Daily Archives: August 16, 2021

It has been 186 years since Charles Darwin collected the samples of the Galapagos Islands species that led to his explanation of how the diversity of life on Earth has evolved and forever changed the way we understand the world.

During his five-week stay on the islands, Darwin collected dozens of samples, including one small, light brownish-grey snake on Floreana Island. That sample, now at the Natural History Museum in London, was the basis for describing a new species, the Galapagos (Floreana) racer.

The species disappeared from Floreana but can still be found on two satellite islands. Now, UC Merceds evolutionary biology and conservation genetics Professor Danielle Edwards and her research group are the first scientists to propose genetically sequencing Darwins original Galapagos racer sample. Edwards recently got a 202021 Research Publication Grant in Engineering, Medicine, and Science Grant through the American Association of University Women (AAUW) to study Galapagos snakes.

We want to effect ecosystem restoration on Floreana Island by finding an evolutionary replacement in the ecosystem for that species. To do this, we will sequence Darwin's specimen to understand the identity of the specimen relative to the populations present on other islands. We can then use the closest genetic relative to repopulate Floreana Island with this key predator, Edwards said. "We will also use this dataset to understand the extent of species diversity across the archipelago with the most extensive sampling to date."

Anecdotally, racer snakes have fallen prey to cats and other invasive species across the islands. Edwards and her team will use genetic data gathered from samples collected by Galapagos National Park and many international collaborators, along with specimens from museums, to assess population size changes in racer populations and see if populations are in decline, or stable, informing future conservation management efforts by the Galapagos National Park.

Its a huge effort, involving an international team of collaborators, and we do not yet know if we will be able to get DNA from the specimen Darwin collected, Edwards said. But we wont know unless we try, and results from other similar studies on museum specimens have shown promising results.

The Galapagos is a prime place to undertake conservation and rewilding research, because the park has an extensive record of incorporating rigorous science into practices with many partner organizations, she said.

Edwards will lead a team including colleagues from the United States, United Kingdom, Ecuador and New Zealand, and the Galapagos National Park and Island Conservation to develop the project. They have been collecting samples for the past five years to try and identify novel species on the islands and expand sampling to include all known islands and islets where snakes occur.

The findings of this research will provide a detailed picture of how snakes evolved across the islands. The snakes are the last reptile group to have their historical movements across the islands studied. They are believed to be the most mobile of the terrestrial vertebrates and are therefore more likely to provide insights into connections among islands, Edwards said. This project will also provide a source population for Floreana rewilding and a detailed assessment of the genetic health of snake populations across the islands.

Edwards, who joined the Department of Life and Environmental Sciences in the School of Natural Sciences in 2015, focuses most of her research on how the environment impacts the evolution of ecological niche, phenotype and behavior in reptiles, mostly in the context of how these changes lead to the development of new species. She applies this research to inform conservation management strategies for endangered and vulnerable reptiles and amphibians.

But this isnt her first genetics project in the Galapagos. In 2015, a team she was part of revealed that it had identified a novel species of Galapagos giant tortoise. Edwards conducted much of the population genetics analyses, using repeat fingerprinting markers like those used in forensic research, that let the team distinguish between two closely related species. She has been involved in projects in the Galapagos and Australia, applying genomic techniques to understanding biodiversity and implementing conservation management over the past 20 years.

Working in the Galapagos is a dream come true for an evolutionary biologist, herpetologist and conservation biologist, she said. I feel privileged and excited to work on a project based in the crucible of evolutionary thought with a specimen Darwin himself collected.

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Edwards Eager to Expand Father of Evolution's Work | Newsroom - UC Merced University News

New Zealands South Island was once covered in thick forest, the trees breaking like a dark green tide around the grassy mountaintops. After the arrival of Maori settlers about 750 years ago, some hillsides were cleared of their trees by humans using fire, and the foliage has not returned. For the organisms living in these forests, their habitat changed nearly overnight from sheltered woodland to exposed, windy grasslands.

