About

Marc Ostermeier, professor of chemical and biomolecular engineering at Johns Hopkins University, is known for his work in protein engineering, synthetic biology, and protein evolution. He is a faculty member of the Johns Hopkins Chemistry-Biology Interface Program and the Program in Molecular Biophysics. His research group seeks insight into the principles of natural evolution and the ability to design novel proteins and cells using directed evolution. Ostermeier's innovations in directed evolution technologies have led to the development of novel proteins such as protein switches that instruct cancer cells to produce their own anti-cancer drugs and proteins that precisely modify the DNA of live cells. His lab was an early developer of deep mutational scanning, a technique for quantifying the effects of thousands of mutations in a single experiment, which has enriched understanding of how mutations shape protein evolution. Ostermeier has been recognized as a fellow of the American Association for the Advancement of Science and the American Institute for Medical and Biological Engineering, and he is a recipient of a National Science Foundation CAREER Award. He holds seven patents for his work.

Research topics

• Biology
• Genetics
• Computer Science
• Medicine
• Evolutionary biology
• Cell biology
• Computational biology

Selected publications

• Environmental and mutational modulation of collateral fitness effects informs their mechanisms

  Molecular Biology and Evolution · 2026-04-18

  articleOpen accessSenior author

  Fitness effects of mutations that do not arise from changes in a protein's ability to perform its physiological functions (called collateral fitness effects or CFEs) are an understudied aspect of fitness landscapes. We have previously systematically measured the CFEs of all possible single amino acid substitutions in four proteins and found the frequency of deleterious mutations to vary by two orders of magnitude. Of these proteins, TEM-1 β-lactamase had the highest frequency, and deleterious mutations caused TEM-1 aggregation. Here, we systematically measured TEM-1 collateral fitness landscapes in environments and situations expected to alter protein aggregation or protein stability. We found a moderate correlation between deleterious CFEs and predicted thermodynamic stability effects in TEM-1's α-domain. Empirically, we found that the frequency and magnitude of deleterious CFEs can be reduced by altering the growth environment to disfavor aggregation (i.e. reducing the growth temperature or shifting to minimal media) or by stabilizing TEM-1 (via the M182T mutation or the addition of the β-lactamase inhibitor avibactam to the growth medium). However, although raising the growth temperature to favor aggregation exacerbated deleterious CFEs of many mutations, many mutations' effects were reduced. Furthermore, although reductions in CFEs occurred with reductions in TEM-1 aggregation for some mutants, for many mutants, they did not. We propose that mutational destabilization exposes protein motifs that can cause deleterious CFEs, but that these motifs and those that cause aggregation are not necessarily the same motifs.

  PublisherDOI

• Environmental and mutational modulation of collateral fitness effects informs their mechanisms

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-01-23

  articleOpen accessSenior authorCorresponding

  Abstract Fitness effects of mutations that do not arise from changes in a protein’s ability to perform its physiological functions (called collateral fitness effects or CFEs) are an understudied aspect of fitness landscapes. We have previously systematically measured the CFEs of all possible single amino acid substitutions in four proteins and found the frequency of deleterious mutations to vary by two orders of magnitude. Of these proteins, TEM-1 β-lactamase had the highest frequency, and deleterious mutations caused TEM-1 aggregation. Here, we systematically measured TEM-1 collateral fitness landscapes in environments and situations expected to alter protein aggregation or protein stability. We found a moderate correlation between deleterious CFEs and predicted thermodynamic stability effects in TEM-1’s α-domain. Empirically, we found that the frequency and magnitude of deleterious CFEs can be reduced by altering the growth environment to disfavor aggregation (i.e. reducing the growth temperature or shifting to minimal media) or by stabilizing TEM-1 (via the M182T mutation or the addition of the β-lactamase inhibitor avibactam to the growth medium). However, although raising the growth temperature to favor aggregation exacerbated deleterious CFEs of many mutations, many mutations’ effects were reduced. Furthermore, although reductions in CFEs occurred with reductions in TEM-1 aggregation for some mutants, for many mutants they did not. We propose that mutational destabilization exposes protein motifs that can cause deleterious CFEs, but that these motifs and those that cause aggregation are not necessarily the same motifs.

