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Research Advances on the Consolidated Bioprocessing of

Lignocellulosic Biomass

Zhongye Li , Pankajkumar R. Waghmare , Lubbert Dijkhuizen ,

Xiangfeng Meng , Weifeng Liu

PII: S2667-3703(24)00002-X
DOI: https://doi.org/10.1016/j.engmic.2024.100139
Reference: ENGMIC 100139

To appear in: Engineering Microbiology

Received date: 28 July 2023

Revised date: 26 January 2024
Accepted date: 29 January 2024

Please cite this article as: Zhongye Li , Pankajkumar R. Waghmare , Lubbert Dijkhuizen ,
Xiangfeng Meng , Weifeng Liu , Research Advances on the Consolidated Bioprocessing of Lignocel-
lulosic Biomass, Engineering Microbiology (2024), doi: https://doi.org/10.1016/j.engmic.2024.100139

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Highlights:

 Lignocellulosic biomass is an abundant resource for the production of

bioproducts

 The high cost of cellulase production is the main hurdle for biorefinery industry

 CBP is an alternative approach to classical biorefinery for biochemical production

 Bacteria, fungi and their co-culture are used for CBP lignocellulosic biorefinery
 Research Advances on the Consolidated Bioprocessing of Lignocellulosic Biomass

Zhongye Lia, Pankajkumar R. Waghmare a, Lubbert Dijkhuizen b, c, Xiangfeng

Menga, *, Weifeng Liua

a
State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong

University, No.72 Binhai Road, Qingdao 266237, China

b
CarbExplore Research BV, Groningen, The Netherlands
c
Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB),

University of Groningen, Groningen, The Netherlands

*
Correspondence should be addressed to X. Meng. E-mail: [email protected]; Phone: +86

0532 58632405; Fax: +86 0532 58631501

Abbreviations:

AECC: alkali-extracted deshelled corn cobs; AFEX: ammonia fiber explosion; AI: artificial

intelligence; BGLs: β-glucosidases; CBB: Calvin-Benson-Bassham; CBHs:

cellobiohydrolases; CBM: carbohydrate-binding modules; CBP: consolidated bioprocessing;

CBS: consolidated bio-saccharification; CMC: carboxymethyl cellulose; DMCC: Direct

microbial conversion of biomass with CO2 fixation; ED: Entner-Doudoroff; EGs:

endoglucanases; FAEE: fatty acid ethyl ester; FPS: farnesyl pyrophosphate synthase; ILs:

ionic liquids; Ldh: L-lactate dehydrogenase; LPMOs: lytic polysaccharide monooxygenases;

MCC: microcrystalline cellulose; ML: machine learning; NRPs: non-ribosomal peptides;

PASC: phosphoric acid swollen cellulose; PEFA: polyol esters of fatty acids; PGASO:
promoter-based gene assembly and simultaneous overexpression; PHA:

polyhydroxyalkanoate; Pta: phosphotransacetylase; PYC: pyrogenic carbon; RAC:

regenerated amorphous cellulose; rTCA: reduced tricarboxylic acid cycle; SCFAs:

short-chain fatty acids; SECS: steam-exploded corn stover; TAL: triacetic acid lactone; XI:

xylose isomerase; XK: xylulose kinase.

Abstract

Lignocellulosic biomass is an abundant and renewable bioresource for the production of

biofuels and biochemical products. The classical biorefinery process for lignocellulosic

degradation and conversion comprises three stages, i.e., pretreatment, enzymatic

saccharification, and fermentation. However, the complicated pretreatment process, high cost

of cellulase production, and insufficient production performance of fermentation strains have

restricted the industrialization of biorefinery. Consolidated bioprocessing (CBP) technology

combines the process of enzyme production, enzymatic saccharification, and fermentation in

a single bioreactor using a specific microorganism or a consortium of microbes and

represents another approach worth exploring for the production of chemicals from

lignocellulosic biomass. The present review summarizes the progress made in research of

CBP technology for lignocellulosic biomass conversion. In this review, different CBP

strategies in lignocellulosic biorefinery are reviewed, including CBP with natural

lignocellulose-degrading microorganisms as the chassis, CBP with biosynthetic

microorganisms as the chassis, and CBP with microbial co-culturing systems. This review

provides new perspectives and insights on the utilization of low-cost feedstock

lignocellulosic biomass for production of biochemicals.

Keywords: Lignocellulosic biomass, Biorefinery, Consolidated bioprocessing, Biochemicals

1. Introduction

Biomass is an abundant and renewable resource on earth and is divided into two types:

food-based biomass resources and lignocellulosic biomass resources. The first generation of

biofuels is produced from food-based resources (corn starch, sugarcane sugar, and sunflower

oil et al.), which created the issue of the competition with food production. In contrast, the

second generation of biofuels is produced from non-food biomass such as lignocellulosic

biomass derived for instance from crop residues, forest residues and municipal solid waste.

The overall amount of lignocellulosic biomass exceeds 145 billion tons per year worldwide

[1]. Given the abundance and environmental friendliness of lignocellulosic biomass [2], it has

the potential to be converted into biofuels, such as bioethanol, and high-value-added

biochemicals [3]. The utilization of lignocellulosic biomass feedstocks facilitates the

transition from a linear to a circular economy, thus meeting global sustainability requirements

[4]. However, only 3 % of lignocellulosic biomass is effectively utilized [5], and further

research is required for the efficient utilization of lignocellulosic biomass.

The recalcitrant nature of lignocellulosic biomass, which is caused by structural

complexity and heterogeneity, is a major obstacle to its decomposition and utilization.

Lignocellulose is composed of three major components: cellulose, hemicellulose, and lignin,

which account for more than 90 % of the total plant cell wall content. Cellulose, formed by

the polymerization of glucosyl units with β-1,4 glycosidic bonds, is the most abundant

biopolymer on the earth [6]. The linear cellulose chains further form a highly crystalline

microfibrillar structure through strong intra- and intermolecular hydrogen bonds [7, 8], which

poses significant challenges for its efficient degradation. Hemicellulose are heteropolymers

with a certain degree of branching, consisting of different hexoses (galactose, mannose,

rhamnose, fucose), pentoses (xylose, arabinose) and glucuronic acid [9]. The chemical

structure and content of hemicelluloses vary significantly within different biomass types of
the same plant as well as among different plants [10]. For example, xylose is the main

component of hemicelluloses in the cell walls of grasses and broadleaf trees, whereas

mannose is the main component of hemicelluloses in the cell walls of cork and coniferous

trees [11, 12]. The diversity and heterogeneity of hemicellulose are also the main obstacles to

its degradation and utilization. Lignin is a complex, non-crystalline, three-dimensional

reticulated phenolic polymer, and its main role is to provide structural support and to form a

natural, impermeable barrier against microbial attack and oxidative stress [13]. In plants,

cellulose, hemicellulose, and lignin form a supramolecular system in which lignin acts as a

binder for cellulose and enhances the mechanical strength of the plant cell wall. In general,

the complex composition of lignocellulosic biomass poses a major obstacle for the efficient

separation of carbohydrates from lignin and their subsequent utilization, limiting the

development of lignocellulose biorefinery technologies and significantly reducing the

efficiency of biofuels and high value-added chemicals production. The main approaches for

lignocellulose biomass biorefinery are the classical three-stage biorefinery process and the

consolidated bioprocessing (CBP) technology, among which CBP combines the sugar

production and fermentation in a single step to produce various bioproducts from

lignocellulose biomass. The present review summarizes three different CBP strategies,

including CBP construction with lignocellulose-degrading microorganisms as the chassis,

CBP construction with biosynthetic microorganisms as the chassis, and the construction of

CBP with microbial co-cultures. This review provides insights and new perspectives on the

utilization of lignocellulose as feedstock for the production of biochemicals.

2. The classical three-stage biorefinery process

The classical lignocellulosic biorefinery process can be divided into three separate steps:

pretreatment, enzymatic saccharification, and fermentation (Fig. 1).

