Available online at www.sciencedirect.
com
ScienceDirect
Chassis engineering for microbial production of
chemicals: from natural microbes to synthetic
organisms
Jingyi Liu1, Xia Wu1, Mingdong Yao2,3,4, Wenhai Xiao2,3 and
Jian Zha1
Chassis provides a setting for the expression of heterologous
pathway genes, which often requires extensive engineering to
achieve complete functions. Traditionally, chassis engineering
relies on gene deletion/overexpression for the regulation of
precursor/cofactor supply and product transportation, which has
generated thousands of high-performance strains. With the
development of synthetic biology, chassis modifications have
expanded to the synthesis of artificial cellular machineries,
creating synthetic cells for the biosynthesis of bioproducts. In this
review, we will discuss the development of chassis engineering
technologies, termed the first-generation and second-
generation technologies, and their applications in the creation of
chassis for the production of valued-added chemicals, with an
emphasis on synthetic chassis and their applications and
potential. The development of chassis engineering technologies
will advance rational design and construction of customized
chassis for the manufacturing of target bioproducts.
Addresses
1
School of Food and Biological Engineering, Shaanxi University of
Science and Technology, Xi’an, Shaanxi 710021, China
2
Frontier Science Center for Synthetic Biology and Key Laboratory of
Systems Bioengineering (Ministry of Education), School of Chemical
Engineering and Technology, Tianjin University, Tianjin 300072, China
3
SynBio Research Platform, Collaborative Innovation Center of
Chemical Science and Engineering, Tianjin 300072, China
4
Frontier Technology Research Institute, Tianjin University, Tianjin
301700, China
Corresponding authors: Xiao, Wenhai (wenhai.xiao@tju.edu.cn),
Zha, Jian (zhajian1985@sust.edu.cn)
Current Opinion in Biotechnology 2020, 66:105–112
This review comes from a themed issue on Tissue, cell and pathway
engineering
Edited by Li Tang, Peng Xu and Haoran Zhang
For a complete overview see the Issue and the Editorial
Available online 29th July 2020
https://doi.org/10.1016/j.copbio.2020.06.013
0958-1669/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
In microbial production of value-added chemicals, selec-
tion and engineering of chassis microorganisms is
www.sciencedirect.com
practically indispensable, as the introduced heterologous
pathways usually cannot function properly in wild-type
chassis [1,2]. The commonly used chassis include Escher-
ichia coli, Saccharomyces cerevisiae, Yarrowia lipolytica, Bacil-
lus subtilis, Corynebacterium glutamicum, cyanobacterium
Synechocystis, and so on. (Table 1) [3,4]. The traditional
approach of introducing heterologous pathways or mod-
ules into chassis microorganisms involves integration of
original or engineered genetic parts from different organ-
isms. At present, the understanding of chassis microor-
ganisms is still limited, and much remains to be explored
in the functions and properties of heterologous pathways
or modules, making it challenging to achieve customized
design and construction of optimal chassis [5,6].
In practical applications, selection of a chassis for the bio-
synthesis of a target product relies on the physiological
nature of the microbe, including its tolerance to heat and
high concentrations of products, abundance of key intracel-
lular precursors, expression conditions of heterologous path-
way enzymes, and so on (Table 2) [7,8]. Moreover, the
availability of genomic sequences and genetic modification
tools is also highly considered. Following chassis selection,
genetic optimization of chassis strains can be conducted to
achieve functional expression of pathway enzymes, supply
sufficient precursors or cofactors, balance cascade pathway
reactions, enhance product transport, and so on [9,10]. The
rapid development of synthetic biology and various enabling
technologies, such as next-generation sequencing, func-
tional genomics, genome editing, and gene circuits, provides
new ways to create engineered chassis for the production of
useful chemicals [11,12]. In this review, we will discuss the
current strategies to modify chassis microorganisms for the
production of chemicals of interest, including the traditional
approaches of genetic modifications and the emerging tech-
niques using synthetic biology methodology, such as bot-
tom-up reconstruction of cellular machineries.
Engineered natural microorganisms as
chassis
The early stage of chassis engineering mainly includes
selection of suitable organisms for functional expression
of pathway enzymes, regulation of precursor/cofactor
supply, and transport of substrates or target products,
which is achieved via intervention of gene expression
in the chosen chassis, such as deletion/overexpression of
certain genes and upregulation/downregulation of
Current Opinion in Biotechnology 2020, 66:105–112
106 Tissue, cell and pathway engineering

