Biology subjects

Adam P Arkin

Publications and source records attributed to Adam P Arkin.

6 recordsLinked to original sources

bioRxiv2016

Programming mRNA decay to modulate synthetic circuit resource allocation

Synthetic biomolecular networks embedded in host-cells compete with cellular processes for limited intracellular resources. Resource utilization is a major variable that impacts synthetic circuit behavior. Here we show that intracellular resources could be diverted from cellular operations to a synthetic circuit by programming global mRNA decay using the sequence-dependent endoribonuclease MazF. Synthetic circuit genes were protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and the metabolic flux of a high-value metabolite gluconate were significantly enhanced using this genome-scale control strategy. Proteomics measurements discovered a key host translation factor in need of protection to optimize the resource redistribution activity. A dynamic computational model demonstrated that the MazF mRNA-decay feedback loop achieved proportional control of MazF levels in an optimal operating regime. RNA-seq time-series measurements of MazF-induced cells elucidated the dynamic shifts in the transcript abundance and discovered regulatory design elements that could be used to expand the control mechanisms of the MazF resource allocator. Together, these results demonstrated that manipulation of resource allocation is a tunable parameter that can be used to redirect resources away from cellular processes to synthetic circuits to enhance target functions.

Synthetic Biology↗

bioRxiv2016

QTL-guided metabolic engineering of a complex trait

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this \"genetic blueprint\", and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait in S. cerevisiae, as well as other organisms.

Bioengineering↗

bioRxiv2016

Deep Annotation of Protein Function across Diverse Bacteria from Mutant Phenotypes

SummaryThe function of nearly half of all protein-coding genes identified in bacterial genomes remains unknown. To systematically explore the functions of these proteins, we generated saturated transposon mutant libraries from 25 diverse bacteria and we assayed mutant phenotypes across hundreds of distinct conditions. From 3,903 genome-wide mutant fitness assays, we obtained 14.9 million gene phenotype measurements and we identified a mutant phenotype for 8,487 proteins with previously unknown functions. The majority of these hypothetical proteins (57%) had phenotypes that were either specific to a few conditions or were similar to that of another gene, thus enabling us to make informed predictions of protein function. For 1,914 of these hypothetical proteins, the functional associations are conserved across related proteins from different bacteria, which confirms that these associations are genuine. This comprehensive catalogue of experimentally-annotated protein functions also enables the targeted exploration of specific biological processes. For example, sensitivity to a DNA-damaging agent revealed 28 known families of DNA repair proteins and 11 putative novel families. Across all sequenced bacteria, 14% of proteins that lack detailed annotations have an ortholog with a functional association in our data set. Our study demonstrates the utility and scalability of high-throughput genetics for large-scale annotation of bacterial proteins and provides a vast compendium of experimentally-determined protein functions across diverse bacteria.

Microbiology↗

bioRxiv2016

A Comparison of the Costs and Benefits of Bacterial Gene Expression

To study how a bacterium allocates its resources, we compared the costs and benefits of most of the proteins in Escherichia coli K-12 during growth in minimal glucose medium. Proteins that are important for fitness are usually highly expressed, and 95% of these proteins are expressed at above 13 parts per million (ppm). Conversely, proteins that do not measurably benefit the host tend to be weakly expressed, with a median expression of 13 ppm. In aggregate, genes with no detectable benefit account for 31% of protein production, or about 22% if we correct for genetic redundancy. Although some of the apparently unnecessary expression could have subtle benefits in minimal glucose medium, the majority of the burden is due to genes that are important in other conditions. We propose that over 10% of the cells protein is \"on standby\" in case conditions change.

Systems Biology↗

bioRxiv2014

Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

Synthetic Biology↗

bioRxiv2014

The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20

Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions.

Genetics↗