Biology subjects

Simmons, B. A.

Publications and source records attributed to Simmons, B. A..

15 recordsLinked to original sources

bioRxiv2025

Binary vector origin predictably determines Agrobacterium-mediated transformation outcome across eukaryotic kingdom

Agrobacterium-mediated transformation (AMT) is the primary means of genetic engineering in plants and many fungi, but the factors that control transformation outcomes--efficiency, transgene insertion number, and transgene integrity--remain poorly characterized. Although transformation outcomes dictate an events potential utility in both industrial and academic contexts, AMT remains largely unoptimized for these metrics. Here, we systematically analyze the impact of the transgene-harboring binary vector on transformation outcomes across plant and fungal species. Through a comparison of different plasmid origin of replication (ORI) families and engineered copy number variants, our results reveal that the ORI family--not plasmid copy number--dictates T-DNA insertion number, backbone inclusion, and transformation efficiency, while plasmid copy number tuning alters efficiency without changing ORI family-specific signatures. Independent of plasmid copy number across kingdoms, the most widely used pVS1 ORI-based vectors (e.g. pCambia) result in significantly more insertions per transformant and high levels of transgene silencing compared to the less-utilized pSa ORI family, which enriches for more uniform single insertion events. Furthermore, we demonstrate that ORI-dependent transformation outcomes in yeast predictably reflect those in Arabidopsis. Together, these results lay the foundation for future binary vector design aimed at achieving more predictable, controllable, and optimized transformation outcomes across diverse eukaryotic hosts.

plant biology↗

bioRxiv2023

A murky ruling threatens the fate of millions of US wetlands

For decades, federal protections were extended to wetlands adjacent to navigable "waters of the US" by the Clean Water Act. In its Sackett v. EPA ruling, however, the US Supreme Court redefined the meaning of "adjacent," eliminating protections to wetlands without a continuous surface connection to these waters (i.e., geographically isolated wetlands). Yet it remains unclear how this continuous surface test will work in reality, where ecological connectivity often extends beyond physical connectivity. Here, we estimate that 22-36% of US wetlands could be considered geographically isolated, depending upon how isolation is defined on the ground, with wetlands in the Midwest and Northeast at greatest risk of losing protection. Stable state-level protections are urgently needed to secure the countrys wetlands from further pollution and destruction. One-Sentence SummaryOver 3 million wetlands could be considered "geographically isolated" and lose extended protections from the Clean Water Act.

ecology↗

bioRxiv2023

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum using statistical design of experiments

BackgroundThe industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance and commodity chemical. We have previously reported serendipitous production of TMP in C. glutamicum strains. In the present study, we demonstrate TMP production in C. glutamicum type strain ATCC13032 via the expression of a heterologous TMP pathway in a defined medium followed by statistical design of experiments to understand the effect of the media composition on TMP production. ResultsThe C. glutamicum strain engineered to overexpress acetolactate synthase and alpha-acetolactate decarboxylase from Lactococcus lactis produced [~]0.8 g/L TMP in CGXII minimal medium supplemented with 40 g/L glucose in 24-deep well plates. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v/v) hydrolysates derived from ionic liquid pretreated sorghum biomass. A screen for improvements in media composition on TMP titer was conducted using fractional factorial design that identified glucose and urea as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g/L TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol/mol) from 80 g/L glucose and 11.9 g/L urea in shake flask batch cultivation. ConclusionsWe engineered the industrially relevant host, C. glutamicum for targeted production of TMP by heterologous expression of pathway proteins. We demonstrated the capability of the engineered strain for growth and TMP production utilizing real world carbon streams such as hydrolysates. We further identified glucose and urea as the key minimal media components significantly affecting TMP production using statistical media optimization.

synthetic biology↗

bioRxiv2023

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc via CRISPRi led to faster growth and uptake of glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatics making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatics present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

microbiology↗

bioRxiv2023

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para-coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para-coumarate minimal medium. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/532821v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f0754aorg.highwire.dtl.DTLVardef@a45d24org.highwire.dtl.DTLVardef@df0596org.highwire.dtl.DTLVardef@1e7e140_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

