Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels Sung Kuk Lee, Howard Chou, Timothy S Ham, Taek Soon Lee, and Jay D Keasling ABSTRACT The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels. INTRODUCTION Alternative transportation fuels are in high demand owing to concerns about climate change, the global petroleum supply, and energy security [ 1,2]. Currently, the most widely used biofuels are ethanol generated from starch (corn) or sugar cane and biodiesel produced from vegetable oil or animal fats [3 ]. However, ethanol is not an ideal fuel molecule in that it is not compatible with the existing fuel infrastructure for distribution and storage owing to its corrosivity and high hygroscopicity [1,4 ]. Also, it contains only about 70% of the energy content of gasoline. Biodiesel has similar problems (URL: http:// www.bdpedia.com/biodiesel/alt/alt.html): it cannot be transported in pipelines because its cloud and pour points are higher than those for petroleum diesel (petrodiesel), and its energy content is approximately 11% lower than that of petrodiesel. Furthermore, both ethanol and bio-diesel are currently produced from limited agricultural resources, even though there is a large, untapped resource of plant biomass (lignocellulose) that could be utilized as a renewable source of carbon-neutral, liquid fuels [ 5]. Microbial production of transportation fuels from renew-able lignocellulose has several advantages. First, the production is not reliant on agricultural resources commonly used for food, such as corn, sugar cane, soybean, and palm oil. Second, lignocellulose is the most abundant biopolymer on earth. Third, new biosynthetic pathways can be engineered to produce fossil-fuel replacements, including short- chain, branched-chain, and cyclic alcohols, alkanes, alkenes, esters and aromatics. The development of cost-effective and energy-efficient processes to convert lignocellulose into fuels is hampered by significant roadblocks, including the lack of genetic engineering tools for native producer organisms (non-model organ-isms), and difficulties in optimizing metabolic pathways and balancing the redox state in the engineered microbes [ 6]. Furthermore, production potentials are limited by the low activity of pathway enzymes and the inhibitory effect of fuels and byproducts from the upstream biomass processing steps on microorganisms responsible for producing fuels. Recent advances in synthetic biology and metabolic engineering will make it possible to overcome these hurdles and engineer microorganisms for the cost- effective production of biofuels from cellulosic biomass. In this review, we examine the range of choices available as potential biofuel candidates and production hosts, review the recent methods used to produce biofuels, and discuss how tools from the fields of metabolic engineering and synthetic biology can be applied to produce transportation fuels using genetically engineered micro-organisms. Liquid fuels and alternative biofuel molecules An understanding of what makes a good fuel is important in order to retool microorganisms to produce more useful alternative biofuels. The best fuel targets for the near term will be molecules that are already found in or similar to components of fossil-based fuel in order to be compatible with existing engines (spark ignition engine for gasoline, compression ignition engine for diesel fuel, and gas turbine for jet fuel). There are several relevant factors to consider when designing biofuel candidates ( Table 1). Energy contents, the combustion quality described by octane or cetane number, volatility, freezing point,

/pdf/metabolic-engineering-of-microorganisms-for-biofuels-1atyuc9cwo.pdf

Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSn, GC-MSn, CE-MSn), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).

/pdf/microbial-metabolomics-past-present-and-future-methodologies-284djr0b8x.pdf

Microbial metabolomics: past, present and future methodologies

Bathyarchaeota, formerly known as the Miscellaneous Crenarchaeotal Group, is a phylum of global generalists that are widespread in anoxic sediments, which host relatively high abundance archaeal communities. Until now, 25 subgroups have been identified in the Bathyarchaeota. The distinct bathyarchaeotal subgroups diverged to adapt to marine and freshwater environments. Based on the physiological and genomic evidence, acetyl-coenzyme A-centralized heterotrophic pathways of energy conservation have been proposed to function in Bathyarchaeota; these microbes are able to anaerobically utilize (i) detrital proteins, (ii) polymeric carbohydrates, (iii) fatty acids/aromatic compounds, (iv) methane (or short chain alkane) and methylated compounds, and/or (v) potentially other organic matter. Furthermore, bathyarchaeotal members have wide metabolic capabilities, including acetogenesis, methane metabolism, and dissimilatory nitrogen and sulfur reduction, and they also have potential interactions with anaerobic methane-oxidizing archaea, acetoclastic methanogens and heterotrophic bacteria. These results have not only demonstrated multiple and important ecological functions of this archaeal phylum, but also paved the way for a detailed understanding of the evolution and metabolism of archaea as such. This review summarizes the recent findings pertaining to the ecological, physiological and genomic aspects of Bathyarchaeota, highlighting the vital role of this phylum in global carbon cycling.

