Metabolic engineering applications to renewable resource utilization

Abstract

Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra.

Introduction

In addition to the magnificent aesthetic value of the planet’s flora, biomass represents a useful and valuable resource to man. The value of the biomass contents is related to the chemical and physical properties of the large molecules of which it is made. For millennia humans have exploited the solar energy stored in the chemical bonds of biomass by burning it as fuel and eating plants for the nutritional energy of their sugar and starch content. More recently, in the past few hundred years, humans have exploited fossilized biomass in the form of coal. This fossil fuel is the result of very slow chemical transformations that convert the sugar polymer fraction into a chemical composition that resembles the lignin fraction. Thus, the additional chemical bonds in coal represent a more concentrated source of energy as fuel. After 1920, petroleum increasingly became the fossil fuel of choice, and after World War II petrochemicals began to dominate the synthetics market. Today, 65% of our clothing is made from oil, as are virtually all of our inks, paints, dyes, pharmaceuticals, plastics, and hundreds of intermediate chemicals. It is fair to characterize the 20^th century as the hydrocarbon century. Because it takes millions of years to convert biomass into coal, fossil fuels are not renewable in the time frame over which we use them. Plant biomass is the only foreseeable sustainable source of organic fuels, chemicals, and materials, and at the end of the 20^th century, there appears to be significant efforts to make the new century one that is based on renewable carbohydrates.

Many different biomass feedstocks can be used for the production of fuels and chemicals. These include various agricultural residues (corn stalks, wheat straws, potato or beet waste), wood residues (leftovers from harvested wood, and unharvested dead and diseased trees), specifically grown crops (hybrid poplar, black locust, willow, silver maple, sugarcane, sugarbeet, corn, and sweet sorghum), and waste streams (municipal solid waste, recycled paper, baggasse from sugar manufacture, corn fiber, and sulfite waste). The chemical composition of biomass varies among species, but biomass consists of ∼25% lignin and ∼75% carbohydrate polymers (cellulose and hemicellulose) [1]. Cellulose is a high-molecular weight linear glucose polysaccharide, with a degree of polymerization (DP) in the range of 200–2000 kDa (4000–8000 glucose molecules connected with β-1,4 links). Cellulose is very strong and its links are broken by cellulase enzymes. The cellulases can be separated into two classes, endoglucanases and cellobiohydrolases [2]. Cellobiohydrolases hydrolyze the cellulose chain from one end, whereas an endoglucanase hydrolyzes randomly along the cellulose chain. Hemicellulose, on the other hand, is a rather low-molecular weight heteropolysaccharide (DP < 200, typically β-1,3 links), with a wide variation in both structure and composition. Commonly occurring hemicelluloses are xylans, arabino-xylan, gluco-mannan, galacto-glucomann, and so on. In contrast to cellulose, which is crystalline, strong, and resistant to hydrolysis, hemicellulose has a random, amorphous structure with little strength. It is easily hydrolyzed by dilute acid or base, but nature provides an arsenal of hemicellulase enzymes for its hydrolysis 3, 4. The cellulose fraction of biomass is typically high (25–60%), whereas the hemicellulose fraction is typically in the range of 10–35%. The monomeric composition of lignocellulosic material can vary widely depending on the biomass source (see Table 1). In general, the carbohydrate fraction is made up primarily of the hexose sugar glucose (with small amounts of the hexoses galactose and mannose); however, the pentose fraction is rather significant: xylose 5–20% and arabinose 1–5%. Xylose is second only to glucose in natural abundance and it is the most copious sugar in the hemicellulose of hardwoods and crop residues.

The conversion of both cellulose and hemicellulose for production of fuel ethanol is being studied intensively with a view to develop a technically and economically viable bioprocess. Ethanol is a versatile transportation fuel that offers high octane, high heat of vaporization, and other characteristics that allow it to achieve higher efficiency use in optimized engines than gasoline. Ethanol is low in toxicity, volatility, and photochemical reactivity, resulting in reduced ozone formation and smog compared to conventional fuels. Researchers at the National Renewable Energy Laboratory estimate that the United States potentially could convert 2.45 billion metric tons of biomass to 270 billion gallons of ethanol each year, which is approximately twice the annual gasoline consumption in the United States. Increased use of bioethanol, also used as a hydrogen fuel source for fuel cells, could become a vital part of the long-term solution to climate change.

