Metabolic engineering of Methanosarcina acetivorans for lactate production from methane.

We previously demonstrated anaerobic conversion of the greenhouse gas methane into acetate using an engineered archaeon that produces methyl-coenzyme M reductase (Mcr) from unculturable microorganisms from a microbial mat in the Black Sea to create the first culturable prokaryote that reverses methanogenesis and grows anaerobically on methane. In this work, we further engineered the same host with the goal of converting methane into butanol. Instead, we discovered a process for converting methane to a secreted valuable product, L-lactate, with sufficient optical purity for synthesizing the biodegradable plastic poly-lactic acid. We determined that the 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum is responsible for lactate production. This work demonstrates the first metabolic engineering of a methanogen with a synthetic pathway; in effect, we produce a novel product (lactate) from a novel substrate (methane) by cloning the three genes for Mcr and one for Hbd. We further demonstrate the utility of anaerobic methane conversion with an increased lactate yield compared to aerobic methane conversion to lactate. Biotechnol. Bioeng. 2017;114: 852-861. © 2016 Wiley Periodicals, Inc.

Computational de novo design of antibodies binding to a peptide with high affinity.

Antibody drugs play a critical role in infectious diseases, cancer, autoimmune diseases, and inflammation. However, experimental methods for the generation of therapeutic antibodies such as using immunized mice or directed evolution remain time consuming and cannot target a specific antigen epitope. Here, we describe the application of a computational framework called OptMAVEn combined with molecular dynamics to de novo design antibodies. Our reference system is antibody 2D10, a single-chain antibody (scFv) that recognizes the dodecapeptide DVFYPYPYASGS, a peptide mimic of mannose-containing carbohydrates. Five de novo designed scFvs sharing less than 75% sequence similarity to all existing natural antibody sequences were generated using OptMAVEn and their binding to the dodecapeptide was experimentally characterized by biolayer interferometry and isothermal titration calorimetry. Among them, three scFvs show binding affinity to the dodecapeptide at the nM level. Critically, these de novo designed scFvs exhibit considerably diverse modeled binding modes with the dodecapeptide. The results demonstrate the potential of OptMAVEn for the de novo design of thermally and conformationally stable antibodies with high binding affinity to antigens and encourage the targeting of other antigen targets in the future. Biotechnol. Bioeng. 2017;114: 1331-1342. © 2017 Wiley Periodicals, Inc.

In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens.

Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

Extension of shelf life of tomato (Solanum lycopersicum L.) by using a coating of polyhydroxybutyrate-carboxymethyl cellulose-pectin-thymol conjugate.

This study targets explicitly finding an alternative to petroleum-based plastic films that burden the environment, which is a high priority. Hence, polymeric films were prepared with carboxymethyl cellulose (CMC) (4%), pectin (2%), and polyhydroxybutyrate (PHB) (0.5%) with different concentrations of thymol (0.3%, 0.9%, 1.8%, 3%, and 5%) and glycerol as a plasticizer by solution casting technique. The prepared films were tested for mechanical, optical, antimicrobial, and antioxidant properties. Film F5 (CMC + P + PHB + 0.9%thymol) showed an excellent tensile strength of 15 MPa, Young's modulus of 395 MPa, antioxidant activity (AA) (92%), rapid soil biodegradation (21 days), and strong antimicrobial activity against bacterial and fungal cultures such as Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Aspergillus niger, and Aspergillus flavus. The thymol content increase in films F6 (1.8%), F7 (3%), and F8 (5%) displayed a decrease in mechanical properties due to thymol's hydrophobicity. For shelf life studies on tomatoes, F2, a film without thymol (poor antimicrobial and antioxidant activities), F5 (film with superior mechanical, optical, antimicrobial, and antioxidant properties), and F7 (film with low mechanical properties) were selected. Film F5 coatings on tomato fruit enhanced the shelf life of up to 15 days by preventing weight loss, preserving firmness, and delaying changes in biochemical constituents like lycopene, phenols, and AA. Based on the mechanical, optical, antimicrobial, antioxidant, and shelf life results, the film F5 is suitable for active food packaging and preservation. PRACTICAL APPLICATION: The developed active biodegradable composite can be utilized as a coating to extend the shelf life of fruits and vegetables. These coatings are easy to produce and apply, offering a sustainable solution to reduce food waste. On an industrial scale, they can be applied to food products, ensuring longer freshness without any technical challenges.