Revealing Pentose Catabolism in Pseudomonas putida

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (strain M2), while the third isolate represents a new species in the group (strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPRi system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion. Originality-Significance StatementMembers of the Pseudomonas putida group are intensively studied for their role in plant growth promotion and biomass conversion. Despite this interest, the scope of pentose oxidation, key sugars in plant biomass, in this group is not known. Here, we report targeted isolation of members of the P. putida group that grow by xylose and arabinose oxidation. Using a combined genomic and proteomic approach, we identify gene products involved in pentose oxidation and identify conditionally essential genes for xylose oxidation using a CRISPRi gene repression approach. This work describes a targeted isolation and analysis strategy that may applied for many microbial groups of industrial and agricultural interest.

microbiology↗

bioRxiv2022

Mapping the risks of China's global coastal development to marine socio-ecological systems

Rapid coastal development continues to jeopardize the integrity of marine socio-ecological systems. China is now the largest bilateral creditor in the world, committing nearly half a trillion US dollars to overseas development finance since 2008. Meanwhile, there are growing concerns over the impacts of this boom in Chinese development finance on marine systems. Here, we quantify the risks of coastal development projects financed by China to marine biodiversity and coastal Indigenous communities. Ports present the greatest impact risks to marine systems, in terms of both magnitude and area at risk, with power plants, roads, and other facilities presenting relatively high localized risks. Risks are most prominent in Africa and the Caribbean, with coastal Indigenous communities in Western and Central Africa particularly vulnerable to the potential negative impacts of development. All projects present some risk to threatened marine species and potential critical habitats, but few present high risks to nearby marine protected areas. Most projects present additional risks to ecosystems that are already under increasing human pressures, but some are likely to introduce new risks to relatively intact ecosystems. "Bluing" future coastal development projects in Chinas overseas development finance portfolio will require more social and environmental safeguards, higher standards for host-country impact assessments, and greater integration of land-sea risk mitigation and management approaches.

ecology↗

bioRxiv2022

Assessing Comparative Microbiome Performance in Plant Cell Wall Deconstruction Using Multi-omics-Informed Network Analysis

Plant cell walls are interwoven structures recalcitrant to degradation. Both native and adapted microbiomes are particularly effective at plant cell wall deconstruction. Studying these deconstructive microbiomes provides an opportunity to assess microbiome performance and relate it to specific microbial populations and enzymes. To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from time-dependent gene expression identified key deconstructive groups within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmanimonadetes populations. Functional analysis of gene expression demonstrated that the microbiomes proceeded through successional stages that are linked to enzymes that deconstruct plant cell wall polymers. This combination of network and functional analysis highlighted the importance of celluloseactive Actinobacteria in differentiating the performance of these microbiomes.

microbiology↗

bioRxiv2021

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress

Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

systems biology↗

bioRxiv2021

Delivering on Biden's 2030 conservation commitment

On January 27, 2021, President Biden signed an executive order, Tackling the Climate Crisis at Home and Abroad, committing the United States to various goals within his campaigns major climate policy, the Biden Plan for a Clean Energy Revolution and Environmental Justice. Included in this executive order is a commitment to "conserving at least 30 percent of [the United States] lands and oceans by 2030." This ambitious conservation target signals a promising direction for biodiversity in the United States. However, while the executive order outlines several goals for climate mitigation, the 30x30 target remains vague in its objectives, actions, and implementation strategies for protecting biodiversity. Biodiversity urgently needs effective conservation action, but it remains unclear where and what this 30% target will be applied to. Achieving different climate and biodiversity objectives will require different strategies and, in combination with the associated costs of implementation, will lead to different priority areas for conservation actions. Here, we illustrate what the 30% target could look like across four objectives reflective of the ambitious goals outlined in the executive order. We compile several variations of terrestrial protected area networks guided by these different objectives and examine the trade-offs in costs, ecosystem representation, and climate mitigation potential between each. We find little congruence in priority areas across objectives, emphasizing just how crucial it will be for the Biden administration to develop clear objectives and establish appropriate performance metrics from the outset to maximize both conservation and climate outcomes in support of the 30x30 target. We discuss important considerations that must guide the administrations conservation strategies in order to ensure meaningful conservation outcomes can be achieved over the next decade.

