Effective bioconversion of fungal-spoiled starchy food waste into fermentable sugars using fungi-degrading, artificial amylosomes.

Fungi-degrading artificial amylosomes were newly developed consisting of fungi-degrading enzyme (NAG), starch-degrading enzymes and a scaffold protein. Amylosome scaffolds containing starch-binding proteins (SbpCbpA and CCSbpCbpA) were highly bound to starch and fungal-spoiled food waste. Amylosomes showed an average of 1.43-fold higher reducing sugar production from starch. 2.00-fold α-amylase in amylosomes increased reducing sugar production from amylose by an average of 1.50-fold. At 70°C for 6 hours, SbpCbpA and CCSbpCbpA maintained an average activity of 56.42% compared to the control (38.37%). The enzyme mixture and amylosomes with NAG showed an average 1.31-fold increase in glucose production in response to fungal-spoiled food waste compared to samples without NAG; in particular, CCSbpCbpA with NAG produced 62.44 ± 0.03 mM glucose (2.55-fold of the enzyme mixture without NAG). This research strategy can be applicable to the starch and fungal-spoiled food waste saccharification in an ecofriendly manner, leading to sugar production in industrial fields.

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.

Surface display of enzyme complex on Corynebacterium glutamicum as a whole cell biocatalyst and its consolidated bioprocessing using fungal-pretreated lignocellulosic biomass.

A novel whole cell biocatalyst using fungal-pretreated lignocellulosic biomass was developed by displaying the enzyme complex consisting of N-acetylglucosaminidase (cNAG) and endoglucanse E (cCelE) on Corynebacterium glutamicum, hereafter called mNC. mNC showed a maximum 4.43-fold cNAG and 2.40-fold cCelE activity compared to single enzyme-secreting C. glutamicum. mNC also showed the highest efficiency of sugar production in various types of cellulose and fungal-pretreated biomass. The growth of mNC was 5.06-fold higher than that of the control. Then, the ability of mNC to produce a valuable chemical was confirmed. mNC overexpressing isopropanol biosynthesis genes showed a maximum titer of 218.9 ± 11.73 mg/L isopropanol and maintained high efficiency for isopropanol production in the recycling test, which was 90.07 ± 4.12 % during 4 cycles. This strategy can be applied to the direct saccharification of fungal-pretreated lignocellulosic biomass efficiently leading to the production of valuable products in various industrial fields.

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.

Ecofriendly Synthesis of l-Carnosine in Metabolically Engineered Corynebacterium glutamicum by Reinforcing Precursor Accumulation.

Biobased processes to minimize environmental pollutants have attracted much attention. l-Carnosine has been produced by chemical synthesis, and as an alternative to this method, we newly developed engineered Corynebacterium glutamicum synthesizing l-carnosine. To develop the strain, the pentose phosphate pathway (PPP) was enhanced by attenuating flux to nonoxidative PPP. Enhanced PPP strengthened the histidine pathway and produced 5.0 g/L l-histidine and 3.9 mg/L l-carnosine. Then, the histidine synthetic pathway was reinforced by overexpressing HisG and Rel. This pathway reduced feedback inhibition by l-histidine and strengthened the flux of the histidine pathway; thus, it produced 552.20 mg/g DCW l-histidine. As a result, enhancement of the PPP accumulates more l-histidine than the histidine pathway; thus, the PPP was further enhanced by pgi gene alteration. For sufficient ß-alanine products, PanD was overexpressed and produced 99.17 mg/L l-carnosine. The final strain, Car15, which consolidated all three pathways, produced 323.26 mg/L l-carnosine via fed-batch fermentation. Finally, we confirmed the antioxidant and antiglycation effects of biologically synthesized l-carnosine, and the biologically synthesized l-carnosine showed inhibitory activity similar to that of commercial l-carnosine. Consequently, this study suggested a new biosynthetic process for l-carnosine and showed potential as a treatment for metabolic disorders through the assessment of its functions.

Animal-free heme production for artificial meat in Corynebacterium glutamicum via systems metabolic and membrane engineering.

Recently, heme has attracted much attention as a main ingredient that mimics meat flavor in artificial meat in the food industry. Here, we developed Corynebacterium glutamicum capable of high-yield production of heme with systems metabolic engineering and modification of membrane surface. The combination of two precursor pathways based on thermodynamic information increased carbon flux toward heme and porphyrin intermediate biosynthesis. The co-overexpression of genes involved in a noncanonical downstream pathway and the gene encoding the transcriptional regulator DtxR significantly enhanced heme production. The overexpression of the putative heme exporters, knockout of heme-binding proteins, modification of the cell wall by chemical treatment, and reduction of intermediate UP III substantially improved heme secretion. The fed-batch fermentation showed a maximum heme titer of 309.18 ± 16.43 mg l-1, including secreted heme of 242.95 ± 11.45 mg l-1, a yield on glucose of 0.61 mmol mol-1, and productivity of 6.44 mg l-1h-1, which are the highest values reported to date. These results demonstrate that engineered C. glutamicum can be an attractive cell factory for animal-free heme production.