Tolerance and transcriptional analysis of Corynebacterium glutamicum on biotransformation of toxic furaldehyde and benzaldehyde inhibitory compounds.

Furaldehydes and benzaldehydes are among the most toxic inhibitors from lignocellulose pretreatment on microbial growth and metabolism. The bioconversion of aldehyde inhibitors into less toxic alcohols or acids (biotransformation) is the prerequisite condition for efficient biorefinery fermentations. This study found that Corynebacterium glutamicum S9114 demonstrated excellent tolerance and biotransformation capacity to five typical aldehyde inhibitors including two furaldehydes: 2-furaldehyde (furfural), 5-(hydroxymethyl)-2-furaldehyde, and three benzaldehydes: 4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin), and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde). Transcription levels of 93 genes hypothesized to be responsible for five aldehydes biotransformation were examined by qRT-PCR. Multiple genes showed significantly up-regulated expression against furaldehydes or benzaldehydes. Overexpression of CGS9114_RS01115 in C. glutamicum resulted in the increased conversion of all five aldehyde inhibitors. The significant oxidoreductase genes responsible for each or multiple inhibitors biotransformation identified in this study will serve as a component of key gene device library for robust biorefinery fermentation strains development in the future biorefinery applications.

Tolerance improvement of Corynebacterium glutamicum on lignocellulose derived inhibitors by adaptive evolution.

Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24 h, and finally a stably evolved C. glutamicum was obtained after 128 days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4 g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications.

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60-70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.