Biotechnology Advances 74 (2024) 108396

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Cordyceps militaris: A novel mushroom platform for metabolic engineering

Jiapeng Zeng a, b, 1 , Yue Zhou a, b, 1 , Mengdi Lyu a, b , Xinchang Huang a, b , Muyun Xie c ,
Mingtao Huang d, ** , Bai-Xiong Chen c, ** , Tao Wei a, b, *
a
Institute of Food Biotechnology & College of Food Science, South China Agricultural University, Guangzhou, Guangdong 510642, China
b
Research Center for Micro-Ecological Agent Engineering and Technology of Guangdong Province, Guangzhou 510642, China
c
School of Bioengineering, Zunyi Medical University, Zhuhai, Guangdong 519090, China
d
School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510641, China

A R T I C L E I N F O A B S T R A C T

Keywords: Cordyceps militaris, widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of
Cordyceps militaris bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This
Metabolic network review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals
Genome editing
and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks,
Cell factory
Metabolic engineering
expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to
Synthetic biology further research on C. militaris across various fields, including computational biology, existing DNA elements,
and genome editing approaches. This review aims to describe specific and promising opportunities for the in-
depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability
of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic
biology development are illustrated.

1. Introduction antitumour, anti-inflammatory, and antiviral functions (Jin et al., 2018;

Radhi et al., 2021; Yoon et al., 2018; Zeng et al., 2023; Zhang et al.,
Cordyceps militaris is an ascomycete that is valued for its medicinal 2022). Therefore, the costs of the raw materials as well as the standard
and nutritional properties. It has been widely used in Asia for centuries, continue to increase. Currently, COR production relies mainly on
both as a dietary supplement and in pharmaceutical applications. chemical and biological synthesis methods. Structural analysis will
Several bioactive compounds are contained in C. militaris, including facilitate the use of chemical synthetic methods to easily obtain the final
cordycepin (COR), pentostatin (PTN), carotenoids, ergothioneine, cor­ product, but raw material costs and environmental pollution, which are
dycepic acid and polysaccharides (Das et al., 2010). In recent years, obstacles to producing COR, should be considered during industrial
there has been improvement in the understanding of the molecular production (Wang et al., 2022). Currently, commercial COR is isolated
mechanisms underlying the effects of high-value chemical compounds mainly from the fruiting bodies of C. militaris. Due to the high time cost
(Chen et al., 2022a; Dong et al., 2013b; Masuda et al., 2007; Thananusak and low yield of traditional cultivation and fermentation approaches,
et al., 2020; Xia et al., 2017). The market demand for both the fruiting currently, COR production is far from meeting market demands; there­
body and the bioactive ingredients of this species has rapidly increased, fore, the combination of genetic modification methods and optimized
leading to the development and optimization of multiple methods for fermentation approaches is needed (Aman et al., 2000; Wang et al.,
artificial cultivation (Masuda et al., 2006). 2022). The heterologous expression of COR by engineered yeast, ach­
COR is one of the most valuable compounds found in C. militaris and ieved via module engineering and protein engineering and resulting in a
has multiple bioactive functions; it is structurally similar to adenosine yield of 4362.54 mg/L after 168 h of fed-batch fermentation, has
but lacks a 3′-hydroxyl group. Its bioactive functions include anticancer, attracted increasing academic and industrial attention (Duan et al.,

* Corresponding author at: Institute of Food Biotechnology & College of Food Science, South China Agricultural University, Guangzhou, Guangdong 510642,
China.
** Corresponding authors.
E-mail addresses: huangmt@scut.edu.cn (M. Huang), baixiong@zmu.edu.cn (B.-X. Chen), weitao@scau.edu.cn (T. Wei).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.biotechadv.2024.108396
Received 14 March 2024; Received in revised form 14 June 2024; Accepted 18 June 2024
Available online 19 June 2024
0734-9750/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
J. Zeng et al. Biotechnology Advances 74 (2024) 108396

Table 1
Beneficial value-added compounds production.
Compound Strain Fermentation method Working volume Yield Reference

C. militaris NBRC 9787 surface/static culture 100 mL 2500 mg/L (Masuda et al., 2007)
C. militaris NBRC 9787 mutant G81–3 surface/static culture 150 mL 14,300 mg/L (Masuda et al., 2014)
C. militaris 34164 solid-state fermentation 20 g 814.60 mg/g (Adnan et al., 2017)
Cordycepin
C. militaris submerged fermentation 50 mL 596.59 mg/L (Fan et al., 2012)
C. militaris 14014 two-step shaking and static fermentation 50 mL 2620 mg/L (Tang et al., 2015)
C. militaris ATCC 26848 submerged fermentation 100 mL 2214.5 mg/L (Shih et al., 2007)
Pentostatin C. militaris C01 solid-state fermentation 30 g 1.31 mg/g dw (Wu et al., 2023)
C. militaris C01 solid-state fermentation 30 g 2.25 mg/g dw (Wu et al., 2022)
Adenosine
natural C. militaris submerged fermentation 200 mL 5.05 mg/g dw (Gu et al., 2007)
C. militaris CM-04 two-step shaking and static fermentation 100 mL 1.22 mg/g dw (Zheng et al., 2019)
C. militaris TBRC6039 two-step shaking and static fermentation 75 mL 1.47 mg/g dw (Thananusak et al., 2020)
Carotenoids
C. militaris strain 40 (CGMCC 3.16322) two-stage culture / 4.41 mg/g dw (Yang et al., 2020b)
C. militaris ZA10-C4 stationary culture 100 mL 13.77 mg/g dw (Lin et al., 2021)

