Biology subjects

Bottoms, S.

Publications and source records attributed to Bottoms, S..

5 recordsLinked to original sources

bioRxiv2023

Bioconversion of Glucose-Rich Lignocellulosic Wood Hydrolysates to 3-Hydroxypropionic Acid and Succinic Acid using Engineered Saccharomyces cerevisiae

Background3-hydroxypropionic acid (3-HP) and succinic acid (SA) were announced as two of the top twelve value-added platform chemicals from biomass out of a group of over 300 potential compounds that could be made from biomass in a government report in 2004 (Werpy and Petersen, 2004) and in an updated report in 2010 (Bozell and Petersen, 2010). The screening criteria used in the report classified 3-HP and SA as direct petroleum replacement building block chemicals. 3-HP is a precursor to several high-value compounds, such as acrylic acid, 1,3-propanediol, acrylamide, and methyl acrylates, that ultimately end up in products such as fibers, contact lenses, diapers, fabric coatings, and other super absorbent polymers (SAPs). SA is a high-value platform chemical used in polyester production and a precursor for nylon and other bioplastics. Additionally, these reports identified pathways to building block compounds from sugars. Yeast fermentations were identified in these reports as a preferred potential pathway to 3-HP and SA production from sugars because of yeasts natural low pH tolerance. ResultsThe laboratory strain Saccharomyces cerevisiae BY4741 was engineered to produce either 3-HP or SA. These yeasts can convert fermentable sugars from glucose-rich lignocellulosic hardwood feedstocks into organic acid products such as 3-HP and SA under low pH conditions using exponential fed-batch cultivation strategies. Glucose-rich wood sugars provided a better growth environment for the engineered yeast strains, increasing production titers by 6.1 and 6.5 times for SA and 3-HP, respectively. ConclusionsThis study shows the potential of locally produced glucose-rich wood sugars to increase the production of platform chemicals necessary in the production of biobased polymers by engineered yeast cell factories.

microbiology↗

bioRxiv2023

Conversion of aromatic compounds from fractionated industrial hydrolysis lignin by Pseudomonas putida and environmental strains

BackgroundThe utilization of Pseudomonas putida was explored in this study as a promising approach for lignin valorization. To this end, dry hydrolysis lignin was used as a feedstock for the first time. Hydrolysis lignin is a product of the enzymatic hydrolysis and separation of cellulose and hemicellulose from the lignin backbone in diverse lignocellulosic sources. Various fractionation techniques were applied to obtain lignin monomers and multimers in solution for use as a growth medium for P. putida, whose tolerance of inhibitory phenolic compounds distinguishes it from most bacteria. ResultsPhysiological evaluations revealed that Pseudomonas putida strains KT2440 and PaW85 exhibited broad pH tolerance ranges, with robust growth observed at elevated pH levels. Batch fermentations using hydrolysis lignin (HL) solutions showed complete consumption of sugars within 24 hours, demonstrating the viability of fractionated HL as a substrate for P. putida cultivation. HPLC analysis of HL monomer concentrations during simulated fed-batch fermentation revealed rapid catabolism of catechol and increased CCMA concentration, followed by stabilization, indicating that CCMA is synthesized more quickly than degraded when the initial catechol concentration is high. Filtered alkaline HL fractionations yielded more than twice as much catechol as unfiltered fractionations. Screening of indigenous bacterial strains isolated from various soil and water samples (CELMS Collection, website http://eemb.ut.ee) identified five new candidate strains for CCMA production, two for PCA production, and three for vanillic acid production. ConclusionsThe novel use of fractionated hydrolysis lignin as a growth medium shows potential for lignin valorization and chemical production. Filtered alkaline fractionation yields more catechol and is superior for cis,cis-muconic acid production; however, unfiltered fractionations may be more suitable for other compounds and upscaling. Further investigation of screened strains could reveal more efficient enzymes, which could be optimized and transformed into P. putida in future research.

microbiology↗

bioRxiv2019

Identifying and engineering ancient variants of enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion ("indel") events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time. Author summaryMassive sequencing projects expose the extent of natural, genetic diversity. Here, we describe a method with capacity to perform ancestor sequence reconstruction from data sets in excess of 10,000 sequences, poised to recover ancestral diversity, including the evolutionary events that determine present-time biological function and structure. We introduce a novel strategy for suggesting "indel variants" that are distinct from, but can be explored alongside, substitution variants for creating ancestral libraries. We demonstrate how indels can be used as building blocks to form "hybrid ancestors"; based on this strategy, we synthesise ancestor variants, with varying enzymatic activities, for wide-ranging applications in the biotechnology sector.

bioinformatics↗

bioRxiv2017

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

BackgroundGamma valerolactone (GVL) is a promising technology for degradation of biomass for biofuel production; however, GVL has adverse toxicity effects on fermentative microbes. Using a combination of chemical genomics and chemical proteomics we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast.\n\nResultsChemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate phenolic acids to vinyl derivatives, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain.\n\nConclusionsWe found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol content in cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates

bioengineering↗

bioRxiv2016

Inhibition of microbial biofuel production in drought stressed switchgrass hydrolysate

BackgroundInterannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during three years with significantly different precipitation profiles, representing a major drought year (2012) and two years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strains of Saccharomyces cerevisiae and Zymomonas mobilis. A chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates.\n\nResultsWhile most corn stover and switchgrass hydrolysates were readily fermented, growth of S. cerevisiae was completely inhibited in hydrolysate generated from drought stressed switchgrass. Based on chemical genomics analysis, yeast strains deficient in genes related to protein trafficking within the cell were significantly more resistant to the drought year switchgrass hydrolysate. Detailed biomass and hydrolysate characterization revealed that switchgrass accumulated greater concentrations of soluble sugars in response to the drought and these sugars were subsequently degraded to pyrazines and imidazoles during ammonia-based pretreatment. When added ex situ to normal switchgrass hydrolysate, imidazoles and pyrazines caused anaerobic growth inhibition of S. cerevisiae.\n\nConclusionsIn response to the osmotic pressures experienced during drought stress, plants accumulate soluble sugars that are susceptible to degradation during chemical pretreatments. For ammonia-based pretreatment these sugars degrade to imidazoles and pyrazines. These compounds contribute to S. cerevisiae growth inhibition in drought year switchgrass hydrolysate. This work discovered that variation in environmental conditions during the growth of bioenergy crops could have significant detrimental effects on fermentation organisms during biofuel production. These findings are relevant to regions where climate change is predicted to cause an increased incidence of drought and to marginal lands with poor water holding capacity, where fluctuations in soil moisture may trigger frequent drought stress response in lignocellulosic feedstocks.

microbiology↗