Metabolic Engineering: Methodologies and Applications.

Metabolic engineering aims to improve the production of economically valuable molecules through the genetic manipulation of microbial metabolism. While the discipline is a little over 30 years old, advancements in metabolic engineering have given way to industrial-level molecule production benefitting multiple industries such as chemical, agriculture, food, pharmaceutical, and energy industries. This review describes the design, build, test, and learn steps necessary for leading a successful metabolic engineering campaign. Moreover, we highlight major applications of metabolic engineering, including synthesizing chemicals and fuels, broadening substrate utilization, and improving host robustness with a focus on specific case studies. Finally, we conclude with a discussion on perspectives and future challenges related to metabolic engineering.

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture.

To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA-MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA-MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.

Controlling circuitry underlies the growth optimization of Saccharomyces cerevisiae.

Microbial growth emerges from coordinated synthesis of various cellular components from limited resources. In Saccharomyces cerevisiae, cyclic AMP (cAMP)-mediated signaling is shown to orchestrate cellular metabolism; however, it remains unclear quantitatively how the controlling circuit drives resource partition and subsequently shapes biomass growth. Here we combined experiment with mathematical modeling to dissect the signaling-mediated growth optimization of S. cerevisiae. We showed that, through cAMP-mediated control, the organism achieves maximal or nearly maximal steady-state growth during the utilization of multiple tested substrates as well as under perturbations impairing glucose uptake. However, the optimal cAMP concentration varies across cases, suggesting that different modes of resource allocation are adopted for varied conditions. Under settings with nutrient alterations, S. cerevisiae tunes its cAMP level to dynamically reprogram itself to realize rapid adaptation. Moreover, to achieve growth maximization, cells employ additional regulatory systems such as the GCN2-mediated amino acid control. This study establishes a systematic understanding of global resource allocation in S. cerevisiae, providing insights into quantitative yeast physiology as well as metabolic strain engineering for biotechnological applications.

Understanding the Excited-State Relaxation Mechanisms of Xanthophyll Lutein by Multi-configurational Electronic Structure Calculations.

The contradictory behaviors in light harvesting and non-photochemical quenching make xanthophyll lutein the most attractive functional molecule in photosynthesis. Despite several theoretical simulations on the spectral properties and excited-state dynamics, the atomic-level photophysical mechanisms need to be further studied and established, especially for an accurate description of geometric and electronic structures of conical intersections for the lowest several electronic states of lutein. In the present work, semiempirical OM2/MRCI and multi-configurational restricted active space self-consistent field methods were performed to optimize the minima and conical intersections in and between the 1Ag-, 2Ag-, 1Bu+, and 1Bu- states. Meanwhile, the relative energies were refined by MS-CASPT2(10,8)/6-31G*, which can reproduce correct electronic state properties as those in the spectroscopic experiments. Based on the above calculation results, we proposed a possible excited-state relaxation mechanism for lutein from its initially populated 1Bu+ state. Once excited to the optically bright 1Bu+ state, the system will propagate along the key reaction coordinate, i.e., the stretching vibration of the conjugated carbon chain. During this period of time, the 1Bu- state will participate in and forms a resonance state between the 1Bu- and 1Bu+ states. Later, the system will rapidly hop to the 2Ag- state via the 1Bu+/2Ag- conical intersection. Finally, the lutein molecule will survive in the 2Ag- state for a relatively long time before it internally converts to the ground state directly or via a twisted S1/S0 conical intersection. Notably, though the photophysical picture may be very different in solvents and proteins, the current theoretical study proposed a promising calculation protocol and also provided many valuable mechanistic insights for lutein and similar carotenoids.

Directed Evolution: Methodologies and Applications.

Directed evolution aims to expedite the natural evolution process of biological molecules and systems in a test tube through iterative rounds of gene diversifications and library screening/selection. It has become one of the most powerful and widespread tools for engineering improved or novel functions in proteins, metabolic pathways, and even whole genomes. This review describes the commonly used gene diversification strategies, screening/selection methods, and recently developed continuous evolution strategies for directed evolution. Moreover, we highlight some representative applications of directed evolution in engineering nucleic acids, proteins, pathways, genetic circuits, viruses, and whole cells. Finally, we discuss the challenges and future perspectives in directed evolution.

