Scholars@Duke
Michael D Lynch

Michael D Lynch

W. H. Gardner, Jr. Associate Professor of Biomedical Engineering
Biomedical Engineering

Selected Publications

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Journal Article Metabolic engineering · September 2024 Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic ... Full text Cite

2-Stage microfermentations.

Journal Article Metabolic engineering communications · June 2024 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 thousan ... Full text Cite

Phase separation methods for protein purification: A meta-analysis of purification performance and cost-effectiveness.

Journal Article Biotechnology journal · April 2024 Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production. To identify barriers, we analyzed the purification performance and economics of 290 phase s ... Full text Cite

"Multiagent" Screening Improves Directed Enzyme Evolution by Identifying Epistatic Mutations.

Journal Article ACS synthetic biology · May 2022 Enzyme evolution has enabled numerous advances in biotechnology and synthetic biology, yet still requires many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which is caused by epis ... Full text Cite

Integrated autolysis, DNA hydrolysis and precipitation enables an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral and clinical-stage anti-COVID-19 candidate.

Journal Article Biochemical engineering journal · April 2022 Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral curr ... Full text Cite

A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.

Journal Article The CRISPR journal · February 2022 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 t ... Full text Cite

Integrated Autolysis, DNA Hydrolysis and Precipitation Enables an Improved Bioprocess for Q-Griffithsin, a Broad-Spectrum Antiviral and Clinical-Stage anti-COVID-19 Candidate.

Journal Article bioRxiv · January 3, 2022 UNLABELLED: Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum an ... Full text Link to item Cite

The past, present, and future of enzyme-based therapies.

Journal Article Drug discovery today · January 2022 Enzyme-based therapeutics (EBTs) have the potential to tap into an almost unmeasurable amount of enzyme biodiversity and treat myriad conditions. Although EBTs were some of the first biologics used clinically, the rate of development of newer EBTs has lagg ... Full text Cite

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Journal Article Bio-protocol · January 2022 Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions ... Full text Cite

Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements.

Journal Article Bio-protocol · January 2022 Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine l ... Full text Cite

Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli.

Journal Article BioTechniques · November 2021 Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bio ... Full text Cite

Two-stage dynamic deregulation of metabolism improves process robustness & scalability in engineered E. coli.

Journal Article Metabolic engineering · November 2021 We report that two-stage dynamic control improves bioprocess robustness as a result of the dynamic deregulation of central metabolism. Dynamic control is implemented during stationary phase using combinations of CRISPR interference and controlled proteolys ... Full text Cite

Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity.

Journal Article Nature communications · August 2021 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 ... Full text Cite

A critical review on the progress and challenges to a more sustainable, cost competitive synthesis of adipic acid

Journal Article Green Chemistry · May 7, 2021 Adipic acid is a key organic diacid intermediate used in nylon manufacturing. It is primarily produced by an industrial process that can form nitrous oxide as a byproduct. Nitrous oxide has a 300-fold higher global warming potential than carbon dioxide, an ... Full text Cite

The bioprocess TEA calculator: An online technoeconomic analysis tool to evaluate the commercial competitiveness of potential bioprocesses.

Journal Article Metabolic engineering · May 2021 Techno-economic analysis connects R&D, engineering, and business. By linking process parameters to financial metrics, it allows researchers to understand the factors controlling the potential success of their technologies. In particular, metabolic and biop ... Full text Cite

BioSamplr: An open source, low cost automated sampling system for bioreactors.

Journal Article HardwareX · April 2021 Autosampling from bioreactors reduces error, increases reproducibility and offers improved aseptic handling when compared to manual sampling. Additionally, autosampling greatly decreases the hands-on time required for a bioreactor experiment and enables sa ... Full text Cite

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

Journal Article Metabolic engineering · March 2021 We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoic ... Full text Cite

Escherichia coli Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability.

Journal Article ACS synthetic biology · January 2021 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 exp ... Full text Cite

“Multi-Agent” Screening Improves the Efficiency of Directed Enzyme Evolution

Journal Article · 2021 Enzyme evolution has enabled numerous advances in biotechnology. However, directed evolution programs can still require many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which in ... Full text Cite

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

Journal Article · 2021 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 ... Full text Cite

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

Journal Article · 2021 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 t ... Full text Cite

Optimization of Phosphate Limited Autoinduction Broth for 2-Stage Heterologous Protein Expression in E. coli

Journal Article · 2021 Autoinducible, 2-stage protein expression leveraging phosphate inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented biore ... Full text Cite

Instant Taq: Rapid Autoinducible Expression and Chromatography-free Purification of Taq polymerase

Journal Article · 2021 DNA modifying enzymes are ubiquitous reagents in synthetic biology. Producing these enzymes often requires large culture volumes, purified nucleases and chromatographic separations to make enzymes of necessary quality. We sought to leverage synthetic biolo ... Full text Cite

CRISPR-Cas "Non-Target" Sites Inhibit On-Target Cutting Rates.

