Improving single cell protein yields and amino acid profile via mutagenesis: review of applicable amino acid inhibitors for mutant selection

Received: February 4th, 2021 ; Accepted: April 24th, 2021 ; Published: May 18th, 2021 ; Correspondence: [email protected] cell protein (SCP) is a good alternative for substituting plant and animal derived dietary proteins, since SCP production is more environmentally friendly, consumes less water, requires smaller land areas and its effect on climate change is much less pronounced than it is in the case of agriculturally derived proteins. Another advantage of SCP is that it is possible to use a wide variety of biodegradable agro-industrial by-products for the cultivation of SCP producing microorganisms. However, to make single cell protein technology more widely available and improve its economic viability in such markets as animal and fish feed industries, it is necessary to improve the protein yields and amino acid profiles in microorganism strains capable of using agro-industrial by-products. One way to improve the strains used in the process is to create and select SCP-rich mutants. In this review authors propose a novel approach to create SCP-rich mutants with improved total protein content and essential amino acid profiles. In this approach amino acid inhibitors are used to create selective pressure on created mutants. It is expected that mutants with the most pronounced growth would either have higher total protein content, increased essential amino acid concentrations or both, when cultivated on selective plates containing one or multiple amino acid inhibitors. This paper reviews the most suitable groups of amino acid inhibitors that could be used for selection of new strains of SCP-producing microorganisms

Reduced Mitochondrial Membrane Potential is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo- ATPase

Background: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. Methodology/Principal Findings: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. Conclusions/Significance: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential

Research for rural development 2003 International scientific conference proceedings

Available from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

Adaptation giving resistance to ISM reduces the mitochondrial membrane potential and ISM uptake capacity.

<p>(A) Mean mitochondrial membrane potential (ΔΨm) with SEM of Tb427WT trypanosomes incubated with or without 0.5 μM ISM for 3 or 5 hours. ΔΨm is recorded in arbitrary units (A.U.) of fluorescence, and flow cytometer is calibrated at the start of the experiment to have exactly 50% of cells with a value of 100 A.U. and over for the drug-free cells (control at t = 0 h). ***, P-value <0.001 (Student’s t-test, n≥3) of the ISM-treated cells relative to their untreated controls. (B) Mean ΔΨm with SEM of ISM resistant cell lines compared to the parental Tb427WT. Valinomycin was used as a positive control. ***, P-value <0.001 (Student’s t-test, n≥3) relative to WT control. (C) Graph showing the means (and SEM) of 3 independent experiments measuring uptake of ISM by the parental Tb427WT and ISMR1 cell lines. For each of the experiments single cell cultures of either Tb427WT or ISMR1 were incubated in 10 μM ISM, which were sampled in triplicate at the indicated times for the determination of intracellular ISM. (D) Graph showing the means (and SEM) of 3 independent experiments showing uptake and efflux of ISM by the Tb427WT and resistant ISMR15 strains. After incubation with 10 μM ISM for 60 minutes, the cells were centrifuged, washed and resuspended in medium without drug. The intracellular concentration of ISM was monitored in triplicate periodically before and after the removal of drug from the medium. (E) Inhibition of ISM uptake in the presence of potential inhibitors. Tb427WT trypanosomes were incubated for 20 minutes with 10 μM ISM in the absence (Control) or presence of 50 μM enrofloxacin, verapamil, TFP, PCP, CCCP, oligomycin or 1 μM valinomycin. Data presented are mean intracellular ISM concentration with SEM; ***, P-value <0.001 (Student’s t-test, n≥3) relative to untreated controls.</p

Resistance profile of ISM-adapted cell lines and the parental cell line Tb427WT.

<p>EC₅₀ values are given as mean of at least 5 independent determinations and SEM. The resistance factor (RF) is the ratio of the EC₅₀ values of the adapted strain and the wild-type control. Statistical significance was determined using a two-tailed unpaired t-test.</p

ISM resistant cell lines have lost their kinetoplasts.

<p>(A) Fluorescence microscopy of DAPI-stained Tb427WT and ISMR1 and ISMR15 cell lines. The white arrowhead indicates the kinetoplast, which is absent in ISMR1 and ISMR15. The white scale bar represents a length of 5 μm. (B) Electrophoresis gel of PCR products of kinetoplast-encoded genes and nuclearly encoded actin. Genomic DNA was extracted from the parental Tb427WT strain as well as from the ISM resistant ISMR1 and ISMR15 strains and subjected to PCR amplification using primers specific for the gene fragments stated (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004791#pntd.0004791.s004" target="_blank">S1 Table</a>). (C) Like frame B but using primers specific for minicircles, using the TbAT1 gene as a positive control for a nuclearly-encoded single copy gene [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004791#pntd.0004791.ref062" target="_blank">62</a>].</p

Characterisation of S284* mutation in ATP synthase subunit γ identified in ISM resistant ISMR1 and ISMR15 trypanosomes.

<p>Tb427WT trypanosomes were genetically manipulated to replace one allele of subunit γ with either a version that contained the S284* mutation or another wild type copy as a control, and then cultured in the normally lethal concentration of either 20 nM ISM or ethidium bromide (Panels A-D). (Panels A and B) Growth of the genetically modified trypanosome strains in the presence of ISM (A) or ethidium bromide (B) alongside the parental Tb427WT cell line. (C) Fluorescence microscopy of DAPI-stained S284* subunit γ expressing trypanosomes before and after 7 days exposure to ISM or ethidium bromide. The white arrowhead indicates the kinetoplast (absent in the drug-treated cells); the white scale bar represents 5 μm. (D) The mitochondrial membrane potential (ΔΨm) of S284* subunit γ expressing trypanosomes, treated and untreated with ISM or ethidium bromide (EtBr) for 7 days compared to an otherwise isogenic strain expressing non-mutated subunit γ and the parental Tb427WT cell line. Data presented is the mean with SEM; **, P-values <0.01; ***, P-values <0.001 (Student’s t-test, n≥3), relative to Tb427WT unless otherwise indicated. (E) The <i>in vivo</i> virulence of the ISM resistant ISMR1 and ISMR15 cell lines as well as the S284* subunit γ expressing strain with the parental Tb427WT as a control. Five mice were inoculated intraperitoneally for each trypanosome strain and monitored daily for parasitaemia, with those mice reaching terminal parasitaemia euthanized. Data presented shows percentage of surviving mice in each cohort.</p

Drug sensitivity profile of Tb427WT cells expressing either the wild-type F₁ subunit γ gene, or the same gene with mutation S284^*, either exposed to ethidium bromide or ISM, or not exposed to any trypanocide.

<p>All data are the mean of three independent experiments and SEM.</p