Carbon-Phosphorus Lyase—the State of the Art Abstract Organophosphonates are molecules that contain a very chemically stable carbon-phosphorus (C-P) bond. Microorganisms can utilize phosphonates as potential source of crucial elements for their growth, as developed several pathways to metabolize these compounds. One among these pathways is catalyzed by C-P lyase complex, which has a broad substrate specifity; therefore, it has a wide application in degradation of herbicides deposited in the environment, such as glyphosate. This multi-enzyme system accurately recognized in Escherichia coli and genetic studies have demonstrated that it is encoded by phn operon containing 14 genes (phnC-phnP). The phn operon is a member of the Pho regulon induced by phosphate starvation. Ability to degradation of phosphonates is also found in other microorganisms, especially soil and marine bacteria, that have homologous genes to those in E. coli. Despite the existence of differences in structure and composition of phn gene cluster, each of these strains contains phnGHIJKLM genes necessary in the C-P bond cleavage mechanism. The review provides a detailed description and summary of achievements on the C-P lyase enzymatic pathway over the last 50 years.

Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails Abstract In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.

Highly Efficient Extraction of Ferulic Acid from Cereal Brans by a New Type A Feruloyl Esterase from Eupenicillium parvum in Combination with Dilute Phosphoric Acid Pretreatment Abstract Feruloyl esterase (FAE) is a critical enzyme in bio-extraction of ferulic acid (FA) from plant cell wall. A new FAE (EpFAE1) encoding gene was isolated from Eupenicillium parvum and heterologously expressed in Pichia pastoris cells. Based on phylogenetic tree analysis, the protein EpFAE1 belongs to type A of the seventh FAE subfamily. Using methyl ferulate as substrate, the optimum temperature and pH for the catalytic activity of EpFAE1 were 50 °C and 5.5, respectively. The enzyme exhibited high stability at 50 °C, in a wide pH range (3.0–11.0), or in the presence of 2 M of NaCl. Together with the endo-xylanase EpXYN1, EpFAE1 released 72.32% and 4.00% of the alkali-extractable FA from de-starched wheat bran (DSWB) or de-starched corn bran (DSCB), respectively. Meanwhile, the substrates were pretreated with 1.75% (for DSWB) or 1.0% (for DSCB) of phosphoric acid (PA) at 90 °C for 12 h, followed by enzymatic hydrolysis of the soluble and insoluble fractions. The release efficiencies of FA were up to 84.64% for DSWB and 66.73% for DSCB. Combined dilute PA pretreatment with enzymatic hydrolysis is a low-cost and highly efficient method for the extraction of FA from cereal brans.

Nitrate Metabolism Decreases the Steroidal Alcohol Byproduct Compared with Ammonium in Biotransformation of Phytosterol to Androstenedione by Mycobacterium neoaurum Abstract In biotransformation of phytosterol to 4-androstene-3,17-dione (AD) by Mycobacterium, the steroidal alcohol (such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one, HBC) was a main byproduct. To weaken the accumulation of this byproduct in sterol biotransformation, ammonium was substituted by nitrate as nitrogen resource. The nitrate was utilized by Mycobacterium and led to metabolic flux shift towards AD production. The ratio of AD/HBC increased maximally from 2.1 to 5.5 and AD production increased correspondently. In the meanwhile, the nitrate metabolism resulted in the decreased intracellular redox level (NADH/NAD+) maximally by 59.5% and a slight descent tendency with the increase of the nitrate concentrations. It indicated that the nitrate utilization effectively decreased the steroidal alcohol production by regulating intracellular redox level in sterol biotransformation. These results gave an insight into the mechanism of the steroidal alcohol formation in sterol biodegradation and provided a simple strategy to regulate the metabolic distribution.

Correction to: Two Novel Acetylesterases from Pantoea dispersa : Recombinant Expression, Purification, and Characterization The original version of this article unfortunately contained a mistake. Under Materials and Methods heading, Bacterial Strains sub-heading, the correct name of the used strain is “FEI4 65” and not “FzEI4 65.”

