Minimal cells comprise only the genes and biomolecular machineries required for basic life. During the past decade, over 100 genomes of archaea that form the third domain of life (Brochier-Armanet et al., 2011) have been sequenced. In fact, their genome data provide insights into the evolution of key central metabolisms, which are directly correlated with the minimal functionality for cell viability and adaptation under extreme environments. Intriguingly, the euryarchaea Picrophilus torridus, which thrives optimally at 60°C and pH 0.7, has 1.55 Mb genome (1535 ORFs), the smallest genome among non-parasitic aerobic microorganisms (Futterer et al., 2004). In particular, an exceptionally high ratio (5.6:1) of secondary over ATP-consuming primary transport systems represents a highly relevant strategy for the adaptation of this organism to its extremely acidic environment. Although Picrophilus torridus has several distinct gene traits in energy metabolism at low pH, not only all genes required for the Embden–Meyerhof–Parnas (EMP) pathway but also a complete set of genes for the oxidative tricarboxylic acid (TCA) cycle are present. Currently, there is a variety of genome data available for other archaea and bacteria, such as Thermoplasma acidophilum (1.56 Mb; Ruepp et al., 2000), Archaeoglobus profundus (1.56 Mb; von Jan et al., 2010), Prochlorococcus marinus (1.75 Mb; Dewall and Cheng, 2011), and Methanocaldococcus jannaschii (1.74 Mb; Bult et al., 1996), of which genome size is less than 2 Mb. Thus, these microorganisms seem to be “closer” to the minimal genome for life, which will also provide the fundamental basis of minimal gene sets for the construction of a genome-minimized platform cell (Figure 1).If this is the case, can we design and construct an engineered platform cell to perform our wanted tasks such as biofuel production? If so, then can we selectively choose which genes are friends or foes for the high productivity between cell mass and biofuels? Prior to considering this issue, we need to categorize essential sets of genes for cellular viability even with imperfect accuracy.

Genes involved in informational processes (not only DNA replication, but also transcription and translation represented by rRNA, tRNA, or structural RNA genes) plus protein folding and processing with strain-dependent metabolisms, are essential. In addition, a complex array of functional systems, including those for membrane transport, energy conversion, the synthesis of vitamins, and nucleic acid precursors, is indispensable for maintaining cellular integrity. Notable examples are the marine cyanobacteria Prochlorococcus, which is the smallest known oxygen-evolving photoautotroph (Chisholm et al., 1988). Although the high light-adapted ecotype MED4 strain has a genome of 1.66 Mb that codes for 1717 proteins, its low light-adapted counterpart contains a larger genome of 2.4 Mb (2275 genes; Rocap et al., 2003). Remarkably, the comparative analysis with their genomes revealed that only 1350 genes are common, whereas the remaining genetic loci are quite different, implying that the variable genes appear to be a consequence of a selective process favoring the bacterial adaptation to their environments (Dufresne et al., 2003, 2005; Rocap et al., 2003). Indeed, the photoheterotrophic marine bacterium Pelagibacter ubique has the smallest genome (1.3 Mb) of any cell known for a free-living microorganism (Giovannoni et al., 2005). Its genome coding for 1354 ORF shows the nearly complete absence of non-functional or redundant DNA, with very short intergenic regions, and the lack of pseudogenes and phage genes, reflecting an adaptive strategy that resembles the highly successful marine unicellular cyanobacteria in its simple metabolism and small genome size.