Pretreatment of the feedstock is required for effective enzymatic saccharification of

lignocellulose. The pretreatment of lignocellulose aims to separate various components of

biomass (especially removing lignin from cellulose and hemicellulose) and also disrupts the

hydrogen bonds and van der Waals interactions of cellulose microfibrils, and thus helps

loosen the rigid lignocellulosic biomass structure [14]. A wide range of pretreatment

techniques, including physical, chemical, and biological pretreatment methods, has been

developed to disrupt the structure of lignocellulosic biomass. Physical pretreatment

techniques are primarily used to reduce the particle size and increase the specific surface area

of lignocellulose through mechanical crushing [15] and high-temperature hydrothermolysis

treatment [16]. In recent years, irradiation methods such as microwave and ultrasonic

techniques have been widely used to release intracellular cellulose in a short time under

high-energy radiation [17]. However, these physical pretreatment methods usually require

huge energy input and thus cannot be easily implemented on a large scale [18]. Chemical

pretreatment methods utilize special chemicals to disrupt the structure of lignocellulose by

selectively dissolving the specific components of lignocellulose. Chemical pretreatment

techniques (e.g., acid, alkali, oxidation, and organic solvent pretreatment) can effectively

increase the biomass surface area and improve lignocellulose degradation [19, 20]. However,

both physical and chemical pretreatment methods generate toxic and inhibitory compounds

like furfural and hydroxymethylfurfural, which interfere with enzymatic saccharification and

fermentation [21-23]. Thus, a detoxification step becomes necessary before using pretreated

biomass for saccharification and fermentation. In addition, toxic liquid effluent released from

pretreatment and detoxification also causes severe environmental pollution [24]. The

increased cost required for detoxification and waste disposal limits its large-scale industrial

application [25]. Compared to physical and chemical pretreatment methods, biological

pretreatment methods have the advantages of lower operating costs, higher yields, and fewer

inhibitory by-product formation but are less efficient and time-consuming. Biological
pretreatment techniques require selected microorganisms or enzymes to degrade lignin and

hemicellulose. For example, white rot, brown rot, and soft rot fungi are capable of secreting

lignin peroxidase, manganese peroxidase, and laccase, which can effectively degrade lignin

[26]. There are also pretreatment technologies that combine several physical, chemical, and

biological methods [21, 22]. Physico-chemical pretreatment includes a combination of

physical and chemical methods such as steam explosion, liquid hot water, CO2 explosion, and

ammonia fiber explosion (AFEX) [27]. Physicochemical pretreatment methods remove lignin

at a higher rate and efficiently disrupt the cellulose polymer by reducing cellulose

crystallinity [28]. For example, the steam explosion pretreatment method can effectively

reduce cellulose crystallinity and enhance glucose yield from enzymatic hydrolysis; therefore,

this method is considered the most cost-effective pretreatment method [29, 30]. In addition,

supercritical fluids pretreatment is also considered an economical and environmentally

friendly process that can replace the conventional pretreatment processes [31].

The pretreatment process loosens the rigid and complex structure of the lignocellulosic

biomass, which facilliates the enzymatic saccharification of cellulose and hemicellulosic

polysaccharides by lignocellulolytic enzymes. In the enzymatic saccharification stage, the

efficient hydrolysis of cellulose requires the synergistic action of cellobiohydrolases (CBHs),

endoglucanases (EGs), and β-glucosidases (BGLs) [21, 32, 33]. Among these, the CBHs

move processively along the cellulose chains and release cellobiose units from either the

reducing ends or non-reducing ends, while the EGs randomly hydrolyze internal glycosidic

bonds within the cellulose chain generating oligosaccharides of different lengths and thus

increasing the acting sites for CBHs; finally the BGLs hydrolyze cellobiose into glucose [34].

The enzyme cost and catalytic efficiency of the lignocellulolytic enzymes determine the

cost-effectiveness and feasibility of the overall biorefinery process [35, 36]. The enzymatic

degradation of hemicellulose in lignocellulosic biomass is carried out by hemicellulase

enzymes including xylanase, mannanase, and arabinosidase. In addition to the hydrolytic

enzymes, recently discovered lytic polysaccharide monooxygenases (LPMOs) and several

other oxidoreductases also play important roles in the efficient degradation of lignocellulosic

biomass [37, 38].

Enzymatic saccharification of the pretreated lignocellulosic biomass yields a series of

monosaccharides, which can be used for downstream fermentation to produce the desired

products, mainly biofuels such as bioethanol. In the study by Zhao et al. (2019), corn stover

pretreated with Na2CO3 and H2O2 was enzymatically hydrolyzed by a lignocellulolytic

enzyme cocktail composed of CBHs, EGs, BGLs, and xylanases. The resulting hydrolysate

was fermented by a highly efficient ethanol-producing Saccharomyces cerevisiae WXY12

strain, yielding 46.87 g/L of bioethanol with a 27.4 % theoretical conversion rate [39].

Hemicellulose is also used for producing functional sugars, like oligosaccharides, xylose,

arabinose, and chemicals such as xylitol, acetic acid, and furfural [40-42]. One of the key

challenges for the efficient production of biofuels and chemicals through biorefinery is to

simultaneously ferment all the released pentoses and hexoses into the target products [43].

Although significant progress has been made in fermenting both pentoses and hexoses using

engineered microorganisms, some challenges still need to be addressed, such as the

production of by-products and poor product tolerance of the strains. Therefore, the

fermentation performance of selected microbial strains also restricts the industrialization of

biorefineries.

In the current stage, several demonstration plants have been established with the

three-stage biorefinery technology. However, the operational costs, particularly those of

cellulases, are still found to be high in practice [44]. The cost of cellulase production has been

reduced to about 10–20 US $ per kg of the enzyme [45], but enzymatic saccharification of

lignocellulosic biomass is still the main limiting step due to the low cellulase activity and
high cost of cellulase production. Thus, lignocellulosic biomass biorefineries are not yet

available for large-scale industrial applications and still need to be continuously improved.

3. Consolidated bioprocessing technology

Consolidated bioprocessing (CBP) is considered another approach worth exploring for

the production of biofuels and high-value-added chemicals from lignocellulose feedstock.

CBP is characterized by combining sugar production and fermentation in a single step (Fig.

1), which accomplishes cellulase production and secretion, lignocellulose whole-component

hydrolysis, and biosynthesis of chemicals in one bioreactor [46]. Thus, the CBP approach

reduces the cellulase production cost along with the elimination of separate enzymatic

hydrolysis, which would significantly decrease the overall biorefinery process cost. Therefore,

CBP represents another avenue worth exploring for lignocellulose biorefinery [47].

CBP with a single microbial strain as the chassis has been extensively studied due to its

relatively well-defined metabolism [48, 49]. A rapid growth rate, extensive substrate

utilization, high product productivity, and robust resistance are required as a microbial CBP

chassis. In the monomicrobial systems, CBP strains must be able to degrade lignocellulosic

feedstocks and synthesize the desired products. Microorganisms with these properties are

rarely found in nature, therefore CBP strains need to be constructed either with natural

lignocellulose-degrading microorganisms as the chassis or microorganisms with biosynthetic

pathways as the chassis [50].

3.1 Construction of CBP strains with lignocellulose-degrading microorganisms as the

chassis

In nature, various bacterial and fungal microorganisms have been found to be capable of

degrading lignocellulose. However, these lignocellulose-degrading microorganisms generally

lack the metabolic pathways for the synthesis of the desired products. Therefore, the
construction of CBP with lignocellulose-degrading microorganisms as the chassis requires

the expression of product synthesis pathways and metabolic engineering to enhance their

synthesis (Table 1). Simultaneously maintaining a high level of lignocellulose degradation

capacity while increasing the productivity of the target product are the main challenges of this

strategy.

3.1.1 Lignocellulose-degrading bacteria as the chassis for CBP construction

Due to the rapid proliferation and strong tolerance to various environmental conditions,

bacteria have received extensive attention in lignocellulose degradation [51]. Most of the

lignocellulose-degrading bacteria are anaerobic, such as Clostridium, Ruminococcus,

Pseudomonas, Bacillus, Proteus, and Serratia [52-54]. These anaerobic

lignocellulose-degrading bacteria generally weave lignocellulolytic enzymes into a complex

structure called cellulosomes [55, 56], which consists of two main parts: a catalytically active,

multi-enzyme subunit with a dockerin domain and a non-catalytic scaffolding protein with a

cohesin domain [57]. Cellulases are assembled into a multi-enzyme complex by specific

binding of the dockerin domain to the cohesin domain on the scaffold protein, which can be

further anchored onto the cell surface. In addition, scaffold proteins contain

carbohydrate-binding modules (CBM) that bind specifically to cellulose, leading to a targeted

effect on substrates [58, 59]. Thus, the effective synergistic degradation of lignocellulosic

biomass is achieved through a combination of spatial proximity of different types of

cellulases (proximity effect) and targeting of the multi-enzyme complex to the substrate

(targeting effect). Cellulosomes thus have a highly ordered spatial structure that allows

multiple synergistic effects between enzyme and enzyme, enzyme and cell, and enzyme and

substrate. Therefore, cellulosomes are superior to the display of free cellulases directly on the

cell surface in terms of cellulose degradation ability.