2022; Song et al., 2023; Tan et al., 2023). PTN, also known as 2′-deox­ mycelial state. This method requires only simple medium sterilization
ycoformycin, is an inhibitor of adenosine deaminase that prevents the and culture inoculation, eliminating the need for complicated mainte­
deamination of COR in C. militaris (Xia et al., 2017). Additionally, it is nance throughout the fermentation process. Moreover, many high-value
used as a pharmaceutical anticancer drug in the treatment of chronic compounds can be obtained directly from the fermented liquid medium,
lymphocyte leukaemia and hairy cell leukaemia (Dillman, 2004; Dill­ reducing the time costs and the need for cumbersome operations. Raw
man et al., 2007). Carotenoids are a class of organic pigments and are agricultural materials such as rice, wheat, and potato can be directly
present in greater amounts in C. militaris than in other known mush­ utilized as media without the need for expensive alternatives. Therefore,
rooms. Among carotenoids extracted and isolated from C. militaris, liquid fermentation, especially surface/static culture, is the most
lutein, zeaxanthin and four cordyxanthins were identified (Wang et al., commonly employed approach for the overproduction of high-value
2018). In addition to imparting colour, carotenoids also possess bioac­ chemicals (Chen et al., 2020). Furthermore, fermentation conditions,
tive functions that can benefit organisms, such as antioxidant activity, including temperature and the composition of ingredients, have been
anti-inflammatory properties, and skin protection functions (Baswan optimized (Le et al., 2009; Mao and Zhong, 2006). The optimal tem­
et al., 2021; Kabir et al., 2022; Young and Lowe, 2018). Various poly­ perature range is 20 to 30 ◦ C, close to room temperature, thereby
saccharides are contained in C. militaris, which are composed mainly of reducing temperature control costs.
mannose, glucose, and galactose in varying molar ratios. These poly­ During solid-state cultivation, C. militaris undergoes morphological
saccharides have been shown to have various health benefits and changes that result in the formation of fruiting bodies, which are of
pharmacological activities, including immunomodulatory, antioxidant, commercial interest. Compared to liquid fermentation, solid-state
antitumour, and anti-inflammatory effects, in numerous animal and cultivation requires a longer duration for fruiting body formation, and
clinical experiments (Zhang et al., 2019). Therefore, industrial devel­ high-value compounds are obtained from the fruiting bodies. Therefore,
opment and technological iteration for controlling the costs of high- high-quality fruiting bodies are commonly acquired through solid-state
value compounds and nutrients are crucial. cultivation with growth cycles under optimal light wavelengths and
In recent years, the rapid development of synthetic biology and light-darkness rhythms (Dong et al., 2013a).
metabolic engineering models has led to the reconstruction of organisms
such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis and 2.2. Beneficial value-added compounds
Corynebacterium glutamicum as cell factories for the production of
industrially oriented high-value chemical compounds (Gu et al., 2018; Numerous value-added compounds, including COR, PTN, caroten­
Heider and Wendisch, 2015; Hong and Nielsen, 2012; Yang et al., oids, ergothioneine, cordycepic acid, and Cordyceps polysaccharides
2020a). Considering the chassis advantages of C. militaris and the (CPs), can be obtained from C. militaris. Here, we summarize the yields
continuous development of molecular toolkits, the potential and value of these value-added compounds obtained via different fermentation
of metabolic engineering of this species were demonstrated. methods to further demonstrate the natural advantages of applying
C. militaris as a chassis cell for the production of these compounds. None
2. Chassis advantages of the strains listed in Table 1 were genetically modified, and the
fermentation yields of the original or mutant strains were determined
2.1. Fermentation advantages under optimized fermentation conditions, which shows the great
metabolic advantages of C. militaris.
Due to the large demand for the fruiting bodies of C. militaris and its Due to the individual differences among strains, the titres of the
high-value compounds, artificial cultivation and fermentation ap­ above products vary considerably. Despite the availability of the model
proaches have been comprehensively developed. In this regard, methods strain CM01, it has not been widely used in academic research or in­
used in C. militaris have been described, among which surface/static dustrial production because it has not been modified to obtain an
fermentation has been shown to have advantages over other methods for excellent chassis cell for metabolic engineering. Strains that are not
high-value compound production, while solid-state cultivation can be genetically modified but are still able to produce high titres of secondary
used for fruiting body formation. The artificial cultivation of C. militaris metabolites are currently favoured for industrial applications. Other
was first achieved without a mature perithecium by De Bary in 1867 fermentation methods involving various additions or optimization of
(Petch, 1936). For over a century, various methods, such as liquid cul­ carbon‑nitrogen sources for compound production will not be further
ture, submerged culture, surface/static culture, and two-step shaking- described here. To date, these fermentation strategies have been scaled
static culture, have been developed to overproduce high-value com­ up to the ton scale without the need for complex medium pretreatment
pounds such as COR analogues (Qin et al., 2019). These strategies can be or sophisticated equipment such as fed-batch fermenters. When
primarily categorized into two types: liquid fermentation for substrate leveraging classic metabolic engineering, it is advantageous to utilize
fermentation and solid-state cultivation for fruiting body formation. C. militaris as a metabolic engineering platform due to its naturally high
Liquid fermentation is utilized to cultivate C. militaris in the initial adenosine flux, low fermentation cost, and practicality. In summary, due

