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1. Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

   https://doi.org/10.1038/s41467-022-35021-x  

   King, Edward ; Maxel, Sarah ; Zhang, Yulai ; Kenney, Karissa C. ; Cui, Youtian ; Luu, Emma ; Siegel, Justin B. ; Weiss, Gregory A. ; Luo, Ray ; Li, Han ( November 2022 , Nature Communications)

   Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN⁺) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN⁺-based reducing power. This is based on an orthogonal, NMN⁺-dependent glycolytic pathway inEscherichia coliwhich can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN⁺. With a throughput of >10⁶variants per iteration, the growth selection discovers aLactobacillus pentosusNADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme’s global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme’s access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.
2. Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

   https://doi.org/10.1038/s41467-022-32727-w  

   Zhang, Linyue ; King, Edward ; Black, William B. ; Heckmann, Christian M. ; Wolder, Allison ; Cui, Youtian ; Nicklen, Francis ; Siegel, Justin B. ; Luo, Ray ; Paul, Caroline E. ; et al ( August 2022 , Nature Communications)

   Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase inEscherichia colito exclusively rely on the reduced NMN⁺(NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺.
3. Co‑cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates

   https://doi.org/10.1186/s13068-021-02083-w  

   Brown, Jennifer L. ; Swift, Candice L. ; Mondo, Stephen J. ; Seppala, Susanna ; Salamov, Asaf ; Singan, Vasanth ; Henrissat, Bernard ; Drula, Elodie ; Henske, John K. ; Lee, Samantha ; et al ( December 2021 , Biotechnology for Biofuels)

   Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungusCaecomyces churrovisand the methanogenMethanobacterium bryantii(not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated inC. churrovisacross a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome ofC. churroviswas obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus.C. churrovispossess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative toC. churrovismonoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation ofmore »genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of theC. churrovisstrain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

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4. Paper-Supported High-Throughput 3D Culturing, Trapping, and Monitoring of Caenorhabditis Elegans

   https://doi.org/10.3390/mi11010099  

   Tahernia, Mehdi ; Mohammadifar, Maedeh ; Choi, Seokheun ( January 2020 , Micromachines)

   We developed an innovative paper-based platform for high-throughput culturing, trapping, and monitoring of C. elegans. A 96-well array was readily fabricated by placing a nutrient-replenished paper substrate on a micromachined 96-well plastic frame, providing high-throughput 3D culturing environments and in situ analysis of the worms. The paper allows C. elegans to pass through the porous and aquatic paper matrix until the worms grow and reach the next developmental stages with the increased body size comparable to the paper pores. When the diameter of C. elegans becomes larger than the pore size of the paper substrate, the worms are trapped and immobilized for further high-throughput imaging and analysis. This work will offer a simple yet powerful technique for high-throughput sorting and monitoring of C. elegans at a different larval stage by controlling and choosing different pore sizes of paper. Furthermore, we developed another type of 3D culturing system by using paper-like transparent polycarbonate substrates for higher resolution imaging. The device used the multi-laminate structure of the polycarbonate layers as a scaffold to mimic the worm’s 3D natural habitats. Since the substrate is thin, mechanically strong, and largely porous, the layered structure allowed C. elegans to move and behave freely in 3Dmore »and promoted the efficient growth of both C. elegans and their primary food, E. coli. The transparency of the structure facilitated visualization of the worms under a microscope. Development, fertility, and dynamic behavior of C. elegans in the 3D culture platform outperformed those of the standard 2D cultivation technique.« less
5. Quality Assessment for Large-Scale Industrial Software Systems: Experience Report at Alibaba

   https://doi.org/10.1109/APSEC48747.2019.00028  

   Zhi, Chen ; Deng, Shuiguang ; Yin, Jianwei ; Fu, Min ; Zhu, Hai ; Li, Yuanping ; Xie, Tao ( December 2019 , Quality Assessment for Large-Scale Industrial Software Systems: Experience Report at Alibaba)

   To assure high software quality for large-scale industrial software systems, traditional approaches of software quality assurance, such as software testing and performance engineering, have been widely used within Alibaba, the world's largest retailer, and one of the largest Internet companies in the world. However, there still exists a high demand for software quality assessment to achieve high sustainability of business growth and engineering culture in Alibaba. To address this issue, we develop an industrial solution for software quality assessment by following the GQM paradigm in an industrial setting. Moreover, we integrate multiple assessment methods into our solution, ranging from metric selection to rating aggregation. Our solution has been implemented, deployed, and adopted at Alibaba: (1) used by Alibaba's Business Platform Unit to continually monitor the quality for 60+ core software systems; (2) used by Alibaba's R&D Efficiency Unit to support group-wide quality-aware code search and automatic code inspection. This paper presents our proposed industrial solution, including its techniques and industrial adoption, along with the lessons learned during the development and deployment of our solution.