Table 1

Biosynthesis of diverse chemicals in various chassis microorganisms

Chassis Engineering strategy Product Titer Reference

E. coli Regulation of precursor malonyl-CoA supply, Baicalein 23.6 mg/L [46]
optimization of the expression of flavone 6-
hydroxylase from plant Scutellaria baicalensis
E. coli Coupling of cell growth with glutarate Glutarate 54.5 g/L [47]
biosynthesis via the biosynthesis of glutamate
and NADPH
E. coli Engineering of an artificial membrane vesicle b-Carotene 12.7 mg/g DCW [20]
efflux system for decreased intracellular
accumulation
S. cerevisiae Overexpression of key genes for increased Geraniol 1.68 g/L [48]
supply of the precursor geranyl diphosphate
S. cerevisiae Enhancement of zymosterol supply by 7-Dehydrocholesterol 1.07 g/L [49]
overexpressing eight endogenous genes and
blocking the competing pathway
Y. lipolytica Blockage of the pathways competing for Campesterol 453 mg/L [50]
precursor ergosta-5,7-dienol
Y. lipolytica Increase in acetyl-CoA supply, decoupling of Fatty acid ethyl esters 142.5 mg/L FAEE, 23.3 mg/L [51]
nitrogen starvation from lipogenesis (FAEE), fatty alkanes, fatty fatty alkanes, 2.15 g/L fatty
alcohols, and free fatty alcohols, 9.67 g/L free fatty
acids acids
Y. lipolytica Elevation in the supply of the precursor malonyl- Taxifolin 110.5 mg/L [52]
CoA and pathway balancing
B. subtilis Regulation of metabolic flux of glucose and N-Acetylneuraminic acid 2.18 g/L [53]
increase in N-acetylglucosamine supply
B. subtilis Redistribution of key intermediates and Hyaluronic acid 1.5 g/L (Mw 1.5 MDa) [54]
enhancement of metabolic flux
C. glutamicum Overexpression of native biosynthetic genes of Cyanidin-3-O-glucoside 41.7 mg/L [55]
UDP-glucose
C. glutamicum Deletion of endogenous aromatic catabolism Stilbene 158 mg/L [56]
pathways
Synechocystis PCC Overexpression of DXS and GGPPS from plant Manoyl oxide 0.45 mg/g DCW [57]
6803 Coleus forskohlii to increase GGPP availability
Synechocystis PCC Enhanced flux of shikimate pathway for the Caffeic acid 47.4 mg/g DCW [58]
6803 supply of aromatic amino acids
Streptomyces Deletion of the whole oxytetracycline gene Chlortetracycline 3.80 g/L [59]
rimosus 461 cluster
Streptomyces Deletion of native polyketide pathways for Botryococcene 212 mg/L [60]
reveromyceticus accumulation of acetyl-CoA and malonyl-CoA

expression levels. These technologies, considered as the
first generation of chassis engineering technologies,
include precursor engineering, cofactor engineering, tol-
erance engineering, and translational system engineering
(Figure 1).
The most frequently used engineering strategy is termed
‘push-pull-block’ (Figure 1), in which the native supply of
precursors is optimized (push), the desired pathway and
product secretion/storage are enhanced (pull), and the
competing pathways are inhibited (block) [10,13]. Dur-
ing the optimization of precursor supply, dynamic control
can be applied, which employs biosensors to indicate
precursor levels and to control gene expression in precur-
sor biosynthesis [14,15]. This helps to create smart chassis
that can provide optimal levels of precursors compatible
with the dynamic change in cell metabolism, which is
beneficial for the production of the desired chemicals.
Additionally, the activity of many pathway enzymes is
dependent on cofactors for electron transfer, such as
NADPH for reductive reactions (Figure 1). Chassis engi-
neering methods for improved cofactor supply and pro-
blems encountered in this process are similar to those for
precursor supply [16]. Given that cofactors are essential
for cell growth and basic metabolism, their supply and
consumption need to be balanced, through which cell
growth and biosynthesis of target products can be coupled
to achieve high production titers [16].
Optimized expression and efficient function of heterolo-
gous pathways often lead to the accumulation of target
products inside chassis cells, which can be harmful or
even devastating to cell growth and metabolism [17]. To
relieve such adverse effects, the chassis can be engi-
neered for either higher tolerance to or more efficient
export of the products. For both approaches, microbial
cell membrane is the focus (Figure 1). The composition of
lipid bilayers directly affects membrane permeability and
cell tolerance to chemicals, hence alterations in the lipid
components can lead to higher production titers and

