The statistics of directed evolution: From library generation to high throughput screens

Directed evolution has been recognized as a powerful approach to creating enzymes and cells with desirable properties, such as growth on a new substrate, thermotolerance, or even novel reaction mechanisms. The directed evolution approach is to generate diversity through genetic mutations and screen the diversity for mutants that have improved properties compared to the parent. This diversification and screening can be repeated to generate mutants with superior properties. While the concept is straightforward, the devil is in the details. How should I create diversity (error prone PCR, mutagenic primers, transposons…)? How much diversity do I need (103, 106, 107, more??). If my high throughput screen (HTS) does not find an improved mutant, does that mean an improved mutant did not exist in the library? Or could it mean my HTS failed to isolate it? How do I identify single cells with 20% improvement, when cell-to-cell variability is more than 10 fold larger? In this workshop, we will start to answer several of these questions. We will review approaches for generating and quantifying diversity. We will develop a Bayesian framework for understanding noise due to cell-to-cell variability and be able to predict the expected enrichment achievable in fluorescence-activated cell sorting (FACS) and other HTS screens. We will discuss common pitfalls in generating libraries and screening them. If you do directed evolution on enzymes or cells, if you use FACS, droplet-based HTS, or even plate-based screening, this workshop will provide you with fundamental tools to add rigor to your directed evolution efforts and improve your likelihood of success

Inverse Metabolic Engineering of Synechocystis PCC 6803 for Improved Growth Rate and Poly-3-hydroxybutyrate Production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems.Singapore-MIT Alliance (SMA

Forward and inverse metabolic engineering strategies for improving polyhydroxybyrate production

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.Includes bibliographical references (p. 165-174).Forward metabolic engineering (FME) is a rational approach to cellular engineering, relying on an understanding of the entire metabolic network to direct perturbations for phenotype improvement. Conversely, inverse metabolic engineering (IME) uses a global, combinatorial approach to identify genetic loci that are important for a given phenotype. These two approaches complement each other in a strain improvement program. FME and IME approaches were applied to poly-3-hydroxybutyrate (PHB)production in Synechocystis PCC6803 [IME] and recombinant E. coli [FME] in this thesis.IME was appropriate for Synechocystis, where metabolic regulation of the native PHB pathway was not well understood. A high throughput screening method was established by developing a staining protocol that quantitatively related nile red fluorescence to PHB content, while maintaining cell viability for both organisms. This was combined with fluorescence activated cell sorting (FACS) to screen for high PHB mutants. A Synechocystis insertion mutagenesis library was screened to identify gene disruptions that increased PHB. Two gene disruptions in proline biosynthesis and an unknown function were identified and characterized.An analogous IME study in E. coli did not find increased PHB mutants, but suggested an FME approach on the PHB pathway. Systematic overexpression of the pathway revealed phaB, acetoacetyl-CoA reductase, limited PHB flux. Beyond this, whole operon overexpression led to even higher PHB fluxes.In a nitrogen-limited chemostat, PHB flux did not change with dilution rate. Unlike prior pleiotropic perturbations, these systematic experiments could clearly conclude that the flux control is in the PHB pathway. At high PHB flux, growth rate was extremely hindered and was accompanied by PHB plasmid genetic instability and rapid PHB productivity loss.(cont.) Tandem gene duplication (TGD) was developed to slow productivity loss caused by "allele segregation," a fast process that propagates a DNA mutation to all copies of a plasmid. By placing the many copies in tandem, rather than on individual plasmids, allele segregation could be avoided, increasing stability significantly.These methods and results should support PHB engineering in higher photosynthetic organisms and better E. coli PHB production in batch or continuous culture.TGD is a broadly applicable technique for high level recombinant expression.by Keith E. J. Tyo.Ph.D

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases

Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

Background: Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. Results: The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. Conclusions: We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the development of new vaccine candidates with reduced restrictions by regulatory agencies, using the successful experience with other yeast-based VLP vaccines commercialized worldwide