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids.

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs) as key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the composition of microbially synthesized FFAs is still limited, particularly, for producing medium-chain fatty acids (MCFAs). This is mainly due to the lack of high-throughput approaches for FFA analysis to engineer enzymes with desirable product specificity. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. In particular, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and a higher m/z peak ratio at 730 and 758 was used as an indication for improved MCFA production. This colony-based method can be performed at a rate of ~2 s per sample, representing a substantial improvement over gas chromatography-MS (typically >30 min per sample) as the gold standard method for FFA detection. To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified nine missense mutations that resulted in improved MCFA production relative to the wild-type strain. Colony-based MALDI-ToF MS screening provides an effective approach for engineering microbial fatty acid compositions in a high-throughput manner.

Screening and characterization of apical membrane antigen 1 interacting proteins in Eimeria tenella.

Avian coccidiosis is a widespread and economically significant disease of poultry. It is an enteric disease caused by several protozoan Eimeria species. Eimeria belongs to the phylum Apicomplexa, which exhibits an unusual mechanism of host cell invasion. During invasion of host cells, the protein apical membrane antigen 1 (AMA1) is essential for invasion of Toxoplasma gondii and Plasmodium. Contrary to the roles of AMA1 during host cell invasion in T. gondii and Plasmodium, the precise functions of Eimeria AMA1 (EtAMA1) are unclear. In order to study the functions of EtAMA1, a yeast two-hybrid cDNA library was constructed from E. tenella sporozoites. The EtAMA1 ectodomain was cloned into the pGBKT7 vector to construct the bait plasmid pGBKT7- EtAMA1. Autoactivation and toxicity of the bait protein in yeast cells were tested by comparison with the pGBKT7 empty vector. Expression of the bait protein was detected by western blots. The bait plasmid pGBKT7-EtAMA1 was used to screen yeast two-hybrid cDNA library from E. tenella sporozoites. After multiple screenings with high-screening-rate medium and exclusion of false-positive plasmids, positive preys were sequenced and analyzed using BLAST. We obtained 14 putative EtAMA1-interacting proteins including E. tenella acidic microneme protein2 (EtMIC2), E. tenella putative cystathionine beta-synthase, E. tenella Eimeria-specific protein, four E. tenella conserved hypothetical proteins (one in the serine/threonine protein kinase family) and seven unknown proteins. Gene Ontology analysis indicated that two known proteins were associated with metabolic process, pyridoxal phosphate binding and protein phosphorylation. Functional analysis indicated EtMIC2 was implicated in parasite motility, migration, recognition and invasion of host cells. The data suggested that EtAMA1 may be important during host cell invasion, but also involved in other biological processes.

Downregulating hypoxia-inducible factor-2α improves the efficacy of doxorubicin in the treatment of hepatocellular carcinoma.

The hypoxic microenvironment inside solid tumors, including hepatocellular carcinoma (HCC), is a major cause of tumor resistance to chemotherapy. The recently identified hypoxia-inducible factor (HIF)-2 executes the hypoxia response. Its expression feature and transcriptional targets indicate a possible dominance of HIF-2 in regulating genes in HCC. The aim of the present study was to determine whether transfection of siRNA targeting HIF-2α could enhance the efficacy of doxorubicin, the most commonly used drug in the treatment of HCC. Transfection of HIF-2 siRNA into human HCC cells downregulated the expression of HIF-2α, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-α, and cyclin D1, but had little effect on the expression of HIF-1α, fms-related tyrosine kinase-1 (Flt-1), the glucose transporter (GLUT)-1, and lactate dehydrogenase A (LDHA). Doxorubicin itself only downregulated VEGF expression. Furthermore, HIF-2 siRNA inhibited proliferation, induced cell cycle arrest at the G(0)/G(1) phase, and acted synergistically with doxorubicin to inhibit the growth of human HCC cells in vitro. Transfection of HIF-2 siRNA also downregulated tumoral expression of HIF-2α, VEGF, TGF-α, and cyclin D1 in vivo, and acted synergistically with doxorubicin to suppress the growth of HepG2 tumors established in immunodeficient mice by inhibiting cell proliferation, tumor angiogenesis and microvessel perfusion. The results of the present study suggest that targeting HIF-2α with siRNA warrants investigation as a potential strategy to enhance the efficacy of doxorubicin in the treatment of HCC.

PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction.

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments. PlasmidMaker should greatly expand the potential of synthetic biology.

macroMS: Image-Guided Analysis of Random Objects by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Mass spectrometry imaging is well-suited to characterizing sample surfaces for their chemical content in a spatially resolved manner. However, when the surface contains small objects with significant empty spaces between them, more efficient approaches to sample acquisition are possible. Image-guided mass spectrometry (MS) enables high-throughput analysis of a diverse range of sample types, such as microbial colonies, liquid microdroplets, and others, by recognizing and analyzing selected location targets in an image. Here, we describe an imaging protocol and macroMS, an online software suite that can be used to enhance MS measurements of macroscopic samples that are imaged by a camera or a flatbed scanner. The web-based tool enables users to find and filter targets from the optical images, correct optical distortion issues for improved spatial location of selected targets, input the custom geometry files into an MS device to acquire spectra at the selected locations, and finally, perform limited data analysis and use visualization tools to aid locating samples containing compounds of interest. Using the macroMS suite, an enzyme mutant library of Saccharomyces cerevisiae and nL droplet arrays of Escherichia coli and Pseudomonas fluorescens have been assayed at a rate of ∼2 s/sample.

Optically guided mass spectrometry to screen microbial colonies for directed enzyme evolution.

Directed evolution is a well-established and powerful tool for enzyme engineering, which consists of iterative rounds of creating and screening a library of variants. In many cases, the ability to characterize these variants in high-throughput remains a bottleneck. In addition, profiling of desired candidates becomes even more challenging when engineering multiple enzymes in a biochemical pathway. In this chapter, we describe a label-free, high-throughput method for the engineering of multistep enzymatic reactions in bacterial colonies via optically guided matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). This method is able to detect products, reactants, and byproducts with high sensitivity and accuracy. We demonstrate its effectiveness in two applications related to natural product biosynthesis, including facile creation of analog of the peptidic antibiotic plantazolicin and rapid profiling of congeners of rhamnolipid. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. In both cases, improved MS acquisition efficiency and information-rich insights were obtained through this technique on large populations of colonies at a rate of 1-2.5s per colony. This method should be generally applicable to high-throughput phenotyping of microbial libraries from a wide range of enzymatic reactions.

A revised Ladevèze criteria for carbon fiber reinforced laminated plates.

All parameters of revised Ladevèze failure criterion in Table 1 were determined based on mechanical tests which mainly include quasi-static tensile experiments, quasi-static compressive experiments, quasi-static tensile cyclic loading experiments, the dynamic tensile experiments, dynamic compressive experiments, quasi-static and dynamic inter-laminar shear experiments. The quasi-static experiments were performed using an electronic universal testing machine with a maximum load capacity of 10KN, and the split Hopkinson pressure bar (SHPB) and split Hopkinson tension bar (SHTB) were employed in dynamic experiments. In addition, the parameters of traditional orthogonal anisotropic model and cohesive layer for the laminated plates are listed in Table 2.

A model for the impact of FFPE section thickness on gene copy number measurement by FISH.

