Biology subjects

Lynch, M. D.

Publications and source records attributed to Lynch, M. D..

26 records · Page 2Linked to original sources

bioRxiv2020

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions. HighlightsO_LIDecreases in NADPH pools lead to increased NADPH fluxes C_LIO_LIPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo. C_LIO_LIDynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH flux C_LIO_LIXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock. C_LI

bioengineering↗

bioRxiv2020

The E. coli Cas1/2 endonuclease complex reduces CRISPR/Cascade guide array stability

CRISPR based interference has become common in various applications from genetic circuits to dynamic metabolic control. In E. coli the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of protospacer sequences from guide arrays utilized for dynamic silencing. We report that unstable guide arrays are due to expression of the Cas1/2 endonuclease complex. A cas1 deletion improves guide array stability. We propose a model wherein basal Cas1/2 endonuclease activity results in the loss of protospacers from guide arrays. Subsequently, mutant guide arrays can be amplified through selection. Replacing a constitutive promoter driving Cascade complex expression with a tightly controlled inducible promoter improves guide array stability, while minimizing leaky gene silencing. HighlightsO_LICas1/2 endonuclease complex mediates CRISPR/Cascade protospacer loss in E. coli C_LIO_LITightly controlled Cascade operon expression increases guide array stability. C_LI

synthetic biology↗

bioRxiv2020

Dynamic control over feedback regulation improves stationary phase fluxes in engineered E. coli.

We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve stationary phase biosynthesis in engineered E. coli. Specifically, we report the impact of dynamic control over two enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in -ketoglutarate, which is an inhibitor of sugar transport, resulting in increased stationary phase glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. The combined reduction in citrate synthase and glucose-6-phosphate dehydrogenase, leads to greatly enhanced stationary phase metabolism and the improved production of citramalic acid enabling titers of 126{+/-}7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a "central" metabolic pathway in stationary phase E. coli, which coupled with ferredoxin reductase comprise a pathway whose physiologic role is maintaining NADPH levels. HighlightsO_LIDynamic reduction in -keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake. C_LIO_LIDynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux. C_LIO_LIPyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions. C_LIO_LIProduction of citramalate to titers 126 {+/-} 7g/L at > 90 % of theoretical yield. C_LI

bioengineering↗

bioRxiv2020

CRISPR/Cas "non-target" sites inhibit on-target cutting rates

CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA. The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity. HighlightsO_LIThe pool of non-target PAM sites inhibit Cas9/gRNA on-target activity C_LIO_LInon-target PAM inhibition is dose dependent C_LIO_LInon-target PAM inhibition is a function of gRNA sequence C_LIO_LInon-target PAM inhibition is a function of Cas9 levels C_LI

biochemistry↗

bioRxiv2020

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology. HighlightsO_LICharacterization of the impact of media on promoter activity C_LIO_LIIdentification of promoters robust to environmental variables C_LIO_LIIdentification of promoters whose expression scale from microtiter plates to bioreactors C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/920280v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@94f035org.highwire.dtl.DTLVardef@264edaorg.highwire.dtl.DTLVardef@102ee30org.highwire.dtl.DTLVardef@163d16e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

synthetic biology↗

bioRxiv2020

Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high throughput cultivation in microtiter plates and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion. HighlightsO_LIAutoinduction of both cell lysis and nucleotide hydrolysis C_LIO_LI>90 % lysis and DNA degradation C_LIO_LIStrains are stable to lysis in the absence of phosphate depletion. C_LI

synthetic biology↗

bioRxiv2019

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.

We report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of [~]30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/-0.7 g/L ([~]270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach [~] 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation. HighlightsO_LIStationary phase protein expression results in high titers. C_LIO_LIAutoinduction by phosphate depletion enables protein titers from 2-8 g/L. C_LIO_LIAutoinduction has been validated from 384 well plates to instrumented bioreactors. C_LI

synthetic biology↗

bioRxiv2019

The Effects of Gene Flow from Unsampled \"Ghost\" Populations on the Estimation of Evolutionary History under the Isolation with Migration Model

Unsampled or extinct ghost populations leave signatures on the genomes of individuals from extant, sampled populations, especially if they have exchanged genes with them over evolutionary time. This gene flow from ghost populations can introduce biases when estimating evolutionary history from genomic data, often leading to data misinterpretation and ambiguous results. Here we assess these biases while accounting, or not accounting for gene flow from ghost populations under the Isolation with Migration (IM) model. We perform extensive simulations under five scenarios with no gene flow (Scenario A), to extensive gene flow to- and from- an unsampled ghost population (Scenarios B, C, D, and E). Estimates of evolutionary history across all scenarios A-E (effective population sizes, divergence times, and migration rates) indicate consistent a) under-estimation of divergence times between sampled populations, (b) over-estimation of effective population sizes of sampled populations, and (c) under-estimation of migration rates between sampled populations, with increased gene flow from the unsampled ghost population. Without accounting for an unsampled ghost, summary statistics like FST are under-estimated, and{pi} is over-estimated with increased gene flow from the ghost. To show this persistent issue in empirical data, we use a 355 locus dataset from African Hunter-Gatherer populations and discuss similar biases in estimating evolutionary history while not accounting for unsampled ghosts. Considering the large effects of gene flow from these ghosts, we propose a multi-pronged approach to account for the presence of unsampled ghost populations in population genomics studies to reduce erroneous inferences.

evolutionary biology↗