Novel α-Amylase Inhibitor Hemi-Pyocyanin Produced by Microbial Conversion of Chitinous Discards.

α-Amylase inhibitors (aAIs) have been applied for the efficient management of type 2 diabetes. The aim of this study was to search for potential aAIs produced by microbial fermentation. Among various bacterial strains, Pseudomonas aeruginosa TUN03 was found to be a potential aAI-producing strain, and shrimp heads powder (SHP) was screened as the most suitable C/N source for fermentation. P. aeruginosa TUN03 exhibited the highest aAIs productivity (3100 U/mL) in the medium containing 1.5% SHP with an initial pH of 7-7.5, and fermentation was performed at 27.5 °C for two days. Further, aAI compounds were investigated for scaled-up production in a 14 L-bioreactor system. The results revealed a high yield (4200 U/mL) in a much shorter fermentation time (12 h) compared to fermentation in flasks. Bioactivity-guided purification resulted in the isolation of one major target compound, identified as hemi-pyocyanin (HPC) via gas chromatography-mass spectrometry and nuclear magnetic resonance. Its purity was analyzed by high-performance liquid chromatography. HPC demonstrated potent α-amylase inhibitory activity comparable to that of acarbose, a commercial antidiabetic drug. Notably, HPC was determined as a new aAI. The docking study indicated that HPC inhibits α-amylase by binding to amino acid Arg421 at the biding site on enzyme α-amylase with good binding energy (-9.3 kcal/mol) and creating two linkages of H-acceptors.

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol.

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

Microbial and enzymatic conversion of levulinic acid, an alternative building block to fermentable sugars from cellulosic biomass.

Levulinic acid (LA) is an important chemical building block listed among the top 12 value-added chemicals by the United States Department of Energy, and can be obtained through the hydrolysis of lignocellulosic biomass. Using the same approach as in the catalytic production of LA from biomass, catalytic methods to upgrade LA to higher value chemicals have been investigated. Since the discovery of the catabolic genes and enzymes in the LA metabolic pathway, bioconversion of LA into useful chemicals has attracted attention, and can potentially broaden the range of biochemical products derived from cellulosic biomass. With a brief introduction to the LA catabolic pathway in Pseudomonas spp., this review summarizes the current studies on the microbial conversion of LA into bioproducts, including the recent developments to achieve higher yields through genetic engineering of Escherichia coli cells. Three different types of reactions during the enzymatic conversion of LA are also discussed. KEY POINTS: • Levulinic acid is an alternative building block to sugars from cellulosic biomass. • Introduction of levulinic acid bioconversion with natural and engineered microbes. • Initial enzymatic conversion of levulinic acid proceeds via three different pathways. • 4-Hydroxyvalerate is one of the target chemicals for levulinic acid bioconversion.

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from L-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from L-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on L-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. KEY POINTS: • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine.

Biofuel production from straw hydrolysates: current achievements and perspectives.

Straw is an agricultural residue of the production of e.g. cereals, rapeseed or sunflowers. It includes dried stalks, leaves, and empty ears and corncobs, which are separated from the grains during harvest. Straw is a promising lignocellulosic feedstock with a beneficial greenhouse gas balance for the production of biofuels and chemicals. Like all lignocellulosic materials, straw is recalcitrant and requires thermochemical and enzymatic pretreatment to enable access to the three major biopolymers of straw-the polysaccharides cellulose and hemicellulose and the polyaromatic compound lignin. Straw is used for commercial ethanol and biogas production. Considerable research has also been conducted to produce biobutanol, biodiesel and biochemicals from this raw material, but more research is required to establish them on a commercial scale. The major hindrance for launching industrial biofuel and chemicals' production from straw is the high cost necessitated by pretreatment of the material. Improvements of microbial strains, production and extraction technologies, as well as co-production of high-value compounds represent ways of establishing straw as feedstock for the production of biofuels, chemicals and food.

Anti-α-Glucosidase Activity by a Protease from Bacillus licheniformis.

Anti-α-glucosidase (AAG) compounds have received great attention due to their potential use in treating diabetes. In this study, Bacillus licheniformis TKU004, an isolated bacterial strain from Taiwanese soil, produced AAG activity in the culture supernatant when squid pens were used as the sole carbon/nitrogen (C/N) source. The protein TKU004P, which was isolated from B. licheniformis TKU004, showed stronger AAG activity than acarbose, a commercial anti-diabetic drug (IC50 = 0.1 mg/mL and 2.02 mg/mL, respectively). The molecular weight of TKU004P, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. High-performance liquid chromatography (HPLC) analysis showed that TKU004P may be a protease that demonstrates AAG activity by degrading yeast α-glucosidase. Among the four chitinous sources of C/N, TKU004P produced the highest AAG activity in the culture supernatant when shrimp head powder was used as the sole source (470.66 U/mL). For comparison, 16 proteases, were investigated for AAG activity but TKU004P produced the highest levels. Overall, the findings suggest that TKU004P could have applications in the biochemical and medicinal fields thanks to its ability to control the activity of α-glucosidase.