  PublisherOA PDFDOI

• Deep Mutational Scanning of the AAV <i>rep</i> Gene to Assess Effects on DNA Packaging in Expi293F Suspension Culture

  Biotechnology and Bioengineering · 2025-08-04 · 2 citations

  articleSenior authorCorresponding

  The adeno-associated virus (AAV) holds great potential as a gene delivery vector for emerging gene therapies. However, AAV's production is currently limited by factors such as low viral titers, inefficient capsid packaging, and producer cell toxicity. We sought to address the issue of low titers through deep mutational scanning of the AAV rep gene, which is involved in AAV replication, transcription regulation, and packaging. After generating a library of all single codon substitutions at approximately 300 sites in the gene, we characterized the mutational effects on packaged virus particles in a high-throughput replication competition assay in Expi293F cells suspension culture. The resulting values of the enrichment (a metric of packaging efficiency of native cargo) correlated moderately with those of a previous study in HEK293 adherent cells. However, difference in the range and distribution of values between the two studies as well as experimental variability between replicas in both data sets complicated assessment of cell line differences in mutational effects. We determined the ability of select mutants to alter the packaging of a synthetic DNA cargo into the AAV capsid but did not observe statistically significant improvements in either Expi293F or HEK293 cells. Our study adds to the growing evidence that beneficial effects of mutations in rep are highly dependent on the nature of the packaged DNA.

  PublisherDOI

• Thermostability Enhancement of GH 62 α-<scp>l</scp>-Arabinofuranosidase by Directed Evolution and Rational Design

  Journal of Agricultural and Food Chemistry · 2024-02-14 · 10 citations

  articleOpen access

  GH 62 arabinofuranosidases are known for their excellent specificity for arabinoxylan of agroindustrial residues and their synergism with endoxylanases and other hemicellulases. However, the low thermostability of some GH enzymes hampers potential industrial applications. Protein engineering research highly desires mutations that can enhance thermostability. Therefore, we employed directed evolution using one round of error-prone PCR and site-saturation mutagenesis for thermostability enhancement of GH 62 arabinofuranosidase from Aspergillus fumigatus. Single mutants with enhanced thermostability showed significant ΔΔG changes (<−2.5 kcal/mol) and improvements in perplexity scores from evolutionary scale modeling inverse folding. The best mutant, G205K, increased the melting temperature by 5 °C and the energy of denaturation by 41.3%. We discussed the functional mechanisms for improved stability. Analyzing the adjustments in α-helices, β-sheets, and loops resulting from point mutations, we have obtained significant knowledge regarding the potential impacts on protein stability, folding, and overall structural integrity.

  PublisherOA PDFDOI

• Fitness and Functional Landscapes of the<i>E. coli</i>RNase III Gene<i>rnc</i>

  Molecular Biology and Evolution · 2023-02-27 · 7 citations

  articleOpen accessSenior author

  How protein properties such as protein activity and protein essentiality affect the distribution of fitness effects (DFE) of mutations are important questions in protein evolution. Deep mutational scanning studies typically measure the effects of a comprehensive set of mutations on either protein activity or fitness. Our understanding of the underpinnings of the DFE would be enhanced by a comprehensive study of both for the same gene. Here, we compared the fitness effects and in vivo protein activity effects of ∼4,500 missense mutations in the E. coli rnc gene. This gene encodes RNase III, a global regulator enzyme that cleaves diverse RNA substrates including precursor ribosomal RNA and various mRNAs including its own 5' untranslated region (5'UTR). We find that RNase III's ability to cleave dsRNA is the most important determinant of the fitness effects of rnc mutations. The DFE of RNase III was bimodal, with mutations centered around neutral and deleterious effects, consistent with previously reported DFE's of enzymes with a singular physiological role. Fitness was buffered to small effects on RNase III activity. The enzyme's RNase III domain, which contains the RNase III signature motif and all active site residues, was more sensitive to mutation than its dsRNA binding domain, which is responsible for recognition and binding to dsRNA. Differential effects on fitness and functional scores for mutations at highly conserved residues G97, G99, and F188 suggest that these positions may be important for RNase III cleavage specificity.

