Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Comprehensive genetic study of the insulin resistance marker TG:HDL-C in the UK Biobank.

Insulin resistance (IR) is a well-established risk factor for metabolic disease. The ratio of triglycerides to high-density lipoprotein cholesterol (TG:HDL-C) is a surrogate marker of IR. We conducted a genome-wide association study of the TG:HDL-C ratio in 402,398 Europeans within the UK Biobank. We identified 369 independent SNPs, of which 114 had a false discovery rate-adjusted P value < 0.05 in other genome-wide studies of IR making them high-confidence IR-associated loci. Seventy-two of these 114 loci have not been previously associated with IR. These 114 loci cluster into five groups upon phenome-wide analysis and are enriched for candidate genes important in insulin signaling, adipocyte physiology and protein metabolism. We created a polygenic-risk score from the high-confidence IR-associated loci using 51,550 European individuals in the Michigan Genomics Initiative. We identified associations with diabetes, hyperglyceridemia, hypertension, nonalcoholic fatty liver disease and ischemic heart disease. Collectively, this study provides insight into the genes, pathways, tissues and subtypes critical in IR.

Segmentation and evaluation of pathway module efficiency: Quantitative approach to monitor and overcome evolving bottlenecks in xylose to ethanol pathway.

Engineering microbes that can efficiently ferment xylose to ethanol is critical to the development of renewable fuels from lignocellulosic biomass. To accelerate the strain optimization process, a method termed Segmentation and Evaluation of Pathway Module Efficiency (SEPME) was developed to enable rapid and iterative identification and removal of metabolic bottlenecks. Using SEPME, the overall pathway was segmented into two modules: the upstream xylose assimilation pathway and the downstream pentose phosphate pathway, glycolysis, and fermentation. The efficiencies of both modules were then quantified to identify the rate controlling module, followed by analyses of control coefficients, reaction rates, and byproduct concentrations to narrow down targets within the module. SEPME analysis revealed that as the strain was engineered with increasing xylose-to-ethanol yields, the bottlenecks shifted within a module and across the two modules. Guided by SEPME, these bottlenecks were removed one by one, and a strain approaching the theoretical ethanol yield was obtained.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering.

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins ß actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Elucidating structure-performance relationships in whole-cell cooperative enzyme catalysis.

Cooperative enzyme catalysis in nature has long inspired the application of engineered multi-enzyme assemblies for industrial biocatalysis. Despite considerable interest, efforts to harness the activity of cell-surface displayed multi-enzyme assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. In this study, we developed a quantitative approach to whole-cell biocatalyst characterization enabling a comprehensive study of how yeast-surface displayed multi-enzyme assemblies form. Here we show that the multi-enzyme assembly efficiency is limited by molecular crowding on the yeast cell surface, and that maximizing enzyme density is the most important parameter for enhancing cellulose hydrolytic performance. Interestingly, we also observed that proximity effects are only synergistic when the average inter-enzyme distance is > ~130 nm. The findings and the quantitative approach developed in this work should help to advance the field of biocatalyst engineering from trial and error to rational design.

Nanocapsules embedded in microparticles for enhanced oral bioavailability and efficacy of Lopinavir as an anti-AIDS drug.

Lopinavir (LPV), an efficient drug for HIV infection treatment, was incorporated into biodegradable PLGA nanocapsules (NCs) embedded in microparticles (MCPs) using the spray-drying technique in an attempt to bypass the P-gp efflux and protect the drug from CYP3A pre-systemic metabolism without ritonavir (RTV). SEM observations confirmed the formation of NCs and their entrapment in the MCPs. LPV-loaded NCs and free LPV were released from the MCPs at pH of 7.4 as evidenced by in vitro release studies. Results obtained from rat studies showed a two-fold higher bioavailability of LPV following oral administration of the optimal formulation than Kaletra®, the marketed drug, showing that when properly entrapped, LPV can be effectively protected from CYP degradation in the gut as well as from the liver following systemic absorption. It was also shown that serum derived from rats following LPV oral administration in two formulations and Kaletra® significantly decreased the multiplication of HIV-1 in cultured SupT1 cells. Furthermore, the LPV formulations markedly restricted the titre of infectious HIV-1 production compared with Kaletra® confirming the improved antiviral activity of LPV delivered in the rat blood circulation by the nanocapsules embedded in microparticle formulations.

Inhibition of APOBEC3G activity impedes double-stranded DNA repair.

The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition.

Structure-based studies on the metal binding of two-metal-dependent sugar isomerases.

Two-metal-dependent sugar isomerases are important in the synthesis of rare sugars. Many of their properties, specifically their metal dependency, have not been sufficiently explored. Here we used X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and electron paramagnetic resonance spectroscopy to investigate the molecular determinants of the metal-binding affinity of l-rhamnose isomerase, a two-Mn(2+) -dependent isomerase from Bacillus halodurans (BHRI). The crystal structure of BHRI confirmed the presence of two metal ion-binding sites: a structural metal ion-binding site for substrate binding, and a catalytic metal ion-binding site that catalyzes a hydride shift. One conserved amino acid, W38, in wild-type BHRI was identified as a critical residue for structural Mn(2+) binding and thus the catalytic efficiency of BHRI. This function of W38 was explored by replacing it with other amino acids. Substitution by Phe, His, Lys, Ile or Ala caused complete loss of catalytic activity. The role of W38 was further examined by analyzing the crystal structure of wild-type BHRI and two inactive mutants of BHRI (W38F and W38A) in complex with Mn(2+) . A structural comparison of the mutants and the wild-type revealed differences in their coordination of Mn(2+) , including changes in metal-ligand bond length and affinity for Mn(2+) . The role of W38 was further confirmed in another two-metal-dependent enzyme: xylose isomerase from Bacillus licheniformis. These data suggest that W38 stabilizes protein-metal complexes and in turn assists ligand binding during catalysis in two-metal-dependent isomerases. STRUCTURED DIGITAL ABSTRACT: BHRI and BHRI bind by x-ray crystallography (View interaction).

