Engineering topics

Donohue, Timothy J. (ORCID:0000000187382467)

Publications and source records attributed to Donohue, Timothy J. (ORCID:0000000187382467).

9 records

DOE OSTI2025

RB-TnSeq barcode abundance data sets for Novosphingobium aromaticivorans grown on the β-5-linked aromatic dimer dehydrodiconiferyl alcohol

ABSTRACT A randomly barcoded transposon insertion sequencing (RB-TnSeq) library of Novosphingobium aromaticivorans DSM12444 was grown in media containing either glucose or the β-5-linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) as the sole carbon source. The cultures were grown to saturation and then sequenced, yielding the barcode abundance data sets presented here.

Metz, Fletcher↗

DOE OSTI2024

Effect of Temperature and pH on Microbial Communities Fermenting a Dairy Coproduct Mixture

Organic-rich industrial residues can serve as renewable feedstocks for the generation of useful products by microbial fermentation. We investigated fermenting communities enriched in a mixture of ultra-filtered milk permeate (UFMP) and acid whey from cottage cheese (CAW), two dairy coproducts rich in lactose. To evaluate how operational pH and temperature affect microbial communities and fermentation products, we operated 12 bioreactors for 140 days, each fed a 1:1 mixture of UFMP and CAW at either 35 °C or 50 °C and at either a pH of 4.8 or 5.5. The bioreactors operated at a pH of 4.8 resulted in the incomplete conversion of lactose, while those operated at a pH of 5.5 consistently fermented lactose, primarily into lactic, acetic, and hexanoic acids. The metagenomic analyses revealed that microbial communities obtained at a pH of 5.5 were dominated by lactic acid-producing organisms. Additionally, an inverse relationship was found between the abundance of chain elongating organisms and lactic acid accumulation, with 50 °C reducing the abundance of these organisms and enhancing lactic acid yields. We conclude that the pH and temperature are important determinants of the fermentation of dairy coproducts with a pH of 5.5 and 50 °C yielding the most promising results for lactic acid production. Additional research is required to better understand the factors affecting functional consistency of the process.

Biotechnology & Applied Microbiology↗

DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a mu ltiplex, r andomized C RISPR i nterference s equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii . Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Ellis, Nicole A. (ORCID:0000000195928415)↗

DOE OSTI2022

The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR ( C ell en velope K inase and R egulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo , and identify genes that are directly and indirectly controlled by CenKR in Rb . sphaeroides . Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb . sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

Lakey, Bryan D. (ORCID:0000000332795242)↗

DOE OSTI2022

iNovo479: Metabolic Modeling Provides a Roadmap to Optimize Bioproduct Yield from Deconstructed Lignin Aromatics by Novosphingobium aromaticivorans

Lignin is an abundant renewable source of aromatics and precursors for the production of other organic chemicals. However, lignin is a heterogeneous polymer, so the mixture of aromatics released during its depolymerization can make its conversion to chemicals challenging. Microbes are a potential solution to this challenge, as some can catabolize multiple aromatic substrates into one product. Novosphingobium aromaticivorans has this ability, and its use as a bacterial chassis for lignin valorization could be improved by the ability to predict product yields based on thermodynamic and metabolic inputs. In this work, we built a genome-scale metabolic model of N. aromaticivorans, iNovo479, to guide the engineering of strains for aromatic conversion into products. iNovo479 predicted product yields from single or multiple aromatics, and the impact of combinations of aromatic and non-aromatic substrates on product yields. We show that enzyme reactions from other organisms can be added to iNovo479 to predict the feasibility and profitability of producing additional products by engineered strains. Thus, we conclude that iNovo479 can help guide the design of bacteria to convert lignin aromatics into valuable chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Redundancy in Aromatic O -Demethylation and Ring-Opening Reactions in Novosphingobium aromaticivorans and Their Impact in the Metabolism of Plant-Derived Phenolics

ABSTRACT Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds, followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM 12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O -demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolic compounds by N. aromaticivorans . The results showed the involvement of DesA in O -demethylation of syringic and vanillic acids, LigM in O- demethylation of vanillic acid and 3-MGA, and a new O- demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring-opening dioxygenase involved in 3-MGA, PCA, and GA metabolism and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O -demethylation of 3-MGA to GA. We predict that this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

Biotechnology & Applied Microbiology↗

DOE OSTI2020

A bacterial biosynthetic pathway for methylated furan fatty acids

Not Available

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope

ABSTRACT Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides , loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

59 BASIC BIOLOGICAL SCIENCES↗