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 Chassis engineering for bioproduction Liu et al. 107

Table 2

Unique properties of representative natural chassis in metabolic engineering of chemical production

Trait Chassis
E. coli S. cerevisiae Y. lipolytica B. subtilis C. glutamicum Synechocystis
Substrate Broad carbon General capability of Very broad substrate Broad carbon sources Broad carbon Inorganic
suitability sources utilizing carbon sources, suitability, including including glucose, sources carbon
including such as glucose, glucose, xylose, xylose, glycerol, and so including sources, such
glucose, fructose, sucrose, acetate, fatty acids, on glucose, as NaHCO3,
xylose, maltose, ethanol, alcohols, and so on sucrose, CO2
glycerol, and and so on aromatics, and
so on so on
Availability of Very Very sophisticated tools Limited tools for Limited tools for Limited tools for Limited tools
genetic tools sophisticated for genetic manipulation genetic engineering genetic engineering genetic for genetic
tools for engineering engineering
genetic
manipulation
CRISPR genome Available Available Available Available Available Available
editing
Tolerance to Moderate Strong tolerance to Very robust and strong Moderate tolerance to Strong tolerance Moderate
harsh chemicals tolerance to ethanol, acids, and other tolerance to harsh harsh stimuli to aromatics in tolerance to
harsh stimuli toxic compounds such stimuli, such as low pH lignocellulosic harsh stimuli
as inhibitors in biomass hydrolysates
hydrolysates
Precursor Pyruvate, Acetyl-CoA, isoprenoid Abundant acetyl-CoA N-Acetylglucosamine, Amino acid 2-
availability acetyl-CoA, precursors, and so on and malonyl-CoA phosphoenolpyruvate, derivatives Oxoglutarate,
malonyl-CoA, and so on. ADP-glucose
and so on
Transcription IPTG, lactose, Galactose Erythritol IPTG, xylose, sucrose, Benzoate, IPTG IPTG, copper
induction arabinose mannitol ions
system
Unique cellular None Vacuole, Golgi Endoplasmic None Carboxysomes None
compartment apparatus, peroxisome, reticulum, peroxisome, with anabolic
for P450 mitochondrion, Golgi apparatus, function
expression endoplasmic reticulum mitochondrion

productivities of certain chemicals [18]. Trans-mem- Fully synthetic chassis

brane transport relies on transporters or vesicles. Activa-
tion of native efflux transporters or expression of heterol-
ogous transporters in the chassis can help to pump out the
newly synthesized chemicals [19], whereas engineering of
the formation of outer membrane vesicles is effective in
improving the excretion of some hydrophobic products
[20]. More detailed engineering strategies of natural
chassis for microbial production have been extensively
discussed in some recent reviews [8,10].
Synthetic microorganisms as chassis
Recently, the fast development of synthetic biology has
stimulated the reconstruction of cellular machineries to
create new chassis for understanding the origin of life, the
complexity of microorganisms, and the production of new
proteins and value-added chemicals. By complying with
the central dogma, which operates microbial metabolism,
synthetic biologists have reprogramed some cellular
machineries, such as genomes and codons, and have
created synthetic microorganisms which can serve as
smart chassis in metabolic engineering. These achieve-
ments open up a new era of chassis engineering, termed
the second-generation engineering (Figure 2).
Advances in genetics, genome sequencing and DNA
synthesis/assembly make it possible to redesign and
artificially synthesize whole genomes (Figure 2). Up to
date, three bacterial genomes have been synthesized, that
is, the 582-kb Mycobacterium genitalium genome and the
1.1-Mb Mycoplasma mycoides genome, which can be trans-
planted into empty Mycoplasma capricolum and function to
generate live M. mycoides cells [21,22]. Recently, a fully
synthetic E. coli with recoded 4-Mb genome has been
created, in which the codons TCG and TCA are replaced
with AGC and AGT, respectively, and the stop codon
TAG is substituted by TAA, creating a E. coli with 61
codons [23]. The synthetic E. coli holds unprecedented
potential in incorporating three different noncanonical
amino acids (ncAAs) simultaneously whereas only one
type of ncAAs can be inserted at once in natural E. coli via
the stop codon TAG (Figure 3).
Additionally, a M. mycoides strain with a minimal genome
(532 kb) was developed with only essential genes, and
around half of the original genes in the genome was
removed [24]. Such microorganisms, although not
widely used yet, may have some exceptional advantages,

