De Novo Computational Design of a Lipase with Hydrolysis Activity towards Middle-Chained Fatty Acid Esters.

Innovations in biocatalysts provide great prospects for intolerant environments or novel reactions. Due to the limited catalytic capacity and the long-term and labor-intensive characteristics of mining enzymes with the desired functions, de novo enzyme design was developed to obtain industrial application candidates in a rapid and convenient way. Here, based on the catalytic mechanisms and the known structures of proteins, we proposed a computational protein design strategy combining de novo enzyme design and laboratory-directed evolution. Starting with the theozyme constructed using a quantum-mechanical approach, the theoretical enzyme-skeleton combinations were assembled and optimized via the Rosetta "inside-out" protocol. A small number of designed sequences were experimentally screened using SDS-PAGE, mass spectrometry and a qualitative activity assay in which the designed enzyme 1a8uD1 exhibited a measurable hydrolysis activity of 24.25 ± 0.57 U/g towards p-nitrophenyl octanoate. To improve the activity of the designed enzyme, molecular dynamics simulations and the RosettaDesign application were utilized to further optimize the substrate binding mode and amino acid sequence, thus keeping the residues of theozyme intact. The redesigned lipase 1a8uD1-M8 displayed enhanced hydrolysis activity towards p-nitrophenyl octanoate-3.34 times higher than that of 1a8uD1. Meanwhile, the natural skeleton protein (PDB entry 1a8u) did not display any hydrolysis activity, confirming that the hydrolysis abilities of the designed 1a8uD1 and the redesigned 1a8uD1-M8 were devised from scratch. More importantly, the designed 1a8uD1-M8 was also able to hydrolyze the natural middle-chained substrate (glycerol trioctanoate), for which the activity was 27.67 ± 0.69 U/g. This study indicates that the strategy employed here has great potential to generate novel enzymes exhibiting the desired reactions.

In-situ co-immobilization of lipase, lipoxygenase and L-cysteine within a metal-amino acid framework for conversion of soybean oil into higher-value products.

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products.

A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials.

Biomaterials with excellent biocompatibility, mechanical performance, and self-recovery properties are urgently needed for tissue regeneration. Inspired by barnacle cement and spider silk, we genetically designed and overexpressed a fused protein (cp19k-MaSp1) composed of Megabalanus rosa (cp19k) and Nephila clavata dragline silk protein (MaSp1) in Pichia pastoris. The recombinant cp19k-MaSp1 exhibited enhanced adhesion capability beyond those of the individual proteins in both aqueous and non-aqueous conditions. cp19k-MaSp1 protein fiber scaffolds prepared through electrospinning have adequate hydrophilicity compared to cp19k and MaSp1 protein fiber scaffolds, and offer improved overall porosity compared to MaSp1 protein fiber scaffolds. The cp19k-MaSp1 protein fiber scaffolds showed excellent proteolytically stable properties because of only 9.6 % depletion after incubation in a biodegradation solution for 56 d. The cp19k-MaSp1 protein fiber scaffolds present remarkably high extreme tensile strength (112.7 ± 11.6 MPa) and superior ductility (438.4 ± 43.9 %) compared with cp19k (34.4 ± 8.1 MPa, 115.4 ± 32.7 %) and MaSp1 protein fiber scaffolds (65.8 ± 9.3 MPa, 409.6 ± 23.1 %), also 68.4 % of tensile strength was recovered by incubation in K+ buffer after multiple stretches, which create a favorable cell adhesion, growth, and proliferation environment for human umbilical vein endothelial cells (HUVECs). The improved biocompatibility, extensive adhesion, mechanical strength, and self-recovery properties make the bioinspired synthetic cp19k-MaSp1 a potential candidate for biomedical tissue reconstruction.

Design of a genetically programmed barnacle-curli inspired living-cell bioadhesive.

In nature, barnacles and bacterial biofilms utilize self-assembly amyloid to achieve strong and robust interface adhesion. However, there is still a lack of sufficient research on the construction of macroscopic adhesives based on amyloid-like nanostructures through reasonable molecular design. Here, we report a genetically programmed self-assembly living-cell bioadhesive inspired by barnacle and curli system. Firstly, the encoding genes of two natural adhesion proteins (CsgA and cp19k) derived from E. coli curli and barnacle cement were fused and expressed as a fundamental building block of the bioadhesive. Utilizing the natural curli system of E. coli, fusion protein can be delivered to cell surface and self-assemble into an amyloid nanofibrous network. Then, the E. coli cells were incorporated into the molecular chain network of xanthan gum (XG) through covalent conjugation to produce a living-cell bioadhesive. The shear adhesive strength of the bioadhesive to the surface of the aluminum sheet reaches 278 â€‹kPa. Benefiting from living cells encapsulated inside, the bioadhesive can self-regenerate with adequate nutrients. This adhesive has low toxicity to organisms, strong resistance to the liquid environment in vivo, easy to pump, exhibiting potential application prospects in biomedical fields such as intestinal soft tissue repair.

Design of a Genetically Programmed Biomimetic Lipase Nanoreactor.

Alternative to the traditionally independent production of lipase, chemical synthesis of nano-carriers, and then preparing nanoimmobilized enzymes, we exploit a yeast genetically programmed virus biomimetic lipase nanoreactor in a sustainable manner. The nanoreactor biogenesis process integrated lipase production, protein component (coat-protein subunit and scaffold protein) production, self-assembly of protein components, and the encapsulation of lipase into protein nanocages using a simple process. It included overexpression of nanocage components, coat-protein subunits, and fused lipase-scaffold proteins and subsequent spontaneous self-assembly and encapsulation based on the specific interaction between the coat-protein subunit and the scaffold protein fused in the target lipase enzyme. The genetically programmable lipase nanoreactor showed improved stability under various harsh conditions, and was validated in fatty acid methyl ester synthesis with 86% yield at a high concentration of waste cooking oil (200 mM), which demonstrates the robustness and feasibility of the lipase nanoreactor in biodiesel production.

First report of a cross-kingdom pathogenic bacterium, Achromobacter xylosoxidans isolated from stipe-rot Coprinus comatus.

Coprinus comatus is an edible mushroom widely cultivated in China as a delicious food. Various diseases have occurred on C. comatus with the cultivated area increasing. In this study, the pathogenic bacterium JTG-B1, identified as Achromobacter xylosoxidans by 16S rDNA and nrdA gene sequencing, was isolated from edible mushroom Coprinus comatus with serious rot disease on its stipe. A. xylosoxidans has been confirmed as an important opportunistic human pathogenic bacterium and has been isolated from respiratory samples from cystic fibrosis. It is widely distributed in the environment. Here, we first report that fungi can also serve as a host for A. xylosoxidans. We confirmed that it can cross-kingdom infect between animals (mice) and fungi (C. comatus). The results of pathogenicity tests, physiological, biochemical and genotyping analysis of A. xylosoxidans from different hosts suggested that different strain of A. xylosoxidans may have pathogenicity differentiation. A. xylosoxidans not only is pathogenic to C. comatus but also may threaten human health.