Clostridium thermocellum is an anaerobic, thermophilic Gram-positive bacterium that

has been widely used for lignocellulosic biomass conversion due to its natural

cellulose-degrading capacity, which is comparable to commercially available cellulases [60,

61]. It usually grows at high temperatures (50–60 ℃) and thus exhibits a higher

lignocellulosic biomass degradation efficiency than mesophilic bacteria. Particularly, the

enzymes produced by C. thermocellum have a strong tolerance to harsh conditions, such as

the presence of phenolic compounds produced during the pretreatment of lignocellulose. In

addition, C. thermocellum has a natural ability to produce ethanol. Therefore, C.

thermocellum has great potential to be developed as a CBP chassis. Argyros et al. (2011)

deleted the genes encoding L-lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) in

C. thermocellum by an established reverse selection system to eliminate the production of the

by-products acetate and lactate, thereby increasing the flux towards ethanol. The engineered

strain was further improved by adaptive evolution of 2000 h. Using Avicel as the carbon

source, the engineered strain was shown to produce 5.6 g/L ethanol, which is a 4.2-fold

increase compared to that of the wild-type strain [62]. C. thermocellum itself cannot produce

butanol and isobutanol. Lin et al. (2015) used different promoters to drive the expression of

isobutanol biosynthetic genes in C. thermocellum, and the engineered strain produced 5.4 g/L

of isobutanol from Avicel under optimized conditions, reaching 41 % of the theoretical yield

[63]. In addition to isobutanol, Tian et al. (2019) engineered a C. thermocellum strain to

convert Avicel to produce 357 mg/L of butanol by heterologously expressing the enzymes for

butanol production, key enzyme engineering, and supplying additional ethanol [64]. Garcia et

al. (2020) designed an advanced genome-scale metabolic model for C. thermocellum. This

model offers a more comprehensive and accurate representation of the organism's metabolism

by integrating genetic, genomic, and metabolic data from various sources. It not only

supports metabolic flux simulations but also serves as a system-level framework for data

integration. Using this model, these authors studied C. thermocellum's redox metabolism and
identified the significance of NADPH as a cofactor, offering insights into potential

engineering targets for improving the production of reduced products, such as ethanol, in C.

thermocellum [65].

Another promising CBP chassis strain belonging to the genus Clostridium is C.

phytofermentans ATCC 700394. Among the sequenced genomes of Clostridium spp., its

genome encodes the largest number of lignocellulolytic enzymes [66], which can degrade

cellulose and hemicellulose into fermentable sugars. Moreover, unlike C. thermocellum,

which is unable to consume xylose, C. phytofermentans can consume almost all types of

sugars present in lignocellulose and produce ethanol and acetate as the main products [66, 67].

Using corn stover pretreated with AFEX with a particle size of 0.5 mm as the feedstock, Jin

et al. (2011) showed that C. phytofermentans ATCC 700394 produced 2.8 g/L of ethanol

after 10 days of fermentation under optimum conditions [30 ℃, 5 % (v/v) inoculum, and

initial pH 7.0] [68]. Bao et al. (2019) engineered Clostridium cellulovorans by introducing

three different aldehyde/alcohol dehydrogenase genes bdhB, adhE1, and adhE2 from

Clostridium acetobutylicum for the production of ethanol and n-butanol. Co-expression of

adhE1 and bdhB in C. cellulovorans enhanced n-butanol production as compared to ethanol,

and the highest butanol/ethanol ratio of 7.0 and 5.6 (g/g) was achieved through fermentation

by using glucose and cellulose, respectively [69]. For further improvement in n-butanol

production, Wen et al. (2019) developed an evolved strain by integrated metabolic and

evolutionary engineering. The engineered C. cellulovorans strain produced n-butanol by

utilizing alkali-extracted deshelled corn cobs (AECC), achieving the highest titer of 3.47 g/L

[70].

3.1.2 Lignocellulose-degrading fungi as the chassis for CBP construction

Cellulases used in industrial applications are mainly produced by filamentous fungi [71],

such as Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei,

Humicola insolens, and Myceliophthora thermophila [72]. Among them, T. reesei has a high

cellulase secretion capacity, and the industrial T. reesei mutant strains are reported to produce

up to 100 g/L cellulases [73]. However, a CBP strain requires both strong

lignocellulose-degrading capabilities and efficient product synthesis pathways. Although T.

reesei can metabolize all types of monosaccharides present in lignocellulose, its ethanol yield

is low, and it also produces other undesired by-products, such as acetic acid and lactic acid

[74]. However, due to the widespread use of T. reesei in commercial enzyme production, the

well-established large-scale fermentation techniques, and the availability of genetic

manipulation tools [75], it is considered a promising CBP chassis strain. The major challenge

of T. reesei as a CBP strain is that the expression of cellulase and glycolysis-related genes is

susceptible to be inhibited by hypoxic conditions [76, 77], which is essential for ethanol

production. In addition, the transcription of cellulase genes is also inhibited in the presence of

ethanol, which is another challenge that still needs to be addressed. Nevertheless, many

studies have used T. reesei as a CBP chassis strain to produce biochemicals such as ethylene,

xylitol, and erythritol from lignocellulose. However, in most cases, their product yields are

far from commercial requirements. Chen et al. (2010) utilized three strong promoters, the

cbh1 promoter from T. reesei, the glyceraldehyde-3-phosphate dehydrogenase (gpd)

promoter from Aspergillus nidulans, and the 3-phosphoglycerate kinase I (pgk1) promoter

from T. reesei to drive the expression of the ethylene synthase gene efe from Pseudomonas

syringae pv. Glycinea in T. reesei QM9414; 14 transformants achieved ethylene production

from wheat straw. The highest ethylene production rate was observed with T. reesei

transformant C30-3 using the pgk1 promoter, reaching a value of 4,012 nL/h/L [78]. Blocking

the downstream metabolism of xylitol in the T. reesei D-xylose metabolic pathway enabled

the strain to produce xylitol from hemicellulose. Dashtban et al. (2013) deleted the xylitol

dehydrogenase gene (xdh1) and the L-arabinitol-4-dehydrogenase gene (lad1) of the T. reesei
D-xylose metabolic pathway, which resulted in the synthesis of xylitol. Using sodium

hydroxide and organic solvent ethanol pretreated barley straw as the carbon source with a

replenishment of 2 % D-xylose, the yield of xylitol reached 13.22 g/L [79].

T. reesei Rut-C30, a hyper-cellulosic mutant strain, has a higher capacity for cellulase

production. Furthermore, in the presence of lignocellulose, Rut-C30 exhibits a pellet-like

morphology in the early stages of fermentation [80] and can grow in a dense pellet form

rather than in an extended mycelial form by the addition of the surfactant Triton X-100 [81],

thus achieving an even higher enzyme production. Compared to the strain QM9414 from the

Natick pedigree, Rut-C30 also shows a greater ethanol tolerance [82]. Jovanovic et al. (2014)

overexpressed the erythritol reductase gene (err1) of T. reesei Rut-C30, and the resulting

engineered strain produced about 10-fold higher amounts of erythritol (5 mg/L) than the

wild-type strain using wheat straw pretreated with an alkaline organic solvent process as the

feedstock [83]. However, this yield was still far from that of erythritol production from

glucose and thus warrants further studies.

The thermophilic fungi M. thermophila, belonging to the genus Myceliophthora, is

capable of secreting a large amount of thermostable cellulase [84], which can efficiently

degrade lignocellulose, including cellulose and hemicellulose. Notably, the growth rates of M.

thermophila with cellulose and glucose as carbon sources are almost identical. Its cellulose

utilization rate is about 5 times that of T. reesei and 2.5 times that of C. thermocellum [85].

Moreover, the optimal growth temperature of M. thermophila is 45 ℃ [86], which is close to

the optimal catalytic temperature of cellulases (50 ℃), thus making M. thermophila an ideal

CBP chassis for simultaneous lignocellulose decomposition and product biosynthesis. Using

M. thermophila as the chassis, Li et al. (2020) constructed a CBP platform, which directly

transformed unpretreated lignocellulose to malic acid and succinic acid in one step without

the addition of additional lignocellulolytic enzymes [87]. In this study, a reduced

tricarboxylic acid cycle (rTCA) pathway was established, and a malic acid transporter was

introduced, which enabled the recombinant strain to produce malic acid from lignocellulose

corncob. Subsequently, the rTCA pathway was further enhanced by overexpression of the

phosphoenolpyruvate carboxylase gene (ppc) and the malate dehydrogenase gene (mdh). In

addition, CO2 fixation was enhanced by heterologous expression of the HCO3- transport

protein gene (bicA) and carbonic anhydrase gene (ca) from Synechococcus sp. PCC7002.