  PublisherOA PDFDOI

• Genes Vary Greatly in Their Propensity for Collateral Fitness Effects of Mutations

  Molecular Biology and Evolution · 2023 · 13 citations

  Senior authorCorresponding
  • Biology
  • Genetics
  • Cell biology

  Mutations can have deleterious fitness effects when they decrease protein specific activity or decrease active protein abundance. Mutations will also be deleterious when they cause misfolding or misinteractions that are toxic to the cell (i.e., independent of whether the mutations affect specific activity and abundance). The extent to which protein evolution is shaped by these and other collateral fitness effects is unclear in part because little is known of their frequency and magnitude. Using deep mutational scanning (DMS), we previously found at least 42% of missense mutations in the TEM-1 β-lactamase antibiotic resistance gene cause deleterious collateral fitness effects. Here, we used DMS to comprehensively determine the collateral fitness effects of missense mutations in three genes encoding the antibiotic resistance proteins New Delhi metallo-β-lactamase (NDM-1), chloramphenicol acetyltransferase I (CAT-I), and 2″-aminoglycoside nucleotidyltransferase (AadB). AadB (20%), CAT-I (0.9%), and NDM-1 (0.2%) were less susceptible to deleterious collateral fitness effects than TEM-1 (42%) indicating that genes have different propensities for these effects. As was observed with TEM-1, all the studied deleterious aadB mutants increased aggregation. However, aggregation did not correlate with collateral fitness effects for many of the deleterious mutants of CAT-I and NDM-1. Select deleterious mutants caused unexpected phenotypes to emerge. The introduction of internal start codons in CAT-1 caused loss of the episome and a mutation in aadB made its cognate antibiotic essential for growth. Our study illustrates how the complexity of the cell provides a rich environment for collateral fitness effects and new phenotypes to emerge.

  PublisherOA PDFDOI

• Fitness and functional landscapes of the <i>E. coli</i> RNase III gene <i>rnc</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2022-11-02

  preprintOpen accessSenior authorCorresponding

  Abstract How protein properties such as protein activity and protein essentiality affect the distribution of fitness effects (DFE) of mutations are important questions in protein evolution. Deep mutational scanning studies typically measure the effects of a comprehensive set of mutations on either protein activity or fitness. Our understanding of the underpinnings of the DFE would be enhanced by a comprehensive study of both for the same gene. Here, we compared the fitness effects and in vivo protein activity effects of ∼4,500 missense mutations in the E. coli rnc gene. This gene encodes RNase III, a global regulator enzyme that cleaves diverse RNA substrates including precursor ribosomal RNA and various mRNAs including its own 5’ untranslated region (5’UTR). We find that RNase III’s ability to cleave dsRNA is the most important determinant of the fitness effects of rnc mutations. The DFE of RNase III was bimodal, with mutations centered around neutral and deleterious effects, consistent with previously reported DFE’s of enzymes with a singular physiological role. Fitness was buffered to small effects on RNase III activity. The enzyme’s RNase III domain (RIIID), which contains the RNase III signature motif and all active site residues, was more sensitive to mutation than its dsRNA binding domain (dsRBD), which is responsible for recognition and binding to dsRNA. Differential effects on fitness and functional scores for mutations at highly conserved residues G97, G99, and F188 suggest that these positions may be important for RNase III cleavage specificity.

