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At least 235 records · Page 13

Theoretical Modeling and Computer Simulations for the Origins and Evolution of Reproducing Molecular Systems and Complex Systems with Many Interactive Parts

Our research effort has produced nine publications in peer-reviewed journals listed at the end of this report. The work reported here are in the following areas: (1) genetic network modeling; (2) autocatalytic model of pre-biotic evolution; (3) theoretical and computational studies of strongly correlated electron systems; (4) reducing thermal oscillations in atomic force microscope; (5) transcription termination mechanism in prokaryotic cells; and (6) the low glutamine usage in thennophiles obtained by studying completely sequenced genomes. We discuss the main accomplishments of these publications.

Liang, Shoudan↗

Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

Non-NASA Center↗

Identification of functional non-coding variants associated with orofacial cleft

Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example, IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line, revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays, which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A, and TP63. For two SNPs near IRF6 and one near FOXE1, we engineer the genome of induced pluripotent stem cells, differentiate the cells into embryonic oral epithelium, and discover allele-specific effects on the levels of effector gene expression, and, in two cases, the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis.

Kumari, Priyanka↗

Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

36 MATERIALS SCIENCE↗

Analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets?

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.

59 BASIC BIOLOGICAL SCIENCES↗

Rational Modulation of Plant Root Development Using Engineered Cytokinin Regulators

Achieving precise control over quantitative developmental phenotypes is a key objective in plant biology. Recent advances in synthetic biology have enabled tools to reprogram entire developmental pathways; however, the complexity of designing synthetic genetic programs and the inherent interactions between various signaling processes remains a critical challenge. Here, we leverage Type-B response regulators to modulate the expression of genes involved in cytokinin-dependent growth and development processes. We rationally engineered these regulators to modulate their transcriptional activity (i.e., repression or activation) and potency while reducing their sensitivity to cytokinin. By localizing the expression of these engineered transcription factors using tissue-specific promoters, we can predictably tune cytokinin-regulated traits. As a proof of principle, we deployed this synthetic system in Arabidopsis thaliana to either decrease or increase the number of lateral roots. The simplicity and modularity of our approach makes it an ideal system for controlling other developmental phenotypes of agronomic interest in plants.

Cell signaling↗

Deletion analysis of the itaconic acid biosynthesis gene cluster components in Aspergillus pseudoterreus ATCC32359

The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster (IA cluster) by a clone-based transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), mitochondrial organic acid transporter (mttA), and MFS (Major Facilitator Superfamily) type transporter (mfsA). In this research, we performed EST (Expressed Sequence Tag) analysis and systematic gene deletions to investigate the role of those genes in IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that was distinct from neighbouring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA, and mfsA genes in the cluster are essential for IA production. Deletion of any of them will either completely abolish the IA production or dramatically decrease amount of IA produced. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, the cadA deletion strain is a novel microorganism host for production of aconitic acid and other value-added chemicals from lignocellulosic biomass.

Deng, Shuang↗

Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair

Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

59 BASIC BIOLOGICAL SCIENCES↗

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit + progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

60 APPLIED LIFE SCIENCES↗

Engineering of holocellulase in biomass-degrading fungi for sustainable biofuel production

Biofuels, such as bioethanol, are a clean and sustainable form of energy and have emerged as a viable alternative to fossil fuels. Plant biomass is an important raw material for the production of clean and renewable energy. The holocellulose contained in the composition of plants may be broken down into simple sugars, such as glucose, which are fermented by yeast to produce bioethanol. The conversion of glucose polymers into fermentable sugars is accomplished by enzymes known as holocellulases, which are produced by lignocellulolytic fungi. These enzymes act synergistically for the efficient degradation of cellulose polymers, and the fine and coordinated regulation of this process is performed by transcription factors (TFs). TFs are regulatory proteins that bind to the promoter region of their target genes (CAZymes, sugar transporters, signaling proteins, other TFs, etc.) to induce or repress their transcription. This review aims to understand the main regulatory mechanisms involved in plant biomass degradation by the most studied lignocellulolytic fungi Trichoderma sp., Aspergillus sp., Penicillium sp., and Neurospora crassa. In this context, the most studied TFs related to holocellulose degradation and genetic modification of TFs or promoters as a valuable tool to improve enzyme production for biotechnological purposes have been discussed. Furthermore, this review enables the expansion of knowledge on the regulation of the cellulolytic system of filamentous fungi and the application of this knowledge to the improvement of numerous bioproducts. Engineering TFs and promoters may yield more efficient strains that may be active in plant biomass hydrolysis. In this way, the technological processes for obtaining ethanol from lignocellulose may become more commercially viable.

09 BIOMASS FUELS↗

MAL33 drives natural variation in maltose metabolism in Saccharomyces eubayanus

Maltose is one of the most abundant sugars in brewer’s wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F 2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33 ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18’s own MAL33 ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. Here, we hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

evolutionary plasticity↗

HAND transcription factors cooperatively specify the aorta and pulmonary trunk

Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A Novel Signaling Pathway Connects Thiamine Biosynthesis, Bacterial Respiration, and Production of the Exopolysaccharide Amylovoran in Erwinia amylovora

Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria

The Forkhead or Fox gene family encodes putative transcription factors. There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans. Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ. Here, we extend this analysis to invertebrates, using available sequences from D. melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes. Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes. We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D. melanogaster, A. gambiae or C. elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage. Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes.

NASA Program Fundamental Space Biology↗

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

NASA Discipline Regulatory Physiology↗

Probing the limits of genetic recoding using multi-omics-guided evolution

Engineering the genetic code—by reassigning multiple of the 64 natural codons—enables making organisms resistant to all viruses, preventing genetic information exchange, and allowing the biosynthesis of genetically encoded unnatural polymers. However, synonymous codon replacement—recoding—is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using genome synthesis, directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple synthetic Escherichia coli genomes. We construct six partially recoded E. coli strains bearing up to 45.8% of a synthetic genome with a deleterious 57-codon genetic code. As our analyses revealed widespread defects—including unassigned codons in Syn61 and Syn57—we apply multi-omics to revise our genome design and mitigate defects. Using multi-omics, we show that recoding induces transcriptional and translational changes leading to fitness defects under hundreds of conditions. Finally, we develop a multi-omics-guided evolution strategy that rapidly restores fitness, enabling genome synthesis with radical changes.

Nyerges, Akos [Harvard Medical School, Boston, MA ↗

Convergent evolution of aerobic fermentation through divergent mechanisms acting on key shared glycolytic genes

As the tree of life becomes increasingly accessible to molecular investigations, describing mechanisms underlying evolutionary convergence and constraint will be crucial to understanding diversification. The lineage including the model yeast Saccharomyces cerevisiae evolved aerobic fermentation in part through an ancient whole genome duplication and retention of glycolytic genes. To evaluate glycolytic rates across diverse yeasts, we developed and deployed an extracellular acidification rates (ECAR) assay on 299 species that span more than 400 million years of evolution and identified a clade in the genus Saturnispora that convergently evolved aerobic fermentation. Through comparative genomics and transcriptomics, we found that several glycolytic genes had higher expression and novel cis-regulatory elements in aerobically fermenting Saturnispora species. When the transcription factor required for their activation was deleted in Saturnispora dispora, the mutants had reduced glycolytic rates and increased respiration. Intriguingly, many of the upregulated genes are orthologous to duplicated glycolytic genes in S. cerevisiae. These divergent genetic mechanisms affecting the same set of genes suggest that there are strong evolutionary constraints on how aerobic fermentation can arise.

Horianopoulos, Linda C. [Great Lakes Bioenergy Res↗