Consequently, the engineered strain produced 83.3 g/L of malic acid from 75 g/L of Avicel in

shake flasks with a yield of 1.11 g/g, which was 1.26 times higher than that of the parental

strain. The production of succinic acid reached 15.4 g/L, which is the highest level ever

reported using lignocellulose as feedstock. Li et al. (2021) constructed a novel biorefinery

system, DMCC (Direct microbial conversion of biomass with CO2 fixation), in M.

thermophila through metabolic engineering by incorporating two CO2 fixation modules, i.e.,

the pyruvate carboxylase (PYC) module and Calvin-Benson-Bassham (CBB) pathway. The

metabolic-engineered M. thermophila CP-51 strain showed an increase in the malic acid titer

by 40 %, 10 %, and 7 %, in xylose, glucose, and cellulose, respectively, as compared to the

parent strain. Using lignocellulosic feedstock, the M. thermophila CP-51 strain with the

DMCC system obtained a malic acid yield of up to 0.53 g/g. This study suggested that a

constructed DMCC system can produce 1 ton of malic acid from 1.89 t of raw lignocellulosic

feedstock by fixing 0.14 t atmospheric CO2 [88].

3.2 Construction of CBP using microorganisms with biosynthetic pathways as the

chassis

Introducing genes encoding lignocellulolytic enzymes into microorganisms with

biosynthetic pathways represents another avenue for CBP construction (Table 2). The main

challenge of this strategy is to achieve the efficient heterologous expression of cellulases or

hemicellulases to accomplish the conversion of lignocellulose. CBP construction using

microorganisms with biosynthetic pathways as the chassis has been applied to many different

microorganisms, including Escherichia coli [89], Z. mobilis [90], Klebsiella oxytoca [91], and

S. cerevisiae [92-94].

3.2.1 Bacteria with biosynthetic pathways as the chassis for CBP construction

E. coli is the most common bacterial host organism for recombinant protein production

and also a commonly used chassis for CBP. Bokinsky et al. (2011) conducted innovative

CBP studies using engineered E. coli and ionic liquids (ILs) pretreated lignocellulose. They

engineered E. coli strains by heterologous expression of an intracellular cellulase gene (cel)

from Bacillus sp. D04 and a xylanase gene (xyn10B) from Clostridium stercoranium. The

recombinant E. coli strain grew well in ILs-pretreated willow jelly, eucalyptus, and yard

waste without exogenous addition of lignocellulolytic enzymes. Further introduction of the

biofuel biosynthetic pathway into this engineered E. coli strain achieved the production of 71

mg/L fatty acid ethyl ester (FAEE), 28 mg/L n-butanol, and 1.7 mg/L pinene using

ILs-pretreated lignocellulose biomass [89].

B. subtilis is a most representative industrial microorganism with many valuable

characteristics such as low nutrient requirements, fast growth rate, easy cultivation, high

protein secretion capacity, and is a biosafe strain [95-97]. B. subtilis is widely used in aerobic

fermentation for the production of enzymes (e.g., nattokinase, α-amylase) [98], vitamins [99],

antibiotics [100], pyrimidine nucleoside [101], hyaluronan [102], etc. Under anaerobic

conditions, B. subtilis can produce two predominant products, lactic acid and 2,3-butanediol,

in addition to acetic acid. B. subtilis 168 encodes a secreted EG of glycoside hydrolase family

5 (BsCel5) and an intracellular BGL, but lacks a CBH [103]. Due to the insufficient

expression level of EG in B. subtilis, it cannot grow on cellulose. Therefore, heterologous

expression of the key cellulase genes is required to enable B. subtilis to produce chemicals

from various lignocellulose biomass [104]. Zhang et al. (2011) enabled B. subtilis to grow on
regenerated amorphous cellulose (RAC) and well-pretreated lignocellulose without the

addition of other organic nutrients (e.g., yeast extract, peptone, amino acids) by

overexpressing the endogenous EG gene (cel5) in a non-cellulose-utilizing B. subtilis.

Subsequently, the specific activity of BsCel5 on RAC was improved by two rounds of

directed evolution, and the expression and secretion levels of BsCel5 in B. subtilis were also

enhanced to improve the lignocellulose-degradation capacity of the B. subtilis strain. In

addition, the authors deleted the α-acetyl lactate synthase gene (alsS) involved in the

2,3-butanediol biosynthetic pathway in the recombinant B. subtilis strain to eliminate the

synthesis of the minor product 2,3-butanediol and aiming to increase the yield of the major

product lactate. The resulting strain produced 3.1 g/L of lactate from RAC, achieving a yield

of 60 % of the theoretical maximum [105, 106].

The production of the chiral lactic acid monomer by fermentation using inexpensive

lignocellulosic biomass instead of starchy feedstock will reduce the production cost of

biodegradable plastic polylactic acid. However, many inhibitors (e.g., furfural and phenolics)

produced during lignocellulose pretreatment can inhibit the growth and metabolism of lactic

acid-producing strains, which will result in lower lactic acid production [107]. Pediococcus

acidilactici strains have been shown to be highly tolerant to inhibitors [108], and are also

shown to be capable of metabolizing all the lignocellulose-derived sugars (glucose, xylose,

mannose, galactose, and arabinose) for lactic acid production [109]. Therefore, P. acidilactici

has great potential as a chassis strain for lactic acid production from lignocellulose. Qiu et al.

(2022) significantly improved the tolerance of P. acidilactici XH11 to four typical aldehyde

inhibitors (5-hydroxymethylfurfural, furfural, vanillin, and 4-hydroxybenzaldehyde) through

a long-term adaptive evolutionary strategy, which allowed the strain to produce 61.9 g/L of

D-lactic acid from undetoxified acid-pretreated corncob slurry [110].

K. oxytoca has the natural ability to metabolize cellobiose and cello-triose, and would
thus be a potential CBP chassis after metabolic engineering. Zhou et al. (2001)

heterologously expressed ethanol synthetic enzymes (pdc, adhB) from Z. mobilis and

endoglucanase genes (celY, celZ) from Erwinia chrysanthemi in K. oxytoca M5A1, resulting

in a recombinant strain that secreted over 20,000 U·L-1 of extracellular EG, which is more

than 10 times the level of enzyme production previously reported for S. cerevisiae as well as

other engineered bacterial strains during fermentation for ethanol production. Combined with

its ability to metabolize cellobiose and cello-triose, the recombinant strain was able to

directly convert amorphous cellulose into 4.67 g/L of ethanol without the addition of

cellulases from other organisms, achieving 76 % of the theoretical yield [111].

Z. mobilis has a unique Entner-Doudoroff (ED) metabolic pathway and exhibits

"uncoupled growth" [112, 113], which means that its cells can consume sugar rapidly,

regardless of its need for growth. This bacterium has a high glucose uptake and catabolism

rate, which is 5 times faster than yeast [114], however, it does not have the natural ability to

metabolize the pentoses released during the hydrolysis of lignocellulosic biomass [90]. Z.

mobilis also has a wide pH range tolerance (pH 3.5–7.5) [115] as well as high glucose (400

g/L) and notable ethanol (16 % v/v) tolerance [116], making it a promising bioethanol

producer [117, 118]. Z. mobilis has now been engineered to metabolize all types of major

biomass sugars [112, 119]; heterologous cellulases have also been expressed in Z. mobilis to

endow it with the ability to degrade lignocellulose [120, 121]. In addition, He et al. (2021)

achieved the heterologous expression of ethylene synthase from P. syringae pv. phaseolicola

in Z. mobilis. With further modifications of the central carbon metabolism, the ZM532-efe

strain achieved an ethylene yield of 5.8 nmol/OD600/mL using enzymatically hydrolyzed corn

straw as the sole carbon source [122]. This study demonstrated the potential of Z. mobilis as a

CBP chassis, after further introduction of lignocellulolytic enzymes.

3.2.2 Fungi with biosynthetic pathways as a chassis for CBP construction

 S. cerevisiae is a well-known industrial host due to its high tolerance to low pH, high

temperature, and various inhibitors [123]. Moreover, versatile genetic manipulation tools

have been developed for S. cerevisiae which facilitate the assembly of biosynthetic pathways

involving multiple genes [124]. Therefore, S. cerevisiae is considered to be a promising

chassis strain for the consolidated bioprocessing of lignocellulose.

