Cell-free Lysate Research Articles

An Efficient Gas Chromatography–Mass Spectrometry Approach for the Simultaneous Analysis of Deoxynivalenol and Its Bacterial Metabolites 3-keto-DON and 3-epi-DON

Deoxynivalenol (DON) is one of the major toxic secondary metabolites produced by Fusarium fungi in cereal grains. Among the many promising strategies of DON detoxification are the microbial and enzymatic ones, which transform DON to nontoxic DON metabolites. Thus, proper analytical methods are needed for those DON metabolites. In this study, a robust gas chromatography-mass spectrometry (GC-MS) procedure was developed and validated for the simultaneous analysis of DON and two of its bacterial metabolites, 3-keto-DON and 3- epi-DON. The procedure involves a straightforward vacuum drying and derivatization step before the subsequent GC-MS analysis. Following the optimized protocol, DON and these two metabolites were separated on a capillary column within 15 min. The linear ranges for the these compounds were 10 to 2,000 ng mL-1 with correlation coefficients >0.99. For DON, 3- epi-DON, and 3-keto-DON, the limits of detection were 0.8, 3.0, and 0.05 ng mL-1, and the limits of quantification were 2.6, 10.0, and 1.0 ng mL-1, respectively. For all three compounds, the obtained relative standard deviation was 1.2 to 5.5%, and the recovery rates were 89.5 to 103.6%. The developed method was further validated by analyzing DON metabolites resulting from the biotransformation of DON initiated by cell-free lysates of the bacterium Devosia mutans 17-2-E-8. The developed protocol was sensitive, precise, accurate, and robust for the determination of DON, 3- epi-DON, and 3-keto-DON in liquid media and potentially other complex matrices without interference from other compounds.

Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER.

Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and direct usage of the permeabilized cells for the determination of metabolic enzyme activities in vivo.

Evaluation of Biotechnological Potential of Novel Mercury Tolerant Strain of Klebsiella Pneumonia

In order to investigate the effect of elevated mercury pollution on antibiotic tolerance and enzyme activities of human pathogens, we isolated a novel K. pneumoniae strain from the soil of a metallurgical dump yard that was capable of tolerating 250 μM HgCl2. Increasing concentration of Hg2+ in growth medium led to increased tolerance of the strain to ampicillin that was accompanied by increased secretion of extracellular polymeric substance (EPS). These results indicate that Hg2+-induced increased synthesis of EPS might be a key factor for increased tolerance of K. pneumoniae to ampicillin. The strain exhibited high catalase, alkaline phosphatase, mercuric reductase, amylase and lipase activity. Presence of 250 μM HgCl2 in growth medium increased mercuric reductase activity to 3 fold, however, no significant alteration in catalase, alkaline phosphatase and lipase was observed. The strain retained 86% amylase activity when grown in presence of 250 μM Hg2+. However, cell free lysate of the bacterium exhibited sensitivity towards Hg2+ leading to 30 to 40% decrease of these enzyme activities at 250 μM Hg2+. These results show that the bacterium possesses cellular mechanism to protect its enzymes from exogenous HgCl2. Our study reveals that increased heavy metal in the environment is a key factor leading to cross resistance of K. pneumoniae against membrane-targeted antibiotics ampicillin. Further, it’s a novel mercury-tolerant strain of K. pneumoniae isolate that exhibits high biotechnological potential for simultaneous bioremediation of mercury and production of industrially important enzymes.

Cell-Free Synthetic Biology for Pathway Prototyping.

Engineering biological systems for the production of biofuels and bioproducts holds great potential to transform the bioeconomy, but often requires laborious, time-consuming design-build-test cycles. For decades cell-free systems have offered quick and facile approaches to study enzymes with hopes of informing cellular processes, mainly in the form of purified single-enzyme activity assays. Over the past 20 years, cell-free systems have grown to include multienzymatic systems, both purified and crude. By decoupling cellular growth objectives from enzyme pathway engineering objectives, cell-free systems provide a controllable environment to direct substrates toward a single, desired product. Cell-free approaches are being developed for prototyping and for biomanufacturing. In prototyping applications, the idea is to use cell-free systems to test and optimize biosynthetic pathways before implementation in live cells and scale-up. We present a detailed method for the generation of crude lysates for cell-free pathway prototyping, mix-and-match cell-free metabolic engineering using preenriched lysates, and cell-free protein synthesis driven cell-free metabolic engineering. The cell-free synthetic biology methods described herein are generalizable to any biosynthetic pathway of interest and provide a powerful approach to building pathways in crude lysates for the purpose of prototyping. The foundational principle of the presented approach is that we can construct discrete metabolic pathways through modular assembly of cell-free lysates containing enzyme components produced by overexpression in the lysate chassis strain or by cell-free protein synthesis (in vitro production). Overall, the modular and cell-free nature of our pathway prototyping framework is poised to facilitate multiplexed, automated study of biosynthetic pathways to inform systems-level cellular design.

Quantitative Proteomics for Xenopus Embryos II, Data Analysis.

