Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin ( l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin ( d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

Simultaneous directed evolution of coupled enzymes for efficient asymmetric synthesis of l-phosphinothricin.

The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

Both symbionts and environmental factors contribute to shape the microbiota in a pest insect, Sogatella furcifera.

Introduction: Bacterial symbionts are prevalent in arthropods globally and play a vital role in the fitness and resistance of hosts. While several symbiont infections have been identified in the white-backed planthopper Sogatella furcifera, the impact of environmental factors on the microbiota within S. furcifera remains elusive. Methods: In this study, a total of 142 S. furcifera individuals from 18 populations were collected from 14 locations across six countries (China, Thailand, Myanmar, Cambodia, Vietnam, and Laos) analyzed with 2bRAD-M sequencing, to examine the effects of symbionts on the microbiota in the S. furcifera population, as well as the vital effects of environmental factors on the bacterial communities. Results and discussion: Based on the results, in S. furcifera, the presence of symbionts Wolbachia and Cardinium negatively influenced the abundance of other bacteria, including Enterobacter, Acinetobacter, and Lysinibacillus, while Wolbachia infection significantly decreased the diversity of the microbial community. Moreover, several environmental factors, including longitude, latitude, temperature, and precipitation, affected the abundance of symbionts and microbiota diversity in S. furcifera. These results collectively highlight the vital role of Wolbachia in S. furcifera microbiota, as well as the intricate effects of environmental factors on the bacterial communities of S. furcifera.

[Pollution Characteristics and Emission Coefficient of Volatile Organic Compounds from Auto/motorcycle Parts & Accessories Manufacturing in Zhejiang Province].

The status of volatile organic compound (VOC) treatment in auto/motorcycle parts & accessories manufacturing in Zhejiang province was analyzed based on data from a survey investigating 70 concerned enterprises conducted in 2015. The pollution characteristics were further explored and the emission coefficient of VOCs released from this industry was preliminarily calculated by analyzing the survey data of 56 of the typical enterprises that had been screened further. The results showed that about two thirds of the enterprises had been equipped with collection and treatment facilities, but most of those facilities were running abnormally. Solvent-based materials were used commonly in this industry, and the main VOCs pollutants were xylene, butyl acetate, cyclohexanone, acetic ether, and toluene. The VOCs emission coefficient of auto/motorcycle parts & accessories manufacturing in Zhejiang was 414 g·(thousand yuan)-1, whereas the emission coefficient of the enterprises producing parts & accessories for only auto was 294 g·(thousand yuan)-1. As for the the enterprises producing parts & accessories for only motorcycle and for both auto and motorcycle, the emission coefficient reached 715 g·(thousand yuan)-1.