Augmenting Fremyella diplosiphon Cellular Lipid Content and Unsaturated Fatty Acid Methyl Esters Via Sterol Desaturase Gene Overexpression Abstract Cyanobacteria have immense prospective as a platform for renewable energy; however, a major barrier in achieving optimal productivity is the low lipid yield. Fremyella diplosiphon, a model cyanobacterium, is an ideal biofuel agent due to its desirable fatty acid methyl esters (FAMEs). To enhance lipid content, we overexpressed the sterol desaturase (SD) gene in F. diplosiphon B481 wild type by genetic transformation. This effort resulted in a transformant (B481-SD) with a 64-fold increase in the SD gene at the mRNA transcript level, with no loss in growth and pigmentation. The transformant was persistently grown for over 32 generations indicating long-term stability and vitality. We observed 27.3% and 23% increases in total lipid content and unsaturated FAMEs respectively in B481-SD transesterified lipids with methyl octadecadienoate as the most abundant unsaturated component. In addition, we detected an 81% increase in FAME composition in the transformant compared with the wild type. Theoretical physical and chemical properties confirmed a FAME profile with very high cetane number (65.972–67.494) and oxidative stability (50.493–18.66 h) in the engineered strain. Results of the study offer a promising approach to augment F. diplosiphon total lipid content and unsaturated FAMEs, thus paving the way to enhance biofuel capacity of the organism.

Characteristics of White-rot Fungus Phlebia brevispora TMIC33929 and Its Growth-Promoting Bacterium Enterobacter sp. TN3W-14 in the Decolorization of Dye-Contaminated Water Abstract The dye decolorization potential of the white-rot fungus Phlebia brevispora TMIC33929 when grown alone or in coculture with its growth-promoting bacterium Enterobacter sp. TN3W-14 was evaluated in low nitrogen liquid medium at different pHs. Axenic fungus removed a similar amount of Congo red and crystal violet at pH 4.5 and 7.0, respectively. The bacterium alone achieved only slightly better decolorization of crystal violet than the fungus at pH 9.0. Compared with axenic fungus, cocultures provided no increased crystal violet removal but achieved higher removal of crystal violet in mixed dye at all pHs, and the best-mixed dye decolorization at pH 9.0. Unlike bacterial growth on dyes, growth of fungal mycelia was not inhibited by the dyes at all pH but the cocultures gave comparably higher mycelial growth.

Exploration of the Tolerance Ability of a Cell-Free Biosynthesis System to Toxic Substances Abstract Cell-free synthetic biology has become a robust technology platform for synthesizing proteins and chemicals in recent years. Cell-free synthetic biology system activates biological machinery without the use of living cells, which opens new opportunities for the addition or synthesis of toxic substances at high concentrations in biological systems. Although it is generally accepted that an in vitro cell-free synthesis system has a high tolerance ability to toxic substances, there is a lack of relevant data to support this view. To explore particular tolerance ability, a range of different surfactants, lipids, materials, biofuels, and chemical drugs were selected for testing their effects in Escherichia coli–based cell-free protein synthesis system. The results showed the limit concentrations of different toxic substances. Moreover, the results demonstrated that the tolerance ability of a cell-free system is much higher than that of a cell system. This study further provides a rationale for the synthesis of toxic biopharmaceuticals, biochemicals, and biofuels by using cell-free systems.

Crude Oil Biodegradation by Newly Isolated Bacterial Strains and Their Consortium Under Soil Microcosm Experiment Abstract Bioremediation has been attracting researchers’ attention to develop as a technique to remove the pollution of crude oil in the environment. However, more or stronger novel strains capable of crude oil removal are still required. In this study, the potential of five newly isolated bacterial strains for crude oil abatement was evaluated in the liquid medium and contaminated soil individually and as a mixed consortium. Raoultella ornithinolytica strain PS exhibited the highest ability and degraded up to 83.5% of crude oil. Whereas Bacillus subtilis strain BJ11 degraded 81.1%, Acinetobacter lwoffii strain BJ10 degraded 75.8%, Acinetobacter pittii strain BJ6 degraded 74.9%, and Serratia marcescens strain PL degraded only 70.0% of crude oil in the liquid media. The consortium comprising the above five strains degraded more than 94.0% of crude oil after 10 days of incubation in the liquid medium. The consortium degraded more than 65.0% of crude oil after 40 days incubation even in the contaminated soil. The five crude oil degrading strains, especially their consortium, exhibited a high capability to break down a wide range of compounds in crude oil including straight-chain alkanes, branched alkanes, and aromatic hydrocarbons. These strains, especially as consortia, have good potential of application in the remediation of crude oil–contaminated environments.

Purification and Characterization of Cellulase from Obligate Halophilic Aspergillus flavus (TISTR 3637) and Its Prospects for Bioethanol Production Abstract A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55 kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9 units (U) mg−1 with Vmax and Km values of 37.87 mol min−1 mg−1 and 3.02 mg mL−1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60 °C, and 200 g L−1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2 mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60 °C, and 150 g L−1 NaCl, whereas the commercial cellulase had a very low activity.