S. cerevisiae has a high capacity for ethanol production, whereas its capability to express

heterologous cellulases is often poor [125], limiting its application as a CBP chassis. Davison

et al. (2019) heterologously co-expressed the β-glucosidase gene (bglI) from

Saccharomycopsis fibuligerabeta and the endoglucanase gene (egII) from T. reesei in the

cellulase hypersecretory strain S. cerevisiae YI13. The resultant recombinant strain was able

to convert 56.5 % of the cellulose present in pretreated corn cobs into glucose and produce

4.05 g/L of ethanol via fermentation [93]. Although S. cerevisiae cannot take up

cello-oligosaccharides, some fungi can take up and assimilate oligosaccharides via the

cellodextrin transporter [126]. To improve the efficiency of cellulose degradation by S.

cerevisiae, Yamada et al. (2013) co-expressed the EG gene egII and the CBH gene cbhII

from T. reesei, the BGL gene bglI from Aspergillus aculeatus, and the cellodextrin

transporter gene cdtI from Neurospora crassa in S. cerevisiae. The engineered strain

produced 4.3 g/L of ethanol from phosphoric acid swollen cellulose (PASC) following 72 h

of fermentation, achieving 37 % of the theoretical yield, which was 1.7 times higher than that

of the strain expressing only cellulase (2.5 g/L) [94]. Arnthong et al. (2022) disrupted the

gene encoding the cell wall mannoprotein (cwp2) and the cell wall-associated secretory

glycoprotein gene (ygp1), respectively, in S. cerevisiae, and the activity of BGL in the

corresponding mutant strains was increased by 63 % and 24 %, respectively, compared to the

original strain BGL-6_Kl. The ethanol production from cellobiose by the ygp1-deficient

strain was increased by 59 % to 11.3 g/L compared to BGL-6_Kl [92]. This study
demonstrated the important role of synergistic optimization and proteins related to cell wall

function in improving the production of biobased products, via yeast strains, from

lignocellulose. Recently, inspired by cellulosomes, synthetic biologists began to display

designed cellulosomes onto yeast cell surfaces which can efficiently depolymerize cellulose

and hemicellulose components of lignocellulosic biomass in an energy-limited environment

[127]. Nevertheless, one of the drawbacks of S. cerevisiae is its inability to metabolize xylose.

Numerous efforts have been devoted to the metabolic engineering of S. cerevisiae to ferment

xylose. For this, we direct the reader to the excellent review by Qiu et al. (2023) [43].

Notably, Chen et al. (2023) identified xylose isomerase (XI) by big data mining and

constructed four S. cerevisiae strains that can efficiently utilize xylose. The developed S.

cerevisiae CRD5HS strain achieved an ethanol titer of 85.95 and 94.76 g/L from pretreated

corn stover and corn cob, respectively, without detoxification or washing the pretreated

biomass [128].

Pichia pastoris is one of the most commonly used hosts for the eukaryotic expression of

heterologous proteins due to its high level of heterologous protein expression [129, 130], its

fast growth rate, and strong pH adaptability (pH 3.0–7.0); it is also less susceptible to ethanol

accumulation and suitable for large-scale high-density fermentation [131, 132]. Additionally,

Pichia pastoris one of the few yeasts that can ferment common sugars present in biomass (i.e.,

glucose and xylose) [133]. Therefore, P. pastoris is an attractive chassis for CBP construction.

However, natural P. pastoris produces little or no cellulases and hemicellulases, and only a

few strains can directly ferment xylan to ethanol [134]. Thus, P. pastoris has been genetically

modified to enhance its lignocellulosic biomass degradation capacity. Dong et al. (2020)

constructed mini-cellulosomes on the cell surface of P. pastoris and used the engineered

yeasts to directly convert carboxymethyl cellulose (CMC) to ethanol with a titer of 5.1 g/L. In

addition to this, P. pastoris with mini-cellulosomes was lyophilized as composite cellulases

without affecting enzyme activity, which has great potential for industrial applications [135].

Chang et al. (2013) also isolated a Kluyveromyces marxianus KY3 strain, which can

metabolize both hexoses and pentoses for ethanol production [136]. It has also been shown

that the K. marxianus strain KY3 exhibits high heat resistance, a high growth rate, a wide

growth temperature and pH range, as well as a broad substrate profile and efficient

heterologous protein expression capacity [137]. In addition, the authors developed a

technique called "promoter-based gene assembly and simultaneous overexpression

(PGASO)" which was employed to simultaneously integrate five cellulase genes (cbhII, cbhI,

egIII, eglA, npabgs), one cellodextrin transporter gene (cdtI), and one selection marker gene

(kanMX) into the genome of the KY3 strain. The resultant strain KR7 was shown to convert

microcrystalline cellulose (MCC) to 0.6 g/L of ethanol, which was a 2.5-fold increase in yield

compared to the control strain [136]. Although the yield of ethanol obtained in K. marxianus

was low compared to yeast strains of other genera so far, it has great potential as a new CBP

chassis strain.

Rhodosporidium toruloides, an oil-producing yeast belonging to Basidiomycota, is a

promising chassis for the conversion of lignocellulose into biobased products [138]. R.

toruloides has the ability to grow to high cell densities with diverse substrates and is also

resistant to strong osmotic stresses [139]. Furthermore, R. toruloides also showed strong

tolerance to inhibitors present in lignocellulose hydrolysates and is capable of utilizing all

types of monosaccharides commonly found in lignocellulose biomass feedstocks for growth

[140]. Significant progress has been made in the development of genetic manipulation tools

for R. toruloides, laying an important foundation for a wide range of bioengineering

applications [141]. R. toruloides has been engineered to produce a variety of bioproducts.

Geiselman et al. (2020) constructed a heterologous synthetic pathway for non-native

diterpene ent-kaurene in R. toruloides, and achieved the synthesis of ent-kaurene. The supply
of the precursor geranylgeranyl diphosphate was found to be the limiting factor for the

synthesis of ent-kaurene. The exploration and introduction of a more effective farnesyl

pyrophosphate synthase (FPS) and the balanced expression of FPS and ent-kaurene synthase

enabled the engineered strain to produce 1.4 g/L of ent-kaurene from corn stover hydrolysate

in a 2 L bioreactor [142]. R. toruloides is also employed for the production of heterologous

non-ribosomal peptides (NRPs). Wehrs et al. (2019) heterologously expressed the indigoidine

synthase gene (BpsA) from Streptomyces lavendulae and the 4’-phosphopantetheinyl

transferase gene (sfp) from Bacillus subtilis in R. toruloides, which resulted in the production

of 2.9 g/L of blue pigment indigoidine from a sorghum lignocellulosic hydrolysate [143]. In

addition to NRPs, Otoupal et al. (2022) achieved the production of the polyketide product

triacetic acid lactone (TAL, 2.0 g/L) in R. toruloides by heterologously expressing the

codon-optimized 2-pyrone synthase gene (2-ps) from Gerbera hybrida using sorghum straw

hydrolysates. Further implementation of the strain in a one-pot separation-free process, which

carried out the pretreatment, saccharification, and fermentation, enabled the production of 3.9

g/L TAL in a 2 L bioreactor from sorghum straw hydrolysates, which represents the highest

titer of TAL obtained from lignocellulose biomass [144]. These studies highlight the potential

of R. toruloides as a CBP chassis for the conversion of lignocellulose into bio-based products

by further introducing heterologous lignocellulolytic enzyme genes into its genome.

The lipid-producing fungus Mucor circinelloides is a model organism for the study of

lipid accumulation and lipid production. M. circinelloides can metabolize a variety of sugars

(e.g., glucose and xylose) present in lignocellulose hydrolysates, making it one of the ideal

microorganisms for the conversion of lignocellulose into functional lipids [145]. Zhang et al.

(2021) increased xylose consumption and lipid production by overexpressing the genes

encoding xylose isomerase (XI) and xylulose kinase (XK) in M. circinelloides. Compared to

the control strain, the fatty acid content of the two constructed strains (Mc-XI and Mc-XK)
increased by 19.8 % and 22.3 %, respectively. In addition, the uptake of xylose from corn

stover hydrolysate by the engineered strains was significantly increased by 71.5 % (Mc-XI)

and 68.8 % (Mc-XK), respectively. Using the corn stover hydrolysates as feedstock, the

engineered strain achieved a production of 2.17–2.28 g/L of lipid in a 2 L bioreactor [106].