Since the forests burned, little winged insects called stoneflies have changed as well, researchers have found. In a kind of evolutionary pivot over the course of just a handful of centuries, the stoneflies living above the tree line have lost the ability to fly, suggesting that human-made changes to an ecosystem, such as deforestation, can radically reshape the bodies of its inhabitants. The discovery was published in the journal Biology Letters.

Charles Darwin noticed that insects on islands have a curious tendency to be flightless, perhaps because flying is dangerous when you are tiny and winds are strong. In New Zealand, scientists had found flightless stoneflies on many different mountains, said Jon Waters, a professor at the University of Otago in New Zealand who is an author of the new paper. It was not initially clear why whether there was something about the altitude that favored a flightless form, or if there was something else going on.

To answer the question, he and his colleagues collected stoneflies at five sites, walking up through the forests onto the bald crests of the mountains. They caught insects as they went up the slopes, recording their locations. Looking at all the data, they were surprised to find a very clear trend.

We found that there was this amazing transition from winged populations to flightless populations as you go up, said Waters. Wherever we looked, that correlation was linked to where the trees stopped, not a particular altitude.

Because the switch happened at the tree line, rather than at a specific elevation, it suggests that the exposed situation above the trees has favored flightlessness in stoneflies. Perhaps, as in the case of Darwins island insects, wind on the heights makes flying a liability.

It is possible that in some places, even before the forests burned, there were already flightless stoneflies that simply expanded their territory after the fires. A genetic analysis of the stoneflies showed that three of the five populations the researchers looked at were quite different from their lowland winged brethren, implying that they may have been evolving on their own for a while.

The other two, however, had smaller differences, suggesting the change might be recent recent enough to be since humans arrived on the island.

The apparent swiftness of the change recalls the case of the peppered moth, whose coloration shifted from light to dark as air pollution from the Industrial Revolution in England darkened the trees it lived on; light moths, of course, were more visible to predators in their changed environment. It does not take millennia for animal populations to be altered by natural selection, these cases show.

You go into the trees, and youve suddenly gone into a different population. Its almost like magic that evolution seems to be working so clearly and so effectively over a short distance in some of these cases and a short time frame, Waters said.

Now the researchers are looking deeper into the genetics of the stoneflies to understand what it is that changes as the insects lose the ability to fly. The details may reveal whether stoneflies apparent flexibility arises from new mutations, or whether their flightlessness draws on variations that already existed in their ancestral populations and was just waiting for the right moment to take over.

This article originally appeared in The New York Times.

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When insects lost their homes, evolution clipped their wings - The Indian Express

Restricting the evolutionary space of BL21(DE3) for the production of toxic proteins

In DE3 strains, the strong lacUV5 promoter drives expression of T7 RNAP, and it was previously shown that homologous recombination between lacUV5 and the weaker wild-type lac promoter is a dominating event that within hours of introducing a toxic gene leads to tolerance in BL21(DE3)15. To prevent this, we first genetically restricted BL21(DE3), creating a lacIlacZ variant by deleting exactly the part of the native lac locus that shared homology with the DE3 locus (Fig.1). In addition, this should prevent the expression of lacY, encoding the lactose permease, allowing a more uniform, concentration-dependent entry of IPTG into all cells in bacterial populations16.

a Illustration showing the experimental set-up used to restrict the evolutionary space for BL21(DE3) to overcome protein production toxicity on three levels: (upper panel) genetic restriction was accomplished by deleting a part of the BL21(DE3) genome that frequently recombines and lowers the T7 RNAP expression, (middle panel) protein production was coupled to both fluorescence and antibiotic resistance to prevent the formation of non-producing mutants and (lower panel) a spatiotemporally structured environment of ageing bacterial colonies was designed in order to identify strong phenotypic mutants. b Schematic of the workflow to produce layered agar plates that allow diffusion of the antibiotic ampicillin (AMP)and the inducer IPTG to allow sufficient time for colony formation on the surface of the top agar layer before toxic protein production is induced.