www.sciencedirect.com Current Opinion in Biotechnology 2020, 66:105–112

108 Tissue, cell and pathway engineering

Figure 1

Tolerance Cofactor
engineering engineering

Precursor
engineering
Current Opinion in Biotechnology

The first-generation strategies to engineer natural chassis as hosts for the production of value-added chemicals.

Figure 2

Proteins with
noncanonical
amino acids

Unnatural
base pair

Synthetic chassis

Current Opinion in Biotechnology

The second-generation engineering strategies using synthetic biology to develop synthetic chassis.

such as highly efficient utilization of nutrients, high-level
production of foreign pathway enzymes or protein pro-
ducts, and faster cell growth [24,25]. Additionally, min-
imal genomes are valuable in investigating the role of
essential genes in response to environmental variations.
Besides the artificial synthesis of bacterial genomes, the
synthesis of artificial S. cerevisiae chromosomes has also
been carried out [26,27–31]. The whole project, termed
the Sc2.0 project, was initially proposed by Dr Jef Boeke
and has grown into an international collaborative project

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 Chassis engineering for bioproduction Liu et al. 109

among global research institutes (http://syntheticyeast.
org). As of this year, the international team has success-
fully synthesized six chromosomes. The design of new
yeast chromosomes incorporates three principles, includ-
ing replacement of all TAG stop codons by TAA, removal
of unstable elements such as tRNAs and transposons,
and incorporation of PCR tags and loxP sites [29]. The
new yeasts with synthetic chromosomes can be adopted
as a useful chassis in metabolic engineering. Upon
incorporation of loxP sites, the synthetic chromosome
can be easily modified with Cre recombinase-mediated
genome recombination, termed ‘SCRaMbLE’, providing
thousands of chassis strains in a single attempt, and can be
used to identify gene loci that are related to the function
of heterologous pathways [32] (Figure 3). The synthetic
yeast chassis have been used in the production of
betulinic acid, b-carotene, and violacein, and in the
improvement of xylose uptake and cell tolerance to alkali
[33,34–36].
Partially synthetic chassis with artificial
cellular architectures
Conventional biotechnology is built on the base-pairing
rule with the ATGC alphabet, which in turn limits further
advancement of the technology in the invention of smart
chassis for the production of novel compounds. To
expand genetic codes, a family of unnatural base pairs
(UBPs), such as dPTMO-dTPT3 and dMTMO-dTPT3,
was introduced into expressed genes, and can be repli-
cated and, with modifications of tRNAs, translated into
proteins with ncAAs (Figure 2) [37]. The creation of
new codes can expand genetic codons for the orthogonal
incorporation of ncAAs into target proteins or pathway
enzymes, which can be used as novel protein drugs or for
site-specific protein modifications [38].
Beyond the creation of new genetic codes to incorporate
ncAAs, reprogramming of translational codons to encode
ncAAs is an alternative (Figure 2). Codon expansion uses
an engineered and orthogonal aminoacyl-tRNA synthe-
tase-tRNA pair to direct the incorporation of ncAAs into
proteins at desired sites. The new codon is generally an
unassigned (usually the amber stop codon UAG) or reas-
signed codon following synonymous codon compression
schemes [39]. More than 100 unnatural amino acids have
been incorporated into proteins in numerous organisms,
such as E. coli and S. cerevisiae [40].
Recently, the artificial redesign has been expanded to
artificial ribosomes (Figure 2). The goal is to develop an
independent, or orthogonal, translation system dedicated
to producing one or a few target proteins, whereas the
wild-type ribosomes continue to synthesize genome-
encoded proteins to maintain cell viability. The artifi-
cially synthesized ribosome, Ribo-T, consists of cova-
lently tethered subunits, where core 16S and 23S ribo-
somal RNAs form a single chimeric molecule [41,42].
The Ribo-T with optimized design is able to synthesize a
diverse set of proteins, and can incorporate multiple
ncAAs into synthesized polypeptides.
Moreover, a mirror-image transcriptional system has been
constructed [43,44]. A mutant of thermostable Sulfolobus
solfataricus P2 DNA polymerase IV (Dpo4) was con-
structed to contain mere D-amino acids. This enzyme
is able to amplify a 120-bp L-DNA fragment, which