Bathyarchaeota: globally distributed metabolic generalists in anoxic environments.

Summary
Investigations of the biogeochemical roles of benthic Archaea in marine sediments are hampered by the scarcity of cultured representatives. In order to determine their metabolic capacity, we reconstructed the genomic content of four widespread uncultured benthic Archaea recovered from estuary sediments at 48% to 95% completeness. Four genomic bins were found to belong to different subgroups of the former Miscellaneous Crenarcheota Group (MCG) now called Bathyarchaeota: MCG-6, MCG-1, MCG-7/17 and MCG-15. Metabolic predictions based on gene content of the different genome bins indicate that subgroup 6 has the ability to hydrolyse extracellular plant-derived carbohydrates, and that all four subgroups can degrade detrital proteins. Genes encoding enzymes involved in acetate production as well as in the reductive acetyl–CoA pathway were detected in all four genomes inferring that these Archaea are organo-heterotrophic and autotrophic acetogens. Genes involved in nitrite reduction were detected in all Bathyarchaeota subgroups and indicate a potential for dissimilatory nitrite reduction to ammonium. Comparing the genome content of the different Bathyarchaeota subgroups indicated preferences for distinct types of carbohydrate substrates and implicitly, for different niches within the sedimentary environment.

Genomic evidence for distinct carbon substrate preferences and ecological niches of Bathyarchaeota in estuarine sediments

Recent determination of genome sequences for a wide range of bacteria has made in-depth knowledge of prokaryotic metabolic function essential in order to give biochemical, physiological, and ecological meaning to the genomic information. Clearly describing the important metabolic processes that occur in prokaryotes under different conditions and in different environments, this advanced text provides an overview of the key cellular processes that determine bacterial roles in the environment, biotechnology, and human health. Prokaryotic structure is described as well as the means by which nutrients are transported into cells across membranes. Glucose metabolism through glycolysis and the TCA cycle are discussed, as well as other trophic variations found in prokaryotes, including the use of organic compounds, anaerobic fermentation, anaerobic respiratory processes, and photosynthesis. The regulation of metabolism through control of gene expression and control of the activity of enzymes is also covered, as well as survival mechanisms used under starvation conditions.

/pdf/bacterial-physiology-and-metabolism-59sorzkb9j.pdf

Bacterial Physiology and Metabolism

The Escherichia coli K-12 strain TG1 was grown at 28 °C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0·044 to 0·415 h−1. The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0·3 h−1. At dilution rates between 0·3 h−1 and 0·4 h−1, a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0·4 h−1. An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0·4 h−1, close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.

/pdf/metabolic-flux-analysis-of-escherichia-coli-in-glucose-3v2i24n704.pdf

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, d- and l-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached. In the downshift experiment, the adenylate energy charge behaved inversely; no by-products were formed, indicating a tight coupling of anabolism and catabolism. Both dilution rate shifts were accompanied by an instantaneous increase of cAMP, presaging the subsequent changes in metabolic pathway utilization. Intracellular key metabolites of the Embden–Meyerhof–Parnas (EMP) pathway were measured to evaluate the metabolic perturbation during the upshift. Fructose 1,6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP) increased rapidly after the upshift, while glyceraldehyde 3-phosphate decreased. It is concluded that this imbalance at the branch-point of FDP induces the methylglyoxal (MG) pathway, a low-energy-yielding bypass of the lower EMP pathway, through the increasing level of DHAP. MG pathway activation after the upshift was simulated by restricting anabolic rates using a stoichiometry-based metabolic model. The metabolic model predicted that low-energy-yielding catabolic pathways are utilized preferentially in the transient after the upshift. Upon severe dilution rate upshifts, an oscillatory behaviour occurred, apparent through long-term oscillations of respiratory activity, which started when the cytotoxic compound MG reached a threshold concentration of 1·5 mg l−1 in the medium.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour

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Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour