constrain metabolic models further, allowing more [467_TD$IF]efficient
engineering strategies in cell factory design.

Systems Biology and Scaling

Even when systems biology is exploited fully to retrofit cell metabolism, bridging the gap
between a promising laboratory-scale cell factory (titers up to 5 g/l) and its industrial scale
counterpart (50 g/l) can be a formidable task. Nonetheless, to commercialize a given bio-
process, transitioning to the industrial scale is an essential stage. Industrial scale fermentations
typically involve medium volumes greater than 1000 l (and exceeding 100 000 l for commodity
chemicals and biofuels), necessitating constant mixing to obtain a homogenous environment, a
challenge that increases alongside scale. Imperfect mixing can result in spatial fluctuations of
medium composition, translating to temporal variation in the environment to which cells are
exposed. Such delays in mass and heat transfer lead to gradient formations within the
bioreactor for a variety of components including substrates, products, potentially toxic end-
and byproducts, dissolved oxygen, temperature, pH, and carbon dioxide. Therefore, unlike
their laboratory-scale equivalents, [42_TD$IF]microbes fermenting at the industrial scale undergo constant
changes in their microenvironment and regular feast/famine cycles, resulting in dynamic
metabolic behavior and heterogeneity in biomass, productivity, and yield [62].

Two ways in which systems biology can support strains through this bottleneck is (i) by multi-omic
characterization, and (ii) integrating fluid dynamics and cell metabolism kinetics. Multi-scale
comparative omic characterization can address phenotypic changes arising from scaling by
pinpointing differences in metabolism for the same cell factory in small-scale batch culture
compared to chemostats which begin to simulate industrial conditions. Computational platforms
can also be used to combine transcriptomic, proteomic, and metabolomic data from the same
fermentation run to understand how extracellular conditions affect cell phenotype. Metabolomic
data [425_TD$IF]in particular can be used to establish kinetic models that inform on pathway activity and the
abundance of precursors, byproducts, and target chemicals [63]. 13C-based metabolic flux
analysis can additionally be used to determine how cell physiology changes with cultivation scale.
For example, in 13[426_TD$IF]C fed Penicillium chrysogenum storage metabolites were found to fluctuate as
cells were subjected to feast and famine cycles [64]. Recent advances in genome-scale metabolic
modeling have also allowed the integration of such metabolic information with bioprocessing-
specific parameters such as oxygenation conditions, substrate usage, and cofactor generation,
enabling cell activity to be more precisely characterized [65,66].

Computational fluid dynamics (CFD) is another promising tool to predict volume-related

performance. Based on numerical analysis (e.g., using Navier–Stokes equations) and fluid
mechanics, CFD helps to predict fluid flow, heat transfer, [427_TD$IF]and mass transfer, and therefore
possible rate-limiting steps at different cultivation scales [67]. By coupling CFD simulations of
fluid flow with multi[428_TD$IF]-scale analyses, the bioprocess scale-up [429_TD$IF]can be rationally approached [62].
For example, CFD was recently employed to help [430_TD$IF]design scale-down simulations in which both
the dynamic extracellular conditions within a large-scale bioreactor as well as microorganism
response[431_TD$IF], in terms of substrate uptake, are considered [68]. By solving challenges of scaling

Trends in Biotechnology, Month Year, Vol. xx, No. yy 9

 TIBTEC 1551 No. of Pages 13

and outlining the dynamic metabolic response, it will eventually be possible to design and Outstanding Questions
simulate cell behavior in bioprocessing as systematically as one would in the automotive How can we improve the contribution
industry. of models to the design phase of
DBTL?

Concluding Remarks How can we improve high-throughput

Since the first proof-of-principle [432_TD$IF]industrial strains were proposed more than a century ago the physiological characterization ([435_TD$IF]make
panel of chemicals produced by microorganisms has continued to expand. Improved platform cheaper and less time-consuming) to
develop DBTL?
strains, alternative host [43_TD$IF]microorganisms, increased substrate range, and advances in engi-
neering and computational capabilities have all contributed to improved output. Successful
How can we reduce the work [436_TD$IF]for cre-
production strains at industrial scale, however, seem to be dominated by microbes which ating synthetic genomes?
naturally overproduce the compound of interest, indicating there is still room to develop the use
of heterologous enzymes [69]. However, as technological advances are made in synthetic How can we elucidate the regulatory
biology, ME, and systems biology, cells that have been significantly retrofitted for non-native structures that coordinate
metabolism?
chemical production are beginning to demonstrate commercially competitive titers [2].

One major bottleneck in bioprocessing is that investment is not linear with scale. For example,
costs can be difficult to estimate because of uncertainties in oil prices, government policies, and
unclear costs in scaling up production [70]. In recent years the emergence of systems biology
has improved cell factory development, allowing an increasing number of chemicals to be

Key Figure
The Contribution of Systems Biology to Bioprocessing

Microbial Metabolic engineering

Renewable cell factory
feedstock

Chemical Systems biology

of interest R1 R2 R3 Rn
−1 1 0 . A
1 0 1 . B
S= 0 −1−1 .. C
1 0 1 D
. . . . n
S•v = 0

S ca
le u p
Bioprocessing

Figure 4. Using systems biology in concert with metabolic engineering, optimal cell factories can be built for bioproces-
sing-mediated chemical production. These factories, once finalized, can synthesize chemicals with high titer, rate, and
yield (TRY[401_TD$IF]), at low cost at an industrial scale.

10 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1551 No. of Pages 13

produced in response to commercial demand (Figure 4, Key Figure). To further improve the
efficiency of cell factory construction, metabolic engineers need to progress through fewer and
faster cycles of the design, build, test, and learn cycle (Figure 3). This can be achieved by
beginning with an optimal design strategy using in silico predictive models. Because current
genome-editing tools have allowed exponential advances in strain construction, the rate-
limiting steps of cell factory development predominantly occur in the test and learn phases
of DBTL (see Outstanding Questions). For example, although large datasets can lead to
meaningful biological insight, the time taken to interpret them remains considerable and
can be biased by sample preparation as well as by the analytical methods used for measure-
ment and data processing. To improve this, more systematic [43_TD$IF]and precise analytical
approaches could be integrated into the omic analysis workflow to expedite learning. Future
work should also be invested in the analysis of more host organisms, ideally to acquire in vivo
absolute values for RNA, proteins, and metabolites under dynamic conditions. In addition, the
various regulatory structures orchestrating metabolic output should be more clearly charac-
terized. During the test phase, new synthetic biology and evolutionary engineering methods,
such as biosensors and ALE respectively, can also be implemented more routinely to identify
top-performing candidates in high throughput, thereby reducing the number of strains for multi-
omic analysis [19,21].

It is clear that systems biology approaches have become an integrated part of cell factory
development for any new bioprocess. However, we foresee that systems biology in particular
will benefit cell factory development when there are major demands on TRY because here it is
necessary to approach close to maximum theoretical yields, and this will require major rerouting
of metabolic fluxes in the cell. For other processes, different systems biology approaches
discussed here may also be applicable, a good case in point being the improvement of
recombinant protein production by gaining better insight into the protein secretory pathway.
In conclusion, systems biology has evolved to become an indispensable approach for next-
generation bioprocessing. By continuing to advance this field, the efficiency and economic
competitiveness of bioprocessing efforts will likely also increase as well, making it possible to
address the growing demand for fuels and chemicals via greener and more sustainable
production processes.

Supplemental Information
Supplemental information associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.
tibtech.2017.08.011.

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