Next, similar to a previous study12, we chose the E. coli membrane protein insertase YidC coupled to GFP to serve as a model protein to investigate stress caused by membrane protein overproduction as it formerly was shown to have a strong negative fitness effect on E. coli17,18. The GFP fusion comes in handy for phenotypic restriction because it allows visual screening for mutants that still produce the fusion protein. We further restricted the evolutionary solution space by introducing a hairpin structure in the expression plasmid that couples YidC-GFP to the expression of a -lactamase gene19, providing resistance to ampicillin (Fig.1). This way, the formation of non-producing populations should be minimised in the presence of the antibiotic.

Finally, in contrast to the previous studies11,13,20, we aimed at introducing the protein production stress after bacterial colonies had established on plates. This spatiotemporally different approach should allow the formation of a large number of bacteria in a state of dormancy and in a structured environment, which previously was shown to constitute a unique evolutionary environment21. Furthermore, we speculated that mutants would be easy to identify as fluorescent secondary colonies, so-called papillae, outgrowing from the initially established colonies. To this end, two-layered agar plates were poured, allowing slow IPTG and ampicillin diffusion from the bottom to the top layer to grant sufficient time for colony formation before YidC-GFP production was induced (Fig.1).

During incubation for 1 week at 37C, we observed several fluorescent papillae (Fig.1) that were restreaked to confirm the fluorescent and ampicillin-resistant phenotype. Based on the fluorescence phenotype, the most promising mutants were isolated and cured of the YidC-GFP plasmid using a mild and simple CRISPR-based approach to plasmid curing22. These strains were subsequently retransformed with the original YidC-GFP plasmid to ensure that mutations leading to tolerance were localised on the genome and not on the expression plasmid. A single clone (Evo21(DE3)) was chosen for further characterisation.

To benchmark Evo21(DE3), we compared its ability to produce the YidC-GFP-fusion protein to the non-evolved BL21(DE3) wild-type strain as well as the derivative Mt56(DE3) previously described to be optimised for YidC-GFP overproduction12. The plasmid pLysS was utilised to limit basal T7 RNAP expression23 in BL21(DE3) and Evo21(DE3). Co-expression of pLysS in Mt56(DE3) is not relevant due to the greatly reduced polymerase activity in this strain. In liquid culture, based on GFP fluorescence, Evo21(DE3) expressed significantly (P0.0001) higher amounts of YidC-GFP than BL21(DE3) and Mt56(DE3). Eight hours post induction, yields were 3.6-fold higher in cultures of Evo21(DE3) than in cultures of BL21(DE3) and 2.1-fold higher than in Mt56(DE3) (Fig.2a). Parallel monitoring of the optical density of the cultures showed no major growth impairment for the strains (Supplementary Fig.1).

a Production of a toxic YidC-GFP-fusion protein in Evo21(DE3) compared to the non-evolved BL21(DE3) wild-type strain, the BL21(DE3)lacI/Z ancestor strain, and a previously evolved BL21(DE3) derivative Mt56(DE3). On the illustrated expression vector (pYidC), YidC-GFP production is translationally coupled to ampicillin resistance. b Fluorescence fold change between Evo21(DE3) and BL21(DE3), producing a library of 24 proteins of the E. coli inner membrane proteome C-terminally fused to GFP. c gfp expression levels in Evo21(DE3) and control strains with and without co-expression of the helper plasmid pLysS. d Western blot showing the expression of a camelid-derived single-chain antibody (nanobody) in Evo21(DE3) and control strains. Samples were normalised to cell density before loading. e Production of YidC under the control of a PrhaBAD promoter allowing titration of expression l-rhamnose. All fluorescence values displayed are normalised to OD at 600nm. Error bars indicated represent the average squared deviation from the mean (SD) of three biologically independent samples (n=3).

To assess whether the Evo21(DE3) phenotype was gene-specific, we next investigated the expression of a set of 24 different GFP-fusion proteins selected from an expression vector library of the E. coli inner membrane proteome24. These membrane proteins were selected to cover a wide range of functions, toxicity (previously reported as a change in OD600 upon IPTG addition24), and the number of predicted transmembrane domains (Supplementary Table1). Comparing the fold change of protein production in Evo21(DE3) and BL21(DE3), titre was improved for 19 of the 24 proteinswith a significant fold change of more than 1.5-fold for 14 of them (Fig.2b). The highest improvement was 6.1-fold (P0.001), observed for the protein YihG.