Utilization of Fishery Processing By-Product Squid Pens for α-Glucosidase Inhibitors Production by Paenibacillus sp.

The supernatants (the solution part received after centrifugation) of squid pens fermented by four species of Paenibacillus showed potent inhibitory activity against α-glucosidases derived from yeast (79-98%) and rats (76-83%). The inhibition of acarbose-a commercial antidiabetic drug, used against yeast and rat α-glucosidases-was tested for comparison; it showed inhibitory activity of 64% and 88%, respectively. Other chitinolytic or proteolytic enzyme-producing bacterial strains were also used to ferment squid pens, but no inhibition activity was detected from the supernatants. Paenibacillus sp. TKU042, the most active α-glucosidase inhibitor (aGI)-producing strain, was selected to determine the optimal cultivation parameters. This bacterium achieved the highest aGI productivity (527 µg/mL) when 1% squid pens were used as the sole carbon/nitrogen source with a medium volume of 130 mL (initial pH 6.85) in a 250 mL flask (48% of air head space), at 30 °C for 3-4 d. The aGI productivity increased 3.1-fold after optimization of the culture conditions. Some valuable characteristics of Paenibacillus aGIs were also studied, including pH and thermal stability and specific inhibitory activity. These microbial aGIs showed efficient inhibition against α-glucosidases from rat, yeast, and bacteria, but weak inhibition against rice α-glucosidase with IC50 values of 362, 252, 189, and 773 µg/mL, respectively. In particular, these aGIs showed highly stable activity over a large pH (2-13) and temperature range (40-100 °C). Various techniques, including: Diaoin, Octadecylsilane opened columns, and preparative HPLC coupled with testing bioactivity resulted in isolating a main active compound; this major inhibitor was identified as homogentisic acid (HGA). Notably, HGA was confirmed as a new inhibitor, a non-sugar-based aGI, and as possessing stronger activity than acarbose with IC50, and maximum inhibition values of 220 µg/mL, 95%, and 1510 µg/mL, 65%, respectively. These results suggest that squid pens, an abundant and low-cost fishery processing by-product, constitute a viable source for the production of antidiabetic materials via fermentation by strains of Paenibacillus. This fermented product shows promising applications in diabetes or diabetes related to obesity treatment due to their stability, potent bioactivity, and efficient inhibition against mammalian enzymes.

Microbial bioconversion and processing methods enhance the phenolic acid and flavonoids and the radical scavenging capacity of Smilax china L. leaf.

BACKGROUND: It has been reported that Smilax china L. leaf (SCL) provided various biological functions owing to polyphenols. The objective of the current study was to assess the enhancing effect of processing methods and microbial conversions on phenolic acid and flavonoid content and radical scavenging capacity of SCL for potential applications of diverse food products. RESULTS: Targeted phenolic acid (chlorogenic acid) and flavonoids (piceid and quercetin) were identified in fresh SCL using liquid chromatography-mass spectrometry. The total amount of identified phenolic acid and flavonoids was highest in steamed SCL (12.70 ± 0.12 mg g(-1) on a dry matter basis, dmb). A substantial amount of chlorogenic acid (5.81 ± 0.16 mg g(-1) dmb), piceid (3.96 ± 0.04 mg g(-1) dmb) and quercetin (6.06 ± 0.12 mg g(-1) dmb) were quantified in SCL fermented by Bacillus species, roasted and steamed, respectively (P < 0.05). The oxygen radical absorbance capacity (ORAC) value was greater in microbial fermented SCL than in others, with the exception of Saccharomyces cerevisiae and Aspergillus oryzae. However, vitamin C equivalent antioxidant capacity (VCEAC) was highest in SCL fermented by Aspergillus oryzae. CONCLUSION: Results from our study suggest that the microbial fermentation processing method could improve accessibility to extraction of phenolic acids and flavonoid content and radical scavenging capacity.

Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs.

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

Insight into response mechanism of short-chain fatty acids to refined microbial transformation order of dissolved organic matter ranked by molecular weight during dry anaerobic digestion.

Dynamic transformation of dissolved organic matter (DOM) contributes to short-chain fatty acids (SCFAs) production during anaerobic digestion. However, the impact of refined transformation of DOM ranked by molecular weight (MW) on SCFAs has never been investigated. Results indicated that DOM conversion order was 3500-7000 Da>(MW>14000 Da) > 7000-4000 Da during hydrolysis stage, while it was independent of their MW in acidogenesis phase and followed a low to high MW order during methanogenesis stage. Proteins-like DOMs with different MW were closely related to SCFAs. Eight groups of microorganisms (e.g., Bacillus and Caldicoprobacter) responsible for the conversion of proteins-like DOMs to SCFAs. The possible routes linking environmental properties to microorganisms-proteins-like DOMs-SCFAs connections were constructed. Microbial activity modifications by regulating moisture, pH, NO3--N and NH4+-N can expedite the conversion of proteins-like DOMs to SCFAs. The study emphasizes the importance of MW-classification-based biotransformation of organic waste, offering a potential strategy to enhance anaerobic digestion performance.