Conservation genetics as a management tool: the five best-supported paradigms to assist the management of threatened species

About 50 y ago, Crow and Kimura [An Introduction to Population Genetics Theory (1970)] and Ohta and Kimura [Genet. Res. 22, 201–204 (1973)] laid the foundations of conservation genetics by predicting the relationship between population size and genetic marker diversity. This work sparked an enormous research effort investigating the importance of population dynamics, in particular small population size, for population mean performance, population viability, and evolutionary potential. In light of a recent perspective [J. C. Teixeira, C. D. Huber, Proc. Natl. Acad. Sci. U.S.A. 118, 10 (2021)] that challenges some fundamental assumptions in conservation genetics, it is timely to summarize what the field has achieved, what robust patterns have emerged, and worthwhile future research directions. We consider theory and methodological breakthroughs that have helped management, and we outline some fundamental and applied challenges for conservation genetics

Parental mental health, socioeconomic position and the risk of asthma in children—a nationwide Danish register study

BACKGROUND: Parental mental illness affects child health. However, less is known about the impact of different severities of maternal depression and anxiety as well as other mental health conditions. The objective of this study was to examine the impact of different severities of maternal and paternal mental health conditions on child asthma. METHODS: This nationwide, register-based cohort study included all children in Denmark born from 2000 to 2014. Exposure was parental mental health conditions categorized in three severities: minor (treated at primary care settings), moderate (all ICD-10 F-diagnoses given at psychiatric hospital) and severe (diagnoses of severe mental illness). The children were followed from their third to sixth birthday. Child asthma was identified by prescribed medication and hospital-based diagnoses. Incidence rate ratios were calculated using negative binomial regression analyses. RESULTS: The analyses included 925 288 children; 26% of the mothers and 16% of the fathers were classified with a mental health condition. Exposed children were more likely to have asthma (10.6–12.0%) compared with unexposed children (8.5–9.0%). The three severities of mental health conditions of the mother and the father increased the risk of child asthma, most evident for maternal exposure. Additive interaction between maternal mental health conditions and disadvantaged socioeconomic position was found. CONCLUSION: We found an increased risk of asthma in exposed children, highest for maternal exposure. Not only moderate and severe, but also minor mental health conditions increased the risk of child asthma. The combination of mental health condition and disadvantaged socioeconomic position for mothers revealed a relative excess risk

Author Correction:CRISPR-based transcriptional activation tool for silent genes in filamentous fungi (Scientific Reports, (2021), 11, 1, (1118), 10.1038/s41598-020-80864-3)

The Supplementary Information published with this Article contained errors. In Note S2, the text formatting including green italics, red bold, yellow underline, purple text and blue underline was omitted. The original Supplementary Information file is provided below. These errors have now been corrected in the Supplementary Information file that accompanies the original Article

Mechanism and regulation of sorbicillin biosynthesis by Penicillium chrysogenum

Penicillium chrysogenum is a filamentous fungus that is used to produce β-lactams at an industrial scale. At an early stage of classical strain improvement, the ability to produce the yellow-coloured sorbicillinoids was lost through mutation. Sorbicillinoids are highly bioactive of great pharmaceutical interest. By repair of a critical mutation in one of the two polyketide synthases in an industrial P. chrysogenum strain, sorbicillinoid production was restored at high levels. Using this strain, the sorbicillin biosynthesis pathway was elucidated through gene deletion, overexpression and metabolite profiling. The polyketide synthase enzymes SorA and SorB are required to generate the key intermediates sorbicillin and dihydrosorbicillin, which are subsequently converted to (dihydro)sorbillinol by the FAD-dependent monooxygenase SorC and into the final product oxosorbicillinol by the oxidoreductase SorD. Deletion of either of the two pks genes not only impacted the overall production but also strongly reduce the expression of the pathway genes. Expression is regulated through the interplay of two transcriptional regulators: SorR1 and SorR2. SorR1 acts as a transcriptional activator, while SorR2 controls the expression of sorR1. Furthermore, the sorbicillinoid pathway is regulated through a novel autoinduction mechanism where sorbicillinoids activate transcription

Identification of the decumbenone biosynthetic gene cluster in and the importance for production of calbistrin

Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory

Emerging Genotype (GGIIb) of Norovirus in Drinking Water, Sweden

From May through June 2001, an outbreak of acute gastroenteritis that affected at least 200 persons occurred in a combined activity camp and conference center in Stockholm County. The source of illness was contaminated drinking water obtained from private wells. The outbreak appears to have started with sewage pipeline problems near the kitchen, which caused overflow of the sewage system and contaminated the environment. While no pathogenic bacteria were found in water or stools specimens, norovirus was detected in 8 of 11 stool specimens and 2 of 3 water samples by polymerase chain reaction. Nucleotide sequencing of amplicons from two patients and two water samples identified an emerging genotype designated GGIIb, which was circulating throughout several European countries during 2000 and 2001. This investigation documents the first waterborne outbreak of viral gastroenteritis in Sweden, where nucleotide sequencing showed a direct link between contaminated water and illness