As a first test that the underlying mechanism allowing Evo21(DE3) to produce more toxic protein was not related to a general decrease in the activity of the T7 expression systemas previously observed for the BL21(DE3) derivatives C41/43(DE3), C44/45(DE3) and Mt56(DE3)we Sanger sequenced the T7 RNAP gene, which confirmed an absence of mutations. Next, we compared the expression of two non-toxic soluble proteins, GFP and a camelid-derived nanobody, and both were produced at higher levels in Evo21(DE3) than in BL21(DE3) or Mt56(DE3)both in the presence and absence of pLysS (Fig.2c, d). Similarly, Evo21(DE3) outperformed other strains in the production of seven out of eight different plant-derived cytochrome P450 enzymesa class of enzymes of significant biotechnological interest25 (Supplementary Fig.2). This indicates that the causative mutation in Evo21(DE3) is different from previously isolated BL21(DE3) derivatives and probably does not cause a general decrease in T7 RNAP activity.

Even though the screening for improved protein productivity was performed with the highly efficient T7 system, the ideal mutant strain would be capable of producing more protein independently from the promoter system. To explore if this was the case for Evo21(DE3), we replaced the T7 promoter with the l-rhamnose inducible rhaBAD promoter in the yidC-GFP expression vector, transformed it into Evo21(DE3), and expressed the construct by inducing with different rhamnose concentrations in liquid culture. With concentrations of 5 and 20mM l-rhamnose, Evo21(DE3) produced significantly (P0.0001) more protein than BL21(DE3) and Mt56(DE3) (Fig.2e).

In summary, this initial characterisation shows that the evolved strain can produce a higher titre of a range of different proteins using a T7-system-independent mechanism.

The phenotype of Evo21(DE3) prompted us to sequence the strain using Illumina whole-genome sequencing. Two point mutationsone in the argE and one in the fecB locus (Fig.3a)and an insertion of a mobile IS1 element into the rne gene were identified. Upon reintroduction of the argE or fecB point mutations into BL21(DE3) by oligonucleotide-based recombineering, the YidC-GFP overexpression phenotype was not obtained (data not shown), whereas, when reintroducing the truncation of the rne locus into BL21(DE3) and the Evo21(DE3) parental BL21(DE3) lacIlacZ strain, the YidC-GFP overexpression tolerance phenotype was nearly identical to Evo21(DE3) (Fig.3b). This makes it highly likely that the rne IS1 insertion is the main causative mutation in Evo21(DE3).

a Illustration of mutations in the Evo21(DE3) genome compared to the ancestor BL21(DE3). Whole-genome sequencing of mutant strain Evo21(DE3) revealed two point mutations (grey) and a truncation of the rne gene caused by the transposition of a mobile element IS1 into the locus. The deletion of the genomically shared homology sequence in the BL21(DE3) ancestor strain with the DE3 area (335,401337,123) is annotated. b Production levels of the toxic YidC-GFP-fusion protein in Evo21(DE3) compared to BL21(DE3), as well as BL21(DE3) and the non-evolved ancestor strain BL21(DE3)lacIlacZ after reintroducing the rne truncation by recombineering. Error bars indicated represent the average squared deviation from the mean (SD) of three biologically independent samples (n=3). c Illustration of the E. coli RNA degradosome. N- and C-terminal domain of the membrane-bound essential endonuclease RNase E (blue) and the localisation of associated enzymes PNPase, Rhlb and enolase along the C-terminal non-catalytic scaffolding region are displayed. Mutations of the rne gene in Evo21(DE3), BL21Star(DE3) and rne* gene harboured on pLysS-Max are indicated.

The identified IS1 insertion causes a truncation of the encoded 1061-residue E. coli endoribonuclease RNase E after amino acid 702 and, therefore, a polypeptide lacking the last 359 residues of its C-terminus in Evo21(DE3) (Fig.3c). RNase E is an essential membrane-associated enzyme involved in the maturation of both ribosomal RNA and tRNA, as well as total mRNA decay, and mediates the assembly of a multi-enzyme complex referred to as the RNA degradosome (Fig.3c). It has previously been shown that only the N-terminal half (1529) of RNase E, accommodating the active catalytic domain, is essential for cell growth, and the C-terminal non-catalytic region is mostly disordered and known to function as a scaffold mediating the association of the enzymes PNPase, Rhlb and enolase26,27,28.