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J. Zeng et al. Biotechnology Advances 74 (2024) 108396

Fig. 1. Metabolic network of cordycepin and pentostatin. G6P: glucose-6-phosphate; F6P: fructose-6-phosphate; FDP: fructose-1,6-bisphosphate; T3P2: glycerone
phosphate; T3P1: D-glyceraldehyde 3-phosphate; 13PG: 1,3-bisphospho-D-glycerate; 3PG: 3-phosphoglycerate; 2PG: 2-phospho-D-glycerate; PEP: phosphoenol­
pyruvate; PYR: pyruvate; CIT: citrate; ICT: isocitrate; AKG: 2-oxoglutarate; SUCCOA: succinyl-CoA; SUC: succinate; FUM: fumarate; MAL: (S)-malate; OAA: oxalo­
acetate; FA: fatty acid; PRPP: 5-phosphoribosyl 1-pyrophosphate; PRAM: 5-Phospho-D-ribosylamine; GAR: N(1)-(5-phospho-D-ribosyl)glycinamide; FGAR: N(2)-
formyl-N(1)-(5-phospho-D-ribosyl)glycinamide; FGAM: 2-formamido-N(1)-(5-phospho-D-ribosyl)acetamidine; AIR: 5-amino-1-(5-phospho-D-ribosyl)imidazole;
CAIR: 1-(5-phospho-D-ribosyl)-5-amino-4-imidazolecarboxylate; SAICAR: (2S)-2-[5-amino-1-(5-phospho-beta-D-ribosyl)imidazole-4-carboxamido]succinic acid;
AICAR: 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide; FAICAR: 5-formamido-1-(5-phosphoribosyl)imidazole-4-carboxamide; ASUC: adenylosuccinate.

to its convenience and low cost in large-scale fermentation, C. militaris shown in module 3 (Wongsa et al., 2020). Recently, another potential
demonstrates inherent and substantial potential as a platform for pro­ biosynthetic pathway for COR was discovered (Kuo et al., 2015; Lennon
ducing COR and other high-value chemicals at the grams per litre scale. and Suhadolnik, 1976; Xiang et al., 2014). In module 4, the enzyme
encoded by the nrd gene catalyses the deoxidization of ADP, which re­
3. Metabolic network identification sults in the formation of 3′-dADP, which is then dephosphorylated to 3′-
dAMP. A 5′-nucleotidase catalyses the conversion of 3′-dAMP to the final
The rapid development of omics technologies, including genomics, product COR. Although this biosynthetic pathway has been reported, it
transcriptomics, proteomics, and metabolomics, has sparked the growth conflicts with existing research (Chen et al., 2022a). Therefore, addi­
of synthetic biology. These omics techniques reveal the intricacies of tional experiments are needed to confirm the biosynthetic pathway of
metabolic pathways in C. militaris, enhancing our understanding of the COR. In addition to that of COR, the synthetic pathway of PTN was also
underlying metabolic network and offering solid data support for initial revealed and confirmed because PTN has the same precursor and
rational design. In addition, the establishment of a clarified metabolic biosynthesis-related gene cluster as COR. The HisG domain of Cns3 ca­
network is crucial for the further comprehensive construction of talyses the conversion of adenosine to PTN. To direct metabolic flux
C. militaris cell factories. Here, we present an overview of the current towards the biosynthesis of PTN, the cns1 gene, encoding Cns1, which is
documented biosynthetic pathways for COR and PTN in C. militaris involved in COR biosynthesis, was deleted, leading to COR not being
(Fig. 1). detected and PTN being obtained from the engineered strain (Zou et al.,
We divided the biosynthetic pathways of COR into four modules 2021a).
highlighted separately in green, red, purple, and blue separately in The colony colour of C. militaris may change from white to yellow or
Fig. 1. We define module 1 as the route from PRPP to IMP and module 2 orange under light exposure, which is the result of carotenoid accu­
as the route from IMP to the final product COR. Module 3 was defined as mulation. Carotenoids, including lutein, zeaxanthin, and cordyxanthin,
the route from mRNA degradation to 2′,3′-cAMP and the subsequent are a class of pigments that share the same backbone as terpenoids.
pathway to COR. Module 4 includes ATP, ADP, 3′-dADP, 3′-dAMP, and Although the biosynthetic pathway of carotenoids has been well
COR. Most studies have described the biosynthetic pathways of module described in other organisms, it remains unclear in C. militaris because of
2 (Chen et al., 2020; Duan et al., 2023; Ma et al., 2023; Vongsangnak the absence of several key genes, such as those encoding phytoene
et al., 2017). In 2017, the Cns1–4 cluster in module 2 was knocked out, synthetase and phytoene dehydrogenase. Genes involved in carotenoid
revealing the functions of different genes involved in the biosynthesis of biosynthesis also showed no significant transcriptional differences be­
COR and the heterologous expression of key genes in S. cerevisiae and tween light and dark cultures (Lou et al., 2019; Yang et al., 2016).
M. robertsii, which indicated that the cns1–3 locus was able to produce Additionally, light was confirmed to be a crucial factor in the production
COR (Xia et al., 2017). Recent research also revealed that truncation of of carotenoids through a series of bioreactions of light receptors (Yang
the large cluster of cns1, cns2, and cns3 in module 2 led to COR no longer et al., 2016; Wang et al., 2017; Zhang et al., 2020a). Although the
being detected by HPLC (Chen et al., 2022b). In addition, there is also a detailed biosynthetic network has not been fully elucidated, a putative
pathway by which COR is synthesized through mRNA degradation, as biosynthetic pathway for carotenoids was proposed several years ago.

