Enhancing Phycocyanobilin Production Efficiency in Engineered Corynebacterium glutamicum: Strategies and Potential Application.

Phycocyanobilin, an algae-originated light-harvesting pigment known for its antioxidant properties, has gained attention as it plays important roles in the food and medication industries and has surged in demand owing to its low-yield extraction from natural resources. In this study, engineered Corynebacterium glutamicum was developed to achieve high PCB production, and three strategies were proposed: reinforcement of the heme biosynthesis pathway with the introduction of two PCB-related enzymes, strengthening of the pentose phosphate pathway to generate an efficient cycle of NADPH, and fed-batch fermentation to maximize PCB production. Each approach increased PCB synthesis, and the final engineered strain successfully produced 78.19 mg/L in a flask and 259.63 mg/L in a 5 L bioreactor, representing the highest bacterial production of PCB reported to date, to our knowledge. The strategies applied in this study will be useful for the synthesis of PCB derivatives and can be applied in the food and pharmaceutical industries.

Biodegradation of oxidized low density polyethylene by Pelosinus fermentans lipase.

Polyethylene (PE) exhibits high resistance to degradation, contributing to plastic pollution. PE discarded into the environment is photo-oxidized by sunlight and oxygen. In this study, a key enzyme capable of degrading oxidized PE is reported for the first time. Twenty different enzymes from various lipase families were evaluated for hydrolytic activity using substrates mimicking oxidized PE. Among them, Pelosinus fermentans lipase 1 (PFL1) specifically cleaved the ester bonds within the oxidized carbon-carbon backbone. Moreover, PFL1 (6 µM) degraded oxidized PE film, reducing the weight average and number average molecular weights by 44.6 and 11.3 %, respectively, within five days. Finally, structural analysis and molecular docking simulations were performed to elucidate the degradation mechanism of PFL1. The oxidized PE-degrading enzyme reported here will provide the groundwork for advancing PE waste treatment technology and for engineering microbes to repurpose PE waste into valuable chemicals.

Detoxifying Cyanides Using Cyanase Enzyme Complexes Composed of Carbonic Anhydrase via Irreversible Covalent Bonds.

Cyanase is a possible solution to reduce the environmental impact of cyanide. However, the enzyme's dependence on HCO3- limits its industrial applications. To overcome this problem, carbonic anhydrase is utilized in this study. Three types of Catcher/Tag systems were introduced into the cyanase (psCYN) from Pseudomonas stutzeri and the carbonic anhydrase (hmCA) from Hydrogenovibrio marinus to construct enzyme complexes via irreversible covalent bonds. Initially, a cyanase complex with the aid of scaffolding proteins was designed. The results of cyanase complexes using scaffolding proteins were similar to or inferior to those of the two free enzymes. To address this, the two enzymes were manipulated to form a direct bioconjugation without the need for scaffolding proteins. The two enzymes forming a direct conjugation showed activity more than 2.5 times higher than that of cyanase alone. In conclusion, this outcome will contribute to solving problems related to residual cyanides in food and the environment.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Advancements in Sustainable Plant-Based Alternatives: Exploring Proteins, Fats, and Manufacturing Challenges in Alternative Meat Production.

The rise in plant-based food consumption is propelled by concerns for sustainability, personal beliefs, and a focus on healthy dietary habits. This trend, particularly in alternative meat, has attracted attention from specialized brands and eco-friendly food companies, leading to increased interest in plant-based alternatives. The dominant plant-based proteins, derived mainly from legumes, include soy protein isolates, which significantly impact sensory factors. In the realm of plant-based fats, substitutes are categorized into fat substitutes based on fats and fat mimetics based on proteins and carbohydrates. The production of these fats, utilizing gums, emulsions, gels, and additives, explores characteristics influencing the appearance, texture, flavor, and storage stability of final plant-based products. Analysis of plant-based proteins and fats in hamburger patties provides insights into manufacturing methods and raw materials used by leading alternative meat companies. However, challenges persist, such as replicating meat's marbling characteristic and addressing safety considerations in terms of potential allergy induction and nutritional supplementation. To enhance functionality and develop customized plant-based foods, it is essential to explore optimal combinations of various raw materials and develop new plant-based proteins and fat separation.

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.

Biotechnological Plastic Degradation and Valorization Using Systems Metabolic Engineering.

Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.

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.