Interestingly, a similar truncation of the rne locus, rne131, resulting in an RNase E polypeptide lacking its non-catalytic region (amino acid residues 1584, Fig.3c) was isolated in a screen for suppressors of a temperature-sensitive allele of the mukB gene26. A later study showed that in strains such as BL21(DE3), introducing the rne131 truncation caused a bulk stabilisation of mRNA degradation, including mRNA produced by T7 RNAP29. The rne131 truncation was engineered into the commercially available BL21Star(DE3) with the rationale that stabilising bulk mRNA would result in increased protein production. However, following the same rationale, the commercial strain also comes with a note suggesting that it might be unsuitable for overexpression of toxic genes.

We compared the expression of six different genes that we previously found expressed better in Evo21(DE3) than in BL21(DE3) with expression in BL21Star(DE3) and found expression levels to be highly similar between BL21Star(DE3) and Evo21(DE3) (Fig.4a). This provides an independent confirmation that the phenotype of Evo21(DE3) is caused by the truncation of rne.

a Heterologous production of a variety of non-toxic and toxic GFP-fusion proteins in Evo21(DE3) and BL21Star(DE3), both harbouring different truncations of the rne gene, compared to BL21(DE3). b Expression of the same genes in Evo21(DE3) is compared to expression in BL21(DE3) when co-expressing either the auxiliary plasmid pLysS or pLysS-Max. c Schematic illustration of the plasmid pRNE-GFP designed to report on RNase activity. Promoters controlling rne expression are indicated (P14)49,50. The expression level of the rne gene can be monitored in vivo via GFP fluorescence signal. d Exploration of the pRNE-GFP reporter plasmid in BL21(DE3) derivatives. Different levels of RNase activity can be monitored in the strains. e Titration of yidC expression in Evo21(DE3), BL21Star(DE3) and BL21(DE3) via increasing levels of l-rhamnose and its effect on the rne regulon expression reporting RNase activation in the cell during toxic membrane protein production. Upper half: Fluorescence corresponds to YidC-GFP production levels. Lower half: Cells harbour both the rne reporter plasmid (pRNE-GFP) and pPrhaBAD-YidC controlling yidC expression (no GFP fusion). Fluorescence levels correspond to GFP produced under the control of the rne regulon. f Effect of the pLyS-Max auxiliary plasmid on RNase activity. Plasmids pLysS and pLysS-Max are co-expressed along with pRNE-GFP in BL21(DE3) cells. To repress leaky expression of the rhamnose promoter controlling rne* expression on pLysS-Max, 0.4% glucose was added where indicated. Error bars indicated represent the average squared deviation from the mean (SD) of three biologically independent samples (n=3).

The way Evo21(DE3) was isolated from papillae outgrowing colonies on week-old agar plates, and because dominant rne mutants previously have been observed30, made us speculate that different rne variants could be studied by simple co-expression from a plasmid in the presence of wild-type rne on the genome. To test this idea and to compare different variants of the rne gene at different expression levels, we cloned rne variants in front of the rhaBAD promoter on the pLysS plasmid backbone: full-length rne, the Evo21(DE3) and BL21Star(DE3) truncated versions and a version with a further truncation in the membrane-binding domain of RNase E. However, none of these constructs showed any positive effect on YidC-GFP expression (Supplementary Fig.3).

Serendipitously, we isolated a spontaneously occurred rne mutant, called rne*, and included it in our analysis. We found that rne* provided on the pLysS plasmid (hereafter called pLysS-Max) could increase YidC-GFP production even further than Evo21(DE3) (Fig.4b). The rne* mutation converts the essential31 aspartate residue in position 346 to an asparagine in the so-called DNase I subdomain of RNase E involved in chelating an essential Mg2+ ion. The aspartate residue is believed to act as a general base to activate the attacking water essential for the catalytic activity of the enzyme32. The replacement of Asp-346 with the polar amino acid Asn was previously shown to decrease RNA cleavage by about 25-fold32. The effect of expressing rne* was not gene-specific as the effect was preserved for three out of four other tested genes (Fig.4b). This provides an alternative demonstration that manipulating with rne severely affects recombinant protein production and provides a simple tool, in the form of an auxiliary pLysS-Max plasmid that can be transformed into other strains, to improve protein production titre.