Synthesizing minimal and minimized cells will improve understanding of core biology, accelerate development of biotechnology strains of bacteria, and enable evolutionary optimization of natural and unnatural biopolymers (Jewett and Forster, 2010). Genome reduction is of particular importance to identify non-essential genes for understanding of not only how many genes are essential for cellular viability, but also which genes are necessary for cellular beneficial properties. Reduction and engineering of microbial genome is the fundamental basis of design and development of synthetic minimal platform cells for estimation of the minimal gene set required to sustain growth of microorganisms (Fleischmann et al., 1995; Fraser et al., 1995; Mushegian and Koonin, 1996). By use of comparative genomics, non-essential genes have been sought to reconstruct ancestral life forms (Mushegian and Koonin, 1996; Koonin, 2003) to define, by transposon-mediated disruption study, with Mycoplasma genitalium (Glass et al., 2006), and to validate and compare the minimal gene sets in Bacillus subtilis (Kobayashi et al., 2003; Ara et al., 2007), E. coli (Gerdes et al., 2003; Joyce et al., 2006), Aquifex aeolicus (Deckert et al., 1998), Streptococcus sanguinis (Xu et al., 2011), and Yeast (Kellis et al., 2003, 2004). Although symbiotic archeal organisms apparently have a much smaller genome (Huber et al., 2002; McCutcheon and Moran, 2012), these are out of scope with respect to the minimal gene-set that is necessary and sufficient to support cellular life. Notably, toxin–antitoxin (TA) loci functioning as stress-response elements are not generally essential to basic life (Pandey and Gerdes, 2005). Yet, combined mutations in two or more genes lead to cell death, whereas a single mutation in only one of those genes does not (Tucker and Fields, 2003). Thus, synthetic lethality should be considered when non-essential genes are deleted for genome minimization (Stieber et al., 2008).

Intriguingly, Posfai et al. (2006) successfully reduced the E. coli K-12 genome (up to 15%) to generate multiple-deletion series of strains without physiological compromise. In addition, genome reduction would provide unexpected benefits, such as high electroporation efficiency and accurate propagation of recombinant plasmids. Several laboratory evolution studies have also been carried out to characterize the genetic traits of adaptation to environmental stresses (e.g., thermal adaptation, salt stress tolerance, utilization of unusual substrates, and susceptibility to antibiotics, etc.; Herring et al., 2006; Dhar et al., 2011; Tran et al., 2011). Remarkably, reduction of the metabolic pathways by selective sorting and deletion of aerobic/anaerobic reactions based on the biomass and biofuel productions enabled cells to have theoretically maximal yields of ethanol even with minimized metabolic functionality under anaerobic conditions (Trinh et al., 2006, 2008). To convert the biomass-derived hexoses and pentoses to ethanol at high yields and productivities, an efficient and robust microorganism has been designed and developed by the removal of seemingly unnecessary pathways for the purpose (Trinh et al., 2006). To construct a minimal E. coli cell that is dedicated to producing ethanol, the functional space of the central metabolic network was reduced with eight gene knockout mutations (e.g., poxB, pta etc.) from over 15,000 pathway possibilities to 6 pathway options that support cell function (Trinh et al., 2008). The remaining pathways consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Remarkably, catabolite repression was completely absent during anaerobic growth by the deletion of acetate-producing pathways, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production in the most efficient way. Thus, this study demonstrated that the ethanol yields of engineered strains with minimized metabolic functionality closely matched the theoretical predictions, implying that reduction of non-essential genes could be quite beneficial with respect to economical production yields in synthetic microorganisms.

To date, a wealth of gene disruption and shuttle vector systems has been developed for E. coli as a model system. Simple inactivation of chromosomal genes by the PCR-mediated gene replacement (Datsenko and Wanner, 2000) has greatly facilitated the generation of specific mutants in the functional analysis of the microbial genome. It has been noted that disseminated throughout the genome are mobile DNA elements, which mediate recombination events, such as transposition and horizontal gene transfer, including insertion sequence (IS) elements, transposases, defective phages, integrases, and site-specific recombinases (Frost et al., 2005). To stabilize the genome and streamline metabolism, these elements must be deleted and unwanted functions removed. These unwanted functions include those specific for human hosts or particular environments. Indeed, a Tn5-targeted Cre/IoxP excision system (Yu et al., 2002) and a high-throughput method for the systematic mutagenesis of the genome by Tn5 transposon (Kang et al., 2004) enabled us to create individual E. coli deletion and insertion mutants without loss of normal growths. In particular, a multiplex automated genome engineering (MAGE) technique as a powerful high-throughput genome engineering tool has been developed (Wang et al., 2009a). MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells using allelic replacement to produce combinatorial genomic diversity through an oligonucleotide-directed recombineering technique. In addition, precise manipulation of chromosomes in vivo enables genome-wide codon replacement as well (Isaacs et al., 2011). Further, as described above genome reductions may improve metabolic efficiency and decrease the redundancy among microbial genes and regulatory circuits (Posfai et al., 2006). Therefore, a rational design allows us to attempt to delete genes more extensively while avoiding loss of robustness, which can allow a chassis cell to be incorporated with biofuel-producing synthetic and/or engineered pathways.