The oocytes, embryos, and cell-free lysates of the frog Xenopus laevis have emerged as powerful models for quantitative proteomic experiments. In the accompanying paper (Chapter 13) we describe how to prepare samples and acquire multiplexed proteomics spectra from those. As an illustrative example we use a 10-stage developmental time series from the egg to stage 35 (just before hatching). Here, we outline how to convert the ~700,000 acquired mass spectra from this time series into protein expression dynamics for ~9000 proteins. We first outline a preliminary quality-control analysisto discover any errors that occurred during sample preparation. We discuss how peptide and protein identification error rates are controlled, and how peptide and protein species are quantified. Our analysis relies on the freely available MaxQuant proteomics pipeline. Finally, we demonstrate how to start interpreting this large dataset by clustering and gene-set enrichment analysis.

The E. coli S30 lysate proteome: A prototype for cell-free protein production

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a “black box”. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures.This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.

High substrate specificity of 3,6-anhydro- l -galactose dehydrogenase indicates its essentiality in the agar catabolism of a marine bacterium

Abstract 3,6-Anhydro- l -galactose (AHG) is a rare sugar found in red macroalgae. The key metabolic steps in AHG catabolism involve its oxidation into 3,6-anhydrogalactonate (AHGA), followed by cycloisomerization of AHGA into 2-keto-3-deoxy-galactonate. These steps were recently discovered in a marine bacterium Vibrio sp. strain EJY3. In this study, we characterized NAD(P)+-dependent AHG dehydrogenase (AHGD) involved in the first step of AHG catabolism. AHGD displayed high substrate specificity for AHG, but showed no catalytic activity toward other aldehyde sugars, including D-form of AHG, glucose, and galactose. This high substrate specificity of AHGD may be associated with the unique chemical structure of its substrate AHG. Unlike other common aldehyde sugars, AHG mainly exists in its hydrated form under aqueous conditions. Growth of EJY3 in presence of AHG, agar, and Gelidium amansii but glucose, as the sole carbon source resulted in a significant increase in the AHGD activity of cell-free EJY3 lysates. Amino acid sequence analysis revealed that AHGD is highly homologous to other aldehyde dehydrogenases from agar-degrading bacteria, suggestive of its key role in agar-related metabolism in marine bacteria utilizing red macroalgae. Therefore, AHGD may serve as an important enzyme involved in the bioconversion of red macroalgal biomass to value-added chemicals.

Quorum quenching activity of rhizosphere bacteria against Ralstonia solanacearum

Many Gram-negative bacteria control the expression of virulence factors through quorum sensing (QS) mechanism. Enzymatic degradation of these signalling molecules is an important possible way to interfere with this QS mechanism. Such quorum quenching (QQ) enzymes are widespread in the bacterial world. The Pseudomonas aeruginosa 2apa, Pseudomonas fluorescens, Serratia marcescens and Azospirillum amazonense which are used as biocontrol agent were tested for QQ activity using Chromobacterium violaceum CV026 biosensor strain. Cell-free lysate of P. aeruginosa 2apa exhibited effective N-acyl homoserine lactone (AHL) degrading ability by inhibiting violacein production in biosensor strain. Further when the cell-free lysate was applied to Ralstonia solanacearum, it resulted in inhibition of biofilm formation. The confocal laser scanning microscopic analysis showed decrease in total biomass formation in treated slides. Our study shows the potential of AHL degradation by AHL acylase present in cell-free lysate of P. aeruginosa 2apa and inhibition of biofilm formation in R. solanacearum.

Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.

Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the ‘cytosol’ of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells.

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria.

BackgroundCyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons.ResultsHere we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at −20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system.ConclusionsAn easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

In Vitro Recombinant Expression of Novel Protective Protein Highly Accumulated in Dry State in an Anhydrobiotic Insect

Recent intensive studies showed that ability of several groups of organisms to withstand complete desiccation (anhydrobiosis state) is largely based on the activity of several groups of key proteins, including representatives of intrinsically disordered ones. In the largest known anhydrobiotic animal, the sleeping chironomid, both proteomic and genome-wide/targeted mRNA expression analysis suggest that several groups of the novel proteins greatly contribute to the formation of the molecular shield. Among them, 114 aa-long protein, dryporin. Being one of the most abundant in the desiccated larvae on protein level and showing one of the highest expression on mRNA level, this protein shows lack of the obvious homology with other known proteins. In this study, we successfully produced recombinant dryporin protein using BY-2 cell free lysate and showed the evidence of post-translational modification of the protein in the anhydrobiotic larvae.

Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis.

Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L-1 (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L-1·hr-1. We also demonstrate that this system can be lyophilized and retain biosynthesis capability. Our system catalyzes ∼1250 turnover events for the cofactor NAD+ and demonstrates the ability to rapidly prototype and debug enzymatic pathways in vitro for compelling metabolic engineering and synthetic biology applications.

