Multi‐enzyme display in minicellulosomes.
Lignocellulosic biomass is a promising feedstock to sustainably produce useful biocommodities. However, its recalcitrance to hydrolysis limits its commercial utility. One attractive strategy to overcome this problem is to use consolidated bioprocessing (CBP) microbes to directly convert biomass into chemicals and biofuels. Several industrially useful microbes possess desirable consolidated bioprocessing characteristics, yet they lack the ability to degrade biomass. Engineering these microbes’ surfaces to display cellulases and cellulosome‐like structures could endow them with potent cellulolytic activity, enabling them to be used in CBP. In this chapter, we discuss recent progress in engineering the surfaces of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and lactic acid bacteria. We discuss the techniques used to display cellulases on their surfaces, their recombinantly achieved cellulolytic activities, and current obstacles that limit their utility.
- consolidated bioprocessing
- cell surface display
As concerns over limited petroleum supplies rise, the momentum to produce renewable fuels, chemicals, and other materials from biomass has increased [1–3]. Second‐generation biofuels derived from sustainable feedstocks such as lignocellulosic biomass are attractive, as plant biomass is cheap and highly abundant; over 1 billion tons of lignocellulosic biomass are produced annually in the United States, while an estimated 10–50 billion tons of waste lignocellulose are produced worldwide [4–6]. However, the resistance of lignocellulose to hydrolysis limits its use in biofuel production and has driven the search for new technologies to cost‐effectively exploit this valuable resource [7, 8]. To produce fermentable sugars to generate cellulosic ethanol, conventional industrial processes utilize a multistep procedure to degrade lignocellulose (Figure 1A) [7, 9]. Typically, the biomass is first pretreated with strong acids and high temperatures to remove lignin and to partially degrade its cellulose and hemicellulose components . It is then exposed to purified cellulase enzymes that hydrolyze the remaining polysaccharides into shorter polysaccharides and monosaccharides that can be fermented into ethanol by yeast. Significant effort has been put forth to optimize these steps, including the development of a range of new pretreatment approaches and enzyme cocktails [9–11]. It is generally believed that the cost of converting biomass into useful biocommodities could be greatly reduced by using a consolidated bioprocessing (CBP) microbe, a single microorganism that produces all of the necessary enzymes to degrade lignocellulose and then utilizes the resulting sugars to produce high levels of the biocommodity (Figure 1B) [12–14]. A CBP microbe would decrease costs by reducing the number of processing steps required to generate the final product and avoid the use of costly purified cellulase enzymes that are estimated to contribute $0.68–1.47 to the per gallon cost of cellulosic ethanol . Given their great potential, a number of research groups have sought to develop a lignocellulose‐utilizing CBP microbe using native and recombinant strategies . In the native strategy, product production pathways are engineered into naturally cellulolytic microbes, while in the recombinant strategy, genetically well‐studied microorganisms that may already be capable of producing a desired product are engineered to possess cellulolytic activity. Here, we describe progress towards creating recombinant cellulolytic microbes to convert biomass into useful commodities by engineering their surfaces to display cellulase enzymes.
Lignocellulose is recalcitrant to degradation and requires the action of many different types of enzymes to break it down . It is composed of varying amounts of cellulose (25–55%), hemicellulose (8–30%), and lignin (18–35%) [7, 16]. Cellulose is a polymer of β‐1,4‐linked glucose molecules that can hydrogen‐bond with other cellulose polymers to form both amorphous and crystalline regions . It is synergistically degraded by three types of cellulases: endoglucanases, exoglucanases, and β‐glucosidases . Endoglucanases attack within a cellulose strand to hydrolyze the β‐1,4‐glucosidic bonds, producing new reducing and nonreducing ends that can be further broken down by exoglucanases . The shorter cellodextrin chains that are produced by these enzymes, including the disaccharide cellobiose, are then degraded into glucose monomers by β‐glucosidases . Hemicellulose is a sugar polymer that is composed of a number of different types of pentose and hexose sugars . Xylan is its main component and is the second most abundant polysaccharide in lignocellulose. As compared to cellulose, hemicellulose is more accessible to degradation by a range of enzymes with different substrate specificities, including among others: xylanases, arabinases, and mannanases. Finally, lignin surrounds and blocks enzyme access to cellulose and hemicellulose and is a complex polymer containing a mixture of phenolic compounds linked through radical coupling reactions . A large number of enzymes are needed to degrade it, including peroxidases and laccases .