Further enhancing the lignocellulose-degrading capability of M. circinelloides will enable it

to be a CBP chassis for lipid production from lignocellulose biomass.

3.3 Construction of CBP with microbial co-culturing systems

In nature, the effective degradation of lignocellulose occurs through the synergistic

action of multiple bacteria, fungi, protists, and wood-feeding animals [146]. Inspired by this,

CBP with microbial co-culturing systems is receiving increased attention [47] (Fig. 1, Table

3).

The simultaneous expression of both lignocellulolytic enzymes and biobased product

synthetic enzymes will increase the metabolic burden of a specific microorganism. Unlike a

single microorganism, in microbial co-culture systems, the expression of lignocellulolytic

enzymes and bio-product synthetic enzymes can be accomplished in different

microorganisms, thus being able to relieve the cellular metabolic burden through functional

specialization [147]. In CBP with a microbial co-culture system, the degradation of

lignocellulose is performed by upstream strains, while the production of bio-based chemicals

is achieved by downstream strains. In particular, the rapid consumption of fermentable sugars

by downstream strains can alleviate the substrate inhibition of lignocellulolytic enzymes and

thus facilitate the hydrolysis of lignocellulose by upstream strains [148]. The three main types

of microbial co-culture systems are bacteria and bacteria, fungi and fungi, and fungi and

bacteria co-culture systems. Consequently, the establishment of a stable and efficient

synthetic microbial community for industrial manufacturing is an important research topic.

3.3.1 Co-culturing systems with bacteria and bacteria

 A specific division of labor between cells in a co-culture system is important for

efficient lignocellulose degradation and conversion. In the study by Lu et al. (2020), they

created a co-culture system consisting of the hemicellulase-producing

Thermoanaerobacterium thermosaccharolyticum strain M5 and a succinic acid-producing

Actinobacillus succinogenes strain. Under optimized conditions, this CBP co-culture system

successfully achieved succinic acid production from xylan and unpretreated corn cobs [46].

In this CBP co-culture system, T. thermosaccharolyticum secreted xylanase and β-xylosidase

to degrade xylan to xylose, which was used by A. succinogenes for succinic acid production.

In addition, the rapid consumption of xylose by A. succinogenes also alleviated the inhibition

of the xylanase activity. These two strains thus exhibited a good synergistic effect, making

the whole fermentation process more efficient. By optimizing the fermentation conditions,

such as inoculation time and pH, this CBP co-culture system produced 32.50 g/L and 12.51

g/L of succinic acid from 84 g/L of xylan and 80 g/L of corn cobs, respectively.

The synthesis of polyhydroxyalkanoate (PHA) using lignocellulose biomass is a

sustainable way to achieve the production of bioplastic-based PHA. The highly cellulolytic

strain Streptomyces sp. SirexAA-E can hydrolyze cellulose and hemicellulose but cannot

produce PHA. Kumar et al. (2023) co-cultured Streptomyces sp. SirexAA-E with Priestia

megaterium, which cannot utilize plant polysaccharides for growth but is capable of

producing PHA. Under optimized conditions (30 ℃, pH 7, 5 g/L of Miscanthus biomass, an

inoculation ratio of 1:4 (v/v) of Streptomyces. sp. SirexAA-E and P. megaterium), this

co-culture system produced 40 mg PHA/g of Miscanthus biomass [149].

Microorganisms in a synthetic microbial co-culture system of CBP generally do not

undergo long-term coevolution, and therefore the growth and metabolism of members of the

CBP co-culture system need to be artificially coordinated. Genetic engineering and adaptive

evolution are usually performed to improve the adaptability between members of a CBP
co-culture system. Wen et al. (2020) constructed a dual Clostridium co-culture system

consisting of C. cellulovorans DSM 743B and Clostridium beijerinckii NCIMB 8052, which

can directly utilize AECC to produce n-butanol [150]. In this co-culture system, the

cellulolytic microorganism C. cellulovorans DSM 743B could degrade cellulose into

fermentable sugars and also produce butyric acid, which supported the growth of C.

beijerinckii and its production of butanol. Moreover, the consumption of fermentable sugars

and butyric acid alleviated the feedback inhibition on cellulase activity and toxicity to the

strain, respectively. The production of butanol requires a low ambient pH (pH 4.5–5.5)

conditions; however, at any pH values below 6.4, the cellulolytic C. cellulovorans grows

poorly and thus cannot produce enough fermentable sugars from lignocellulose for the

growth of both strains and the production of butanol. Therefore, the authors further

engineered C. cellulovorans to improve its tolerance to a low pH value. Without pH control,

the engineered co-culture system produced 3.94 g/L of butanol in 83 h, which was 5 times

more than the control under the same conditions. Wen et al. (2022) further introduced the

isobutanol biosynthetic pathway into C. beijerinckii, allowing it to produce both butanol and

isobutanol [151]. After medium optimization, the recombinant C. beijerinckii strain was able

to produce 194 mg/L of isobutanol and 7.16 g/L of butanol from glucose. Overexpression of

acetaldehyde/ethanol dehydrogenase (adhE1), ketoisovalerate decarboxylase (kivD), and

aldehyde reductase (yqhD) in C. cellulovorans enabled the strain to synthesize 156 mg/L of

isobutanol and 1.81 g/L of butanol within 120 h from 39.5 g/L of AECC. Finally, the

co-culture of the above two recombinant strains yielded 1.05 g/L and 6.22 g/L of isobutanol

and butanol, respectively, which were 6.73 and 3.44 times higher than the monoculture.

3.3.2 Co-culturing systems with fungi and fungi

Filamentous fungi display high lignocellulose-degrading ability and exuberant

metabolisms, which provide great potential in the biorefinery of lignocellulose biomass

[152-154]. Therefore, CBP co-culture systems consisting of fungi and fungi have also been

widely studied. A fungal CBP co-culture system consisting of the lignocellulose-degrading

fungus T. reesei and the fumaric acid-producing strain Rhizopus delemar was established by

Scholz et al (2018). In this fungal co-culture, cellulases produced by T. reesei degraded

lignocellulosic biomass to release fermentable sugars, which were immediately converted to

fumaric acids by R. delemar in the same bioreactor. The titer of fumaric acids produced by

this fungal co-culture reached 6.87 g/L using 40 g/L of MCC as the substrate [155]. No

addition of cellulases or expensive supplements such as yeast extract are required in the

above process, which can significantly reduce the production cost. Ustilago maydis has a

strong capacity for the production of itaconic acid. Although U. maydis possesses its own

lignocellulolytic enzymes [156], the cellulase activity of U. maydis is so low that it cannot

efficiently produce itaconic acid directly from lignocellulose. Schlembach et al. (2020)

co-cultured U. maydis with the cellulolytic fungus T. reesei, which can grow in a similar

environment at 30 ℃ under aerobic conditions. In this co-culture system, T. reesei was

responsible for the degradation of lignocellulose, while U. maydis was responsible for the

production of itaconic acid. With a clear division of labor between the two fungi, this

co-culture system produced 33.8 g/L of itaconic acid from 270 g/L of α-cellulose in a

fed-batch fermentation [157].

Fang et al. (2022) designed an microbial co-culture system consisting of T. reesei C10

and an engineered S. cerevisiae strain LGA-1 [158]. T. reesei C10 could produce more

cellulases and thus release more fermentable sugars from the lignocellulose, while the S.

cerevisiae strain LGA-1 was engineered to metabolize cellobiose for the biosynthesis of

D-gluconic acid. This T. reesei-S. cerevisiae co-culture system managed to produce 6.42 g/L

of D-glucaric acid from 50 g/L of steam-exploded corn stover (SECS). Both cellulase

production by T. reesei and D-glucaric acid production by S. cerevisiae were carried out
under aerobic conditions, which simplified the process in commercial applications. Thus, this

T. reesei-S. cerevisiae co-culture system provides a promising CBP platform for the direct

conversion of lignocellulose to D-glucaric acid. However, the yield is not yet sufficient for

industrialization and warrants further investigation.

3.3.3 Co-culturing systems with fungi and bacteria

Regarding the co-culture of fungi and bacteria, Minty et al. (2013) developed a powerful

fungal-bacterial consortium for the conversion of lignocellulose into valuable products [159].

This consortium was composed of two "specialists", the cellulolytic specialist T. reesei

Rut-C30, which secretes cellulases to hydrolyze lignocellulose into soluble sugars, and the

fermentation specialist E. coli NV3 pSA55/69, which metabolizes soluble sugars to

synthesize bio-based products. The E. coli strain was metabolically engineered to produce

isobutanol [160, 161]. The authors used this synthetic fungal-bacterial consortium to achieve

the direct conversion of AFEX-pretreated corn stover to 1.88 g/L of isobutanol, reaching up

to 62 % of the theoretical maximum [159].

