Microbial production of fatty acid-derived fuels and chemicals

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Microbial production of fatty acids and derivative chemicals

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Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages

Arachidonic acid (C20:4) and other unsaturated fatty acids are shown to activate superoxide (O₂^?) production in a cell-free system represented by sonically disrupted guinea pig peritoneal macrophages. The reaction requires a heat-sensitive cellular component and NADPH, is enhanced by flavin adenine dinucleotide (FAD), and is not linked to enzymatic oxidation of the fatty acid. C20:4-elicited O₂^? formation is dependent on the cooperation between a subcellular component sedimentable at 48,000g (probably containing the O₂^?-forming enzyme) and a cytosolic factor. This appears to be the first report of O₂^? generation being elicited in a cell-free system derived from unstimulated cells and supports the idea that unesterified unsaturated fatty acids act as second messengers of O₂^? formation in intact phagocytes.     

Biomass-derived volatile fatty acid platform for fuels and chemicals

The typical biorefinery platforms are sugar, thermochemical (syngas), carbon-rich chains, and biogas platforms, each offering unique advantages and disadvantages. The sugar platform uses hexose and pentose sugars extracted or converted from plant body mass. The thermochemical (syngas) platform entails a chemical or biological conversion process using pyrolysis or gasification of plants to produce biofuels. The carbon-rich chains platform is used to produce biodiesel from long-chain fatty acids or glycerides. In the present work, we suggest a new platform using volatile fatty acids (VFAs) for the production of biofuels and biochemicals production. The VFAs are short-chain fatty acids composed mainly of acetate and butyrate in the anaerobic digestion (AD) process, which does not need sterilization, additional hydrolysis enzymes (cellulose or xylanase), or a high cost pretreatment step. VFAs are easily produced from almost all kinds of biomass with low lignin content (terrestrial, aquatic, and marine biomass) by the AD process. Considering that raw material alone constitutes 40∼80% of biofuel production costs, biofuels made from VFAs derived from waste organic biomass potentially offer significant economical advantage.     

Photosynthetic terpene hydrocarbon production for fuels and chemicals

Photosynthetic hydrocarbon production bypasses the traditional biomass hydrolysis process and represents the most direct conversion of sunlight energy into the next‐generation biofuels. As a major class of biologically derived hydrocarbons with diverse structures, terpenes are also valuable in producing a variety of fungible bioproducts in addition to the advanced ‘drop‐in’ biofuels. However, it is highly challenging to achieve the efficient redirection of photosynthetic carbon and reductant into terpene biosynthesis. In this review, we discuss four major scientific and technical barriers for photosynthetic terpene production and recent advances to address these constraints. Collectively, photosynthetic terpene production needs to be optimized in a systematic fashion, in which the photosynthesis improvement, the optimization of terpene biosynthesis pathway, the improvement of key enzymes and the enhancement of sink effect through terpene storage or secretion are all important. New advances in synthetic biology also offer a suite of potential tools to design and engineer photosynthetic terpene platforms. The systemic integration of these solutions may lead to ‘disruptive’ technologies to enable biofuels and bioproducts with high efficiency, yield and infrastructure compatibility.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

Non-conventional hosts for the production of fuels and chemicals

Biotechnology offers a green alternative for the production of fuels and chemicals using microbes. Although traditional model hosts such as Escherichia coli and Saccharomyces cerevisiae have been widely studied and used, they may not be the best hosts for industrial application. In this review, we explore recent advances in the use of nonconventional hosts for the production of a variety of fuel, cosmetics, perfumes, food, and pharmaceuticals. Specifically, we highlight twenty-seven popular molecules with a special focus on recent progress and metabolic engineering strategies to enable improved production of fuels and chemicals. These examples demonstrate the promise of nonconventional host engineering.     

A novel biochemical route for fuels and chemicals production from cellulosic biomass

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.     

Metabolic engineering of yeast for production of fuels and chemicals

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Photosynthetic production of fuels and chemicals in immobilized systems

Whole cells of algae, cyanobacteria and photosynthetic bacteria entrapped in alginate gels or polyurethane foams can retain their photo-synthetic activities for months and in some cases for years. Such immobilized cells can be used in bioreactors for the production of H₂, NH₃, NADPH₂, carbohydrates, hydrocarbons, etc., with sunlight as the energy source.     

Pyrolysis of triglyceride materials for the production of renewable fuels and chemicals

Conversion of vegetable oils and animal fats composed predominantly of triglycerides using pyrolysis type reactions represents a promising option for the production of renewable fuels and chemicals. The purpose of this article was to collect and review literature on the thermo-chemical conversion of triglyceride based materials. The literature was divided and discussed as (1) direct thermal cracking and (2) combination of thermal and catalytic cracking. Typically, four main catalyst types are used including transition metal catalysts, molecular sieve type catalysts, activated alumina, and sodium carbonate. Reaction products are heavily dependant on the catalyst type and reaction conditions and can range from diesel like fractions to gasoline like fractions. Research in this area is not as advanced as bio-oil and bio-diesel research and there is opportunity for further study in the areas of reaction optimization, detailed characterization of products and properties, and scale-up.     

Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste

ABSTRACT: The considerable increase in biodiesel production worldwide in the last 5 years resulted in astoichiometric increased coproduction of crude glycerol. As an excess of crude glycerol hasbeen produced, its value on market was reduced and it is becoming a waste-stream insteadof a valuable coproduct. The development of biorefineries, i.e. production of chemicals andpower integrated with conversion processes of biomass into biofuels, has been singled out asa way to achieve economically viable production chains, valorize residues and coproducts,and reduce industrial waste disposal. In this sense, several alternatives aimed at the use ofcrude glycerol to produce fuels and chemicals by microbial fermentation have beenevaluated. This review summarizes different strategies employed to produce biofuels andchemicals (1,3-propanediol, 2,3-butanediol, ethanol, n-butanol, organic acids, polyols andothers) by microbial fermentation of glycerol. Initially, the industrial use of each chemical isbriefly presented; then we systematically summarize and discuss the different strategies toproduce each chemical, including selection and genetic engineering of producers, andoptimization of process conditions to improve yield and productivity. Finally, the impact ofthe developments obtained until now are placed in perspective and opportunities andchallenges for using crude glycerol to the development of biodiesel-based biorefineries areconsidered. In conclusion, the microbial fermentation of glycerol represents a remarkablealternative to add value to the biodiesel production chain helping the development ofbiorefineries, which will allow this biofuel to be more competitive.     

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.     

Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development

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Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals

Research towards the commercialization of fungible biofuels has received a great deal of recent interest and investment. To this end the microbial production of fatty acid-derived fuels from sustainable feedstocks is emerging as a viable option with rapid advances from both industry and academia. The manipulation of the fatty acid biosynthesis pathway, especially in Escherichia coli, has been widely studied and several approaches that increase fatty acid production have been identified. However, further advances will be required for the economic large-scale production of fatty acid-derived biofuels. Here we present an overview of fatty acid biosynthesis and its regulation in E. coli from a metabolic engineering viewpoint and offer potential approaches and considerations for the microbial overproduction of custom designed fatty acids for use as biofuels or in the manufacture of oleochemicals.     

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.