The important key technologies required for the successful biological conversion of lignocellulosic biomass to ethanol have been extensively reviewed 5, 6••, 7. Microbial conversion of the sugar residues present in wastepaper and yard trash from US landfills alone could provide more than 400 billion liters of ethanol [8], 10 times the corn-derived ethanol burned annually as a 10% blend with gasoline [9]. The biological process of ethanol fuel production utilizing lignocellulose as substrate requires three steps (Figure 1): firstly, delignification to liberate cellulose and hemicellulose from their complex with lignin; secondly, depolymerization of the carbohydrate polymers (cellulose and hemicellulose) to produce free sugars; and finally, fermentation of mixed hexose and pentose sugars to produce ethanol (see Table 2). The development of a feasible biological delignification process should be possible if lignin-degrading microorganisms (e.g. Phanerochaete chrysosporum and Phlebia radiata), their ecophysiological requirements, and optimal bioreactor design are effectively coordinated. Some thermophilic anaerobes and recently developed recombinant bacteria have advantageous features for direct microbial conversion of cellulose to ethanol — that is, the simultaneous depolymerization of cellulosic carbohydrate polymers with ethanol production. New fermentation technology for converting xylose to ethanol also needs to be developed to make the overall conversion process more cost-effective. The fermentation of glucose, the main constituent of the cellulose hydrolysate, to ethanol can be carried out efficiently. On the other hand, although bioconversion of xylose (the main pentose sugar obtained on hydrolysis of hemicellulose) to ethanol presents a biochemical challenge, especially if it is present along with glucose, it needs to be achieved to make the biomass-to-ethanol process economical. A lot of attention has therefore been focussed on the utilization of both glucose and xylose to ethanol. The economics of the ethanol process is determined by the cost of sugar. The average biomass cost amounts to ∼$0.06 per kg of sugar, or a contribution to the feedstock costs for ethanol production of as low as $0.10 per l.

Applied research in the area of biomass conversion to ethanol in the past 20 years has answered most of the major challenges on the road to commercialization but, as with any new technology, there is still room for performance improvement. Over the past decade, the total cost of ethanol has dropped from more than $1.0 per l to ∼$0.3–0.5 per l, with a projected cost of less than $0.25 per l in the near future. As a number of studies have indicated, efficient utilization of the hemicellulose component of lignocellulosic feedstocks offers an opportunity to reduce the cost of producing fuel ethanol by 25% [10]. This essentially requires the ability to convert all fermentable sugars (i.e. pentoses and hexoses) to product, which dictates the need to develop advanced hexose/pentose-fermenting organisms. As no naturally occurring organism can satisfy all necessary specifications (e.g. high yield, high productivity, wide-substrate range, ethanol tolerance, tolerance to inhibitors present in hydrolysates, and biomass disposal cost), this has to come about by the utilization of modern genetic engineering techniques aimed at organisms that are endowed with most of the desirable properties for such bioprocesses. Along with the introduction of ethanol genes in enteric bacteria (Escherichia coli), parallel efforts were also undertaken to incorporate pentose-metabolizing pathways in natural ethanol producers such as Saccharomyces cerevisiae and Zymomonas mobilis (as discussed later).

Genetic improvements in the microorganism cultures have been made either to enlarge the range of substrate utilization or to channel metabolic intermediates specifically toward ethanol. These contributions represent real significant advances in the field and have also been adequately dealt with from the point of view of their impact on utilization of both cellulose and hemicellulose sugars to ethanol. The bioconversion process of lignocellulosics to ethanol could be successfully developed and optimized by aggressively applying the related novel science and technologies to solve the known key problems of conversion process; for example, simultaneous saccharification and fermentation (SSF), continuous ethanol processes based on flocculent yeast, and continuous end-product removal. The efficient fermentation of xylose and other hemicellulose constituents may prove essential for the development of an economically viable process to produce ethanol from biomass [11].

This review deals with the metabolic strategies to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulose sugars to products that can be derived from the primary metabolism, such as ethanol.