Recording temporal data with minutes resolution into DNA

Recording complex biological signals is a crucial application of synthetic biology and essential for a deeper understanding of biological processes. An ideal “biorecorder” would have the ability to record biological signals over a wide spatial distribution of cells with high temporal resolution. However, the genetically encoded biorecording tools available have very good spatial resolution (cellular level), but currently rely on turning on and off transcription and translation of a protein (e.g., Cas9 or a recombinase) to record the biological signal, making their temporal resolution on the order of hours. Here we introduce a DNA polymerase based biorecorder that can record cationic concentration fluctuations into DNA sequence with a resolution of ~1 minute. We use a template independent DNA polymerase; terminal deoxynucleotidyl transferase (TdT) that randomly incorporates bases onto a single strand of DNA. The preference of base incorporated by TdT changes with the concentration of cations in TdT’s environment. Therefore, by analyzing a strand of DNA that was extended in fluctuating cation concentrations, we can determine the temporal profile of cation concentration from the bases added. Using this method, we can measure a change in Co2+ concentration during a one hour period with an accuracy of 1 min. We also show the approach works for Zn2+ and Ca2+. We will present our methods for optimizing this biorecorder and characterize its performance in vitro. Recording data onto DNA with minutes time resolution could solve many challenging data acquisition problems in neuroscience and developmental biology, and could aid in the use of DNA as a data storage medium

Meta-omic profiling reveals ubiquity of genes encoding for the nitrogen-rich biopolymer cyanophycin in activated sludge microbiomes

Recovering nitrogen (N) from municipal wastewater is a promising approach to prevent nutrient pollution, reduce energy use, and transition toward a circular N bioeconomy, but remains a technologically challenging endeavor. Existing N recovery techniques are optimized for high-strength, low-volume wastewater. Therefore, developing methods to concentrate dilute N from mainstream wastewater will bridge the gap between existing technologies and practical implementation. The N-rich biopolymer cyanophycin is a promising candidate for N bioconcentration due to its pH-tunable solubility characteristics and potential for high levels of accumulation. However, the cyanophycin synthesis pathway is poorly explored in engineered microbiomes. In this study, we analyzed over 3,700 publicly available metagenome assembled genomes (MAGs) and found that the cyanophycin synthesis gene cphA was ubiquitous across common activated sludge bacteria. We found that cphA was present in common phosphorus accumulating organisms (PAO) Ca. ‘Accumulibacter’ and Tetrasphaera, suggesting potential for simultaneous N and P bioconcentration in the same organisms. Using metatranscriptomic data, we confirmed the expression of cphA in lab-scale bioreactors enriched with PAO. Our findings suggest that cyanophycin synthesis is a ubiquitous metabolic activity in activated sludge microbiomes. The possibility of combined N and P bioconcentration could lower barriers to entry for N recovery, since P concentration by PAO is already a widespread biotechnology in municipal wastewater treatment. We anticipate this work to be a starting point for future evaluations of combined N and P bioaccumulation, with the ultimate goal of advancing widespread adoption of N recovery from municipal wastewater

Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing

High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases – Dpo4 and Klenow exo[subscript −] – obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn[superscript 2+] with a misincorporation rate gain of ~2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn[superscript 2+] and Mg[superscript 2+] change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.Damon Runyon Cancer Research FoundationNational Institutes of Health (U.S.)National Science Foundation (U.S.)McGovern Institute for Brain Research at MITMassachusetts Institute of Technology. Media LaboratoryNew York Stem Cell Foundation (Robertson Neuroscience Investigator Award)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Statistical Analysis of Molecular Signal Recording

A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a “molecular ticker tape”, in which an engineered DNA polymerase (DNAP) writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1) the polymerase biochemical parameters, and (2) the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.United States. Defense Advanced Research Projects Agency. Living Foundries ProgramGoogle (Firm)New York Stem Cell Foundation. Robertson Neuroscience Investigator AwardNational Institutes of Health (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Transformative R01 1R01GM104948)National Institutes of Health (U.S.) (Single Cell Grant 1 R01 EY023173)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (CAREER Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS1042134)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

Toward Design-based Engineering of Industrial Microbes

Engineering industrial microbes has been hampered by incomplete knowledge of cell biology. Thus an iterative engineering cycle of modeling, implementation, and analysis has been used to increase knowledge of the underlying biology while achieving engineering goals. Recent advances in Systems Biology technologies have drastically improved the amount of information that can be collected in each iteration. As well, Synthetic Biology tools are melding modeling and molecular implementation. These advances promise to move microbial engineering from the iterative approach to a design-oriented paradigm, similar to electrical circuits and architectural design. Genome-scale metabolic models, new tools for controlling expression, and integrated -omics analysis are described as key contributors in moving the field toward Design-based Engineering. \ua9 2010 Elsevier Ltd. All rights reserved