Biology subjects

Moreb, E. A.

Publications and source records attributed to Moreb, E. A..

11 recordsLinked to original sources

bioRxiv2022

The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of optimal protocols used for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, the development approach described is adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

synthetic biology↗

bioRxiv2021

A meta-analysis of gRNA library screens enables an improved understanding of the impact of gRNA folding and structural stability on CRISPR-Cas9 activity.

CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here we perform a meta-analysis of published CRISPR-Cas9 datasets to better understand the role of secondary structures in inhibiting gRNA activity. We identify two inhibitory structures and provide estimated free energy cutoffs at which they become impactful. Further, we identify the prevalence of these structures in existing datasets. The cutoffs provided help to explain conflicting impacts of free energy values in different datasets as well as providing a guideline for future gRNA designs. HighlightsO_LIClearly define two secondary structures that inhibit CRISPR-Cas9 activity C_LIO_LIProvide free energy calculations and cutoffs at which each structure begins to inhibit activity C_LIO_LIEvaluate impact of these structures in published datasets C_LI

synthetic biology↗

bioRxiv2021

An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. To better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 39 published data sets. We find strong evidence that the genomic context, which can be defined as the DNA content outside of the gRNA/target sequence itself, greatly contributes to differences in gRNA dependent activity. Context underlies variation in activity often attributed to differences in gRNA sequence. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants. HighlightsO_LISpecies-specific genomic context drives variability in gRNA activity in a PAM proximal sequence-dependent manner C_LIO_LIIncreased PAM specificity of Cas9 and/or increased Cas9/gRNA expression reduces the impact of species-specific context C_LIO_LICurrent gRNA prediction algorithms trained on species are not expected to predict activity in another species C_LI

synthetic biology↗

bioRxiv2020

Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in Engineered E. coli.

We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

synthetic biology↗

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↗