Bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories: engineering, metabolic regulations, challenges, and perspectives.

Porphyrins, phycobilins, and their proteins have abundant π-electrons and strongly absorb visible light, some of which bind a metal ion in the center. Because of the structural and optical properties, they not only play critical roles as an essential component in natural systems but also have attracted much attention as a high value specialty chemical in various fields, including renewable energy, cosmetics, medicines, and foods. However, their commercial application seems to be still limited because the market price of porphyrins and phycobilins is generally expensive to apply them easily. Furthermore, their petroleum-based chemical synthesis is energy-intensive and emits a pollutant. Recently, to replace petroleum-based production, many studies on the bioproduction of metalloporphyrins, including Zn-porphyrin, Co-porphyrin, and heme, porphyrin derivatives including chlorophyll, biliverdin, and phycobilins, and their proteins including hemoproteins, phycobiliproteins, and phytochromes from renewable carbon sources using microbial cell factories have been reported. This review outlines recent advances in the bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories developed by various microbial biotechnology techniques, provides well-organized information on metabolic regulations of the porphyrin metabolism, and then critically discusses challenges and future perspectives. Through these, it is expected to be able to achieve possible solutions and insights and to develop an outstanding platform to be applied to the industry in future research.

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.

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.