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Engineering topics

Zhao, Huimin

Publications and source records attributed to Zhao, Huimin.

71 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

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. Furthermore, 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.

47 OTHER INSTRUMENTATION↗
DOE OSTI2020

Unlocking nature’s biosynthetic potential by directed genome evolution

Microorganisms have been increasingly explored as microbial cell factories for production of fuels, chemicals, drugs, and materials. Among the various metabolic engineering strategies, directed genome evolution has emerged as one of the most powerful tools to unlock the full biosynthetic potential of microorganisms. Furthermore we summarize the directed genome evolution strategies that have been developed in recent years, including adaptive laboratory evolution and various targeted genome-scale engineering strategies, and discuss their applications in basic and applied biological research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Stereoconvergent Reduction of Activated Alkenes by a Nicotinamide Free Synergistic Photobiocatalytic System

There is a growing interest in developing cooperative chemoenzymatic reactions to harness the reactivity of chemical catalysts and the selectivity of enzymes for the synthesis of nonracemic chiral compounds. However, existing chemoenzymatic systems with more than one chemical reaction and one enzymatic reaction working cooperatively are rare. Moreover, the application of oxidoreductases in cooperative chemoenzymatic reactions is limited by the necessity of using expensive and unstable redox equivalents such as nicotinamide cofactors. Here we report a lightdriven cooperative chemoenzymatic system comprised of a photoinduced electron transfer reaction (PET) and a photosensitized energy transfer reaction (PEnT) with enzymatic reduction in one-pot to synthesize chiral building blocks of bioactive compounds. As a proof of concept, ene-reductase was directly regenerated by PET in the absence of external cofactors. Meanwhile, enzymatic reduction worked cooperatively with photocatalyst-catalyzed energy transfer that continuously replenished the reactive isomer from the less reactive one. The whole system stereoconvergently reduced E/Z mixtures of alkenes to the enantiopure products. Additionally, enantioselective enzymatic reduction worked competitively with photocatalyst-catalyzed racemic background reaction and side reactions to channel the overall electron flow to the single enantiopure product. As a result, such a light-driven cooperative chemoenzymatic system holds great potential for asymmetric synthesis using inexpensive petroleum or biomass derived alkenes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Data from Stereoconvergent Reduction of Activated Alkenes by a Nicotinamide Free Synergistic Photobiocatalytic System

There is a growing interest in developing cooperative chemoenzymatic reactions to harness the reactivity of chemical catalysts and the selectivity of enzymes for the synthesis of nonracemic chiral compounds. However, existing chemoenzymatic systems with more than one chemical reaction and one enzymatic reaction working cooperatively are rare. Moreover, the application of oxidoreductases in cooperative chemoenzymatic reactions is limited by the necessity of using expensive and unstable redox equivalents such as nicotinamide cofactors. Here, we report a light-driven cooperative chemoenzymatic system comprised of a photoinduced electron transfer reaction (PET) and a photosensitized energy transfer reaction (PEnT) with an enzymatic reduction in one-pot to synthesize chiral building blocks of bioactive compounds. As a proof of concept, ene-reductase was directly regenerated by PET in the absence of external cofactors. Meanwhile, enzymatic reduction worked cooperatively with photocatalyst-catalyzed energy transfer that continuously replenished the reactive isomer from the less reactive one. The whole system stereoconvergently reduced E/Z mixtures of alkenes to the enantiopure products. Additionally, enantioselective enzymatic reduction worked competitively with photocatalyst-catalyzed racemic background reaction and side reactions to channel the overall electron flow to the single enantiopure product. Such a light-driven cooperative chemoenzymatic system holds great potential for asymmetric synthesis using inexpensive petroleum or biomass-derived alkenes.

Catalysis↗
DOE OSTI2020

Photoenzymatic enantioselective intermolecular radical hydroalkylation

Enzymes are increasingly explored for asymmetric synthesis, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis has spurred the discovery of new reactivity from known enzymes. Yet, so far photo-induced enzymatic catalysis has not been used for cross-coupling of two molecules. For instance, intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in stereochemical control of the remote prochiral radical intermediate. Here we show a new-to-nature, visible-light-induced ene-reductase catalysed intermolecular radical hydroalkylation of terminal alkenes with readily available α-halo carbonyl compounds. This method provides an efficient approach to various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99% yield, 99% enantiomeric excess), which otherwise are difficult to access by chemocatalysis. Mechanistic studies suggest that the substrates/ene-reductase complex formation at the enzyme active site triggers the enantioselective photo-induced radical reaction. Our research further expands the reactivity repertoire of biocatalytic, synthetically-useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Biosystems Design by Machine Learning