The attractive strategy described above immediately tempts us to ask questions like “Can we selectively delete the alternative energy transduction pathways as an engineering target?” and “Are minimal sets of genes beneficial in terms of energy efficiency?” This might be true simply because extreme environments already show a variety of extremophiles that can grow optimally with relatively small genome sizes (less than 2 Mb), as discussed above. In fact, their energy metabolisms appear to be designed and adapted to survive under their own specific environments in the most minimal but efficient way. This rationale seems to be supported by several challenging experiments about the effect of the modulation of respiratory chains on fermentation products (Portnoy et al., 2008, 2010). In these studies, a series of aerobic respiratory chains in E. coli were progressively knocked out and aerobically adapted to generate an evolved mutant deficient in three terminal oxidases. Initially, this mutant could not grow on M9 minimal medium containing D-glucose. However, 60-day adaptive evolution on the same medium created E. coli mutants that exhibited the ability to undergo mixed-acid fermentation during aerobic growth and to produce lactate as a fermentation product from D-glucose. Moreover, the removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) yielded an E. coli mutant that exhibits anaerobic physiology even in the presence of oxygen, through the activation of the anoxic redox control transcriptional regulator ArcA (Portnoy et al., 2010). The knockout strain exhibited nearly identical physiological behaviors and produced D-lactate as the sole by-product under oxic and anoxic conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations.

Genome-scale transcriptome analysis, ¹³C tracing experiments, and physiological characterization demonstrated that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool (Portnoy et al., 2010). This result suggests that composition of quinone pool may be tightly coordinated with the activation of ArcB/ArcA regulatory system, concomitantly linked to a major shift in the metabolic flux distribution through the central metabolism in cells (Alexeeva et al., 2000). Therefore, respiratory control and/or modulation could be an efficient engineering strategy for changing the central metabolic flux of cells for the high yields of biofuel production (Fischer et al., 2008; Portnoy et al., 2010). This strategy can be validated by a systems-level characterization of fermentative profiles, using single gene knockouts in E. coli, which are related to redox reactions (Lee and Lee, unpublished data). The data demonstrated that single gene deletion mutations in guaB, pyrD, and serA increased D-lactic acid production. Combined knockouts of guaB, pyrD, serA, fnr, arcA, or arcB further enhanced D-lactate production.

Very recently, the hyperthermophilic archaeon Pyrococcus furiosus with the small genome size of 1.91 Mb, which grows optimally at 100°C, can be engineered to produce important organic chemicals from CO₂ by use of low potential reducing power from H₂ (Keller et al., 2013). For this, five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C were heterologously expressed in Pyrococcus furiosus. The engineered Pyrococcus furiosus strain is able to use H₂ and incorporate CO₂ into 3-hydroxypropionic acid, a chemical building block for the production of acrylic acid, acrylamide, and 1,3-propanediol. Remarkably, this is operated at temperatures that are suboptimal for its growth to minimize the metabolic burden of the engineered microorganisms during chemical production from H₂ and CO₂. Such a strategic operation support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The unique temperature-dependent approach (Basen et al., 2012) that confers on a microorganism the capacity to use CO₂, a reaction that it does not occur naturally circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates.

In contrast to starches and simple sugars derived from sugar cane and corn, lignocellulosic crops are regarded as sustainable and renewable. However, they also have some technical hurdles due to recalcitrance of cellulosic biomass rich in lignin, resistance to enzymatic hydrolysis and the presence of five carbon sugars (Demain et al., 2005). In this respect, consolidated bioprocessing is a promising strategy to overcome biomass recalcitrance by using cellulolytic microorganisms. One of the most closely studied of the cellulolytic microbes, Clostridium thermocellum (Wiegel et al., 1985), is being used for the production of ethanol through the consolidated bioprocessing of plant biomass (Bayer et al., 2008; Lynd et al., 2008). Recently, several genome sequences of thermophilic, plant biomass-degrading members of this genus (Blumer-Schuette et al., 2011) indicate that significant differences in glycoside hydrolase inventories and numbers of carbohydrate transporters exist, which likely relates to variability observed in plant biomass degradation capacity (Dam et al., 2011). In addition, the proteomic analysis of C. phytofermentans, which contains 161 carbohydrate-active enzymes, has been performed to identify hydrolases and metabolic enzymes to engineer microorganisms for improved cellulosic bioconversion (Tolonen et al., 2011). Intriguingly, it has been found that increase in tryptophan and nicotinamide synthesis was entailed with cellulosic fermentation for the production of ethanol, providing novel genetic targets for more efficient conversion of biomass to fuels and biomaterials (Tolonen et al., 2011). Alternatively, degradation of cellulosic biomass by extremely thermophilic bacteria Caldicellulosiruptor strains could have high potential for the production of biofuels (Blumer-Schuette et al., 2011).