Pathway for the Biosynthesis of the Pigment Chrysogine by Penicillium chrysogenum

Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 (chyA) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum. Each of the genes of the chyAcontaining gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido) benzoic acid, which was verified by feeding experiments of a Delta chyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds. IMPORTANCE Penicillium chrysogenum is used in industry for the production of Delta-lactams, but also produces several other secondary metabolites. The yellow pigment chrysogine is one of the most abundant metabolites in the culture broth, next to Delta-lactams. Here, we have characterized the biosynthetic gene cluster involved in chrysogine production and elucidated a complex and highly branched biosynthetic pathway, assigning each of the chrysogine cluster genes to biosynthetic steps and metabolic intermediates. The work further unlocks the metabolic potential of filamentous fungi and the complexity of secondary metabolite pathways

D-xylonat produktion och tolerans mot organiska syror i jäster

Various organic acids have huge potential as industrial platform chemicals. Biotechnological routes of organic acid production are currently being sought, so that fossil resources and petrochemistry could be replaced with renewable resources. Microbial production of organic acids can provide an environmentally sound, sustainable way of producing industrial chemicals, and efficient processes are needed to produce large quantities of acids which are often novel to the production organism. Production of such acids imposes stresses on the organism. These stresses affect the vitality, viability and productivity of the cells in a bioprocess. Understanding the physiology of micro-organisms which have been genetically engineered to produce an organic acid, can make valuable contributions to the development of production organisms for biorefineries, which provide means to convert agricultural and forestry waste into these useful chemicals. Production of D-xylonate, an industrial platform chemical with high application potential, was successfully demonstrated in various yeast species. D-xylonate is produced from D-xylose via D-xylono-γ-lactone that can be hydrolysed to D-xylonate spontaneously or with the aid of a lactonase enzyme. Various ways to improve production of D-xylonate in the yeast Saccharomyces cerevisiae, Kluyveromyces lactis or Pichia kudriavzevii as production organisms were successfully applied. The best D-xylonate production was obtained by expression of the D-xylose dehydrogenase encoding gene xylB from Caulobacter crescentus and the highest D-xylonate titre was achieved with P. kudriavzevii that produced 171 and 146 g D-xylonate l-1, at a rate of 1.4 or 1.2 g l-1 h-1, at pH 5.5 and pH 3, respectively. Production at low pH is desirable as this would make product recovery and process operations more economically feasible. The consequences of D-xylonate production on the physiology of S. cerevisiae were studied in detail, both at population and single-cell level. D-xylonate and D-xylono-γ-lactone were produced and also exported from the cells from the very start of cultivation in D-xylose, even in the presence of D-glucose. There was no apparent preference for export of either compound. However, great amounts of D-xylono-γ-lactone and/or D-xylonate was accumulated inside the cells during the production. The D-xylonolactone lactonase encoding gene xylC was co-expressed with the D-xylose dehydrogenase encoding gene xylB (both genes from C. crescentus). This lead to a significant increase in the D-xylonate production rate compared to cells expressing only xylB and showed that accumulation of D-xylonate and protons releases during hydrolysis, was harmful for the cells. The accumulation of D-xylonate led to acidification of the cytosol, as determined by loss of pHluorin (a pH dependent fluorescent protein) fluorescence, and this loss of fluorescence was faster in cells co-expressing xylC with xylB compared to cells expressing xylB alone. Acidification of the cytosol was shown to correlate with decreased viability of the D-xylonate producing cells and the rate of loss of pHluorin fluorescence and loss in viability was highly dependent on the pH of the production medium. The decrease in vitality and challenges in export of D-xylonate are major obstacles for D-xylonate production by S. cerevisiae. The excellent D-xylonate producer, P. kudriavzevii also accumulated large amounts of D-xylonate and suffered decreased vitality, especially when D-xylonate was produced at low pH. The stress response to weak organic acids is highly dependent on the properties of the acids and the presence of high concentrations of weak organic acids may lead to lost viability. The role of Pdr12, a membrane transporter, in resistance to weak organic acids was studied and found to be highly dependent on the acid. Deletion of PDR12 led to improved tolerance to formic and acetic acids, a feature that makes this modification interesting for micro-organisms used in biorefining of lignocellulosic hydrolysates that commonly contain these acids. Biotechnological