Biosystems such as enzymes, pathways, and whole cells have been increasingly explored for biotechnological applications. Yet, the intricate connectivity and complexity of biosystems pose a major hurdle in designing biosystems with desired 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 non-obvious 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 bioprocess. Lastly, we discuss some limitations of these methods and potential solutions as well as prospects of the combination of ML and biosystems design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems

Abstract Metabolic engineering to divert carbon flux from sucrose to oil in high biomass crop like sugarcane is an emerging strategy to boost lipid yields per hectare for biodiesel production. Sugarcane stems comprise more than 70% of the crops' biomass and can accumulate sucrose in excess of 20% of their extracted juice. The energy content of oils in the form of triacylglycerol (TAG) is more than twofold that of carbohydrates. Here, we report a step change in TAG accumulation in sugarcane stem tissues achieving an average of 4.3% of their dry weight (DW) in replicated greenhouse experiments by multigene engineering. The metabolic engineering included constitutive co‐expression of wrinkled1; diacylglycerol acyltransferase1‐2; cysteine‐oleosin; and ribonucleic acid interference‐suppression of sugar‐dependent1. The TAG content in leaf tissue was also elevated by more than 400‐fold compared to non‐engineered sugarcane to an average of 8.0% of the DW and the amount of total fatty acids reached about 13% of the DW. With increasing TAG accumulation an increase of 18:1 unsaturated fatty acids was observed at the expense of 16:0 and 18:0 saturated fatty acids. Total biomass accumulation, soluble lignin, Brix and juice content were significantly reduced in the TAG hyperaccumulating sugarcane lines. Overcoming this yield drag by engineering lipid accumulation into late stem development will be critical to exceed lipid yields of current oilseed crops.

09 BIOMASS FUELS↗
DOE OSTI2020

Recent Advances in Domesticating Non-model Microorganisms

Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. Yet, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. Furthermore, we highlight some successful applications of non-model microorganisms as microbial cell factories.

synthetic biology↗
DOE OSTI2020

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), key components of crucial nutrients, soaps, industrial chemicals, and fuels. Yet, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method offers an alternative way for engineering microbial fatty acid compositions in a high-throughput manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data from 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.

Conversion↗
DOE OSTI2020

Integrating biocatalysis with chemocatalysis for selective transformations

The integration of biocatalysis with chemocatalysis combines the excellent selectivity of the former with the robust reactivity of the latter and offers many advantages, such as lower cost, higher yield, enhanced selectivity, as well as less waste generation. Despite the challenge of incompatibilities between different classes of catalysts, recent advances in synthetic chemistry and biology provide ample opportunities for multistep cascade transformations that combine biocatalysis and chemocatalysis. In this research, we review recent progress in merging biocatalysis with chemocatalysis, highlighting selected examples of photo-/electricity-driven biotransformations and recently developed strategies for addressing the catalyst incompatibility issue.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

A genetic toolbox for metabolic engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. Yet, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As evidence of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data from A Genetic Toolbox for Metabolic Engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis , which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis .

Conversion↗
DOE OSTI2019

Assembling Reusable Genetic Modules for Efficient Biofuel Production from Marine Macroalgae

This project investigated microbial biodiversity capable of degrading algal polysaccharides to investigate potential for biofuel production. This project uncovered the role of ecological interactions among closely related strains of bacteria necessary for degradation of common polysaccharides, which had not been previously appreciated. In addition new bacterial isolates and genes were identified that can be used in future projects aimed at biofuel production. Finally, these enzymes were characterized for their efficiency under a range of environmental parameters, and encapsulated for future use in genetic engineering studies.

09 BIOMASS FUELS↗
DOE OSTI2019

Photoenzymatic Enantioselective Intermolecular Radical Hydroalkylation: Experimental Crystal Structure Determination

Enzymes are increasingly explored for use in asymmetric synthesis, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis has spurred the discovery of novel reactivity from known enzymes. However, so far photoinduced enzymatic catalysis has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an ‘ene’ reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 percent yield with 99 percent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the ‘ene’ reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Huang, Xiaoqiang↗