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription.

Cloning and characterization of a rhamnose isomerase from Bacillus halodurans.

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an L-rhamnose isomerase (L-RhI). The identified L-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k(cat) of 8,971 min⁻¹ and a k(cat)/K(m) of 17 min⁻¹mM⁻¹ for L-rhamnose. Although L-RhIs have been characterized from several other sources, B. halodurans L-RhI is distinguished from other L-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans L-RhI a good choice for industrial applications. This work describes one of the most thermostable L-RhI characterized thus far.

In silico studies on the substrate specificity of an l-arabinose isomerase from Bacillus licheniformis.

l-Arabinose isomerase (BLAI) from Bacillus licheniformis was found to be active only with l-arabinose, unlike other l-arabinose isomerases (l-AIs) active with a variety of aldoses. Therefore, the differences in molecular interactions and substrate orientation in the active site of l-AIs have been examined and the residue at position 346 is proposed to be responsible for the unique substrate specificity of BLAI.

Crystallization and preliminary X-ray crystallographic analysis of L-rhamnose isomerase with a novel high thermostability from Bacillus halodurans.

L-Rhamnose isomerases catalyze isomerization between L-rhamnose (6-deoxy-L-mannose) and L-rhamnulose (6-deoxy-L-fructose), which is the first step in rhamnose catabolism. L-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 A, beta = 116.0 degrees . Diffraction data were collected to 2.5 A resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V(M) of 3.0 A(3) Da(-1) and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method.

Probing the molecular determinant for the catalytic efficiency of L-arabinose isomerase from Bacillus licheniformis.

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

Alginate immobilization of recombinant Escherichia coli whole cells harboring L-arabinose isomerase for L-ribulose production.

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca(2+) concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L(-1), respectively. The interactions between Ca(2+) concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for L-ribulose production, the productivity was as high as 56.7 g L(-1) h(-1) at 400 g L(-1) substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of L-ribulose, particularly because of its high stability and low cost.

Characterization of an L-arabinose isomerase from Bacillus subtilis.

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k(cat) of 14,504 min(-1) and a k(cat)/K(m) of 121 min(-1) mM(-1) for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.

Enhanced stability of Bacillus licheniformis L-arabinose isomerase by immobilization with alginate.

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were harvested to prepare alginate-immobilized biocatalysts. The operational conditions for immobilization were optimized according to relative activity and the cell leakage of the immobilized cell. The optimal conditions are as follows: alginate concentration, Ca(2+) concentration, cell mass loading, and curing time were 2% (w/v), 0.1 M, 50 g l(-1), and 4 hours, respectively. After immobilization, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The immobilized whole cells harboring BLAI were very stable with 89% residual activity remaining after 33 days of incubation at 50 degrees C and were much more stable than the free enzyme and cells. The results showed that immobilizing whole cells harboring BLAI is suitable for use as a biocatalyst in the production of L-ribulose, largely due to its high stability and low cost.

Immobilization of Bacillus licheniformis L-arabinose isomerase for semi-continuous L-ribulose production.

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 degrees C (free enzyme) to a range between 55 and 65 degrees C. The half life improved from 2 at 50 degrees C to 212 h at 55 degrees C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.

Cloning and characterization of a novel L-arabinose isomerase from Bacillus licheniformis.

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an L-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni-NTA chromatography. The molecular mass of the purified enzyme was estimated to be approximately 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn(2+)or Co(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50 degrees C, respectively, with a k (cat) of 12,455 min(-1) and a k (cat)/K (m) of 34 min(-1) mM(-1) for L-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for L-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of L-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.

Optimization of culture conditions and scale-up to plant scales for teicoplanin production by Actinoplanes teichomyceticus.

This report describes the optimization of culture conditions for teicoplanin production by Actinoplanes teichomyceticus KCCM-10601, an identified high-teicoplanin-producing strain (US 2006/0134757 A1). Among the conditions tested, temperature, pH, and the dissolved oxygen tension (DOT) were key factors affecting teicoplanin production. When the temperature, pH, and DOT were controlled at 34 degrees C, 7.0 and 20-30%, respectively, a dry-cell weight of 42.8 g l(-1) and a teicoplanin production of 2.9 g l(-1) were obtained after 120 h of batch culture, corresponding to a specific teicoplanin content of 67.8 mg g-DCW(-1). Teicoplanin production was scaled-up from a laboratory scale (7-l fermenter) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity (V tip) as a scale-up parameter. Teicoplanin production at the laboratory scale was similar to those at the pilot and plant scales. This is the highest report of pilot- and plant-scale production of teicoplanin.