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Wei, Hui

Publications and source records attributed to Wei, Hui.

14 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli : bioinformatic and experimental approaches

Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO 2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO 2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

B56$δ$ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan’s syndrome mutations

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Continuous multimodal technologies in industrial microbiology: potential for achieving high process performance and agility

The field of industrial microbiology has great potential to tackle the most pressing challenges facing society, but today, this entails much more than classical fermentation technology followed by product upgrading. Methods are needed to confer unit-operation-level functionality and whole-system-level robustness, as well as flexibility. As examples of this concept, we focus here on methodologies often deployed at industrial scale as discrete processes—such as biological, chemical, and electrochemical unit operations—that are also capable of substantial complementarity when combined sequentially and operated continuously. A key and relatively new type of conversion step is made possible by catalyst immobilization methods at both the enzymatic and cellular level. Cases drawn from electro-fermentation for acetoin production and syngas-mediated microbial electrosynthesis of ethanol exemplify the synergy of combined operational approaches. Because modular processes can be treated as a series of reactor elements, considerable flexibility is possible. As discussed, however, not all processes are amenable to this intensification.

09 BIOMASS FUELS↗
DOE OSTI2023

Prospects for engineering Ralstonia eutropha and Zymomonas mobilis for the autotrophic production of 2,3-butanediol from CO 2 and H 2

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO 2 emissions make CO 2 an attractive feedstock for manufacturing chemicals. Moreover, H 2 is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water (scH 2 O) gasification enable sustainable production of molecular hydrogen (H 2 ). We review the recent advances in engineering Ralstonia eutropha, the representative species of "Knallgas" bacteria, for utilizing CO 2 and H 2 to autotrophically produce 2,3-butanediol (2,3-BDO). This assessment is focused on state-of-the-art approaches for splitting H 2 to supply energy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO 2 fixation. Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO 2 and H 2 -utilizing microorganisms, exemplified by Zymomonas mobilis.

42 ENGINEERING↗
DOE OSTI2022

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 ( Snf1 ) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase ( ACL ) and diacylglycerol acyltransferase 1 ( DGA1 ) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

09 BIOMASS FUELS↗
DOE OSTI2021

Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

Cell free biocatalysis is showing promise as a replacement or complement to conventional microbial biocatalysts due to the potential for achieving high yields, titers, and productivities. However, there exist several challenges that need to be addressed before its broader industrial adoption is achieved. New paradigms and innovative solutions are needed to overcome these challenges. In this study we demonstrate high levels of glycerol conversion to 1,3-propanediol using a self-assembling metabolic pathway leveraging the arraying strategy (protein scaffolds) used by thermophilic cellulolytic bacteria to assemble their biomass degrading enzymes. These synthetic metabolons were capable of producing 1,3-PDO at a yield more than 95% at lower glycerol concentration and close to 70% at higher concentrations at a higher productivity rate than the equivalent microbial strain. One of the benefits of our approach is the fact that no enzyme purification is required, and that the assembly of the complex is accomplished in vivo before immobilization, while product formation is conducted in vitro. We also report the recovery of enzymatic activity upon fusion enzymes binding to these protein scaffolds, which could have broader applications when assembling arrayed protein complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass

Abstract Background Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. Results In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. Conclusions We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2021

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metrology of Time-Domain Soft X-Ray Attosecond Pulses and Reevaluation of Pulse Durations of Three Recent Experiments

Attosecond pulses in the soft x-ray (SXR) to water-window energy region offer the tools for creating and studying target-specific localized inner-shell electrons or holes in materials, enabling monitoring or controlling charge and energy flows in a dynamic system on attosecond timescales. Recently, a number of laboratories have reported generation of continuum harmonics in the hundred-electron-volt to kilovolt region with few-cycle long-wavelength mid-infrared lasers. These harmonics have the bandwidth to support pulses with durations of a few to a few-ten attoseconds. But harmonics generated in a gas medium have attochirps that cannot be fully compensated by materials over a broad spectral range; thus, realistically what are the typical shortest attosecond pulses that one can generate? To answer this question, it is essential that the temporal attosecond pulses be accurately characterized. By reanalyzing the soft x-ray attosecond metrology reported in three recent experiments using a newly developed broadband phase retrieval algorithm, we demonstrate here that the generated attosecond pulses in the first two papers have durations of about 60 as, longer than what they have reported. Similarly, the duration from the third experiment is retrieved to be about half of the 322-as upper limit cited in that work. We also introduce the autocorrelation (AC) of the streaking spectrogram. By comparing the ACs from the experiments and from the retrieved SXR pulses, the accuracy of the retrieved results can be directly visualized. Our retrieval method is fast and accurate, and it shall provide a powerful tool for the metrology of the emerging few-ten-attosecond pulses.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2020

Ferrous and Ferric Ion-Facilitated Dilute Acid Pretreatment of Lignocellulosic Biomass under Anaerobic or Aerobic Conditions: Observations of Fe Valence Interchange and the Role of Fenton Reaction

Ferrous ion co-catalyst enhancement of dilute-acid (DA) pretreatment of biomass is a promising technology for increasing the release of sugars from recalcitrant lignocellulosic biomass. However, due to the reductive status of ferrous ion and its susceptibility to oxidation with exposure to atmosphere, its effective application presumably requires anaerobic aqueous conditions created by nitrogen gas-purging, which adds extra costs. The objective of this study was to assess the effectiveness of oxidative iron ion, (i.e., ferric ion) as a co-catalyst in DA pretreatment of biomass, using an anaerobic chamber to strictly control exposure to oxygen during setup and post-pretreatment analyses. Remarkably, the ferric ions were found to be as efficient as ferrous ions in enhancing sugar release during DA pretreatment of biomass, which may be attributed to the observation that a major portion of the initial ferric ions were converted to ferrous during pretreatment. Furthermore, the detection of hydrogen peroxide in the liquors after DA/Fe ion pretreatment suggests that Fenton reaction chemistry was likely involved in DA/Fe ion pretreatments of biomass, contributing to the observed ferric and ferrous interchanges during pretreatment. These results help define the extent and specification requirements for applying iron ions as co-catalysts in DA pretreatments of biomass.

09 BIOMASS FUELS↗
DOE OSTI2020

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

09 BIOMASS FUELS↗