The positive effect of rne truncations such as rne131 on protein production was previously assumed to be due to the stabilisation of the recombinant mRNA29. However, the observation that the rne truncation in Evo21(DE3) leads to tolerance of toxic gene expression suggests a broader role involving balancing of RNA levels more globally.

Autoregulation allows RNase E to continuously adjust its synthesis to that of its substrates by controlling the degradation rate of its own mRNA33,34. This could work as a biosensor for RNase E activity by fusing the promoter and 5end of rne with a genetic reporter, as previously demonstrated with lacZ34. To explore RNase E activity in our evolved strains for recombinant protein production, we constructed a similar RNase E biosensor (pRNE-GFP) using GFP as a reporter (Fig.4c).

We transformed the pRNE-GFP reporter into BL21, BL21(DE3), BL21Star(DE3), Evo21(DE3) and Mt56(DE3) and monitored fluorescence in a microplate reader under conditions similar to the typical protein production scenario. Surprisingly, under these conditions, fluorescence was 14-fold reduced in Evo21(DE3) and sevenfold reduced in BL21Star(DE3) compared to the ancestral BL21(DE3 (Fig.4d). Given that Evo21(DE3) behaves almost identically to BL21Star(DE3) and that the rne131 truncation has been shown to cause a bulk stabilisation of mRNA degradation29, this suggests that low fluorescence from our reporter correlates with increased bulk mRNA stabilisation. Interestingly, fluorescence from the reporter was reduced approximately twofold in BL21 compared to BL21(DE3), suggesting an effect of the DE3 locus itself on RNase E activity in the cell.

Next, we wanted to explore if the expression of YidC-GFP affected RNase E activity in the different strains. To this end, because YidC-GFP cannot be expressed from the T7 promoter in BL21 (no T7 RNA polymerase) or BL21(DE3) (no growth), we expressed it from the rhaBAD promoter construct (Fig.2e) using different concentrations of rhamnose. This showed that YidC-GFP levels could be titrated with rhamnose and confirmed higher expression in BL21Star(DE3) and Evo21(DE3) than in the other strains at high rhamnose concentrations (Fig.4e, upper half).

To monitor fluorescence from the RNase E GFP reporter, we then deleted GFP from the YidC construct and attempted to co-transform it with pRNE-GFP into different strain backgrounds (Fig.4e, lower half). However, we were unable to recover and grow transformants in BL21(DE3) and BL21Star(DE3), suggesting a lethal imbalance in RNA levels caused by the presence of these two plasmids. We were able to recover and grow double transformants in BL21, Evo21(DE3), and Mt56(DE3) and monitor fluorescence as an indication of RNase E activity. Fluorescence increased to high levels upon increasing the concentration of rhamnose in BL21 and Mt56(DE3), but fluorescence levels were at least 2.5-fold lower (at 1mM rhamnose) in Evo21(DE3) and hardly increased upon further rhamnose addition. This suggests that RNase E activity towards bulk mRNA is increased when yidC expression is increased but that the activity is lower in Evo21(DE3) than in the other strains (Fig.4e).

Finally, we explored the effect of the pLysS-Max auxiliary plasmid (harbouring the mutant rne*) on RNase E activity by co-transforming it along with pRNE-GFP into BL21(DE3). As controls, we included a strain co-transformed with pLysS and pRNE-GFP and BL21(DE3) transformed only with pRNE-GFP. Because we previously observed effects on RNase E activity due to the presence of the DE3 locus, we repressed leaky expression of T7 RNAP from the lacUV5 promoter by adding glucose to the medium and titrated rne* levels with rhamnose. In the absence of glucose and the presence of pLysS, we observed an increase in fluorescence which was repressed by expression of rne* (Fig.4f). This shows that the pLysS-Max plasmid can be used to regulate RNase E activity in the cell.