Inhibition of QS-regulated virulence factors in Pseudomonas aeruginosa PAO1 and Pectobacterium carotovorum by AHL-lactonase of endophytic bacterium Bacillus cereus VT96

Abstract Expression of virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum is regulated by quorum sensing signals, predominantly belonging to acyl homoserine lactones (AHLs). Hence, effective quorum quenching mechanism could be a promising strategy to suppress the virulence of these pathogens. The cell-free lysate of endophytic bacterium Bacillus cereus VT96 isolated from Ventilago madraspatana Gaertn., showed significant quorum quenching activity when assayed using biosensor strains. An AHL-lactonase enzyme encoded by aiiA gene, possessing a conserved motif 104HXHXDH109 for zinc metal binding and tyrosine residue was partially purified from the cell-free lysate of B. cereus VT96 and tested against the expression of virulence factors in Pseudomonas aeruginosa PAO1. The enzyme effectively inhibited biofilm formation and production of pyocyanin, rhamnolipid and exopolysaccharides in P. aeruginosa. P. carotovorum was selected as the in vitro model for testing the efficacy of enzyme, where the enzyme effectively reduced the tissue maceration in potato tuber, which evidently demonstrated the control of QS regulated pathogenicity. Therefore, the AHL-lactonase from endophytic strain of B. cereus VT96 could be exploited to control AHL mediated QS dependent virulence factors of pathogens in clinical and biocontrol applications.

Micropollutant degradation via extracted native enzymes from activated sludge

A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the full variety of indigenous enzymatic activity of the activated sludge source material could not be restored, experimental modifications, e.g. different lysate filtration, significantly enhanced specific enzyme activities (e.g. >96% removal of the antibiotic erythromycin). Therefore, the approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins.

Quorum quenching activity in the cell-free lysate of Enterobacter ludwigii isolated from beef and its effect on quorum sensing regulation in Yersinia enterocolitica

A wide range of Gram negative bacteria regulate their phenotypic characteristics through an auto-inducing signaling mechanism called Quorum Sensing (QS).

In vitro effects of infrared A radiation on the synthesis of MMP-1, catalase, superoxide dismutase and GADD45 alpha protein

Harmful influences in the process of photoaging and skin damage are associated with infrared A (IRA) radiation, such as, disturbance of dermal extracellular matrix by up regulation of matrix metalloproteinase-1 (MMP1). Furthermore, DNA damage, induction of cytotoxicity and oxidative stress by decreasing natural antioxidant ability has been reported after acute exposure to IRA. The present study provides additional evidence that IRA radiation response in human skin fibroblasts produces deleterious effects to the cell, such as accelerating aging and weakening of their antioxidant defense mechanism. Human skin fibroblasts were exposed to a non-cytotoxic dose of IRA radiation and cultured for different periods for further collection of cell-free supernatants and lysates, and quantification of MMP-1, catalase, superoxide dismutase, and GADD45a. Our results corroborate previous published data and strongly indicate a negative impact of IRA radiation on the skin physiological by mechanisms involving reduced endogenous antioxidant enzymatic defense, increased MMP-1 and decreased repair process of DNA by reducing GADD45a protein, in cultured human fibroblasts. From a clinical perspective, IRA radiation acts by mechanisms distinct from those observed in ultraviolet radiation indicating the need for developing and making available cosmetics for skin care with properties beyond protection exerted by traditional sunscreens.

Abstract 17412: Paracrine Role of B Lymphocytes in Successful Bone Marrow Cell Therapy for Myocardial Infarction

Introduction: Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) or cell-free BMC lysate substantially improves post-MI cardiac function in rodent experiments. However, clinical trials of BMC therapy have been less successful. While most mouse experiments use young healthy BMC donors, MI patients undergoing autologous cell therapy (i.e., receiving their own BMCs) are older and are post-MI. We have previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, that donor MI induces inflammation leading to changes in BMC composition, and that BMCs from aged or post-MI mice have reduced levels of B cells, which reportedly can be a therapeutic BMC subpopulation. We recently observed that partial depletion of B cells from young healthy donor BMCs reduces the BMCs’ therapeutic effect, mimicking the effects of donor age or MI. Hypothesis: B cells play a crucial role in BMC therapy for MI, and B cell lysate is also therapeutic. Methods: We induced permanent coronary artery ligation MI, and implanted purified CD19+ bone marrow B cells from young healthy donors into the recipient infarct border zone 3 days post-MI via ultrasound guidance. CD3ε+ T cells and vehicle (HBSS) were injected as negative controls. A similar experiment was carried out with B cell lysate and T cell lysate. Results: In all groups, recipient left ventricular ejection fraction (EF) declined uniformly from baseline to day 2 post-MI, before cell injection. Injection of B cells significantly improved day 28 EF (p<.0005 adjusted for multiple comparisons). In contrast, T cells provided little or no benefit relative to HBSS. Notably, B cell lysate, but not T cell lysate, increased EF comparably to intact B cells. *p<.05 in figure. Conclusions: Bone marrow B cells play a crucial role in BMC therapy for MI via a paracrine mechanism. Reduction of bone marrow B cells by age or MI may explain why autologous BMC therapy in patients has not matched the success of rodent experiments.

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol

Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3±0.1g/L/h with a theoretical yield of 71% (0.36gm2,3-BD/g glucose) in batch reactions. Titers reached 82±8g/Lm2,3-BD in a 30h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation.