Genetic sensor-regulators functional in Clostridia.

This study addressed the functionality of genetic circuits carrying natural regulatory elements of Clostridium acetobutylicum ATCC 824 in the presence of the respective inducer molecules. Specifically, promoters and their regulators involved in diverse carbon source utilization were characterized using mCherryOpt or beta-galactosidase as a reporter. Consequently, most of the genetic circuits tested in this study were functional in Clostridium acetobutylicum ATCC 824 in the presence of an inducer, leading to the expression of reporter proteins. These genetic sensor-regulators were found to be transferable to another Clostridium species, such as Clostridium beijerinckii NCIMB 8052. The gradual expression of reporter protein was observed as a function of the carbohydrates of interest. A xylose-inducible promoter allows a titratable and robust expression of a reporter protein with stringency and efficacy. This xylose-inducible circuit was seen to enable induction of the expression of reporter proteins in the presence of actual sugar mixtures incorporated in woody hydrolysate wherein glucose and xylose are present as predominant carbon sources.

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol.

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl-acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.

Simultaneous utilization of glucose and mannose from woody hydrolysate for free fatty acid production by metabolically engineered Escherichia coli.

In this study, the Escherichia coli strain MG1655 with fadD mutant (named as ML103), and MG1655 with fadD and ptsG double mutant (named as ML190) carrying the plasmid with the acyl-ACP thioesterase (TE) from Ricinus communis (pXZ18) or the plasmid with the combination of the TE and the native (3R)-hydroxyacyl-ACP dehydrase (fabZ) (pXZ18Z), produced free fatty acids (FFAs) efficiently using mannose as the sole carbon source. Due to the carbon catabolite repression (CCR) regulation, ML103(pXZ18) utilized glucose and mannose sequentially in the mixed sugar culture, while ML190(pXZ18) and ML190(pXZ18Z), with ptsG mutation, used glucose and mannose simultaneously. The highest total FFA concentration from the mixed sugar culture reached 2.96g/L by ML190(pXZ18Z). Furthermore, the strain ML190(pXZ18Z) can produce 2.86g/L FFAs with a high yield of 0.23g/g using hydrolysate mainly contained glucose and mannose from a commercial plant.

Efficient free fatty acid production from woody biomass hydrolysate using metabolically engineered Escherichia coli.

Four engineered Escherichia coli strains, ML103(pXZ18), ML103(pXZ18Z), ML190(pXZ18) and ML190(pXZ18Z), were constructed to investigate free fatty acid production using hydrolysate as carbon source. These strains exhibited efficient fatty acid production when xylose was used as the sole carbon source. For mixed sugars, ML103 based strains utilized glucose and xylose sequentially under the carbon catabolite repression (CCR) regulation, while ML190 based strains, with ptsG mutation, used glucose and xylose simultaneously. The total free fatty acid concentration and yield of the strain ML190(pXZ18Z) based on the mixed sugar reached 3.64 g/L and 24.88%, respectively. Furthermore, when hydrolysate from a commercial plant was used as the carbon source, the strain ML190(pXZ18Z) can produce 3.79 g/L FFAs with a high yield of 21.42%.