production of D-xylonic acid with yeast clearly has the potential of becoming an industrially applicable process. In order for biotechnological production processes to become economically feasible, biorefinery approaches in which lignocellulosic hydrolysates or other biomass side- or waste streams are used as raw materials need to be employed. This thesis provides new understanding on how production of an organic acid affects the production host and presents novel approaches for studying and increasing the production.Organiska syror har en enorm potential som industriella plattformskemikalier. En bioteknisk produktion av organiska syror kunde ersätta produktionen av motsvarande, oljebaserade kemikalier. En mikrobiell produktion av organiska syror kan utgöra ett miljövänligt, hållbart sätt att producera kemikalier för industrin. För detta behövs effektiva processer och mikroorganismer med kapacitet att producera stora mängder syror. Dessvärre är syrorna ofta okända för produktionsorganismen och därmed medför produktionen av syra stora påfrestningar, vilket leder till stress. Denna stress påverkar vitaliteten, livskraften och produktiviteten hos cellerna i en bioprocess. Genom att förstå fysiologin hos mikroorganismer som är genetiskt manipulerade för att producera en organisk syra, kan nya produktionsorganismer för bioraffinaderier utvecklas. I ett bioraffinaderi kan jord-och skogsbruksavfall omvandlas till användbara kemikalier. D-xylonat, en industriell prekursorkemikalie med stor potential, kan produceras med hjälp av olika jästsvampar. D-xylonat framställs från D-xylos via D-xylono-γ-lakton, som kan hydrolyseras till linjär D-xylonat, spontant eller med hjälp av ett laktonas enzym. I denna studie förbättrades produktionen av D-xylonat märkbart med hjälp av jästerna Saccharomyces cerevisiae, Kluyveromyces lactis eller Pichia kudriavzevii som produktionsorganismer. Den bästa produktionen av D-xylonat erhölls genom att uttrycka xylB, en gen från Caulobacter crescentus som kodar för ett D-xylos dehydrogenas enzym. Den största D-xylonatproduktionen uppnåddes med P. kudriavzevii, som var kapabel att producera 171 eller 146 g D- xylonat l-1, med en hastighet av 1.4 eller 1.2 g l-1 h-1, vid pH 5.5 respektive pH 3. Det är fördelaktigt att producera syra vid ett lågt pH-värde, eftersom det gör uppsamlandet av syran enklare och därmed processen mer ekonomiskt lönsam. Konsekvenserna av D-xylonatproduktionen på S. cerevisiae jästens fysiologi studerades i detalj, både på populations- och encellsnivå. Under produktionen samlades stora mängder av D-xylonat och D-xylono-γ-lakton inuti cellerna. Ändå producerades och exporterades D-xylonat från cellerna från början av produktionsprocessen, även i närvaro av D-glukos. Både D-xylonat och D-xylono-γ-lakton exporterades från S. cerevisiae cellerna och det fanns ingen uppenbar preferens för någondera molekylen. Genom att uttrycka genen som kodar för D-xylonolakton laktonas enzymet, xylC, tillsammans med genen som kodar för D-xylos dehydrogenas enzymet, xylB, fastställdes att ackumulering av linjärt D-xylonat och i hydrolysen frigjorda protoner, var skadligt för cellerna. D-xylonatproduktionen skedde märkbart snabbare i celler som uttryckte både xylB och xylC jämfört med celler som uttryckte endast xylB. Ackumuleringen av D-xylonat ledde till att fluorescensen från pHluorin, ett pH-känsligt fluorescerande protein, försvann. Detta antyder att cellens cytosol försurnade då cellen producerade D-xylonat. Fluorescensen från pHluorin proteinet försvann snabbare i de celler som uttryckte både xylC och xylB, jämfört med de celler som uttryckte endast xylB. Denna försurning av cytosolen visade sig korrelera med en minskad livskraft bland cellerna som producerade D-xylonat och graden av försurning och förminskningen i viabiliteten var starkt beroende av pH-värdet i produktionsunderlaget. En förminskad livskraft och utmaningar i exporten av D-xylonat utgör stora hinder för D-xylonatproduktion med S. cerevisiae. Även i P. kudriavzevii cellerna samlades det stora mängder av D-xylonat och livskraften hos dessa var minskad, speciellt då D-xylonatet producerades vid lågt pH. Stressreaktionerna gentemot svaga organiska syror är starkt beroende av egenskaperna hos syrorna och höga koncentrationer av svaga organiska syror leder till en förlorad livskraft. Vid studier av den roll transportproteinet Pdr12 har i resistensen mot svaga organiska syror, framkom att syrans egenskaper har stor inverkan på cellernas syratolerans. Mikroorganismer med en deleterad PDR12 gen uppvisade en förbättrad tolerans mot myr- och ättiksyra, vilket kan utnyttjas vid bioraffineringen av lignocelluloshydrolysat, som oftast innehåller dessa syror. En bioteknisk produktion av D-xylonsyra med hjälp av jästceller har stor potential att bli en industriellt användbar process. För att biotekniska produktionsprocesser skall kunna bli ekonomiskt möjliga, måste man utveckla bioraffinaderier där lignocellulosahydrolysat eller andra sido- eller avfallsströmmar används som råvaror. Denna avhandling ger ny förståelse för hur produktionen av en organisk syra påverkar produktionsorganismen och presenterar nya metoder för att studera och öka produktionen