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Fig. 2. Putative biosynthetic pathway of carotenoids. IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl diphosphate; GPP: geranyl diphosphate; FPP: farnesyl
pyrophosphate; GGPP: geranylgeranyl pyrophosphate.

Table 2
Available DNA elements in C. militaris.
Name Source Size Type Describe Reference
(bp)

CmLsm3 promoter
C. militaris 547 constitutive promoter Promoter of U6 small nuclear ribonucleoprotein gene (Chen et al., 2018)
(Pcmlsm3)
CmGPD promoter Promoter of glyceraldehyde 3-phosphate
C. militaris 1035 constitutive promoter (Gong et al., 2009)
(Pcmgpd) dehydrogenase gene
AnGpdA promoter Promoter of glyceraldehyde 3-phosphate
A. nidulans 728 constitutive promoter (Xiong et al., 2013)
(PgpdA) dehydrogenase gene
TrpC promoter Promoter of trifunctional tryptophan biosynthesis
A. nidulans 430 constitutive promoter (Zheng et al., 2011)
(PtrpC) gene TrpC, medium strength
CaMV 35S Cauliflower mosaic Promoter of trifunctional tryptophan biosynthesis (Benfey and Chua, 1990;
678 constitutive promoter
promoter virus gene TrpC, medium strength Zhang et al., 2020b)
CmGAPDH Promoter region of the glyceraldehyde-3-phosphate
C. militaris unknow constitutive promoter (Choi et al., 2023)
promoter dehydrogenase gene of KACC43316
nutritional deficiency-related (Bach and Lacroute, 1972;
ura3 C. militaris 878 Coding orotidine 5′-phosphate decarboxylase
selection marker Gong et al., 2009)
Coding two-component osmosensing histidine kinase
CmHk1 C. militaris 3912 negative selection marker (Choi et al., 2023)
(Bos1)
Streptomyces
hph 211 positive selection marker Coding bacterial hygromycin B phosphotransferase (Mullins et al., 2001)
hygroscopicus
Streptomyces
Bar, blpR 552 positive selection marker Phosphinothricin acetyltransferase, positive selection (Chen et al., 2018)
hygroscopicus
Fungicide inhibits the polymerization of tubulin
ben Neurospora crassa 1378 positive selection marker (Lou et al., 2021)
monomers into functional microtubules
Terminator of trifunctional tryptophan biosynthesis
TtrpC Aspergillus nidulans 572 terminator (Chen et al., 2018)
gene TrpC
Agrobacterium
TNOS 253 terminator Terminator of nopaline synthase gene (Lou et al., 2018)
tumefaciens
Terminator of orotidine 5′-phosphate decarboxylase
Tcmura3 C. militaris 502 terminator (Chen et al., 2018)
gene
CaMV poly(A) Cauliflower mosaic (Benfey and Chua, 1990;
175 terminator Terminator for CaMV 35S promoter
signal virus Zhang et al., 2020a)
Origin of replication from Aspergillus and shorten the
AMA1_2.8 Aspergillus niger 2816 origin of replication (Chen et al., 2022b)
length to 2.8 kb
(Dang and Lee, 1988; Ray
c-Myc Homo sapiens 27 signal peptide Nuclear localization signal (NLS)
et al., 2015)