A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry.

The extracellular amylase production level by the moderate halophile Bacillus siamensis F2 was optimized, and the enzyme was biochemically characterized. The culture parameters for NaCl, carbon, nitrogen, pH, and temperature were optimized for high titers of amylase production. Growing B. siamensis F2 cultures in Great Salt Lake-2 medium with additions of (in g/L) NaCl (100), starch (30), yeast extract (2), KNO3 (2), and MgSO4 (1) at pH 8, 30 °C resulted in the maximum amylase production (4.2 U/ml). The amylase was active across a wide range of salinities (0 to 30% NaCl), pH (5.0-10.0), and temperatures (20-70 °C) and showed good stability with surfactants (sodium dodecyl sulfate (SDS) and Triton X-100); hence, it was identified as halo-acid-alkali-tolerant and surfactant stable. Temperature, pH, and salinity were optimal for amylase activity at 50 °C, pH 7, and 5% NaCl, respectively. It also generates amylase by utilizing agricultural wastes like sugarcane bagasse, sweet potato peel, and rice husk. Based on the performance of B. siamensis F2 using agricultural wastes and synthesizing amylase, the current study attempted to produce bioethanol by coculturing with baker's yeast using sugarcane bagasse and sweet potato peel as a substrate, which yielded 47 and 57 g/L of bioethanol, respectively. Besides bioethanol production, amylase secreted by F2 was also employed for juice clarification for better yield and clarity and for softening dough to produce better-quality buns. This novel amylase may have many potential applications in waste valorization, biorefinery sectors, and food industries.

Impact of ultrasonication and blanching as a pre-treatment on quality parameter of dried and rehydrated bitter gourd.

Vegetables are owed to the restriction of seasonal availability and regional abundance; it becomes essential that vegetables are preserved safely during the off-season. These existing demands look for dried products with high nutritional and organoleptic properties similar to fresh products. This study aimed to investigate the effect of ultrasonication and blanching before hot air drying on the quality attributes of bitter gourd (Momordica charantia). Dried samples were rehydrated to find the efficiency of pre-treatment and physicochemical properties. M. charantia slices were pre-treated with ultrasonication and blanched and dried at two different temperatures, 50 °C and 60 °C. M. charantia pre-treated using ultrasonication and dried at 60 °C reduces the drying time to 50% and rehydration time to 40% compared to untreated samples. Physico-chemical analysis of ultrasonicated samples revealed to have better retention of moisture (dried - 3.6%, rehydrated - 88%), Colour ΔE (dried - 9.07, rehydrated - 1.6), ascorbic acid (dried - 513, rehydrated - 310 mg/100 g), phenol (dried - 302, rehydrated - 231 GAE mg/100 g) and ß-carotene (dried - 68 µg/100 g, rehydrated - 39 µg/100 g) compared to blanching.

Purification and characterization of novel halo-acid-alkali-thermo-stable xylanase from Gracilibacillus sp. TSCPVG.

An aerobic xylanolytic moderately halophilic and alkali-tolerant bacterium, Gracilibacillus sp. TSCPVG, produces multiple xylanases of unusual halo-acid-alkali-thermo-stable nature. The purification of a major xylanase from TSCPVG culture supernatant was achieved by hydrophobic and gel permeation chromatographic methods followed by electroelution from preparatory PAGE. The molecular mass of the purified xylanase was 42 kDa, as analyzed by SDS-PAGE, with a pI value of 6.1. It exhibited maximal activity in 3.5 % NaCl and retained over 75 % of its activity across the broad salinity range of 0-30 % NaCl, indicating a high halo-tolerance. It showed maximal activity at pH 7.5 and had retained 63 % of its activity at pH 5.0 and 73 % at pH 10.5, signifying the tolerance to broad acid to alkaline conditions. With birchwood xylan as a substrate, K m and specific activity values were 21 mg/ml and 1,667 U/mg, respectively. It is an endoxylanase that degrades xylan to xylose and xylobiose and had no activity on p-nitrophenyl-ß-D-xylopyranoside, p-nitrophenyl-ß-D-glucopyranoside, p-nitrophenyl acetate, carboxymethylcellulose, and filter paper. Since it showed remarkable stability over different salinities, broad pH, and temperature ranges, it is promising for application in many industries.