Biology subjects

Stefan Bauer

Publications and source records attributed to Stefan Bauer.

3 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↗

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↗