Journal Article The CRISPR journal · December 2020 CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once fo ... Full text Cite

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

Journal Article Biotechnology and bioengineering · September 2020 We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved p ... Full text Cite

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.

Journal Article Biotechnology and bioengineering · September 2020 We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonucleas ... Full text Cite

Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli.

Journal Article ACS synthetic biology · June 2020 A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the evaluation of the media robustness of a small library of E. coli PhoB regulated promoters t ... Full text Cite

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

Journal Article · 2020 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 ... Full text Cite

A Review of the Biotechnological Production of Methacrylic Acid.

Journal Article Frontiers in bioengineering and biotechnology · January 2020 Industrial biotechnology can lead to new routes and potentially to more sustainable production of numerous chemicals. We review the potential of biobased routes from sugars to the large volume commodity, methacrylic acid, involving fermentation based biopr ... Full text Cite

Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phaseE. coli

Journal Article · 2020 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: ... Full text Cite

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

Journal Article · 2020 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 st ... Full text Cite

TheE. coliCas1/2 endonuclease complex reduces CRISPR/Cascade guide array stability

Journal Article · 2020 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 guid ... Full text Cite

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

Journal Article · 2020 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 ... Full text Cite

Low-Cost, Large-Scale Production of the Anti-viral Lectin Griffithsin.

Journal Article Frontiers in bioengineering and biotechnology · January 2020 Griffithsin, a broad-spectrum antiviral lectin, has potential to prevent and treat numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For these indications, the annual demand for Griffithsin could reach billions of doses and affordability ... Full text Cite

BioSamplr: An open source, low cost automated sampling system for bioreactors

Journal Article · 2020 Autosampling from bioreactors reduces error, increases reproducibility and offers improved aseptic handling when compared to manual sampling. Additionally, autosampling greatly decreases the hands-on time required for a bioreactor experiment and enables sa ... Full text Cite

A Technoeconomic Evaluation of the Potential of Industrial Biotechnology for the Competitive Production of Commodity and Bulk Chemicals

Journal Article · 2020 Fermentation based bioprocesses are routinely developed for higher value products; however, the development of bioprocesses for larger volume chemicals has lagged. While electrification of transportation may enable significant reductions in greenhouse gas ... Full text Cite

A review of lipidation in the development of advanced protein and peptide therapeutics.

Journal Article Journal of controlled release : official journal of the Controlled Release Society · February 2019 The use of biologics (peptide and protein based drugs) has increased significantly over the past few decades. However, their development has been limited by their short half-life, immunogenicity and low membrane permeability, restricting most therapies to ... Full text Cite

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

Journal Article · 2019 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 expre ... Full text Cite

Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity.

Journal Article ACS synthetic biology · December 2017 Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for ... Full text Cite

Large-scale bioprocess competitiveness: the potential of dynamic metabolic control in two-stage fermentations

Journal Article Current Opinion in Chemical Engineering · November 1, 2016 Bioprocessing technology offers a potentially promising and more sustainable alternative to many traditional chemical process technologies; however to date this potential has largely not been realized. For large-scale bioprocessing to have larger penetrati ... Full text Cite

Into new territory: improved microbial synthesis through engineering of the essential metabolic network.

Journal Article Current opinion in biotechnology · April 2016 Advances in synthetic biology and metabolic engineering offer the promise of next generation bioprocesses to produce numerous products including specialty and bulk chemicals and even biofuels sustainably from renewable feedstocks. A primary challenge is th ... Full text Cite

A rational microbial design perspective in the design build test era

Conference Metabolic Engineering X · January 1, 2014 Cite

Strategies for the multiplex mapping of genes to traits.

Journal Article Microbial cell factories · October 2013 Rewiring and optimization of metabolic networks to enable the production of commercially valuable chemicals is a central goal of metabolic engineering. This prospect is challenged by the complexity of metabolic networks, lack of complete knowledge of gene ... Full text Cite

Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing.

Journal Article Biotechnology journal · May 2013 Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, wh ... Full text Cite

Identification of a 21 amino acid peptide conferring 3-hydroxypropionic acid stress-tolerance to Escherichia coli.

