No abstract

Saccharomyces cerevisiae was engineered for assembly of minicellulosomes by heterologous expression of a recombinant scaffolding protein from Clostridium cellulovorans and a chimeric endoglucanase E from Clostridium thermocellum. The chimeric endoglucanase E fused with the dockerin domain of endoglucanase B from C. cellulovorans was assembled with the recombinant scaffolding protein. The resulting strain was able to ferment amorphous cellulose [carboxymethyl-cellulose (CMC)] into ethanol with the aid of beta-glucosidase 1 produced from Saccharomycopsis fibuligera. The minicellulosomes assembled in vivo retained the synergistic effect for cellulose hydrolysis. The minicellulosomes containing the cellulose-binding domain were purified by crystalline cellulose affinity in a single step. In the fermentation test at 10 g L(-1) initial CMC, approximately 3.45 g L(-1) ethanol was produced after 16 h. The yield (in grams of ethanol produced per substrate) was 0.34 g g(-1) from CMC. This result indicates that a one-step processing of cellulosic biomass in a consolidated bioprocessing configuration is technically feasible by recombinant yeast cells expressing functional minicellulosomes.

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.

Surfactin is a biological surfactant with numerous potential applications. In this study, Bacillus subtilis was engineered to improve surfactin production by the activation of two competence-stimulating pheromones, ComX and competence and sporulation factor (CSF) to stimulate the transcription of srfA operon. Both signaling factors, encoded by comX and phrC, were successfully overexpressed and subsequently increased surfactin production. Surfactin produced by engineered strains showed functional groups similar to the commercially available surfactin analyzed via Fourier transform infrared spectroscopy (FTIR). Surfactin production in the B. subtilis (pHT43-comXphrC) strain was 6.4-fold greater than in the wild strain, with approximately 135.1 mg/L surfactin produced after 48 h cultivation. To reduce the production costs of surfactin, synthetic wastewater was used, from which the B. subtilis (pHT43-comXphrC) strain produced approximately 140.2 mg/L surfactin. The results obtained demonstrated the production of surfactin from synthetic wastewater, which is beneficial in lowering the overall production costs.