A co-culture system developed by Zuroff et al. (2013) consisting of the

cellulase-producing C. phytofermentans and the cellodextrin-fermenting yeast Candida

molischiana or S. cerevisiae cdt-1 is another example of a system exhibiting division of labor

between cellulolytic and sugar fermentation microorganisms. By controlling the volumetric

transport rate of oxygen, a symbiotic relationship was established between C.

phytofermentans and the yeast species. Both yeasts were able to provide respiratory

protection to the obligatory anaerobic bacterium C. phytofermentans in exchange for soluble

sugars released by lignocellulose hydrolysis. The yeasts were able to convert these soluble

sugars to ethanol, thus enabling direct ethanol production from α-cellulose [162]. However,

the lignocellulose degradation by C. phytofermentans was relatively low under high substrate

loading; thus, additional EGs were added to the co-culture of C. phytofermentans and S.
cerevisiae cdt-1, achieving the conversion of 100 g/L of α-cellulose into approximately 22

g/L of ethanol, which is significantly higher than that of C. phytofermentans (6 g/L) and S.

cerevisiae cdt-1 (9 g/L) monocultures.

The lignocellulose-degradation rate remains the key rate-limiting step in CBP. Therefore,

accelerating the rate of lignocellulose-degradation and increasing the release rate of

fermentable sugars is critical for the application of CBP. In general, fungi have a higher

lignocellulose-degradation capacity than bacteria [163]. However, the distinction in fungal

and bacterial growth conditions, such as temperature, oxygen demand, and pH, is also a

critical issue that needs to be addressed for fungal and bacterial co-culture systems. Shahab et

al. (2018) took advantage of metabolic compartmentalization and spatial structure to

construct a fungal-bacterial co-culture system consisting of the aerobic fungus T. reesei and

the facultative anaerobic bacterium Lactobacilli pentosus in a biofilm reactor, achieving the

synthesis of 34.7 g/L of lactic acid with 5 % (w/w) MCC as the substrate [164]. This is the

first reported production of lactic acid from lignocellulose using microbial co-cultures. In this

co-culture system, aerobic T. reesei formed a biofilm on the surface of an oxygen-permeable,

dense tubular membrane, through which oxygen could diffuse into the fungal biofilm by

locally defined aeration of the tubular membrane. T. reesei consumed oxygen and produced

cellulases and hemicellulases under aerobic conditions, and these cellulolytic enzymes were

secreted into the fermentation slurry, which could effectively degrade lignocellulose and

release soluble sugars. Since all the oxygen was consumed in the biofilm, facultative

anaerobic L. pentosus fermented the different sugars to produce lactic acid under anaerobic

conditions. Further addition of Clostridium tyrobutyricum to the co-culture system of T.

reesei- L. pentosus achieved the production of 196 kg of butyric acid per ton of beechwood

[165]. In addition, replacing C. tyrobutyricum with the specialized anaerobic bacteria

Veillonella criceti and Megasphaera elsdenii, which synthesize various short-chain fatty
acids (SCFAs) using lactic and acetic acid as substrates, respectively, achieved the

conversion of lignocellulose to SCFAs [165]. The utilization of oxygen by T. reesei and L.

pentosus created a lower redox condition in the co-culture system, for the growth of

anaerobic microorganisms, making the co-culture stable.

4. Outlook and Challenges

Although CBP is considered a promising strategy for the production of biofuels and

biochemicals from lignocellulosic biomass in a single bioreactor, CBP still faces some

practical challenges such as the recalcitrant nature of lignocellulosic biomass, the tolerance of

strains to the toxic compounds generated during lignocellulosic degradation, the engineering

of strains for the high yield of various products, and the complexity of the metabolic

interactions between different microorganisms used in consortium. In the monomicrobial

systems, the construction of CBP with natural lignocellulose-degrading microorganisms as

the chassis requires the multiple steps of strain engineering for the introduction of synthetic

pathway and strain metabolic engineering, which requires efficient genomic editing

techniques. This is particular time-consuming for lignocellulose-degrading fungi. The

construction of CBP with biosynthetic microorganisms as the chassis requires the

heterologous expression of a suite of cellulases, hemicellulases and LPMOs to accomplish

the efficient degradation of lignocellulosic biomass, which is also challenging due to the low

expression levels of lignocellulolytic enzymes. Considering that the efficient degradation of

lignocellulose requires enzyme cocktails with multiple proteins and the product biosynthetic

pathway also requires multiple genes, the development of CBP microbial co-culture systems

has strong potential. In this regard, a specific division of labor between strains in the

co-culture systems is critical for effective lignocellulose degradation and product biosynthesis.

Establishing robust synthetic microbial communities with both high

lignocellulose-degradation and bio-product synthesis capabilities is an important future

research direction, which requires an in-depth understanding of the interactions between

enzymes, metabolic pathways, and microorganisms.

As CBP combines multiple steps in a single bioreactor, the switching of bio-product

synthesis is not flexible. Liu et al. (2020) proposed a consolidated bio-saccharification (CBS)

strategy using cellulosomes as biocatalysts and achieved the integration of hydrolytic enzyme

production and saccharification of lignocellulosic biomass [166]. The CBS strategy can

separate the downstream fermentation steps to some extent, providing more flexibility than

the consolidated bioprocessing technology in fermentations for different biochemicals. Liu et

al. integrated the CBS strategy with the fermentation of the deep-sea yeast strain Rhodotorula

paludigena P4R5 for the synthesis of polyol esters of fatty acids (PEFA). The authors further

developed a semi-continuous process without complicated product separation, which resulted

in the production of 41.1 g/L of PEFA from corn cob hydrolysis residues [167]. Recently,

they isolated a thermophilic lactic acid-producing Geobacillus stearothermophilus 2H-3 and

combined it with the developed CBS, achieving the production of 51.36 g/L of lactic acid

from various agricultural wastes, including corn stover, corncob residue, and wheat straw

[168]. In the future, strains used in CBS may be co-cultured with downstream fermentation

strains to develop an efficient co-culture system for the economic production of biochemicals

from lignocellulose.

The recent digital revolution based on machine learning (ML) and artificial intelligence

(AI) tools is transforming many research fields in biotechnology [169, 170] and will also be

helpful for biorefinery development in key enzyme engineering, optimization of degradation

enzyme cocktails, improvement of enzyme production and secretion, strain metabolic

engineering, and the construction of co-culture consortia based on iterative

Design-Build-Test-Learn cycles [171, 172]. Models based on an artificial neural network

have been implemented to predict the sugar yields of inorganic salt-based pretreatments of
lignocellulosic biomass; they exhibited high coefficients of determination (R2 of 0.097),

facilitating the initial screening of lignocellulose bioprocess development [173]. A novel

model for the optimization of enzyme cocktails has been recently established based on

deep-learning methods. With no need for reliance on expert-level prior reaction mechanism

knowledge, the developed model speeded up the optimization and screening of enzyme

cocktails for the efficient degradation of complex lignocellulose substrates [174]. Recently,

Huang et al. (2023) explored the potential of establishing machine learning models to

simulate lignocellulose-biomass-based mixed sugar fermentation, which facilitates strain

comparison, product titer evaluation, and fermentation profile construction. These AI tools in

combination with the developments in synthetic biology and metabolic engineering will

greatly facilitate the development and improvement of CBP to achieve the economical

production of desired products from lignocellulosic biomass [175, 176].

Taken together, CBP combines multiple-steps of biorefinery in a single bioreactor and

represents a promising route to achieve the conversion of lignocellulose into

high-value-added bio-products. Importantly, the CBP technology avoids the addition of

exogenous hydrolytic enzymes, which can significantly reduce the cost of lignocellulose

biorefinery. CBP technologies with cellulolytic or biosynthetic microorganisms, such as

chassis and microbial co-culture systems, show great potential and are expected to be able to

provide alternative pathways worth exploring for lignocellulose biorefinery. In addition, it is

worthwhile to investigate the potential of CBP for the production of fuel and different

industrial chemicals from low-cost renewable lignocellulose biomass with the aid of synthetic

biology and artificial intelligence.

Acknowledgments

This work was supported by grants from the National Key R&D Program of China (No.

2019YFA0905700), the National Natural Science Foundation of China (3190071), the Young

Scholars Program of Shandong University, and the Major Basic Research of Shandong

Provincial Natural Science Foundation (ZR2019ZD19).

