9.6 METABOLIC ENGINEERING AND “OMICS”—ANALYSES OF SOLVENTOGENIC CLOSTRIDIA
9.6.1 Development and Application of Metabolic
In the past 20 years significant genetic engineering technologies were developed to overcome the genetic inaccessibility of solventogenic clostridia species. The major focus of metabolic engineering is to improve butanol tolerance and to increase the final butanol concentration (Ezeji et al., 2010). Further approaches deal with the detailed regulation of the metabolic switch between acidogenesis and solventogene- sis, for example, involved regulators, influencing enzyme activities or blocking whole cellular pathways (Papoutsakis, 2008). Here, pioneering genetic work was done by the laboratory of E.T. Papoutsakis byin vivomethylation of plasmids for overexpres- sion purposes (Mermelstein et al., 1992; Mermelstein and Papoutsakis, 1993) as well as the development of a gene expression reporter system (Tummala et al., 1999). First techniques for gene deletion were developed based on a plasmid integration tech- nology (Green et al., 1996) followed by gene knockdown techniques using antisense RNA constructs (Tummala et al., 2003a,b). The group of P. Soucaille developed a chromosomal manipulation technique for gene deletion by homologous recombina- tion (Soucaille et al., 2007) and a gene knockout technology for clostridia (TargeTron) based on a mobile group II intron system was developed and published in parallel by Heap et al. (2007, 2010) and Shao et al. (2007). Recently, an anhydrotetracycline- inducible gene expression system forC. acetobutylicumwas published and discussed
as useful tool for further strain engineering approaches (Dong et al., 2012). All these efforts markedly influenced the progress of metabolic engineering of solventogenic clostridia, especially targeting the biphasic fermentative metabolism, and are reflected in several published single as well as first double knockout mutants based on different genetic technologies (Table 9.2).
One major goal of metabolic engineering is to diminish the by-product formation of acetone to generate a homo-butanol fermentative producing strain. Therefore, sev- eral groups deleted the respective geneadc, whose product converts acetoacetate to acetone (Jiang et al., 2009; Han et al., 2011; Lehmann et al., 2012a). Interestingly, all adcknockout mutant strains are still able to produce acetone although in significant different amounts (0.34–8.0 g/L). This phenomenon can be explained by a nonenzy- matic decarboxylation of acetoacetate to acetone and CO2. The first acetone-negative phenotype of C. acetobutylicum was published by Lehmann et al. (2012a). Here, the authors generated a acetoacetyl-CoA:acyl-CoA transferase subunit A (ctfA::int) mutant and resulted an acetone-negative phenotype concomitant with an accumula- tion of acetate. In general, the whole enzyme complex CtfA/B is responsible for the reassimilation of the acids acetate and butyrate. Thus, thectfA::int mutant is unable to convert acetate to acetyl-CoA and acetoacetate, the precursor of acetone. However, the ctfA::int mutant is still able to reassimilate butyrate with concomitant butanol synthe- sis, although thectfA::int mutant reaches only the half amount of butanol compared to the wild-type strain. This phenotype suggests thatC. acetobutylicumis able to convert butyrate to butanol independently of CtfAB. Furthermore, the authors suggest that Buk and Ptb convert butyrate to butanol during their reverse reactions (Lehmann et al., 2012a). Another approach to succeed in decreasing the acetone amount is the enzy- matic conversion of acetone to higher-value alcohols. For the first time two research groups documented an engineered isopropanol–butanol–ethanol (IBE)-producing C. acetobutylicum strain without further acetone accumulation. Here, C. aceto- butylicumwas engineered by overexpressing a NADPH-dependent secondary alcohol dehydrogenase ofC. beijerinckiiNRRL B 593 to convert acetone in a further reaction to isopropanol (Dai et al., 2012; Lee et al., 2012).
Further progress was achieved by targeting the acid-formation pathways to exam- ine the detailed role of the respective acids acetate and butyrate for solvent production.
So far, different mutants were investigated that were affected in the acetate pathway (pta or ack) (Green et al., 1996; Zhao et al., 2005; Kuit et al., 2012; Lehmann et al., 2012a) or butyrate pathway (ptb or buk) (Green et al., 1996; Zhao et al., 2005; Lehmann et al., 2012b). Furthermore, first double knockout mutants targeting acetate production and reassimilation (pta::ctfA) or acetate and acetone production (pta::adc) were published (Lehmann et al., 2012a). An overview is given in Table 9.2.
Interestingly, further research elucidated thatC. acetobutylicumis able to grow inde- pendently of the entire butyrate/butanol (C4) metabolic pathway. Here, the authors were able to disrupt the gene of theβ-hydroxybutyryl-CoA dehydrogenase (hbd) and switched the solventogenic organism to a significant ethanol producer (Lehmann and L¨utke-Eversloh, 2011). However, further efforts have to focus on the genetic con- trol of the metabolic switch between acidogenesis and solventogenesis, especially to
TABLE9.2SelectedSingleorDoubleKOMutantsofClostridiumacetobutylicum(C.ac.)orClostridiumbeijerinckii(C.bei.)Targeting BiphasicFermentativeMetabolismandDocumentedFermentationProductsUnderCertainBatchGrowthConditions ParentalstrainAffectedgeneaAffectedpathway(s)Acetone(g/L)Butanol(g/L)Ethanol(g/L)Reference C.ac.WURackAcetyl-phosphate→Acetate5.711.61.6Kuitetal.(2012) C.ac.ATCC824ptaAcetyl-CoA→Acetyl-phosphate3.58.70.6Greenetal.(1996) C.ac.ATCC824ptaAcetyl-CoA→Acetyl-phosphate1.23.3n.d.Zhaoetal.(2005) C.ac.ATCC824ptaAcetyl-CoA→Acetyl-phosphate2.911.81.2Lehmannetal.(2012a) C.ac.ATCC824bukButyryl-CoA→Butyryl-phosphate1.910.50.7Greenetal.(1996) C.ac.ATCC824bukButyryl-CoA→Butyryl-phosphate2.99.3n.d.Zhaoetal.(2005) C.ac.ATCC824ptbButyryl-CoA→Butyryl-phosphate0.1/4.2b3.4/7.8b0.3/32.4bLehmannetal.(2012b) C.ac.ATCC824hbdAcetoacetyl-CoA→ 3-Hydroxybutyryl-CoA1.6/2.5b0.016.2/33.1bLehmannand L¨utke-Eversloh (2011) C.ac.ATCC824adcAcetoacetate→Acetone0.55.50.8Lehmannetal.(2012a) C.ac.EA2018adcAcetoacetate→Acetone0.3412.23.86Jiangetal.(2009) C.bei.NCIMB8052adcAcetoacetate→Acetone8.012.02.0Hanetal.(2011) C.ac.ATCC824ctfAAcetate→Acetyl-CoA Butyrate→Butyryl-CoA0.07.41.0Lehmannetal.(2012a) C.ac.ATCC824pta1::adc21Acetyl-CoA→Acetyl-phosphate 2Acetoacetate→Acetone0.13.00.4Lehmannetal.(2012a) C.ac.ATCC824pta1::ctfA21Acetyl-CoA→Acetyl-phosphate 2Acetate→Acetyl-CoA 2Butyrate→Butyryl-CoA
0.00.70.3Lehmannetal.(2012a) n.d.,novaluesdocumented. aack,acetatekinase;pta,phosphotransacetylase;buk,butyratekinase;ptb,phosphotransbutyrylase;hbd,β-hydroxybutyryl-CoAdehydrogenase;adc,acetoacetate decarboxylase;ctfA,acetoacetyl-CoA:acyl-CoAtransferasesubunitA. bSecondvaluebasedonglucosefed-batchfermentation.
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unravel interplay between initiation of solventogenesis with different cellular mech- anisms of granulose formation, quorum sensing, and/or spore formation (Paredes et al., 2005; Jones et al., 2011; Steiner et al., 2011; Tracy et al., 2011; Steiner et al., 2012).