Isopropanol biosynthesis from crude glycerol using fatty acid precursors via engineered oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Isopropanol is widely used as a biofuel and a disinfectant. Chemical preparation of isopropanol destroys the environment, which makes biological preparation of isopropanol necessary. Previous studies focused on the use of expensive glucose as raw material. Therefore, the microbial cell factory that ferments isopropanol with cheap raw materials will provide a greener way to produce isopropanol. RESULTS: This study converted crude glycerol into isopropanol using Y. lipolytica. As a microbial factory, the active natural lipid and fatty acid synthesis pathway endows Y. lipolytica with high malonyl-CoA production capacity. Acetoacetyl-CoA synthase (nphT7) and isopropanol synthesis genes are integrated into the Y. lipolytica genome. The nphT7 gene uses the accumulated malonyl-CoA to synthesize acetoacetyl-CoA, which increases isopropanol production. After medium optimization, the best glycerol medium was found and resulted in a 4.47-fold increase in isopropanol production. Fermenter cultivation with pure glycerol medium resulted in a maximum isopropanol production of 1.94 g/L. In a crude glycerol fermenter, 1.60 g/L isopropanol was obtained, 82.53% of that achieved with pure glycerol. The engineered Y. lipolytica in this study has the highest isopropanol titer reported. CONCLUSIONS: The engineered Y. lipolytica successfully produced isopropanol by using crude glycerol as a cheap carbon source. This is the first study demonstrating the use of Y. lipolytica as a cell factory to produce isopropanol. In addition, this is also a new attempt to accumulate lipid synthesis precursors to synthesize other useful chemicals by integrating exogenous genes in Y. lipolytica.

Enhanced biodegradation of waste poly(ethylene terephthalate) using a reinforced plastic degrading enzyme complex.

Poly(ethylene terephthalate) (PET) is synthesized via a rich ester bond between terephthalate (TPA) and ethylene glycol (EG). Because of this, PET degradation takes a long time and PET accumulates in the environment. Many studies have been conducted to improve PET degrading enzyme to increase the efficiency of PET depolymerization. However, enzymatic PET decomposition is still restricted, making upcycling and recycling difficult. Here, we report a novel PET degrading complex composed of Ideonella sakaiensis PETase and Candida antarctica lipase B (CALB) that improves degradability, binding ability and enzyme stability. The reaction mechanism of chimeric PETase (cPETase) and chimeric CALB (cCALB) was confirmed by PET and bis (2-hydroxyethyl terephthalate) (BHET). cPETase generated BHET and mono (2-hydroxyethyl terephthalate (MHET) and cCALB produced terephthalate (TPA). Carbohydrate binding module 3 (CBM3) in the scaffolding protein greatly improved PET film binding affinity. Finally, the final enzyme complex demonstrated a 6.5-fold and 8.0-fold increase in the efficiency of hydrolysis from PET with either high crystalline or waste to TPA than single enzymes, respectively. This complex could effectively break down waste PET while maintaining enzyme stability and would be applied for biological upcycling of TPA.

Bio-isopropanol production in Corynebacterium glutamicum: Metabolic redesign of synthetic bypasses and two-stage fermentation with gas stripping.

Isopropanol is a commodity chemical widely used as a biofuel, fuel additive, rubbing alcohol and intermediate in various fields. Here, an engineered Corynebacterium glutamicum overproducing isopropanol was developed. To our knowledge, despite a representative industrial host to produce valuable chemicals, the high-level production of isopropanol in C. glutamicum has never been reported. First, the problem of the inability to produce isopropanol was solved by finding a key factor in its metabolism. The consolidation and modular optimization of synthetic bypasses including succinate and mevalonate bypasses enhanced isopropanol production. Flux redistribution of central metabolism significantly directed the carbon flux toward isopropanol biosynthesis. The final engineered strain produced 10.25 ± 1.12 g/L isopropanol in two-stage fed-batch fermentation with an optimized gas stripping, which is the highest titer, yield and productivity in C. glutamicum. These strategies could be useful for the high-level production of isopropanol in C. glutamicum.

Efficient Synthesis of Food-Derived Antioxidant l-Ergothioneine by Engineered Corynebacterium glutamicum.

l-Ergothioneine (EGT) is a strong antioxidant used in industry, and it is commonly extracted from mushrooms; however, its production is limited. As an alternative, we developed metabolically engineered Corynebacterium glutamicum with reinforced sulfur assimilation and pentose phosphate pathways, which led to the accumulation of 45.0 and 63.2 mg/L EGT, respectively. Additionally, the overexpression of cysEKR resulted in further promoted EGT production in ET4 (66.5 mg/L) and ET7 (85.0 mg/L). Based on this result, we developed the strain ET11, in which all sulfur assimilatory, PP, and l-cysteine synthetic pathways were reinforced, and it synthesized 264.4 mg/L EGT. This study presents the first strategy for EGT synthesis that does not require precursor addition in C. glutamicum, and the production time was shortened. In addition, the synthesized EGT showed high radical scavenging activity (70.7%), thus confirming its antioxidant function. Consequently, this study showed the possibility of EGT commercialization by overcoming the limitations of industrial processes.

Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive.

Taurine is a biologically and physiologically valuable food additive. However, commercial taurine production mainly relies on environmentally harmful chemical synthesis. Herein, for the first time in bacteria, we attempted to produce taurine in metabolically engineered Corynebacterium glutamicum. The taurine-producing strain was developed by introducing cs, cdo1, and csad genes. Interestingly, while the control strain could not produce taurine, the engineered strains successfully produced taurine via the newly introduced metabolic pathway. Furthermore, we investigated the effect of a deletion strain of the transcriptional repressor McbR gene on taurine production. As a result, sulfur accumulation and l-cysteine biosynthesis were reinforced by the McbR deletion strain, which further increased the taurine production by 2.3-fold. Taurine production of the final engineered strain Tau11 was higher than in other previously reported strains. This study demonstrated a potential approach for eco-friendly biosynthesis as an alternative to the chemical synthesis of a food additive.

Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

Comparative analysis of microRNA and mRNA expression profiles in cells and exosomes under toluene exposure.

Recent studies have illustrated the growing importance of exosomes (small extracellular vesicles) and their constituent microRNAs (miRNAs) in the fields of toxicology and pathology. The mechanism of toxicity of toluene, a highly-prevalent and volatile organic compound, is largely unknown. To examine the role of miRNAs in toluene-induced toxicity, we investigated miRNAs and toluene-induced gene expression in HL-60 human promyelocytic leukemia cells and exosomes using microarrays. A total of 54 miRNAs were differentially expressed in HL-60 cell lines exposed to toluene and exosomes from the cells. Four out of the 54 miRNAs (hsa-miR-1290, hsa-miR-718, hsa-miR-3663-3p, and hsa-miR-320c) were subsequently validated by qRT-PCR. Integrated analysis of miRNA and mRNA expression profiles identified 8 miRNA-mRNA correlations. By performing Comparative Toxicogenomics Database analysis, we found that the eight putative target genes of the differentially expressed miRNAs under toluene exposure (EXOSC6, RHOH, GFER, HERC2, GOLGA4, SLC7A11, GCLM, and BACH1) are related to diverse disease categories such as nervous system disease, cancer, cardiovascular disease, and respiratory tract disease. In conclusion, our data demonstrated that miRNA-mRNA networks provide a better understanding of toxicological mechanism caused by environmental pollutants in vitro using HL-60 cells and exosomes.

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil in solvent-free system.

The enzymatic coproduction of biodiesel and glycerol carbonate by transesterification of soybean oil and dimethyl carbonate (DMC) has been studied in a solvent-free system. The effects on biodiesel and glycerol carbonate conversion of reaction conditions including the kind of enzyme, the amount of enzyme, the molar ratio of DMC to soybean oil, the reaction temperature, and water addition were investigated. The optimal conditions for biodiesel and glycerol carbonate were 20% Novozym 435, 10:1 molar ratio of DMC to soybean oil, and 0.7% water addition. Under these conditions, the conversions of 96.4% biodiesel and 92.1% glycerol carbonate have been achieved after 48h.

Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate.

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol-3-phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4-fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC-TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4-fold compared with 3.6% for the wild-type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3-fold improvement, compared with 3.6% for the wild-type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel.

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil and dimethyl carbonate.

The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 °C, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved.

Cellulosomic profiling produced by Clostridium cellulovorans during growth on different carbon sources explored by the cohesin marker.

Clostridium cellulovorans produces large extracellular enzyme complex, called cellulosomes. The diversity of the cellulosomal enzymes, which are secreted by C. cellulovorans that has been cultured on different carbon sources, such as Avicel, xylan, AXP (Avicel-xylan-pectin, 3:1:1) and cellobiose, was explored by two-dimensional gel electrophoresis. To identify the cellulosomal enzymes, we constructed a biomarker using cohesin 6, one of the CbpA cohesins, that was labeled with fluorescence. The major apparent spots were isolated and identified by ESI MS/MS protein sequencing. Fluorescently labeled cohesin clearly showed that the amount of the cellulosomal enzymes was influenced by the available carbon source. EngE, ExgS, EngK, XynB and ManA were most frequently expressed under all conditions. However, EngY was only observed on the AXP culture. We found two novel putative cellulosomal proteins, NC1[GH9] and NC2[GH26], and five unknown proteins, NU1, NU2, NU3, NU4 and NU5. The cohesin biomarker clearly showed different production patterns of the cellulosomal subunits under different culture conditions and revealed novel cellulosomal subunits.