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This pathway commences with the formation of the 20‑carbon com­ plug-and-play components in experiments for metabolic modification
pound geranylgeranyl pyrophosphate (Fig. 2). Glucose undergoes and functional validation of key genes in C. militaris. Therefore, no ex­
glycolysis to transform into acetyl-CoA, which then proceeds through periments have been conducted to quantify the strength of terminators
the mevalonate (MVA) pathway to form the isopentenyl pyrophosphate or for endogenous terminator mining. Nevertheless, terminators are still
(IPP)/dimethylallyl diphosphate (DMAPP) isomer, geranyl diphosphate needed for high-level metabolic engineering to accurately control gene
(GPP), farnesyl pyrophosphate (FPP), and, finally, geranylgeranyl py­ transcription and translation without interrupting the normal expression
rophosphate (GGPP), which serve as substrates for carotenoids. Carot­ of flanking genes.
enoid biosynthetic pathways are often regulated by environmental As genome editing tools continue to advance, multiple genome-
factors, as evidenced by the light-related regulation of carotene pro­ editing systems are anticipated to emerge for use in C. militaris, while
duction in C. militaris. Consequently, the production of carotenoids may metabolic engineering involving multiple genetic manipulations calls
involve numerous inducible elements, which could boost the develop­ for diverse kinds of selection markers. To date, only three selection
ment of DNA elements in synthetic biology. markers, one nutritional deficiency-related selection marker (ura3) and
The biosynthetic pathway for CPs comprises several steps, although two positive selection markers (blpR and hyg), are available, which limits
the complete pathway has not been fully elucidated (Wu et al., 2024). the capacity for multigene editing in cells. Consequently, uncovering
The pathway begins with the synthesis of nucleotide-activated sugars, diverse selection markers and constructing a marker-free genome edit­
which serve as substrates for the initial synthesis of CPs. Specific en­ ing system would facilitate the advancement of synthetic biology in
zymes recognize activated sugars and transfer them to an acceptor C. militaris. However, except the promoters, terminators, and selection
molecule, typically a lipid carrier, to establish the initial glycosidic markers mentioned above, there are few other regulators related to
bond. Following the formation of this bond, additional monosaccharide transcription or expression elements.
units are added to elongate the growing polysaccharide chain, which is The AMA1 origin of replication from Aspergillus niger has been
ultimately exported through various mechanisms. However, due to the applied for CRISPR-based gene editing in C. militaris in recent years. The
complex structure and species specificity of CPs, further research is AMA1 sequence was the only origin of replication applied and reported
needed to identify the compositions of CPs through chromatography, in C. militaris, where AMA1_2.8 was a short version that eliminated the
mass spectrometry, and NMP spectroscopy, which will also contribute to half-inverted repeat of the regular AMA1 element, resulting in ready-to-
the identification of their biosynthetic pathways. lose reproduction without selective pressure (Sarkari et al., 2017). The
To comprehensively evaluate the efficiency of C. militaris, the application of the AMA1 element expands the possibility of editing and
reconstruction of engineered strain should be upgraded from the gene validation in C. militaris. A vector carrying the AMA1 sequence and
metabolic network level to the whole-system level. Genome-scale ori could be used as a shuttle vector in E. coli and C. militaris, which
metabolic network (GEM) is a computational-based biological predic­ would greatly reduce the time and effort needed to conduct gene editing
tion algorithm that has been extensively employed for more than two and epigenetic studies. However, the characterization of AMA1 also
decades for model organisms such as E. coli and S. cerevisiae (Edwards poses some questions, such as how long can cells retain a plasmid pos­
and Palsson, 2000; Förster et al., 2003).To date, the algorithm for sessing the AMA1 sequence during culture, how plasmid loss can be
C. militaris has undergone only two upgrades, with the most recent being determined, and if the plasmid is not lost, will phenomena such as re­
iPC1469, which includes 1469 genes, 1904 metabolic reactions, and sidual resistance or plasmid mutual exclusion in a new round of editing
1229 metabolites (Cheawchanlertfa et al., 2022; Raethong et al., 2020; occur. Another type of mature vector commonly used is those of the
Vongsangnak et al., 2017). Most high-value products, particularly sec­ pCAMBIA series, which can participate in random genome editing
ondary metabolites, remain unexplored, and there is a need to verify the through T-DNA. With the advanced modification of vectors, precise and
consistency between the precursor-predicted results and wet experi­ effective genome editing has become possible.
ments. Moreover, the results of the wet experiments also contribute to In general, the existing DNA elements and vectors available in
the improvement of GEM. In contrast to the rapid development and C. militaris are adequate for simple metabolic pathway modifications but
iteration seen in model organisms, the GEM of C. militaris lacks are insufficient for complex metabolic network modification and
comprehensive omics, enzymatic catalysis, and global limitation data. multifunction system construction. Therefore, identifying novel DNA
Therefore, its development in terms of coverage and accuracy is still far elements usable in C. militaris for further system-level engineering is
from being achieved. critical, and the use of alternative parts of inducible promoters, consti­
tutive promoters, sensors, and response elements can lead to improved
4. Genetic engineering of C. militaris remodelling. A high-throughput screening method was established for
Aspergillus nidulans fungal promoters based on single-cell level fluores­
4.1. DNA elements cence detection by flow cytometry, which can quantitatively indicate
the strength of promoter expression in a more efficient, rapid, and
To utilize C. militaris effectively as a metabolic engineering platform, precise manner (Wei et al., 2023). This study provides a research
diverse DNA elements are crucial. The identified and utilized elements paradigm utilizing a high-throughput approach for subsequent work on
are summarized in Table 2. DNA element discovery and screening in C. militaris, accelerating the
Only five promoters belonging to the constitutive category have been development and application of DNA element engineering.
identified in C. militaris, which is still limited compared to the number in
model organisms. Among these, Pgpd is a strong and relatively large 4.2. Genome editing systems
promoter, while Pcmlsm3, PcmtrpC, and PcaMV are regarded as medium-
strength promoters with a more variable size in the construction of To increase the titre of high-value products and identify the key
gene circuits. However, due to the limitations in the convenience and functional genes associated with fruiting body development, a range of
efficiency of these editing tools, the quantification of promoter strength, genome editing systems have been developed and employed in
which is crucial for rationally controlling metabolic flux in the entire C. militaris.
metabolic network, remains incomplete. The creation of a library
comprising overlapping constitutive promoters represents a promising 4.2.1. Agrobacterium tumefaciens-mediated transformation
paradigm for promoter engineering to modulate gene transcription and Agrobacterium tumefaciens-mediated transformation (ATMT) is a
translation at different levels (Lyu et al., 2022). widely used technique in plant genetic engineering and biotechnology.
Terminators have received less attention than promoters in meta­ Agrobacterium tumefaciens is a plant pathogen that naturally infects
bolic network engineering with minimal modification. Terminators are plants by transferring a small segment of its DNA, known as T-DNA

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Fig. 3. Development timeline of CRISPR system in C. militaris.