Moreover, several attempts to tackle these practical issues to promote biomass waste solutions and biofuel production have been made through metabolic engineering and synthetic biology (Himmel et al., 2007; Vazana et al., 2012). To efficiently degrade crystalline cellulose, artificial enzymatic cellulosome complexes were designed and examined. There are an increasing number of research papers describing the production of designer cellulosomes either in vitro, ex vivo, or in vivo (Vazana et al., 2012). In designer cellulosomes, each enzyme is equipped with a dockerin module that interacts specifically with one of the cohesin modules of the chimeric scaffoldin. Artificial scaffoldins serve as docking backbones and contain a cellulose-specific carbohydrate-binding module that directs the enzymatic complex to the cellulosic substrate, as well as one or more cohesin modules from different natural cellulosomal species, each exhibiting a different specificity that allows the specific incorporation of the desired matching dockerin-bearing enzymes (Demain et al., 2005).

Another interesting approach in algae is that carbohydrates as a primary store of photosynthates can be exploited for biofuels through conversion to alcohols (Santelia and Zeeman, 2011). Algal polysaccharides can be hydrolyzed and then either fermented to ethanol by yeast or used as a heterotrophic carbon source for the production of a variety of biofuels (Harun et al., 2010). These carbohydrate productions in algae could be advantageous for biofuel production through overexpression of key enzymes in starch biosynthesis and secretion of soluble carbohydrates (Work et al., 2010).

To overcome the challenges (e.g., limited supply and land yield, inconsistent performance, and challenging economics) for production of biodiesels derived from plant oils, microbial fatty acid esters production could be an alternative way (Reijnders, 2008; Work et al., 2010; Li et al., 2012). In contrast to chemical refining to obtain designed biodiesels, microbial fatty acid esters can be easily altered in their composition (Mayer and Shanklin, 2007) and degree of saturation through manipulation of key regulators of the fatty acid biosynthesis (Peralta-Yahya et al., 2012) and introduction of foreign genes encoding wax synthase/diacylglycerol acyltransferase from other microbes (Stoveken and Steinbuchel, 2008). In addition, when Arabidopsis fatty acyl-CoA reductases (Rowland et al., 2006), that catalyze the formation of a fatty alcohol from an acyl-CoA, were expressed in E. coli, more favorable short fatty alcohols were synthesized (Doan et al., 2009), indicating that more efficient fatty alcohol-producing enzymes might be paramount for the commercial production of fatty alcohols.

To improve the efficiency of electrical energy in storage, Li et al. (2012) reported a method to store electrical energy as chemical energy in higher alcohols, which used synthetic biology approach for converting electricity and CO₂ to liquid biofuels. They genetically engineered a lithoautotrophic microorganism, Ralstonia eutropha H16, to produce isobutanol and 3-methyl-1-butanol in an electro-bioreactor using CO₂ as the sole carbon source and electricity as the sole energy input. This recent breakthrough technology integrated with electrochemical formate production and biological CO₂ fixation and higher alcohol synthesis, opens the possibility of electricity-driven bioconversion of CO₂ to commercial chemicals.

As an ideal replacement for diesel fuel, fatty acid-derived hydrocarbons such as alkanes are produced directly from fatty acid metabolites in numerous organisms. Although two biochemical routes (decarbonylation vs. reduction of fatty alcohols) are available (Dennis and Kolattukudy, 1992; Wackett et al., 2007), their biosynthetic pathways to alkanes are not fully understood. Recently, Schirmer et al. (2010) performed comparative genomics and subtractive genome analysis with more than 50 genome sequences of cyanobacteria to find out two hypothetical enzymes and propose these as acyl-ACP reductase and aldehyde decarbonylase, responsible for alkane biosynthesis. Subsequently, their co-expression in E. coli enabled in vivo alkane biosynthesis (C13 to C17 mixtures), which is a major step toward the goal of low-cost renewable transportation fuels.