One promising recombinant strategy to create a useful CBP microbe is to engineer it to display a range of enzymes that degrade lignocellulose, thereby allowing it to produce sugars that can be further metabolized by the microorganism into useful chemicals [21–26]. In this approach, the goal was to engineer the microbial surface to effectively mimic the activity of naturally cellulolytic anaerobic bacteria. These microbes have the impressive capacity to adhere to, and degrade, untreated biomass and are typified by the cellulolytic anaerobic bacterium
Microbes displaying cellulosomes are believed to degrade lignocellulose much more efficiently than microbes that degrade biomass by secreting cellulases . There are three main reasons why increased efficiencies may be obtained. First, secreting enzymes presumably imposes a higher metabolic burden upon the microorganism as compared to displaying the enzymes on the cell surface. This is because the secreted enzymes can be lost to the environment with no guarantee that the sugars that they will produce will ultimately be accessible to the microbe for use as nutrients. As a result, larger quantities of the enzymes must be produced if they are to be secreted. For example, aerobic fungi (such as
Microbe‐based CBP technologies are not currently used industrially to produce biocommodities from lignocellulosic biomass. However, a major step towards CBP of starch into ethanol has recently been demonstrated by Lallemand and Mascoma. These companies created TransFerm®, a genetically modified yeast strain that secretes a glucoamylase and that is optimized to produce higher yields of ethanol. Although the cells do not fully degrade starch on their own, they reduce by 20–45% the amount of exogenous enzymes that needs to be added to process starch. At present, microbe‐based CBP of lignocellulose is not being performed industrially, and ongoing research is primarily focused on constructing and identifying microorganisms with optimal cellulolytic and biocommodity production capabilities. Furthermore, detailed cost analyses of CBP versus conventional pretreatment and saccharification approaches have not been reported . This is because the specific costs associated with CBP will vary based on the biocommodity produced, and the microbe and biomass source that is employed. However, the greatest cost savings associated with CBP will likely be obtained by reducing the costs of saccharification. A detailed cost analysis has been performed for cellulosic ethanol production from corn stover using dilute acid pretreatment, enzymatic saccharification, and cofermentation . In this analysis, on‐site production of fungal enzymes was estimated to contribute $0.34 per gallon of ethanol (assuming enzyme loadings of 20 mg enzyme per gram of biomass), which could in principle be eliminated using a CBP microbe. Another analysis by Johnson explored the potential cost savings associated with altering the source of enzyme production from off‐site to on‐site cultivation, specifically on biomass as a primary substrate . The estimated full cost of producing cellulosic ethanol was reduced by 19% if the enzymes were produced on‐site because it eliminated the need for enzyme purification, formulation of the enzyme mixture to preserve stability, and transport. Similar substantial gains could be obtained using microbe‐based CBP.
In this chapter, we review progress towards engineering microbes to display “minicellulosomes,” smaller cellulosome‐like complexes that can degrade biomass (Figure 2B). A list of the microorganisms engineered to display minicellulosomes discussed in this chapter is presented in Table 1. We discuss recent developments in displaying these structures on industrially useful microorganisms, including
|Anchor||Assembly (# enzymes/complex)||Enzymes displayed||References|
|Aga2||β‐Glucosidase 1 (BGL1)|
Cellobiohydrolase II (CBHII)
Endoglucanase II (EGII)
|Aga2||β‐Glucosidase 1 (BGL1)|
Cellobiohydrolase II (CBHII)
Endoglucanase II (EGII)
Cellobiose dehydrogenase (CDH)
Lytic polysaccharide monooxygenase (LPMO)
|α‐Agglutinin||Acetylxylan esterase (AwAXEf)|
|α‐Agglutinin||β‐Glucosidase 1 (BGL1)|
Endoglucanase II (EGII)
|LPXTG from ||Exoglucanase (Cel9E)|
|LPXTG from |
|LPXTG from |
|LPXTG from ||Endoglucanase (Cel6A)|
2. Displaying enzymes on
Significant effort has been put forth to display cellulolytic enzymes on the surface of
2.1. Individual cellulases
Individual cellulases were originally displayed on the cell surface by the Tanaka group . They engineered cells to codisplay individual carboxymethylcellulase (CMCase) and β‐glucosidase (BGL1) enzymes derived from
Yeast displaying cellulases may be industrially useful. For example, strains displaying the BGL1, CBHII, and EGII enzymes produced 43.1 g/L ethanol from 200 g/L liquid hot water pretreated rice straw in 72 h . While supplementation with a purified cellulase cocktail at 10 filter paper units (FPU)/g‐biomass was necessary to achieve this high ethanol yield, a control strain that did not display the enzymes required 10‐fold more added cellulase to produce similar quantities of ethanol. Attractively, the cells displaying the enzymes could be reused in five fermentation cycles without significantly losing their activity . Displaying enzymes also reduces the amount of purified cellulase lost through irreversible adsorption onto crystalline cellulose, facilitating more efficient cellulose degradation and higher ethanol yields . Recently, studies using cellulase displaying cells have further reduced the amount of purified cellulase cocktail that needs to be added to convert biomass into ethanol . These newer generation cells require 44% less commercial enzyme supplementation to degrade pretreated biomass by displaying four enzymes using the Sed1 anchor:
Towards the goal of improving their cellulolytic activity, several studies have engineered cellulase displaying yeast cells to also produce complementary enzymes and transporters that improve cellulose solubilization and utilization. The Kondo group constructed cells that co‐expressed three displayed cellulases, as well as the
Ex vivoassembled minicellulosomes
Several research groups engineered
In 2009, two groups demonstrated
In order to build larger surface structures with higher enzyme densities, the Chen laboratory used an adaptive assembly approach . In this procedure, the minicellulosome is built using two separate scaffoldin pieces: a primary scaffoldin that binds to the catalytic components and an anchoring scaffoldin that is attached to the cell surface and only binds to the primary scaffoldin (Figure 4D). They produced a yeast strain that displayed an Aga2‐fused anchoring scaffoldin that contained two cohesin domains from
In vivoassembled minicellulosome
The need to add enzymes to cells displaying a miniscaffoldin may make the use of
2.4. Hemicellulases and hemicellulosomes
In order to develop xylose‐fermenting strains of yeast, similar strategies have been employed to display hemicellulases and hemicellulosomes on the cell surface. The Kondo laboratory displayed an individual
Clustering hemicellulases within surface displayed complexes leads to improved enzymatic activity. The Silva group developed an
2.5. Artificial cellulosome structures
In order to better control enzyme placement and increase the number of displayed enzymes on the cell surface, several groups have created artificial cellulosomes that are structurally distinct from naturally occurring cellulosomes. These structures use unique protein‐protein interaction domains to tether the enzymes to the scaffoldin instead of naturally occurring cohesin‐dockerin interactions. The Kondo laboratory created an
Escherichia colisurface display of individual cellulases
The model Gram‐negative bacterium
Pilot studies have shown that
4. Displaying cellulases and minicellulosomes in Gram‐positive bacteria
Many species of Gram‐positive bacteria are used industrially to produce biocommodities and have great promise as agents to produce second‐generation biofuels. However, they are not naturally cellulolytic, prompting efforts designed to decorate their surfaces with cellulases. Below, we discuss progress towards creating cellulolytic strains of
Two groups have displayed
4.2. Lactic acid bacteria (LAB)
Gram‐positive lactic acid bacteria (LAB) are widely used in the food industry to ferment sugars into lactic acid . They have potential in biomass processing, as they can utilize pentose and hexose sugars, and some members of this group, namely
Two groups have used sortases to assemble minicellulosomes
In separate studies, the Mizrahi group engineered
Towards the goal of cost effectively converting biomass into useful biocommodities, several research groups have developed creative ways to display cellulases on microbes to endow them with cellulolytic activity. Comparing the biomass degradation efficiencies of different types of recombinant microorganisms is difficult. This is because investigators have measured their activities using a variety of cellulosic substrates that can vary dramatically in their resistance to enzymatic degradation as they differ in their solubility, enzyme accessibility, crystallinity, degree of polymerization, fraction of reducing ends, and the presence of hemicellulose and lignin . Moreover, different methods are frequently used to pretreat biomass and to measure the extent of degradation, which can be reported as enzymatic activity, sugar released, biomass remaining, or product produced [17, 135, 136]. However, despite these qualifiers, the results of studies reported to date enable several major conclusions to be drawn. In particular, they provide convincing evidence that clustering enzymes on the surface within minicellulosomes leads to improved microbial cellulolytic activity by promoting synergistic interactions, and in some instances, by improving enzyme stability . Interestingly, synergistic enzyme interactions can also be obtained by displaying different types of individual enzymes, as long as they are densely clustered and have complementary activities . Because complexed enzyme systems require smaller amounts of enzymes to be produced than secreted enzyme systems to achieve efficient degradation of lignocellulose, the cellulosomal system may be optimal for CBP microorganisms, since conserved energy may be directed towards product production.
Displaying large, enzymatically diverse recombinant minicellulosomes remains a challenging problem, but progress has been made in
A great variety of surface engineering approaches have been developed to create ever more impressive recombinant cellulolytic organisms. However, it is clear that the lignocellulose hydrolysis rates of these recombinant microorganisms needs to be improved if they are to be used in CBP. Future studies may improve their cellulolytic activity by using directed evolution to enhance complex display and stability, by judiciously displaying cellulases that exhibit maximal enzyme synergy, and by devising new methods to stably attach proteins to the cell surface. Genetic engineering of the host will also be critical, enhancing expression, secretion, and display levels, and by eliminating proteins or factors affecting the stability and retention of anchored protein complexes over time. Displayed complexes will also have to be optimized to be maximally active against different types of biomass that have different sugar compositions and structures [138–140]. When these cells are further engineered to produce useful chemicals, their ability to cost‐effectively produce biocommodities from biomass will be an important step towards reducing the world's dependency on oil [141–144].
This material is based on work supported by the U.S. Department of Energy Office of Science, Office of Biological and Environmental Research program under Award Number DE‐FC02‐02ER63421. GL Huang was supported by a Ruth L. Kirschstein National Research Service Award GM007185.
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