ecology↗

bioRxiv2021

The status of current and tentative marine natural World Heritage areas

The 1972 World Heritage Convention (WHC), along with the 1994 Global Strategy, aim to preserve the outstanding universal value of internationally important cultural and natural sites within a "representative, balanced and credible" network of highly-protected areas. Increasing human pressures and shortfalls in representation have been documented across the World Heritage network, particularly in terrestrial and cultural sites, threatening the integrity and primary goals of the WHC. However, the conservation status of current and tentative (i.e., proposed) marine natural World Heritage areas remains relatively unknown. We assessed the extent of recent (2013) and historical (2008-2013) cumulative human impacts and several metrics of representation (country, continent, ecoregion, wilderness, and threatened species) within existing and tentative marine natural World Heritage areas. We found moderate yet increasing cumulative human impacts across most existing sites, and high or very high impacts across the majority of tentative sites. Climate change impacts comprised nearly 75% of impact scores, on average, and differences in land and marine impacts across sites could help prioritise management decisions. Over 75% of marine ecoregions and 85% of threatened species considered in this study have no representation within the existing marine natural World Heritage network. We outline examples of how prioritizing representation across tentative sites for future World Heritage listing could greatly increase these measures. We urge the WHC to adopt quantitative, systematic and transparent evaluations of how current and tentative sites contribute to the overarching goals of maintaining a representative World Heritage network and preserving their outstanding universal values for future generations.

ecology↗

bioRxiv2019

Succession of physiological stages hallmarks the transcriptomic response of fungus Aspergillus niger to lignocellulose

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergilllus niger over its life time to six substrates important for biofuel production.\n\nResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.\n\nConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

microbiology↗

bioRxiv2019

The response of Pseudomonas putida to a complex lignolysate

There is strong interest in the valorization of lignin derived from plant biomass to produce valuable products; however, the structural complexity of this biopolymer has been a major bottleneck to conversion. Chemical pretreatment liberates soluble fractions of lignin that may be upgraded by biological conversion. Here, ionic liquid pretreatment was employed to obtain soluble aromatic-rich fractions from sorghum, which were converted by Pseudomonas putida KT2440, a promising host for bioconversion of aromatics derived from lignin. Growth studies and mutational analysis demonstrated that P. putida growth on these soluble lignin-derived fractions, referred to as lignolysate, was dependent on aromatic monomers derived from lignin (p-coumarate and ferulate), but other, unknown factors in the lignolysate contributed to growth. Proteomic and metabolomic analyses provided evidence that these unknown factors were amino acids and residual ionic liquid. Proteomic measurements indicated a coordinated response in which these substrates were catabolized simultaneously. A cholinium catabolic pathway was identified and deletion of five genes in the pathway abrogated the ability of P. putida to grow on cholinium as a sole carbon source. This work demonstrates that lignolysates obtained through biomass pretreatment contain multiple substrates and conversion strategies for lignin-derived should take this complexity into account.\n\nImportanceLignin is one of the most abundant biopolymers on Earth and is generated as a co-product in the processing of lignocellulosic biomass. Valorization of these residual lignin streams is a promising method to enhance the economic viability of modern lignocellulosic biorefineries. In this study we developed a process to couple chemical depolymerization of lignin and biological conversion using Pseudomonas putida KT2440. Water-soluble and bioavailable lignolysate was obtained from sorghum and further characterized as a growth substrate for P. putida. Proteomic and metabolomic analyses demonstrated that P. putida metabolized other components of the lignolysate beyond monoaromatic compounds, which illuminates how microbes can process complex lignolysates obtained from plants. Understanding the underlying microbial responses in lignolysates will enable the design of rational strategies for lignin valorization.

microbiology↗

bioRxiv2019

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

BackgroundRhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides.\n\nResultsOur data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression strength by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) of potential value for genetic engineering of R. toruloides.\n\nConclusionsWe present a list of robust and constitutive, native promoters to facilitate genetic engineering of R. toruloides. This set of thoroughly characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

synthetic biology↗

bioRxiv2018

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids

Plant biomass is an attractive source of renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the total biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as plant-derived aromatics and amino acids have the potential to improve the efficiency of overall biomass conversion. Pseudomonas putida is a promising host for biomass conversion for its ability to metabolize a wide variety of organic compounds, including aromatics derived from lignin. P. putida was engineered to produce medium chain methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics, 4-hydroxybenzoate (4-HB) and protocatechuate (PCA). Unexpectedly, P. putida methyl ketone production was enhanced 2-to 5-fold compared to sugar controls when Arabidopsis thaliana hydrolysates derived from engineered plants that overproduce 4-HB and PCA, while E. coli production was lowered in these hydrolysates. This enhancement was more pronounced (~7-fold increase) with hydrolysates derived from non-engineered switchgrass (Panicum virgatum L.) suggesting it did not arise from overproduction of 4-HB and PCA. Global proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a 9-fold increase in methyl ketone titer (1.1 g/L).

bioengineering↗