(transfer DNA), into plant cells. In its natural setting, this DNA transfer of resistance are notable concerns during subsequent genome editing.
induces the formation of crown gall tumours on infected plants. Re­
searchers have attempted to adapt this technique for transfer into fungi, 4.2.2. CRISPR-Cas9 technique
with the goal of enabling random or direct gene editing (de Groot et al., The clustered regularly interspaced short palindromic repeats
1998). After coculture of the spores of C. militaris and A. tumefaciens with (CRISPR) and CRISPR-associated protein 9 (Cas9) system is a revolu­
DNA fragments of interest cloned at the T-DNA region in the vector, the tionary gene-editing system. The CRISPR system used in C. militaris has
T-DNA fragment could be easily integrated into the genome of been upgraded several times (Fig. 3).
C. militaris (Zheng et al., 2011). By using this technique, a mutant library In 2018, the CRISPR-Cas9 technique was first used in C. militaris
was constructed to identify genes involved in fruiting body formation (Chen et al., 2018). The Cas9 expression cassette consisted of the newly
and COR production (Zheng et al., 2015). Some genome modifications discovered promoter Pcmlsm3, the terminator Tcmura3, and the cmcas9
and gene identifications were achieved through this technique. The ura3 gene, which was derived from S. pyogenes Cas9 but codon optimized for
gene was deleted through ATMT (Chen et al., 2018). The ATMT tech­ C. militaris. However, the implementation of this system in C. militaris
nique was applied to reveal the essential function of light in fruiting faces several challenges. First, the integration of the Cas9 protein into
body development in C. militaris (Yang et al., 2016). ATMT, a mature the C. militaris genome by ATMT may introduce potential cell toxins,
editing method for simple gene editing and key gene identification, has posing an unnecessary growth burden. Additionally, the integration may
the advantage of a high positivity rate of approximately 90% for random result in a low cutting efficiency because the Cas9 protein is present only
insertion during editing of the genome of C. militaris (Zheng et al., 2011). at the genome transcription and translation levels. Second, the use of a
Despite the efficiency of ATMT being sufficiently high, the decrease in presynthesized sgRNA in vitro may lead to accidental degradation dur­
the positivity rate of homologous integration has raised concerns, ing experimental manipulations. Furthermore, because of susceptibility
particularly in the context of rational design (Lou et al., 2018). In to degradation, sgRNA cannot maintain high concentrations in cells for
addition, evidence has demonstrated that the positivity rate is relatively long periods, affecting its ability to perform its function. Overall, this
low without a selection marker, presenting a trade-off between residual method provides novel insight into genome editing in C. militaris, but the
resistance and success rate (Chen et al., 2018). The culture and collec­ low efficiency of genome editing poses a serious challenge.
tion of spores also require additional steps and time compared to pro­ The efficiency of direct RNP transformation into fungal protoplasts is
toplast preparation. currently low. Therefore, researchers have experimented with addi­
In addition, a split-marker approach was developed to improve the tional chemical reagents to improve the transformation efficiency of
frequency of homologous integration and gene knockout (Lou et al., RNPs. In 2021, the CRISPR-Cas9 technique was improved by adding
2018). A linear cassette and a split-marker deletion cassette were con­ Triton X-100 and prolonging the incubation time of protoplasts, result­
structed and introduced into C. militaris protoplasts using PEG-mediated ing in an impressive 100% editing efficiency in C. militaris. The opti­
transformation. The transformation of split-marker fragments resulted mized RNP-based method has also shown efficient integration of
in a higher efficiency of targeted gene disruption compared to that homologous recombination and gene disruption without the use of
achieved with linear deletion cassettes, because the transformants could foreign DNA as a selection marker (Zou et al., 2021a).
not survive without successful homologous recombination to repair the In 2022, significant advancements in the CRISPR-Cas9 system were
bar resistance gene. An “alive” or “dead” selection approach has been made by two research teams (Chen et al., 2022a; Meng et al., 2022). An
employed to streamline the screening process and stimulate cells to autonomously replicating plasmid with an AMA1 sequence, which has
engage in homologous recombination more effectively. Although this the ability to perform extrachromosomal replication in Aspergillus
method has successfully enhanced the frequency of homologous nidulans, was introduced and constructed into CRISPR-Cas9 system.
recombination, the limited selection marker options and the persistence Furthermore, precisely targeted gene deletion via homology-directed