Journal Article Biotechnology and bioengineering · May 2012 We report the identification of a novel small open reading frame in Escherichia coli. The sORF (called iroK) encodes a 21 amino cid peptide, which when translated confers a 133% (ca. 20 g/L) increase in resistance to 3-hydroxypropionic acid. We show that i ... Full text Cite

Enzyme engineering of malonyl coA reductase (MCR) to improve 3-hydroxypropionate (3HP) production

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · March 25, 2012 Link to item Cite

Production of fungible diesel via fatty acid synthesis from hydrogen and carbon dioxide feedstocks

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · March 25, 2012 Link to item Cite

Novel biological conversion of hydrogen and carbon dioxide directly into biodiesel

Conference ACS National Meeting Book of Abstracts · December 1, 2011 There is increasing pressure to reduce dependence on foreign petroleum sources. As such, the development of green chemistry routes to produce fuels from renewable feedstocks has been the focus of significant research. Traditional bio-refining processes rel ... Cite

Novel biological conversion of hydrogen and carbon dioxide directly into biodiesel

Conference ACS National Meeting Book of Abstracts · August 25, 2011 There is increasing pressure to reduce dependence on foreign petroleum sources. As such, the development of green chemistry routes to produce fuels from renewable feedstocks has been the focus of significant research. Traditional bio-refining processes rel ... Cite

Enzyme evolution of an alpha-keto decarboxylase

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · March 27, 2011 Link to item Cite

Broad-host-range vectors for protein expression across gram negative hosts.

Journal Article Biotechnology and bioengineering · June 2010 Expression of recombinant proteins across a range of different host organisms is often necessary in metabolic engineering applications. Doing so can be facilitated by the use of vectors having origins of replication with a broad-host-range, the option for ... Full text Cite

Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes.

Journal Article Metabolic engineering · May 2010 The understanding and engineering of complex phenotypes is a critical issue in biotechnology. Conventional approaches for engineering such phenotypes are often resource intensive, marginally effective, and unable to generate the level of biological underst ... Full text Cite

Genes restoring redox balance in fermentation-deficient E. coli NZN111.

Journal Article Metabolic engineering · November 2009 The objectives of this study were to improve understanding of the biochemical mechanisms underlying the growth defects resulting from deletion of pflB and ldhA in E. coli (strain NZN111) and identify genes for which overexpression would relieve this growth ... Full text Cite

Parallel mapping of genotypes to phenotypes contributing to overall biological fitness.

Journal Article Metabolic engineering · November 2008 Laboratory selection is a powerful approach for engineering new traits in metabolic engineering applications. This approach is limited because determining the genetic basis of improved strains can be difficult using conventional methods. We have recently r ... Full text Cite

A genomics approach to improve the analysis and design of strain selections.

Journal Article Metabolic engineering · May 2008 Strain engineering has been traditionally centered on the use of mutation, selection, and screening to develop improved strains. Although mutational and screening methods are well-characterized, selection remains poorly understood. We hypothesized that we ... Full text Cite

Genome-scale analysis of anti-metabolite directed strain engineering.

Journal Article Metabolic engineering · March 2008 Classic strain engineering methods have previously been limited by the low-throughput of conventional sequencing technology. Here, we applied a new genomics technology, scalar analysis of library enrichments (SCALEs), to measure >3 million Escherichia coli ... Full text Cite

SCALEs: multiscale analysis of library enrichment.

Journal Article Nature methods · January 2007 We report a genome-wide, multiscale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves (i) growth selections on a mixture of several different plasmid-ba ... Full text Cite

Broad host range vectors for stable genomic library construction.

Journal Article Biotechnology and bioengineering · May 2006 We describe the construction of 36 stable vectors for genomic library construction in gram-negative species. These vectors contain the pBBR1 replicon that has been shown to stably replicate in every gram-negative species tested. The plasmids also contain b ... Full text Cite

Genomics tools for elucidating the function of trait conferring genes

Conference AIChE Annual Meeting, Conference Proceedings · December 1, 2005 We have developed a variety of functional genomics tools useful for the identification and characterization of trait conferring genes in prokaryotes. These include: a series of broad host range vectors for library screening, a system for transposon-based i ... Cite

Identification of organic acid tolerance genes in E. coli for biorefinery applications

Conference AIChE Annual Meeting, Conference Proceedings · December 1, 2005 Synthesis of building block chemicals at high titers can be toxic to host microorganisms. We have developed Mixed Library Parallel Gene Trait Mapping (ML-PGTM) as a technique that identifies the effect of specific genes on the expression of a specific bact ... Cite

Post-genomics approach to understanding E. Coli network architecture.

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · March 13, 2005 Link to item Cite

Mapping phenotypic landscapes using DNA micro-arrays.

Journal Article Metabolic engineering · July 2004 Inverse metabolic engineering is a useful approach for engineering phenotypes in biological systems. The overarching objective of this approach is to combine the power of evolutionary engineering approaches with the precision of constructive metabolic engi ... Full text Cite