Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

<p>Targeted protein degradation is a powerful strategy to address the canonically undruggable proteome. However, current technologies are limited to targets with cytosolically-accessible and ligandable domains. Here, we designed and synthesized conjugates capable of binding both a cell surface lysosome targeting receptor and the extracellular domain of a target protein. These lysosome targeting chimeras (LYTACs) consist of an antibody fused to agonist glycopeptide ligands for the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs enabled a CRISPRi knockdown screen revealing the biochemical pathway for CI-M6PR-mediated cargo internalization. We demonstrated that LYTACs mediate efficient degradation of Apolipoprotein-E4, epidermal growth factor receptor (EGFR), CD71, and programmed death-ligand 1 (PD-L1). LYTACs represent a modular strategy for directing secreted and membrane proteins for degradation in the context of both basic research and therapy. <b></b></p>

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Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽

10.3390/life11010015 ◽

2020 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Radek Kaňa ◽

Gábor Steinbach ◽

Roman Sobotka ◽

György Vámosi ◽

Josef Komenda

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Protein Interactions ◽

Thylakoid Membrane ◽

Protein Complexes ◽

Correlation Spectroscopy ◽

Membrane Microdomains ◽

Fast Diffusion ◽

Light Harvesting Complexes ◽

Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

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Three-dimensional crystallization of membrane proteins

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500004625 ◽

1988 ◽

Vol 21 (4) ◽

pp. 429-477 ◽

Cited By ~ 156

Author(s):

W. Kühlbrandt

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Complexes ◽

Three Dimensional ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography ◽

Membrane Protein Complexes ◽

Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

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High-Molecular-Weight Complexes of RsbR and Paralogues in the Environmental Signaling Pathway of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00892-06 ◽

2006 ◽

Vol 188 (22) ◽

pp. 7885-7892 ◽

Cited By ~ 45

Author(s):

Olivier Delumeau ◽

Chien-Cheng Chen ◽

James W. Murray ◽

Michael D. Yudkin ◽

Richard J. Lewis

Keyword(s):

Bacillus Subtilis ◽

Protein Complexes ◽

Binary Complex ◽

Regulatory Pathway ◽

Large Structure ◽

General Stress Response ◽

Stress Response Genes ◽

Alternative Protein ◽

Reversible Protein Phosphorylation

ABSTRACT Bacillus subtilis has developed an intricate signal transduction cascade to respond to the imposition of a variety of stresses on the cell. Reversible protein phosphorylation and the formation of alternative protein-protein complexes modulate the activity of σB, the RNA polymerase sigma factor subunit responsible for the transcription of the general stress response genes. Some of the regulators of σB, such as RsbR and RsbS, are known to associate in a 25S complex, called the stressosome, that can bind RsbT until RsbT phosphorylates target residues in RsbR and RsbS. To date, the RsbR-RsbS complex appears to be the most upstream component of the σB regulatory pathway. This large structure is thought to play an important role in sensing and/or integrating signals from different physical stresses. The roles of the paralogues of RsbR that are found in B. subtilis remain unclear. We describe here how the RsbR paralogues copurify with RsbR from B. subtilis cell lysates, and we demonstrate in vitro that the paralogues form large complexes either with RsbS or with a prepurified RsbR-RsbS binary complex. We conclude from these biochemical studies that stressosomes in B. subtilis cells contain minimally RsbS and all of the RsbT-phosphorylatable RsbR paralogues.

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Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

Journal of Bacteriology ◽

10.1128/jb.184.7.1998-2004.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1998-2004 ◽

Cited By ~ 47

Author(s):

Takako Murakami ◽

Koki Haga ◽

Michio Takeuchi ◽

Tsutomu Sato

Keyword(s):

Bacillus Subtilis ◽

Membrane Proteins ◽

Vegetative Growth ◽

Fluorescent Protein ◽

Specific Expression ◽

Rich Media ◽

Green Fluorescent ◽

High Level ◽

Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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Reassessing Escherichia coli as a cell factory for biofuel production

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2017.02.010 ◽

2017 ◽

Vol 45 ◽

pp. 92-103 ◽

Cited By ~ 28

Author(s):

Chonglong Wang ◽

Brian F Pfleger ◽

Seon-Won Kim

Keyword(s):

Escherichia Coli ◽

Biofuel Production ◽

Cell Factory ◽

A Cell

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Periprotein lipidomes of Saccharomyces cerevisiae provide a flexible environment for conformational changes of membrane proteins

eLife ◽

10.7554/elife.57003 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Joury S van 't Klooster ◽

Tan-Yun Cheng ◽

Hendrik R Sikkema ◽

Aike Jeucken ◽

Branch Moody ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Small Molecules ◽

Conformational Changes ◽

Direct Detection ◽

Protein Complexes ◽

Lateral Diffusion ◽

Low Ph ◽

Lipid Particles ◽

High Degree

Yeast tolerates a low pH and high solvent concentrations. The permeability of the plasma membrane (PM) for small molecules is low and lateral diffusion of proteins is slow. These findings suggest a high degree of lipid order, which raises the question of how membrane proteins function in such an environment. The yeast PM is segregated into the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP), which have different lipid compositions. We extracted proteins from these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALP-lipid-protein complexes by chromatography and quantitatively analyzed periprotein lipids located within the diameter defined by one SMALP. Phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from this periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Direct detection of PM lipids in the 'periprotein space' supports the conclusion that proteins function in the presence of a locally disordered lipid state.

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Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF

10.1101/2021.12.12.472321 ◽

2021 ◽

Author(s):

Aurelie Guyet ◽

Amirah Alofi ◽

Richard A Daniel

Keyword(s):

Bacillus Subtilis ◽

Cell Envelope ◽

Cellular Level ◽

Penicillin Binding Proteins ◽

Class A ◽

Peptidoglycan Synthesis ◽

A Cell ◽

Antimicrobial Peptide Resistance ◽

Lipoteichoic Acids

In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

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Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699235 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuxiao Xie ◽

Shulin Chen ◽

Xiaochao Xiong

Keyword(s):

Yarrowia Lipolytica ◽

Fold Increase ◽

Genetically Engineered ◽

Cell Factory ◽

Yeast Nitrogen Base ◽

Nitrogen Base ◽

Yeast Extract Peptone Dextrose ◽

Dry Cell Weight ◽

A Cell ◽

Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

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