High-throughput enzyme engineering for commercial-scale production of natural products

The natural products world is unparalleled in its molecular diversity and wide application space. There are however numerous challenges associated with realizing the full potential of these molecules. Amyris has fermentatively produced eight different molecules at commercial scale. This track record is due to investment in advanced tools for strain engineering, high throughput screening, analytics, and bioinformatics. An integrated pipeline encompassing these tools has enabled Amyris to rapidly accelerate the engineering cycle and reduce the number of design-build-test iterations needed for microbial production of any natural product. In this presentation, we will discuss how this infrastructure is now being leveraged for high-throughput enzyme mutagenesis and screening, enabling greater access to natural products and their derivatives. Further, the application of our massive screening infrastructure to enzyme libraries would not be possible without equally sophisticated statistical models and data analysis tools. Scientists at Amyris are accessing ever greater portions of the enzyme sequence space to improve specificity and activity – ultimately enabling sustainable industrial-scale production of natural products. This talk will describe how each aspect of the enzyme engineering pipeline has led to rapid and high-quality screening of hundreds of thousands of mutants for multiple enzymes

High-throughput enzyme engineering for commercial-scale production of natural products

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High-throughput enzyme engineering for commercial-scale production of natural products

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Effect of different rice transplanting patterns on microbial community in water, sediment, and Procambarus clarkii intestine in rice-crayfish system

Although the microbial ecology of integrated rice-crayfish farming systems is receiving increasing attention with the expanding application area in China, the effects of rice transplanting patterns on the microbial community of water, sediment and Procambarus clarkii intestine in rice-crayfish system has yet to be determined. This study explored the microbial community present in water, sediment and intestine samples from three transplant patterns (rice crayfish with wide-narrow row transplanting, rice-crayfish with normal transplanting and pond-crayfish, abbreviated as RC-W, RC, and PC, respectively) using high-throughput sequencing. The results showed that the dominant microbial taxa from sediment, surrounding water, and intestine at phylum level were Proteobacteria, Chloroflexi, Cyanobacteria, Actinobacteria, Bacteroidetes. The patterns of rice transplanting had significant effects on microbial biodiversity and species composition in surrounding water. The OTUs community richness of water under RC group was significantly higher than that of PC group and RC-W group. The OTU relative abundance of top 10 operational taxonomic units had significantly different (p < 0.05) in the water samples from the three groups. The intestinal OTU community richness of Procambarus clarkii in the three groups was positively correlated with the community richness of water. The proximity between intestinal and water samples in PCA diagram indicated that their species composition was more similar. The results also showed that rice transplanting patterns can affect intestinal microbial biodiversity of Procambarus clarkii and the intestinal microbial biodiversity correlated with water bodies. Although the intestinal microbial diversity of crayfish in RC-W group was lower than that in RC group, the relative abundance of potential pathogenic bacteria, such as Vibrio, Aeromonas, in intestine of the crayfish in the RC-W group was significantly decreased under rice wide-narrow row transplanting model. Redundancy analysis revealed that environmental parameters, such as pH, DO, nitrate, which regulate the composition of microbial community structures. This study provides an understanding for microbial response to different rice transplanting pattern in rice-crayfish farming system

MoIVD-Mediated Leucine Catabolism Is Required for Vegetative Growth, Conidiation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae

Isovaleryl-CoA dehydrogenase (IVD), a member of the acyl-CoA dehydrogenase (ACAD) family, is a key enzyme catalyzing the conversion of isovaleryl-CoA to β-methylcrotonyl-CoA in the third reaction of the leucine catabolism pathway and simultaneously transfers electrons to the electron-transferring flavoprotein (ETF) for ATP synthesis. We previously identified the ETF ortholog in rice blast fungus Magnaporthe oryzae (MoETF) and showed that MoETF was essential for fungal growth, conidiation and pathogenicity. To further investigate the biological function of electron-transferring proteins and clarify the role of leucine catabolism in growth and pathogenesis, we characterized MoIVD (M. oryzaeisovaleryl-CoA dehydrogenase). MoIvd is highly conserved in fungi and its expression was highly induced by leucine. The Δmoivd mutants showed reduced growth, decreased conidiation and compromised pathogenicity, while the conidial germination and appressorial formation appeared normal. Consistent with a block in leucine degradation, the Δmoivd mutants accumulated isovaleric acid, grew more slowly, fully lacked pigmentation and completely failed to produce conidia on leucine-rich medium. These defects were largely rescued by raising the extracellular pH, suggesting that the accumulation of isovaleric acid contributes to the growth and conidiation defects. However, the reduced virulence of the mutants was probably due to their inability to overcome oxidative stress, since a large amount of ROS (reactive oxygen species) accumulated in infected host cell. In addition, MoIvd is localized to mitochondria and interacted with its electron receptor MoEtfb, the β subunit of MoEtf. Taken together, our results suggest that MoIVD functions in leucine catabolism and is required for the vegetative growth, conidiation and full virulence of M. oryzae, providing the first evidence for IVD-mediated leucine catabolism in the development and pathogenesis of plant fungal pathogens

Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors

The mechanism of autoprocessing and catalysis in human asparaginase-like protein 1

Differences in metabolic pathways between cancer and normal tissue can lead to a new generation of protease therapeutics. Heterologous enzymes to deplete specific amino acids that are deficient in cancer cells have applications for treating a number of cancers. One successful example is acute lymphoblastic leukemia (ALL) treatment with bacterial L-asparaginase to systematically deplete L-asparagine (L-Asn). However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity and secondary specificity. An alternative strategy using a human version of asparaginase with higher substrate specificity was not available until the recent identification of human asparaginase-like protein 1 (hASRGL1). In order to provide good guidance for hASRGL1 therapeutic engineering, in this dissertation study, we use a combination of structural biology, kinetics, protein engineering and a novel method of differential scanning fluorometry to study the enzyme mechanism of hASRGL1. Autoprocessing is a critical post-translation modification step for hASRGL1 function. It is intertwined with substrate catalysis which fetters the study of hASRGL1 enzyme mechanism. To effectively tackle this problem and to gain a good control over activation of hASRGL1 for therapeutic application, we circumvented this obstacle by a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like, hASRGL1-Thr168Ala variant. In doing so, we revealed that a torsional restraint on the Thr168 side-chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation (Chapter 2). Following that, we further delineated the roles of several critical residues in catalyzing autoprocessing and/or substrate catalysis using the uncoupled system of WT-hASRGL1 and cp-hSARGL1 (Chapter3). Furthermore, we developed a de novo method using differential scanning fluorometry to quantitatively analyze the autoprocessing reaction and, for the first time, identified three distinct molecular complexes during maturation that confirmed the existing of half processed heterotrimer. Finally, we found that the dimer-dimer interface is critical for stabilization of the αβ-βα homodimer and the rest of the autoprocessing reaction through a mutagenesis study.Biochemistr