Fluorescent in situ hybridization (FISH) assays to detect gene amplification such as HER2 or MET in tumors are used for prognosis evaluation and selection of targeted therapies. Although FISH guidelines recommended 4~6 µm FFPE sections, many laboratories use 2~3 µm sections, which is a common practice for H&E staining and immunohistochemistry. A former study concluded that section thickness did not affect FISH results. We found, however, that thinner FFPE sections may lead to false negative results for gene amplification. A mathematic model was constructed and cell-line based controls with known gene copy number were prepared, and the model had a reasonable fit with the experimental data. The model revealed that even when counting the apparently full-sized nuclear images, many of them have partial volumes, which leads to under-estimation of gene copy number. Therefore, improperly thinner sections are prone to give false negative results, and thicker sections give a better approximation to the true value. The discrepancy between this and the former study was discussed. In summary, the model applies generally to FISH/ISH detection of gene copy number, and section thickness is an important parameter to control for precision medicine research, assay development, clinical trials and daily practice in pathology laboratory.

Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.

We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

Alpha-glucosidase inhibition of natural curcuminoids and curcumin analogs.

Natural curcumin (1), demethoxycurcumin (2) and bisdemethoxycurcumin (3) isolated from Curcuma longa (turmeric), and synthetic curcumin analogs (A(1-7), B(1-7), C(1-6) and D(1-7)) were evaluated in vitro for the alpha-glucosidase inhibitory activity via UV and circular dichroism (CD) spectroscopy. The results indicated that natural curcuminoid compound 3 showed a remarkable inhibitory effect with IC(50) of 23.0 microM, and the synthetic compounds A(2), B(2), C(2) and D(2) showed potent inhibitory effects with IC(50) of 2.8, 2.6, 1.6 and 8.2 microM, respectively. Kinetic study exhibited that the mechanism of alpha-glucosidase inhibition of both 3 and C(2) was non-competitive. The structure activity relationship revealed that the ortho dihydroxyl groups could form a more tight interaction with alpha-glucosidase to exert more potential inhibitory activities.

A simple self-assembly method for colloidal photonic crystals with a large area.

Colloidal photonic crystals were fabricated using polystyrene particles (180 nm) and PMMA particles (450 nm), respectively, with a new and simple self-assembly method without special equipment. SEM images indicate that the prepared samples have ordered structures with few defects. The position of the stop-band scale nicely agrees with the particles' size. The sintering process of the PS photonic crystal film was studied with AFM heating system.

Up-regulation of survivin by AKT and hypoxia-inducible factor 1α contributes to cisplatin resistance in gastric cancer.

This study investigated the contribution of survivin and its upstream regulators, AKT and hypoxia-inducible factor 1α (HIF-1α), to the resistance of gastric cancer cells to cisplatin (CDDP). We found that over-expression of survivin increased the resistance of SGC7901 and BGC823 gastric cancer cells to CDDP. Its over-expression abrogated CDDP-induced inhibition of cell proliferation and CDDP-induced cell apoptosis. In contrast, down-regulation of survivin expression using small hairpin RNA (shRNA) vectors and the small-molecule inhibitor YM155, or inhibition of survivin function using a recombinant cell-permeable dominant-negative survivin protein (dNSur9), promoted CDDP-induced apoptosis. CDDP-resistant sub-lines generated from the parental SGC7901 and BGC823 cells by exposure to increasing concentrations of CDDP expressed higher levels of HIF-1α and survivin in response to hypoxia, and higher levels of phosphorylated AKT (pAKT). Specific inhibition of AKT reduced the expression of HIF-1α and survivin, whereas specific inhibition or depletion of HIF-1α reduced survivin expression but had no effect on the expression of phosphorylated AKT. The expression levels of survivin affected the therapeutic efficacy of CDDP in treating gastric tumors in mice. Specific inhibition of survivin, AKT and HIF-1α enhanced the sensitivity of CDDP-resistant cells to CDDP. Specific inhibition of survivin, AKT and HIF-1α synergized with CDDP to suppress the growth of gastric tumors that had been engineered to overexpress survivin. In summary, the results provide evidence that up-regulation of survivin by AKT and HIF-1α contributes to CDDP resistance, indicating that inhibition of these pathways may be a potential strategy for overcoming CDDP resistance in the treatment of gastric cancer.