  PublisherOA PDFDOI

• A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity <i>In Vivo</i> in <i>Escherichia coli</i>

  The CRISPR Journal · 2022-12-09 · 2 citations

  articleSenior authorCorresponding

  Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo assay for RNase III activity that utilizes RNase III's cleavage of the 5′-untranslated region (UTR) of its own messenger RNA. The key molecule in the system is a hybrid guide RNA (gRNA) between the 5′-UTR of RNase III and gGFP, a gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved by RNase III for full GFP repression. Our system uses GFP fluorescence to report on Escherichia coli RNase III activity in culture and on an individual cell basis, making it effective for selecting individual cells through fluorescence-activated cell sorting. Homology between enzymes within the RNase III family suggests this assay might be adapted to measure the activity of other enzymes in the RNase III family such as human Dicer or Drosha.

  PublisherDOI

• Genes vary greatly in their propensity for collateral fitness effects of mutations

  bioRxiv (Cold Spring Harbor Laboratory) · 2022-10-25

  preprintOpen accessSenior authorCorresponding

  Abstract Mutations can have deleterious fitness effects when they decrease protein specific activity or decrease active protein abundance. Mutations will also be deleterious when they cause misfolding or misinteractions that are toxic to the cell (i.e., independent of whether the mutations affect specific activity and abundance). The extent to which protein evolution is shaped by these and other collateral fitness effects is unclear in part because little is known of their frequency and magnitude. Using deep mutational scanning (DMS), we previously found at least 42% of missense mutations in the TEM-1 β-lactamase antibiotic resistance gene cause deleterious collateral fitness effects. Here, we used DMS to comprehensively determine the collateral fitness effects of missense mutations in three genes encoding the antibiotic resistance proteins New Delhi metallo-β-lactamase (NDM-1), chloramphenicol acetyltransferase I (CAT-I), and 2”-aminoglycoside nucleotidyltransferase (AadB). AadB (20%), CAT-I (0.9%), and NDM-1 ( 0.2%) were less susceptible to deleterious collateral fitness effects than TEM-1 (42%) indicating that genes have different propensities for these effects. As was observed with TEM-1 , all the studied deleterious aadB mutants increased aggregation. However, aggregation did not correlate with collateral fitness effects for many of the deleterious mutants of CAT-I and NDM-1 . Select deleterious mutants caused unexpected phenotypes to emerge. The introduction of internal start codons in CAT-1 caused loss of the episome and a mutation in aadB made its cognate antibiotic essential for growth. Our study illustrates how the complexity of the cell provides a rich environment for collateral fitness effects and new phenotypes to emerge.

  PublisherOA PDFDOI

• A bacterial dual positive and negative selection system for dCas9 activity

  PLoS ONE · 2022-06-03 · 3 citations

  articleOpen accessSenior authorCorresponding

  The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.

  PublisherOA PDFDOI

Recent grants

• The fitness landscape of genes and proteins

  NSF · $676k · 2010–2014

• CAREER: Engineering a Protein Molecular Switch

  NSF · $400k · 2003–2008

• NIH Grant F32GM018560

  NIH · $63k

• Alternative pathways in directed evolution

  NSF · $350k · 2014–2018

• UNS: Collaborative Research: Targeted CpG Methylation

  NSF · $300k · 2015–2019

Frequent coauthors

• Christian Limberg

  35 shared

• R.M. Meudtner

  22 shared

• James R. Eshleman

  Johns Hopkins University

  19 shared

• Sidney M. Hecht

  Arizona State University

  18 shared

• Chapman Wright

  15 shared

• Jennifer Tullman

  14 shared

• Nathan Nicholes

  Bioengineering Center

  14 shared

• B. Ziemer

  11 shared

Labs

• Ostermeier LabPI

Education

• Postdoctoral Fellow, Chemistry

  Pennsylvania State University

  2000

• Ph.D., Chemical Engineering

  University of Texas at Austin

  1995

• B.S., Chemical Engineering

  University of Wisconsin Madison

  1990

Awards & honors

• Fellow of the American Association for the Advancement of Sc…
• Fellow of the American Institute for Medical and Biological…
• National Science Foundation CAREER Award