The invention provides a non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid or caprolactam pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective adipate, 6-aminocaproic acid or caprolactam pathway. The invention additionally provides a method for producing adipate, 6-aminocaproic acid or caprolactam. The method can include culturing an adipate, 6-aminocaproic acid or caprolactam producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an adipate, 6-aminocaproic acid or caprolactam pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce adipate, 6-aminocaproic acid or caprolactam.

Microorganisms for the production of adipic acid and other compounds

The invention provides a non-naturally occurring microbial organism having an acetyl-CoA pathway and the capability of utilizing syngas or syngas and methanol. In one embodiment, the invention provides a non-naturally occurring microorganism, comprising one or more exogenous proteins conferring to the microorganism a pathway to convert CO, CO2 and/or H2 to acetyl-coenzyme A (acetyl-CoA), methyl tetrahydrofolate (methyl-THF) or other desired products, wherein the microorganism lacks the ability to convert CO or CO2 and H2 to acetyl-CoA or methyl-THF in the absence of the one or more exogenous proteins. For example, the microbial organism can contain at least one exogenous nucleic acid encoding an enzyme or protein in an acetyl-CoA pathway. The microbial organism is capable of utilizing synthesis gases comprising CO, CO2 and/or H2, alone or in combination with methanol, to produce acetyl-CoA. The invention additionally provides a method for producing acetyl-CoA, for example, by culturing an acetyl-CoA producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme or protein in a sufficient amount to produce acetyl-CoA, under conditions and for a sufficient period of time to produce acetyl-CoA.

Methods and organisms for utilizing synthesis gas or other gaseous carbon sources and methanol

The invention provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided are methods for the production of 4-HB and BDO.

Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors

The invention provides a non-naturally occurring microorganism comprising one or more gene disruptions, the one or more gene disruptions occurring in genes encoding an enzyme obligatory to coupling 1,4-butanediol production to growth of the microorganism when the gene disruption reduces an activity of the enzyme, whereby the one or more gene disruptions confers stable growth-coupled production of 1,4-butanediol onto the non-naturally occurring microorganism. The microorganism can further comprise a gene encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic pathway. The invention additionally relates to methods of using microorganisms to produce BDO.

Methods and organisms for the growth-coupled production of 1,4-butanediol

Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae.

Studies on rat brain acyl-coenzyme A hydrolase (short chain)

15-Azasteroid blockage of cell permeability and mitochondrial respiration.

Amyloid-beta (Aβ1-42) aggregation triggers neurotoxicity and is linked to Alzheimer's disease. Aβ1-42 oligomers, rather than extended fibrils, adhere to the cell membrane, causing cell death. Phosphatidylserine (PS), an anionic phospholipid, is prevalent in neuronal membranes (< 20 molar percentage) and, while isolated to the cytoplasmic leaflet of the membrane in healthy cells, its exposure in apoptotic cells and migration to exoplasmic leaflet is triggered by oxidative damage to the membrane. It is widely believed that PS plays a crucial role in the Aβ peptide interaction in the membranes of neuronal cells. However, due to the complexity of the cell membrane, it can be challenging to address molecular level understanding of the PS-Aβ binding and oligomerization processes. Herein, we use microcavity supported lipid bilayers (MSLBs) to analyse PS and Aβ1-42 binding, oligomer formation, and membrane damage. MSLBs are a useful model to evaluate protein-membrane interactions because of their cell-like dual aspect fluidity, their addressability and compositional versatility. We used electrochemical impedance spectroscopy (EIS) and confocal fluorescence microscopy to compare the impact of Aβ1-42 on simple zwitterioinic membrane, dioleoylphosphatidylcholine (DOPC), with MSLBs comprised of transversally asymmetric binary DOPC and dioleoylphosphatidylserine (DOPS). Monomeric Aβ1-42 adsorbs weakly to the pristine zwitterionic DOPC membrane without aggregation. Using a membrane integrity test, with pyranine trapped within the cavities beneath the membrane, Aβ1-42 exposure did not result in pyranine leakage, indicating that DOPC membranes were intact. When 10 mol% DOPS was doped asymmetrically into the membrane's outer leaflet, oligomerization of Aβ1-42 monomer was evident in EIS and atomic force microscopy (AFM), and confocal imaging revealed that membrane damage, resulted in extensive pyranine leakage from the pores. The effects were time, and DOPS and Aβ1-42 concentration-dependent. Membrane pore formation was visible within 30 minutes, and oligomerization, membrane-oligomer multilayer, and Aβ1-42 fibril formation evident over 3 to 18 hours. In asymmetric membranes with DOPS localized to the lower leaflet, optothermally (laser induced) damage increased local DOPS concentrations at the distal leaflet, promoting Aβ1-42 aggregation.

Role of phosphatidylserine in amyloid-beta oligomerization at asymmetric phospholipid bilayers.

Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane’s admittance and fluidity, as well as integrin’s lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan–Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5β1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3−α5β1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid–lectin (GL–Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

https://pubs.acs.org/doi/pdf/10.1021/acs.jpcb.2c05717

Jack B. Robinson

Papers

Studies on rat brain acyl-coenzyme A hydrolase (short chain)

15-Azasteroid blockage of cell permeability and mitochondrial respiration.

Role of phosphatidylserine in amyloid-beta oligomerization at asymmetric phospholipid bilayers.

Galectin-3 Binding to α5β1 Integrin in Pore Suspended Biomembranes