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Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity | Communications Biology - Nature.com

HOUSTON, TX / ACCESSWIRE / August 16, 2021 / Evolution Petroleum (NYSE American:EPM) ("Evolution" or the "Company") today announced its reserves as determined by its independent reserve engineer, DeGolyer & MacNaughton, for the fiscal year ended June 30, 2021.

Highlights:

Diversified its portfolio through the acquisition of long-life producing assets in the Barnett Shale adding 13.1 million barrels of oil equivalent ("MMBOE") to its reserves

Substantially increased fiscal year-end proved reserves to 23.4 MMBOE from 10.2 MMBOE at fiscal year-end 2020

Positive reserve revisions net of production due primarily to the return to service of the Delhi CO2 supply line and well reactivations at Hamilton Dome

Reserves based on trailing twelve-month commodity prices that are substantially below current prices

Jason Brown, President and Chief Executive Officer, commented, "We were pleased to more than double our fiscal 2021 year-end proved reserves with the acquisition of a non-operated ownership interest in Barnett Shale natural gas assets in May 2021. The associated proved developed producing (PDP) reserves are anchored by a low annual decline of less than 10% and include potential upside through workover opportunities. The acquisition of the Barnett Shale assets helped to diversify our production mix to include natural gas with favorable access to the Gulf Coast markets that offer premium pricing. Additionally, we had positive reserve revisions this year at Delhi and Hamilton Dome, further demonstrating the strength and value of our existing long-life producing assets. We were encouraged by the return to service of the CO2 supply line to Delhi in late 2020 that will allow resumption of normal injection levels and provide meaningful reservoir pressure support for the continued recovery and development of the field's long-life reserves. At Hamilton Dome, the operator was able to return substantially all of the wells shut-in due to the dramatic price drop caused by the pandemic, adding to production and extending the life of the field. Hamilton Dome reserves were also revised upward due to improved year-over-year pricing. We look forward to continuing to benefit from the improved price environment as we evaluate and execute additional opportunities to further grow our asset base and reserves."

Story continues

As of June 30, 2021

Oil

NGL

Natural Gas

Equivalent

MBO

MBL

MMcf

MBOE*

Proved Developed Producing

6,815

6,663

48,571

21,573

92

%

Proved Undeveloped

1,605

209

-

1,814

8

%

Total Proved

8,420

6,872

48,571

23,387

100

%

Probable Developed Producing

2,052

743

-

2,795

85

%

Probable Undeveloped

489

-

-

489

15

%

Total Probable**

2,541

743

-

3,284

100

%

Possible Developed Producing

2,251

314

-

2,565

91

%

Possible Undeveloped

255

-

-

255

9

%

Total Possible**

2,506

314

-

2,820

100

%

* Equivalent reserves based on a ratio of 6 Mcf of natural gas to 1 Bbl of oil.

** Read the section captioned "Cautionary Statement" later in this release addressing reserves.

Proved Reserves

The Company's year ended June 30, 2021 SEC proved reserves were 23.4 MMBOE, up from 10.2 MMBOE at year ended June 30, 2020. Although both Delhi and Hamilton Dome had positive revisions net of production, the majority of the increase was due to the acquisition of non-operated interests in the Barnett Shale in May 2021 that added 48.6 Bcf of natural gas, 4.9 MMBL of natural gas liquids, and 0.1 MMBO of oil proved reserves (13.1 MMBOE).

The SEC pricing (twelve-month first day of the month average realized prices) used in the report for the year ended June 30, 2021 was $49.72 per barrel of oil and $2.46 per MCF of natural gas. For fiscal year ended June 30, 2020, SEC average realized prices were $47.37 per barrel of oil and $2.12 per MCF of natural gas.

Approximately 65% of year ended June 30, 2021 SEC proved reserves were liquids (36% crude oil and 29% NGLs) and 35% natural gas. At fiscal year-end, approximately 92% of proved reserves were classified as proved developed producing and 8% as proved undeveloped.

