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Biosystems such as enzymes, pathways, and whole cells have been increasingly explored for biotechnological applications. However, the intricate connectivity and resulting complexity of biosystems poses a major hurdle in designing biosystems with desirable features. As -omics and other high throughput technologies have been rapidly developed, the promise of applying machine learning (ML) techniques in biosystems design has started to become a reality. ML models enable the identification of patterns within complicated biological data across multiple scales of analysis and can augment biosystems design applications by predicting new candidates for optimized performance. ML is being used at every stage of biosystems design to help find nonobvious engineering solutions with fewer design iterations. In this review, we first describe commonly used models and modeling paradigms within ML. We then discuss some applications of these models that have already shown success in biotechnological applications. Moreover, we discuss successful applications at all scales of biosystems design, including nucleic acids, genetic circuits, proteins, pathways, genomes, and bioprocesses. Finally, we discuss some limitations of these methods and potential solutions as well as prospects of the combination of ML and biosystems design.Triple-negative breast cancer (TNBC) has high rate of metastasis, which is associated with breast cancer stem-like cells (CSCs). Although Taxol (micelle formulation of paclitaxel) is the first line chemotherapy to treat TNBC, it increases CSCs in residual tumors. Abraxane, albumin nanoparticle of paclitaxel, showed lower plasma concentration compared to Taxol in both human and animal models, but it is not clear why Abraxane showed superior efficacy to Taxol in treatment of metastatic breast cancer in human. Adriamycin ic50 In this study, we intend to investigate if Abraxane decreases CSCs for its better efficacy. The results showed that Abraxane showed similar cytotoxicity in SUM149 cells in comparison with Taxol. Although Abraxane showed 3 to 5-fold lower blood drug concentration compared to Taxol, it achieved similar tumor drug concentration and 10-fold higher tumor/plasma ratio in SUM149 xenograft NOD/SCID mouse model. In addition, Abraxane and Taxol showed similar efficacy to shrink the tumor size in orthotopic breast cancer NOD/SCID mouse model. However, Abraxane decreased breast CSCs frequency by 3 to 9-fold, while Taxol increased breast CSCs frequency in orthotopic breast cancer NOD/SCID mouse model. Furthermore, Abraxane increased 3 to 15-fold higher intracellular uptake in both ALDH+ CSCs and differentiated ALDH- cells in comparison with Taxol, which provides a mechanism for Abraxane’s superior efficacy to eliminate CSCs in comparison with Taxol. Our data suggest albumin nanoparticle Abraxane may have a broad implication to enhance drug’s efficacy by eliminating breast cancer stem cells for treatment of metastatic diseases.Synthetic biology is enabling rapid advances in the areas of biomanufacturing and live therapeutics. Dynamic circuits that can be used to regulate cellular resources and microbial community behavior represent a defining focus of synthetic biology, and have attracted tremendous interest. However, the existing dynamic circuits are mostly gene editing-dependent or cell lysis-based, which limits their broad and convenient application, and in some cases, such lysis-based circuits can suffer from genetic instability due to evolution. There is limited research in quorum sensing-assisted CRISPRi, which can function in a gene editing-independent manner. Here, we constructed a series of quorum sensing controlled CRISPRi systems (Q-CRISPRi), which can dynamically program bacteria by using customized sgRNA without introducing cell lysis. We successfully applied Q-CRISPRi circuits to dynamically program gene expression, population density, phenotype, physical property, and community composition of microbial consortia. The strategies reported here represent methods for dynamic cell programming and could be effective in programming industrially and medically important microorganisms to offer better control of their metabolism and behavior.New technologies to target nucleotide diversification in vivo are promising enabling strategies to perform directed evolution for engineering applications and forward genetics for addressing biological questions. Recently, we reported EvolvR-a system that employs CRISPR-guided Cas9 nickases fused to nick-translating, error-prone DNA polymerases to diversify targeted genomic loci-in E. coli. As CRISPR-Cas9 has shown activity across diverse cell types, EvolvR has the potential to be ported into other organisms, including eukaryotes, if nick-translating polymerases can be active across species. Here, we implement and characterize EvolvR’s function in Saccharomyces cerevisiae, representing a key first step to enable EvolvR-mediated mutagenesis in eukaryotes. This advance will be useful for mutagenesis of user-defined loci in the yeast chromosomes for both engineering and basic research applications, and it furthermore provides a platform to develop the EvolvR technology for performance in higher eukaryotes.Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 29 ± 4 μM) through the inhibition of NpsA (KD = 29 ± 4 nM).