In contrast to fermentative alcohols produced from carbohydrates and lipids (Figure 2) like ethanol and butanol, the non-fermentative short-chain alcohols can be generated with protein sources by introducing exogenous transamination and deamination cycles (Huo et al., 2011). In order to develop a process for conversion of mixtures of peptides and amino acids to biofuels or chemicals, carbon skeletons could be provided from 2-keto acids through deamination of amino acids by 2-keto acid decarboxylases, and then to alcohols by alcohol dehydrogenases as shown in Figure 2 (Atsumi et al., 2008). Alternatively, other amino acids could be deaminated to TCA cycle intermediates, which can be directed to pyruvate by malic enzymes or phosphoenolpyruvate carboxykinase. Pyruvate can be further extended to longer keto acids by acetohydroxy acid synthase or isopropylmalate synthase chain elongation pathways (Zhang et al., 2008). Such implementation of synthetic pathways together with rewiring metabolisms consequently enabled us to develop a protein-based process for biorefining and fuel production in potential platform cells such as E. coli, B. subtilis, yeast, and microalgae. Indeed, several E. coli variants that were improved for amino acid utilization were screened, and then isobutanol synthesis pathways were introduced into the cells to yield a strain that grew despite the stress generated by increased fuel production (Huo et al., 2011). Further, deletion of ammonium-assimilation genes, gdhA and glnA, increased the production of alcohols in the presence of the keto acid pathway. Thus, in contrast to previous metabolic engineering of carbon metabolic modules, application of nitrogen metabolic modules can be a significantly considerable alternative strategy in biofuel metabolic engineering.

Biological hydrogen (H₂) is a potentially favorable, renewable, and ideal fuel for future demand in terms of climate change and sustainability. At present, biological H₂ has been produced through three major processes: biophotolysis, photofermentation, and dark fermentation (Oh et al., 2011). In green algae and cyanobacteria, photosynthesis coupled to H₂ evolution requires only water and sunlight (Prince and Kheshgi, 2005). Several cyanobacteria utilize an indirect pathway wherein storage carbohydrates generated through photosynthesis are fermentatively used to produce H₂. Significant improvements for H₂ production yield could be achieved by truncation of light-harvesting complexes and reduction of chlorophyll content in green algae and cyanobacteria (Beckmann et al., 2009; Mitra and Melis, 2010; Srirangan et al., 2011). Impaired cyclic electron transport also results in increased electron flow to the hydrogenase (Kruse et al., 2005a). Further findings of alternative anaerobic pathways (Hemschemeier and Happe, 2011), auxiliary electron transport (Peltier et al., 2010), and other distinct anaerobic H₂ metabolisms (Meuser et al., 2009) could be potential targets to improve H₂ production. Most hydrogenases are extremely oxygen-sensitive, which is one of the greatest challenges facing the establishment of an industrial biophotolysis process. For this reason, either control of O₂ levels (Surzycki et al., 2007) or use of protein engineering should be implemented to create enzymes with greater O₂ tolerance (Fritsch et al., 2011; Goris et al., 2011; Liebgott et al., 2011).

Alternatively, strictly anaerobic extremophiles, such as methanogenic archaea and hyperthermophilic bacteria, could be an attractive option for the production of biological H₂ (Kim et al., 2010). H₂ provides an excellent source of low potential reducing power for growth and biosynthesis of archaea (Sun et al., 2010). Recently, the heterologous expression in E. coli of a functional [NiFe]-hydrogenase from a hyperthermophilic archaea, Pyrococcus furiosus, was performed by employing a novel set of compatible vectors modified with an anaerobically induced E. coli hydrogenase promoter (Hopkins et al., 2011). Together with successful engineering of hydrogenase and nitrogenase (O₂ sensitivity and higher molar production; Stripp et al., 2009; Yamamoto et al., 2009; Liebgott et al., 2010, 2011; Fritsch et al., 2011), development of versatile genetic systems and improvements in such oxygen-sensitive and intricate maturation requiring enzymes enable us to design and exploit the use of novel microorganisms and their constituent hydrogenases for biohydrogen production.