Probable and Possible Reserves

The probable and possible reserves are categories that represent potential recoveries from the CO2 flood developed in the Delhi Field greater than those included in the proved reserves. Consequently, while the probable and possible reserves are 85% and 91% developed, respectively, and require de minimis capital expenditures, they remain less certain of attainment and have more risk of recovery than proved reserves and should not be aggregated with other categories. These categories of reserves reflect the incremental reserves associated with different engineering assumptions with respect to the percentage of original oil in place that can be recovered through CO2 enhanced oil recovery. Probable and possible reserves decreased approximately 1% and 8%, respectively, from the prior year primarily due to the movement of probable and possible reserves to the proved reserves category as a result of a positive change to the proved reserves life at Delhi

Investor Conference Presentation

Evolution today announced that the Company will participate in EnerCom's The Oil & Gas Conference in Denver, Colorado on August 17, 2021. The Company's presentation will be webcasted and is scheduled to be available at 11:40 am Eastern (10:40 a.m. Central) on August 17, 2021. The presentation can be accessed through the EnerCom conference portal for registered participants at https://www.theoilandgasconference.com, or in the investor relations section of the Company's website: http://www.EvolutionPetroleum.com.

About Evolution Petroleum

Evolution Petroleum Corporation is an oil and natural gas company focused on delivering a sustainable dividend yield to its shareholders through the ownership, management, and development of producing oil and natural gas properties onshore in the United States. The Company's long-term goal is to build a diversified portfolio of oil and natural gas assets primarily through acquisition, while seeking opportunities to maintain and increase production through selective development, production enhancement, and other exploitation efforts on its properties. Our largest assets are our interest in a CO2 enhanced oil recovery project in Louisiana's Delhi Field, our interest in a secondary recovery project in Wyoming's Hamilton Dome Field, and our recently acquired interest in the Barnett Shale in Texas. Additional information, including the Company's annual report on Form 10-K and its quarterly reports on Form 10-Q, is available on its website at http://www.EvolutionPetroleum.com.

Cautionary Statement

All forward-looking statements contained in this press release regarding current expectations, potential results and future plans and objectives of the Company involve a wide range of risks and uncertainties. Statements herein using words such as "believe," "expect," "plans," "outlook," "should," "will," and words of similar meaning are forward-looking statements. Although our expectations are based on business, engineering, geological, financial, and operating assumptions that we believe to be reasonable, many factors could cause actual results to differ materially from our expectations and we can give no assurance that our goals will be achieved. These factors and others are detailed under the heading "Risk Factors" and elsewhere in our periodic documents filed with the Securities and Exchange Commission. The Company undertakes no obligation to update any forward-looking statement.

Our reserves as of June 30, 2021 were estimated by DeGolyer & MacNaughton, a global independent reservoir engineering firm. All reserve estimates are continually subject to revisions based on production history, results of additional exploration and development, price changes and other factors. The SEC's current rules allow oil and gas companies to disclose not only proved reserves, but also probable and possible reserves that meet the SEC's definitions of such terms. Estimates of probable and possible reserves by their nature are much more speculative than estimates of proved reserves. These non-proved reserve categories are subject to greater uncertainties and the likelihood of recovering those reserves is subject to substantially greater risk. When estimating the amount of oil and natural gas liquids recoverable from a particular reservoir, probable reserves are those additional reserves that are less certain to be recovered than proved reserves but which, together with proved reserves, are as likely as not to be recovered, generally described as having a 50% probability of recovery. Possible reserves are even less certain and generally require only a 10% or greater probability of being recovered. These three reserve categories have not been adjusted to different levels of recovery risk among these categories and are therefore not comparable and are not meaningfully combined.

Company Contacts:

Jason Brown, President & CEOJBrown@evolutionpetroleum.com(713) 935-0122

Ryan Stash, SVP & CFORStash@evolutionpetroleum.com(713) 935-0122

SOURCE: Evolution Petroleum Corporation

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Acquisition and Positive Revisions Drive Substantial Increase in Evolution Petroleums Proved Reserves for Fiscal Year-End 2021 - Yahoo Finance