The genomes of several solventogenic Clostridia, including gas fermenting species, have been sequenced since 2001 [54, 62, 109, 119, 123, 130], and an array of transcriptomic [100, 116, 121, 131, 132], proteomic [132] and systems analysis [133, 134] are being made increas­ingly available. However, the generation of stable recombinant Clostridia has been severely hindered by the difficulties encountered introducing foreign DNA into cells and a lack of established genetic tools for this genera of bacteria. In comparison to starch-utilizing Clostri­dia, very little information is available for metabolic engineering of acetogens. Although this section describes recent advances in the development of genetic tools for mostly sugar-uti­lizing Clostridia, these techniques are highly relevant and applicable to the closely related acetogenic Clostridia for biofuels or chemical production via gas fermentation.

The ideal microbial catalyst for industrial scale gas fermentation might exhibit the following traits: high product yield and selectivity, low product inhibition, no strain degeneration, as — porogenous, prolonged cell viability, strong aero-tolerance, high biomass density and effi­cient utilization of gas substrates. These can be achieved by directed evolution, random mutagenesis and/or targeted genetic engineering. Traditionally, chemical mutagenesis [135—137] and adaption strategies [138, 139] have been deployed to select for these traits. However, these strategies are limited and often come with the expense of unwanted events.

First attempts of targeted genetic modification of Clostridia were made in the early 1990s by the laboratory of Prof. Terry Papoutsakis [140—142]. While these pioneering efforts relied on use of plasmids for (over)expression of genes in C. acetobutylicum, more sophisticated tools were later developed for a range of solventogenic and pathogenic Clostridia.

Antisense RNA (asRNA) has been employed to down-regulate genes. Here, single stranded RNA binds to a complementary target mRNA and prevents translation by hindering ribo­some-binding site interactions [143]. For instance, this method has been used to knockdown ctfB resulting in production of 30 g/l solvents with significantly suppressed acetone yield in C. acetobutylicum ATCC 824 [144, 145].

Several homologous recombination methods have been developed for integration or knock-out of genes in a range of sugar-utilizing Clostridia. In early stage, knockout mu­tants were almost exclusively generated from single crossover events that could revert back to wild-type [146—152], with stable double crossovers only observed in rare cases [153, 154]. For C. acetobutylicum [155] and cellulolytic C. thermocellum [156] counter selecta­ble markers have been developed to allow more efficient screening for the rare second re­combination event.

ClosTron utilizes the specificity of mobile group II intron Ll. ltrB from Lactoccocus lactis to propagate into a specified site in the genome via a RNA-mediated, retro-homing mecha­nism which can be used to disrupt genes [157]. This technique has initially been devel­oped by InGex and Sigma-Aldrich under the name ‘TargeTron™’ and successfully adapted to a range of solventogenic and pathogenic Clostridia including C. acetobutyli­cum, C. difficile, C. sporogenes, C. perfringens, and C. botulinum [158—160] by the laboratory of Prof. Nigel Minton.

The same laboratory recently also developed another method for integration of DNA into the genome. Termed Allele-Coupled Exchange (ACE), this approach does not employ a counter selective marker to select for the rare second recombination event. Rather, it utilizes the activation or inactivation of gene(s) that result in a selectable phenotype, and asymmetri­cal homology arms to direct the order of recombination events [161]. Remarkably, the whole genome of phage lambda (48.5kb minus a 6kb region) was successfully inserted into the ge­nome of C. acetobutylicum ATCC 824 in three successive steps using this genetic tool. This technique was also demonstrated in C. difficile and C. sporogenes [161].

For reverse engineering, mainly transposon mutagenesis has been utilized. Earlier efforts of transposon mutagenesis were demonstrated in C. acetobutylicum P262 (now: C. saccharobuty — licum [162]), C. acetobutylicum DSM792, C. acetobutylicum DSM1732, and C. beijerinckii NCIM 8052, but issues with multiple transposon insertions per mutant, and non-random distribu­tion of insertion were reported [163, 164]. Recent developments have seen the successful generation of mono-copy random insertion of transposon Tn1545 into cellulolytic C. cellulo — lyticum [165] and mariner transposon Himarl into pathogenic C. difficile [166].

While there is still a lack of some other essential metabolic engineering tools such as efficient inducible promoters, the array of available tools that enabled significant improvements to the ABE process and cellulolytic Clostridia fermentations as summarized in Table 3.

In contrast, to date only a limited number of acetogenic Clostridia have been successfully modified. Pioneering work in this area has been undertaken in the laboratory of Prof. Peter Durre. C. Ijungdahlii, a species that does not naturally produce butanol, was modified with butanol biosynthetic genes (thlA, hbd, crt, bcd, adhE and bdhA) from C. acetobutylicum ATCC 824 resulting in production of up to 2 mM of butanol using synthesis gas as sole energy and carbon source [62]. By delivering a plasmid with acetone biosynthesis genes ctfA, ctfB, adc, and thlA in C. aceticum, production of up to 140 |oM acetone was demonstrated from various gas mixes (80% H/20% CO2 and 67% H2/33% CO2) [169, 170]. Recent patent filings by Lanza — Tech describe the production of butanol as main fermentation product and increased alcohol tolerance in genetically engineered acetogens. Up to 26 mM butanol were produced with ge­netically modified C. ljungdahlii and C. autoethanogenum using steel mill gas (composition 44% CO, 32% N2, 22% CO2, and 2% H2) as the only source of carbon and energy when the butanol biosynthetic genes thlA, hbd, crt, bcd, etfA, and etfB were heterologously expressed [167]. Overexpression of native groESL operon in C. autoethanogenum resulted in a strain that displayed higher alcohol tolerance relative to wild-type when challenged with ethanol [168].

Besides the classical Clostridial butanol pathway (which constitutes genes thlA, crt, hbd, bcd, etfA and etfB; see earlier section), a non-fermentative approach has been described and dem­onstrated in E. coli for branched chain higher alcohol production [182]. This alternative ap­proach requires a combination of highly active amino acid biosynthetic pathway and artificial diversion of 2-keto acid intermediates into alcohols by introduction of two addi­tional genes: broad substrate range 2-keto-acid decarboxylase (kdc) which converts 2-keto acids into aldehydes, followed by Adh to form alcohols [182]. Engineered strains of E. coli have been shown to produce alcohols such as isobutanol, n-butanol, 2-methyl-1-butanol, 3- methyl-1-butanol and 2-phenylethanol via this strategy [182]. For instance, the overexpres­sion of kivD (KDC from Lactococcus lactis), adh2, ilvA, and leuABCD operon, coupled with deletion of ilvD gene and supplementation of L-threonine, increased n-butanol yield to 9 mM while producing 10 mM of 1-propanol [182].An even more remarkable yield of 300 mM isobutanol was achieved through introduction of kivD, adh2, alsS (from B. subtilis), and ilvCD into E. coli [182]. Like butanol, isobutanol exhibits superior properties as a transporta­tion fuel when compared to ethanol [177]. By applying similar strategy into C. cellulolyticum, 8.9 mM isobutanol was produced from cellulose when kivD, yqhD, alsS, ilvC, and ilvD were overexpressed [177]. This result suggests that such non-fermentative pathway is suitable tar­get for metabolic engineering of acetogens for the biosynthesis of branched chain higher al­cohols. Via synthetic biology and metabolic engineering, production of additional potential liquid transportation fuels like farnesese or fatty acid based fuels has successfully been dem­onstrated in E. coli or yeast from sugar [183, 184]. Given the unsolved energetics in aceto — gens, it is unclear if production of such energy dense liquid fuels could be viable via gas fermentation.