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.
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 Table 1. Synthesis of bio-based chemicals using metabolically engineered natural

lignocellulose-degrading microorganisms

Microorganism Engineering approach Substrate Product Referenc

C. Deletion of key genes 19.6 g/L Avicel 5.6 g/L [62]

cellulolyticum M1570 involved in the production ethanol

of byproducts acetic acid

and lactic acid and

adaptive evolution

C. thermocellum CT24 Heterologous expression 33.6 g/L Avicel 5.4 g/L [63]

of the isobutanol isobutanol

biosynthetic pathway

C. thermocellum Heterologous expression 47.5 g/L Avicel 357 mg/L [64]

LL1668 of the n-butanol and 4 g/L n-butanol

biosynthetic pathway ethanol

C. phytofermentans None 0.5 % (w/w) 2.8 g/L [68]

ATCC 700394 AFEX-pretreate ethanol

d corn stover

C. cellulovorans Heterologous expression Cellulose 1.11 g/L [69]

of different butanol and

aldehyde/alcohol 0.20 g/L

dehydrogenases ethanol

C. cellulovorans Heterologous expression AECC 3.47 g/L [70]

of adhE1 and n-butanol

ctfA-ctfB-adc genes from

 C. acetobutylicum ATCC

824 for constructing a

coenzyme A dependent

acetone-butanol-ethanol

pathway, and adaptive

laboratory evolution

T. reesei QM9414 Heterologous expression 2 % (w/v) 4,012 nL/h/L [78]

of the ethylene wheat straw ethylene

biosynthetic pathway

T. reesei Deletion of the xylitol 2 % (w/v) 13.22 g/L [79]

dehydrogenase gene organosolv-pret xylitol

(xdh1) and the reated barley

L-arabinitol-4-dehydrogen straw and 2 %

ase gene (lad1) (w/v) D-xylose

T. reesei Rut-C30 Overexpression of the 1.7 % (w/v) 5 mg/L [83]

erythrose reductases gene alkaline erythritol

(err1) organosolv-pret

reated wheat

straw

M. thermophila JG424 Overexpression of the 75 g/L Avicel 83.3 g/L [87]

PEP carboxylase gene malic acid

(ppc) and the malate and 15.4 g/L

dehydrogenase gene succinic acid

(mdh); heterologous

expression of the HCO3−

 transporter gene (bicA)

and the carbonic

anhydrase gene (ca) from

Synechococcus. sp.

PCC7002

M. thermophila CP-51 Heterologous expression Corncob and 0.53 g/g [88]

of genes encoding CBB CO2 malic acid

cycle enzymes RuBisCO

(cbbM) from

Rhodospirillum rubrum

and PRK (prk) from

Spinacia oleracea;

deletion of the pyruvate

decarboxylase gene (pdc),

lactate dehydrogenase

gene (ldh), and PEP

carboxykinase gene (pck)

 Table 2. Synthesis of bio-based chemicals using microorganisms with biosynthetic pathways

Microorganism Engineering approach Substrate Product Referen

ce

E. coli MG1655 Heterologous expression of 55 g/L 71 mg/L [89]

ΔfadE the intracellular cellulase gene ILs-pretreated FAEE

(cel) from Bacillus. sp. D04 switchgrass

and the xylanase gene

(xyn10B) from C.

stercoranium; heterologous

expression of the FAEE

biosynthetic pathway

E. coli DH1 ΔadhE Heterologous expression of 55 g/L 28 mg/L [89]

the intracellular cellulase gene ILs-pretreated n-butanol

(cel) from Bacillus. sp. D04 switchgrass

and the xylanase gene

(xyn10B) from C.

stercoranium; heterologous

expression of the n-butanol

biosynthetic pathway

E. coli MG1655 Heterologous expression of 55 g/L 1.7 mg/L [89]

the intracellular cellulase gene ILs-pretreated pinene

(cel) from Bacillus. sp. D04 switchgrass

and the xylanase gene

(xyn10B) from C.

stercoranium; heterologous
 expression of the pinene

biosynthetic pathway

B. subtilis Overexpression of the RAC 3.1 g/L [105]

endoglucanase gene (cel5); lactate

deletion of the α-acetyl lactate

synthase gene (alsS) involved

in the biosynthesis of the

minor product 2,3-butanediol

P. acidilactici Long-term adaptive evolution Undetoxified 61.9 g/L [110]

XH11 (111 days) acid-pretreated D-lactic

corncob slurry acid

K. oxytoca SZ21 Heterologous expression of 6.85 g/L 4.67 g/L [111]

the endoglucanase gene (celY, amorphous ethanol

celZ) from E. chrysanthemi; cellulose

heterologous expression of the

ethanol biosynthetic pathway

S. cerevisiae Heterologous expression of Pretreated corn 4.05 g/L [93]

the β-glucosidase gene (bglI) cobs ethanol

from S. fibuligerabeta and the

endoglucanase gene (egII)

from T. reesei

S. cerevisiae Heterologous expression of PASC 4.3 g/L [94]

the endoglucanase gene (egII) ethanol

and the cellobiohydrolase gene

(cbhII) from T. reesei, the

 β-glucosidase gene (bglI) from

A. aculeatus and the

cellodextrin transporter gene

(cdtI) from N. crassa

S. cerevisiae Deletion of the gene encoding 5 % (w/v) 11.3 g/L [92]

cell wall mannoprotein (cwp2) cellobiose ethanol

and cell wall-associated

secretory glycoprotein (ygp1),

respectively

S. cerevisiae Adaptive laboratory evolution Pretreated corn 85.95 and [128]

CRD5HS stover and corn 94.76 g/L

cob ethanol

P. pastoris Construction of CMC 5.1 g/L [135]

mini-cellulosomes on the cell ethanol

surface

K. marxianus KR7 Heterologous expression of MCC 0.6 g/L [136]

five cellulase genes (cbhII, ethanol

cbhI, egIII, eglA, npabgs), one

cellodextrin transporter gene

(cdtI), and one selection

marker gene (kanMX)

R. toruloides Heterologous expression of Corn stover 1.4 g/L [142]

ABFPUB_26 ent-kaurene synthase hydrolysate ent-kauren

R. toruloides Heterologous expression of Unfiltered 2.9 g/L [143]

 indigoidine synthase sorghum indigoidine

hydrolysate

R. toruloides Heterologous expression of Unfiltered 3.9 g/L [144]

2-pyrone synthase sorghum TAL

hydrolysate

M. circinelloides Mc-XI: overexpression of Corn straw 2.17–2.28 g/L [106]

xylose isomerase hydrolysate lipid

Mc-XK: overexpression of prepared by

xylulokinase dilute acid

pretreatment

and enzymatic

hydrolysis
Table 3. Synthesis of bio-based chemicals from lignocellulose using microbial co-culturing

CBP systems

Cellulolytic Biosynthetic Substrate Product Reference

microorganism microorganism

T. A. succinogenes 80 g/L 12.51 g/L [46]

thermosaccharolyticum 130Z unpretreated succinic acid

M5 corn cobs

Streptomyces. sp. P. megaterium 5 g/L 40 mg/g [149]

SirexAA-E Miscanthus PHA

biomass

C. cellulovorans DSM C. beijerinckii 30.1 g/L AECC 3.94 g/L [150]

743B NCIMB 8052 n-butanol

C. cellulovorans DSM C. beijerinckii 55.1 g/L AECC 1.05 g/L [151]

743B NCIMB 8052 isobutanol

and 6.22 g/L

n-butanol

T. reesei R. delemar 40 g/L MCC 6.87 g/L [155]

fumaric acid

T. reesei Rut-C30 U. maydis 270 g/L 33.8 g/L [157]

α-cellulose itaconic acid

T. reesei C10 S. cerevisiae 50 g/L SECS 6.42 g/L [158]

LGA-1C3S2 D-glucaric

acid

T. reesei Rut-C30 E. coli NV3 20 g/L 1.88 g/L [159]

pSA55/69 AFEX-pretreated isobutanol

 corn stover

C. phytofermentans S. cerevisiae cdt-1 100 g/L 22 g/L [162]

α-cellulose ethanol

T. reesei L. pentosus 5 % (w/w) MCC 34.7 g/L [164]

lactic acid

T. reesei L. pentosus and C. Beechwood 196 kg/t [165]

tyrobutyricum butyric acid

Figure 1

Fig. 1 A schematic diagram of the classic three-stage biorefinery process and consolidated

bioprocessing technologies

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
Graphical abstract