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repair was effectively performed, with editing efficiencies of 55.1% and knock-in of genes. The use of transposase may allow parallel and mul­
89% for Cmwc-1 and Cmvvd, respectively. Double gene editing was tiplexed chromosomal integration (Yang et al., 2021). Notably, the
performed simultaneously with an efficiency of 10%. The loss of the article presents a system of CRISPR-associated transposases (CASTs) for
plasmid under nonselective culture conditions allowed recycling of the efficient RNA-guided transposition, with a specific focus on character­
selection marker and avoidance of the adverse effects of the CRISPR- izing the PtrCAST element. The results emphasized that PtrCAST and
Cas9 system. tRNA is a small noncoding RNA that can help release a VchCAST can independently and efficiently insert cargo genes into their
single sgRNA when the sgRNA neighbours tRNAs through a splicing respective target loci without interference, thus indicating their poten­
mechanism for tRNA maturation. In addition, CRISPR-Cas9-TRAMA, tial applicability in orthogonal genome editing. In 2023, a novel RNA-
constructed by Chen et al., shortens the AMA1 sequence to construct a guided endonuclease system, named the Fanzor system, was discov­
leaner and more efficient vector, modifying the endogenic tRNAGly ered in eukaryotes (Saito et al., 2023). This system shares similarities
element and assembling it with the gRNA scaffold in the Cas9 vector to with the CRISPR-Cas system but is more compact, is easier to deliver to
construct a one-step multiple-target gene editing system. Moreover, the cells and tissues, and has more precise genome editing capabilities.
system combines the AMA1 element with the homologous template, Consequently, in the future, Fanzor may emerge as a novel editing
achieving precise multiplex gene editing, large synthetic cluster dele­ system for C. militaris.
tion, and the ability to edit the genome to modify proteins and evaluate
promoter strength, demonstrating the benefit and wider application of
this system in genomic studies. Despite advancements, several chal­ 5.2. Chassis cell robustness
lenges remain in implementing further genome editing with existing
CRISPR-Cas9 systems. First, the vectors and DNA elements being utilized The robustness of a constructed cell factory encompasses the ability
are still lengthy and jumbled, constraining the flexibility of system of the engineered microbial system to maintain stable and efficient
construction and genome editing. Second, the frequency of homologous production of desired compounds in the face of various challenges and
recombination remains relatively low in multigene editing, and poten­ perturbations. Therefore, robustness is a crucial factor for the successful
tial off-target effects must be carefully considered. Finally, an effective industrial implementation of cell factories and can be evaluated through
and inducible promoter is needed to control Cas9 expression blockade determination of genetic stability, environmental stress tolerance,
after successful genome modification, thereby safeguarding the cell metabolic stability, process robustness, and strain competitiveness. The
from potential cell toxins associated with the Cas9 protein. performance of C. militaris in scale-up fermentation needs to be evalu­
ated and analysed. Compared with classic model organisms, C. militaris
5. Improvement in cell factory construction has a drawback in terms of strain robustness in that it can easily
degenerate after repeated culture. Some results have indicated that the
In addition to the fermentation advantages, clearly elucidated degeneration of C. militaris has adverse effects on not only the produc­
metabolic network, DNA elements, and mature genome editing tools tion of fruiting bodies but also COR production, both of which are core
mentioned above, there are also some key factors to ensure that the host quality indicators of the strain products (Zhang et al., 2023). Thus, strain
serves as a metabolic platform for the production of high-value products degeneration needs to be considered when constructing a cell factory,
from the laboratory to the industrial scale. These include effective and and culture parameters, nutrient supplements, and genetic modifica­
precise multigene editing tools, the robustness of the chassis cell, the tions need to be optimized and precisely controlled. Fortunately, the
construction of a chassis cell with clear and efficient editing mecha­ molecular mechanisms of strain degeneration have been gradually
nisms, optimized growth conditions, nutrient availability, fermentation studied, and some candidate genes have been primarily selected as
parameters, scale-up parameters and enzyme engineering for high yields markers of degeneration, which could help in screening degraded strains
of products. at an early stage.

5.1. Efficacy and precision of multigene editing tools

5.3. Chassis cell assisted by a repair mechanism
The PAM is a short nucleotide motif of 3 bp (NGG) responsible for
recognition by the Cas9 protein located at the 3′ end of the target DNA, In addition to the efficiency and convenience of genome editing
which consequently limits the flexibility of gRNA design in C. militaris. tools, the repair systems and mechanisms in C. militaris play a significant
To address this limitation, it is essential to explore and engineer Cas9 role in the editing success rate. Genomic DNA cleavage in C. militaris can
variants capable of recognizing and cleaving novel PAM sequences cause a double-strand break (DSB), triggering the repair mechanisms of
(Walton et al., 2020). This approach would expand the scope of genome- nonhomologous end joining (NHEJ) or homologous recombination
targeted editing and involve building a library based on different PAM (HR). NHEJ involves direct ligation of the broken DNA ends, often
sites for Cas9 nucleases. Moreover, the use of directed evolution ap­ resulting in small insertions or deletions at the repair site, while in HR,
proaches to evolve Cas9 nucleases to recognize and cleave target se­ an undamaged homologous DNA sequence is used as a template for
quences without strict PAM requirements can be explored. This would accurate repair. However, of the two repair modalities, NHEJ pre­
entail subjecting the Cas9 protein to rounds of mutagenesis and selection dominates and is driven by two key genes, ku70 and ku80. Research has
to ultimately obtain variants capable of functioning with reduced or demonstrated that knocking out ku70 or ku80 significantly increases the
altered PAM specificity. proportion of HR in DSB repair (Wang et al., 2021a). Research on other
To date, several delivery systems have been improved to enhance the nonmodel organisms, such as Ogataea polymorpha and Monascus ruber,
efficiency and applicability of genome editing. For further studies in has demonstrated that knocking out or knocking down key NHEJ genes
C. militaris, such as for gene function identification, metabolic network is crucial for chassis cell construction and increasing the success rate of
modification, and cell system reconstruction for rational design, multi­ genome editing (Gao et al., 2021; He et al., 2013). In addition, some
plex gene editing in a single round is necessary and will be time-saving. enzymes that improve the efficiency of HR were found in S. cerevisiae.
The CRISPR-Cas9-TRAMA system achieves four-target edits in a single Therefore, heterologous expression of key enzymes in chassis cells can
round with a positivity rate of 7/39 (17.95%), relying on the nonho­ improve the frequency and success rate of homologous repair (Gaines
mologous end joining (NHEJ) repair mechanism in C. militaris. Although et al., 2015; Ji et al., 2020). Therefore, for C. militaris cell factory con­
multitarget gene editing has been achieved, the reliance on NHEJ repair, struction, a chassis cell with truncation of ku70 or ku80 and heterolo­
rather than HDR, presents challenges for subsequent editing, particu­ gous expression of RAD52 would improve the efficiency of HR,
larly in achieving multilocus homologous recombination and multicopy facilitating genome editing with desired modifications.