Figure 3

Exploration of the
origin of life Minimal
genome

Production of proteins
with noncanonical
amino acids
Synthetic Chassis

SCRaMbLE Mirror-image
proteins
Rapid genome rearrangement for
biosynthesis of chemicals
Current Opinion in Biotechnology

Potential applications of synthetic microorganisms in synthetic biology.

www.sciencedirect.com Current Opinion in Biotechnology 2020, 66:105–112

110 Tissue, cell and pathway engineering
encodes E. coli 5S ribosomal RNA, by mirror-image PCR
amplification. The chassis with orthogonal and mirror-
image transcriptional and translational systems might
produce mirror-image proteins with novel functions
[44] (Figure 3).
Conclusions
Chassis engineering is indispensable in microbial produc-
tion of value-added chemicals and bioproducts. In the
first generation of chassis engineering technologies, chas-
sis strains have been extensively engineered to fit the
metabolic requirement of the introduced pathways, such
as sufficient supply of precursors or cofactors, efficient
influx of substrates or efflux of products, and functional
expression of pathway enzymes. The engineering relies
on genetic engineering tools, and more recently, genome-
scale engineering or editing tools [10]. These technolo-
gies, coupled with the design and optimization of biosyn-
thetic pathways, create a number of commercial chassis
strains for the production of many bioproducts. These
technologies still dominate chassis engineering although
extensive modifications are needed before the construc-
tion of an efficient production biosystem.
With the advancement of synthetic biology and enabling
technologies, the second generation of chassis engineer-
ing technologies has emerged. Redesign and reconstruc-
tion of cellular machineries enable the multi-target engi-
neering of chassis in a very short time span. Ever since the
development of synthetic genomes and artificial tran-
scription/translation systems, synthetic microorganisms
have shown their potential in creating chassis libraries
for pathway regulation and optimization, high-titer pro-
duction of target products, incorporation of ncAAs into
proteins, and so on. At present, the applications of syn-
thetic chassis are still very rare, which can be expanded to
customized design with emerging synthetic biology tech-
nologies and increased understanding of life evolution
and the complexity of microorganisms.
The limited application of synthetic chassis is partially
attributed to the complexity of the design and high cost of
chassis construction. For example, one fully synthetic
yeast chromosome can cost several hundred thousand
US dollars besides the complicated design of the genomic
sequence, which is not achievable for most research labs.
Moreover, our understanding of pathway functions and
interactions between cells and introduced genes is still at
infancy, and the current redesign of genomes and other
cellular architectures is based on the original context of
natural chassis, thus making it challenging to accomplish
great strides on the production. These obstacles can be
overcome with a thorough and comprehensive under-
standing of cellular physiology and metabolism, such as
how heterologous pathways work inside cells, how
expressed pathway enzymes interfere with cellular
metabolism, and how the interactions can be balanced
Current Opinion in Biotechnology 2020, 66:105–112
to achieve maximal production. Detailed information of
these cellular behavior can help with the design of syn-
thetic chassis to fit the actual demand in real applications.
It is anticipated that synthetic chassis with synthetic
genomes, recoded genetic codons or other cellular
machineries, open a window for creating artificial micro-
bial carriers for customized applications. For example,
synthetic organisms with recoded genomes can be easily
used for the production of drug proteins with unnatural
amino acids incorporated for in vivo tracking; synthetic
chassis with minimal genomes and reconfigured genomes
can be used for the production of proteins or natural
products with unprecedented titers. Also, chassis engi-
neering can be extended from mono-culture to co-culture
systems for practical applications in complex settings [45].
Conflict of interest statement
Nothing declared.
Acknowledgements
The financial support from National Natural Science Foundation of China
(31900114 and 21676192), the Natural Science Foundation of Shaanxi
Province (2020JQ-702 and 2020JQ-703) and Natural Science Foundation of
Tianjin (17JCYBJC23900) is acknowledged. This work was also supported
by the startup funds from Shaanxi University of Science and Technology
awarded to JZ and XW.
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