The increased efficiency of porphyran hydrolysis by constructing a multifunctional enzyme complex from marine microorganisms.

Porphyran, a polysaccharide composed of red algae, is a source of a multifunctional oligosaccharide material and raw biomass with various physiological activities. The glycolysis of porphyrans into oligosaccharides through various porphyranases is an approach for obtaining high-quality and promising alternative resources. In this study, porphyran was extracted from Porphyra yezoensis and used as a research substrate. We also established an efficient hydrolysis method using an enzymatic complex obtained through cohesin-dockerin interactions that degrade natural polysaccharides. The cohesion-dockerin interaction is designed to genetically bind the dockerin module to the end of an existing enzyme and then attach the cohesin module to obtain a protein complex. The designed protein complex has been shown to further increase the activity on the substrate, which can be considered a useful method to obtain efficient oligosaccharides or monosaccharides through hydrolysis of red algae for bioresources.

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.

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification.

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.

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.

Glucose/Xylose Co-Fermenting Saccharomyces cerevisiae Increases the Production of Acetyl-CoA Derived n-Butanol From Lignocellulosic Biomass.

Efficient xylose catabolism in engineered Saccharomyces cerevisiae enables more economical lignocellulosic biorefinery with improved production yields per unit of biomass. Yet, the product profile of glucose/xylose co-fermenting S. cerevisiae is mainly limited to bioethanol and a few other chemicals. Here, we introduced an n-butanol-biosynthesis pathway into a glucose/xylose co-fermenting S. cerevisiae strain (XUSEA) to evaluate its potential on the production of acetyl-CoA derived products. Higher n-butanol production of glucose/xylose co-fermenting strain was explained by the transcriptomic landscape, which revealed strongly increased acetyl-CoA and NADPH pools when compared to a glucose fermenting wild-type strain. The acetate supplementation expected to support acetyl-CoA pool further increased n-butanol production, which was also validated during the fermentation of lignocellulosic hydrolysates containing acetate. Our findings imply the feasibility of lignocellulosic biorefinery for producing fuels and chemicals derived from a key intermediate of acetyl-CoA through glucose/xylose co-fermentation.

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.

Non-Photosynthetic CO2 Utilization to Increase Fatty Acid Production in Yarrowia lipolytica.

Metabolic engineering of non-photosynthetic microorganisms to increase the utilization of CO2 has been focused on as a green strategy to convert CO2 into valuable products such as fatty acids. In this study, a CO2 utilization pathway involving carbonic anhydrase and biotin carboxylase was formed to recycle CO2 in the oleaginous yeast Yarrowia lipolytica, thereby increasing the production of fatty acids. In the recombinant strain in which the CO2 utilization pathway was introduced, the production of fatty acids was 10.7 g/L, which was 1.5-fold higher than that of the wild-type strain. The resulting strain had a 1.4-fold increase in dry cell mass compared to the wild-type strain. In addition, linoleic acid was 47.7% in the fatty acid composition of the final strain, which was increased by 11.6% compared to the wild-type strain. These results can be applied as an essential technology for developing efficient and eco-friendly processes by directly utilizing CO2.

Efficient utilization of brown algae for the production of Polyhydroxybutyrate (PHB) by using an enzyme complex immobilized on Ralstonia eutropha.

Marine macroalgae are potential renewable feedstocks for valuable biomaterials. Among them, alginate is a primary component in brown algae that can be nonenzymatically converted and enzymatically degraded by alginate lyases to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). Here, we constructed alginolytic enzyme complexes comprising two different alginate lyases for synergistic alginate degradation. The complexes showed good thermostability with 60% of the residual activity at high temperature (60 °C). Furthermore, they produced 0.85 and 0.18 mg/mL DEH from alginate and natural brown algae as substrates, respectively. The enzyme complex successfully decomposed brown algal biomass, resulting in a 3.15-fold improvement in DEH when compared to free enzymes. The Ralstonia eutropha strain with alginolytic enzyme complexes on the cell surface showed higher Polyhydroxybutyrate (PHB) production and produced 2.58 g/L PHB from alginate. After the use of alginate, remaining biomass such as fucoidan and laminaran can also be used in the future for high value ingredients in nutritional, medical device, skincare and dermatological products. These results demonstrate that it is possible to create more efficient strategies for producing biodegradable PHB and functional polysaccharides from brown algal substrates.

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.