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J. Zeng et al. Biotechnology Advances 74 (2024) 108396

Fig. 4. The strategy of synthetic biology in developing C. militaris.

5.4. Enzyme engineering production through overexpression of the cmtf1 and cmtf2 genes. This
work investigated the relationships among energy generation, amino
Enzyme engineering involves the modification of enzymes through acid interconversion, and COR production (Chen et al., 2020). The role
various techniques to improve their properties or create new function­ of ribonucleotide reductases was first verified, and RNR genes were
alities. While laboratory-scale enzyme engineering serves as a proof of expressed in C. militaris (Zhang et al., 2020a). The results showed that
concept for the potential utility and benefits of modified enzymes, the COR concentration was significantly greater harbouring RNRM,
industrial-scale enzyme engineering focuses on achieving cost-effective while the level in RNRL-overexpressing plants did not markedly change
and efficient production processes. However, it has been noted that compared with that in wild-type. Researchers have attempted to over­
there has been not only limited enzyme engineering in C. militaris but express the predicted sterol regulatory element binding protein (SREBP)
also limited research on the intracellular environment, which needs to genes in C. militaris to improve the hypoxia adaptation of fungal cells;
be adapted to enzymes. To establish a more rational and high-value cell consequently, the COR content increased more than 2-fold (Wang et al.,
factory, enzyme engineering, in conjunction with metabolic engineer­ 2021b). The findings revealed the role of SREBPs in growth and bioac­
ing, is essential for modifying the whole cell system. Through the utili­ tive compound synthesis. A strain in which the Cmsnf1 gene was deleted
zation of gene and genome editing tools, the identification, was constructed; this gene plays an important role in penetration of the
modification, and design of enzymes have become feasible. This inte­ insect cuticle, and COR production in this strain increased more than 7-
gration represents a significant addition to metabolic engineering and fold (Wāng et al., 2020). To date, engineered cells have been constructed
serves as a critical step towards industrialization. with truncation of the COR biosynthesis-related gene cluster, cns1, cns2,
and cns3, indicating that this truncation may cause the accumulation of
6. Construction and application of C. militaris cell factories adenosine (Chen et al., 2022b). DASH-type cryptochrome, a photore­
ceptor identified in C. militaris, was deleted, leading to the accumulation
Transforming C. militaris into a cell factory has already provided a of carotenoids and cordycepin at the cost of disabling fruiting body
foundation for scientific work. In this section, the genetically modified elongation (Wang et al., 2017). The carotenoid content of the CmVVD
strains generated for the production of value-added compounds are deletion strains was 6-fold greater than that of the wild type (Zhang
summarized. In 2020, transcriptome analysis of C. militaris revealed two et al., 2020b). Many genes related to cordycepin or carotenoid pro­
key transcription factors that play important roles in doubling COR duction and whole-system regulatory factors have been identified

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J. Zeng et al. Biotechnology Advances 74 (2024) 108396

through knockout and complementation by researchers; however, these Data availability

genes have not been applied in cell factory construction.
The spent mushroom substrate which lignocellulosic waste contrib­ No data was used for the research described in the article.
utes to environmental pollution, was converted to the high-value anti­
cancer drug PTN by C. militaris through heterologous expression of Acknowledgement
cellulase and an optimized composition ratio of a cellulase combination
(Zou et al., 2021b). Additionally, this study investigated the competition This work was supported by the Guangdong Basic and Applied Basic
between the biosynthesis of COR and PTN in C. militaris and demon­ Research Foundation (grant number 2019A1515110609), the Scientific
strated the potential application of synthetic biology for sustainable Research Platform Project of Education Department of Guangdong
waste removal and the biosynthesis of high-value natural products. To Province (grant number 2020KTSCX019), the Foundation for Distin­
increase the number of alternative overexpression sites available for cell guished Young Talents in Higher Education of Guangdong, China (grant
factory construction, the first safe harbour site, the CmSH1 locus, was number 2023KQNCX156), the National Natural Science Foundation of
identified, and a hydrophobic gene was inserted to enhance disease China (grant number 32360787), the Guangdong Basic and Applied
resistance (Liu et al., 2023). The screening, classification, and identifi­ Basic Research Foundation (grant number 2022A1515010057), and the
cation of safe harbour sites inspired researchers to identify more sites Seed Industry Revitalization Project of Rural Revitalization Strategy of
that are stable in the genome, have no negative effect on the host and are Guangdong Province (grant number 2022-WPY-00-006). Biorender (htt
suitable for overexpression. These sites are vital for cell factory con­ ps://biorender.com/) was used for making Figs. 3 and 4.
struction for heterologous gene expression, to achieve a real rational
design in synthetic biology. As the editing performance of the CRISPR References
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