Developing a Nanopore Sequencing Workflow for Protein Engineering Applications.

Sequencing plays a critical role in protein engineering, where the genetic information encoding for a desired mutation can be identified. We evaluated the performance of two commercially available NGS technologies (Illumina NGS and nanopore sequencing) on the available mutant libraries that were either previously constructed for other protein engineering projects or constructed in-house for this study. The sequencing results from Illumina sequencing indicated that a substantial proportion of the reads exhibited strand exchange, which mixed information of different mutants. When nanopore sequencing was used, the occurrence of strand exchange was substantially reduced compared with that of Illumina sequencing. We then developed a new library preparation workflow for nanopore sequencing and were successful in further reducing the incidence of strand exchange. The optimized workflow was successfully used to aid selection of improved alcohol dehydrogenase mutants in cells where their activities were coupled with the cell growth rate. The workflow quantified the enrichment fold change of most mutants in the library (size = 1728) in the growth-based selection passaging. A mutant that was >500% more active than its parent variant was identified based on the fold change data but not with the absolute abundance data (random sampling of the passaged cells), highlighting the usefulness of this rapid and affordable sequencing workflow in protein engineering.

Analysis of Conservation Priorities and Runs of Homozygosity Patterns for Chinese Indigenous Chicken Breeds.

To achieve sustainable development of the poultry industry, the effective conservation of genetic resources has become increasingly important. In the present study, we systematically elucidated the population structure, conservation priority, and runs of homozygosity (ROH) patterns of Chinese native chicken breeds. We used a high-density genotyping dataset of 157 native chickens from eight breeds. The population structure showed different degrees of population stratification among the breeds. Chahua chicken was the most differentiated breed from the other breeds (Nei = 0.0813), and the Wannan three-yellow chicken (WanTy) showed the lowest degree of differentiation (Nei = 0.0438). On the basis of contribution priority, Xiaoshan chicken had the highest contribution to the total gene diversity (1.41%) and the maximum gene diversity of the synthetic population (31.1%). WanTy chicken showed the highest contribution to the total allelic diversity (1.31%) and the maximum allelic diversity of the syntenic population (17.0%). A total of 5242 ROH fragments and 5 ROH island regions were detected. The longest ROH fragment was 41.51 Mb. A comparison of the overlapping genomic regions between the ROH islands and QTLs in the quantitative trait loci (QTL) database showed that the annotated candidate genes were involved in crucial economic traits such as immunity, carcass weight, drumstick and leg muscle development, egg quality and egg production, abdominal fat precipitation, body weight, and feed intake. In conclusion, our findings revealed that Chahua, Xiaoshan, and WanTy should be the priority conservation breeds, which will help optimize the conservation and breeding programs for Chinese indigenous chicken breeds.

Dynamic Expression Profile of Follicles at Different Stages in High- and Low-Production Laying Hens.

Improving the efficiency of hens and extending the egg-laying cycle require maintaining high egg production in the later stages. The ovarian follicles, as the primary functional units for ovarian development and oocyte maturation, play a crucial role in regulating the continuous ovulation of hens. The egg production rate of laying hens is mostly affected by proper follicle growth and ovulation in the ovaries. The objective of this study was to identify the key genes and signaling pathways involved in the development of ovarian follicles in Taihang hens through transcriptome screening. In this study, RNA sequencing was used to compare and analyze the transcriptomes of ovarian follicles at four developmental stages: small white follicles (SWF), small yellow follicles (SYF), F5 follicles, and F2 follicles, from two groups: the high continual production group (H-Group) and the low continual production group (L-Group). A total of 24 cDNA libraries were constructed, and significant differential expression of 96, 199, 591, and 314 mRNAs was detected in the SWF, SYF, F5, and F2 follicles of the H and L groups, respectively. Based on the results of GO and KEGG enrichment analyses, each stage of follicle growth possesses distinct molecular genetic features, which have important effects on follicle development and significantly promote the formation of continuous production traits through the biosynthesis of steroid hormones, cytokine-cytokine receptor interaction, and neuroactive ligand-receptor interaction. Additionally, through STEM analysis, we identified 59 DEGs, including ZP4, KCNH1, IGFs, HMGA2, and CDH1, potentially associated with follicular development within four significant modules. This study represents the first transcriptome investigation of follicles in hens with high and low egg-producing characteristics at four crucial developmental stages. These findings provide important molecular evidence for understanding the regulation of follicular development and its variations.

A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases.

Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat /km value toward (S)-2-octanol compared to its wild type.

A Simple Biosystem for the High-Yielding Cascade Conversion of Racemic Alcohols to Enantiopure Amines.

The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing, requiring simple and efficient solutions. Herein, we report the development of a cascade biotransformation to aminate racemic alcohols. This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize a racemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine, and isopropylamine to recycle PMP and NAD+ cofactors via the reversed cascade reactions. The concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs, an enantioselective BmTA, and isopropylamine. A biosystem containing isopropylamine and E. coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72-99 % yield and 98-99 % ee, providing with a simple and practical solution to this type of reaction.

Directed evolution of Thermomyces lanuginosus lipase to enhance methanol tolerance for efficient production of biodiesel from waste grease.

Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM) is reported. The best double mutant, TLL-S105C/D27R, retained 71% of its original activity after incubation in methanol, showing 30% greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27% higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure. E. coli (TLL-S105C/D27R)-catalyzed biotransformation of waste grease produced biodiesel in 81% yield in 8h, showing improvement over the 67% yield for E. coli (TLL), while retaining 92% productivity after 4 cycles of biotransformation of waste grease. The engineered TLL mutant shows high potential for commercial biodiesel production.

Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease.

A whole-cell based solvent-free system was developed for efficient conversion of waste grease to biodiesel via one-pot esterification and transesterification. By isolation and screening of lipase-producing strains from soil, Serratia marcescens YXJ-1002 was discovered for the biotransformation of grease to biodiesel. The lipase (SML) from this strain was cloned and expressed in Escherichia coli as an intracellular enzyme, showing 6 times higher whole-cell based hydrolysis activity than that of wild type strain. The recombinant cells were used for biodiesel production from waste grease in one-pot reactions containing no solvent with the addition of methanol in several small portions, and 97% yield of biodiesel (FAME) was achieved under optimized conditions. In addition, the whole-cell biocatalysts showed excellent reusability, retaining 74% productivity after 4 cycles. The developed system, biocatalyst, and process enable the efficient, low-cost, and green production of biodiesel from waste grease, providing with a potential industrial application.